OIE Manual of Diagnostic Tests and Vaccines For Terestrial Animals Vol 2 - Ecvine, Leporide, Suine, Caprine

WORLD ORGANISATION FOR ANIMAL HEALTH
MANUAL OF DIAGNOSTIC TESTS AND VACCINES

FOR TERRESTRIAL ANIMALS
(mammals, birds and bees)
Seventh Edition
Volume 2
2012
This Terrestrial Manual has been edited by the

OIE Biological Standards Commission and adopted by
the World Assembly of Delegates of the OIE
Reference to commercial kits does not mean their endorsement by the OIE. All commercial kits should
be validated; tests on the OIE register have already met this condition (the register can be consulted
at: www.oie.int).
OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals

Seventh Edition, 2012
Manual of Recommended Diagnostic Techniques and Requirements for Biological Products:

Volume I, 1989; Volume II, 1990; Volume III, 1991.
Manual of Standards for Diagnostic Tests and Vaccines:
Second Edition, 1992
Third Edition, 1996
Fourth Edition, 2000
Fifth Edition, 2004
Sixth Edition, 2008
Seventh Edition: ISBN 978-92-9044-878-5

Volume 2: ISBN 978-92-9044-880-8
Copyright
World Organisation for Animal Health (OIE), 2012
12, rue de Prony, 75017 Paris, FRANCE
Telephone: 33-(0)1 44 15 18 88
Fax: 33-(0)1 42 67 09 87
Electronic mail: oie@oie.int
http://www.oie.int
All World Organisation for Animal Health (OIE) publications are protected by international copyright law. Extracts
may be copied, reproduced, translated, adapted or published in journals, documents, books, electronic media and
any other medium destined for the public, for information, educational or commercial purposes, provided prior
written permission has been granted by the OIE.
The designations and denominations employed and the presentation of the material in this publication do not
imply the expression of any opinion whatsoever on the part of the OIE concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its frontiers and boundaries.
FOREWORD
The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual) aims to
prevent and control animal diseases, including zoonoses, to contribute to the improvement of
animal health services world-wide and to allow safe international trade in animals and animal
products. The principal target readership is laboratories carrying out veterinary diagnostic tests and
surveillance, along with vaccine manufacturers and users, and regulatory authorities in Member
Countries. The main objective is to provide internationally agreed diagnostic laboratory methods
and requirements for the production and control of relevant vaccines and other biological products.
This ambitious task has required the cooperation of highly renowned animal health specialists from
many OIE Member Countries. The OIE, the World Organisation for Animal Health, received the
mandate from its Member Countries to undertake this task on a global level. The main activities of
the organisation, which was established in 1924, and in 2012 comprised 178 Member Countries,
are as follows:
1.
To ensure transparency in the global animal disease and zoonosis situation.
2.
To collect, analyse and disseminate scientific veterinary information on animal disease control
methods.
3.
To provide expertise and encourage international solidarity in the control of animal diseases.
4.
Within its mandate under the WTO (World Trade Organization) Agreement on Sanitary and
Phytosanitary Measures (SPS Agreement), to safeguard world trade by publishing health
standards for international trade in animals and animal products.
5.
To improve the legal framework and resources of national Veterinary Services.
6.
To provide a better guarantee of the safety of food of animal origin and to promote animal
welfare through a science-based approach.
The Terrestrial Manual, covering infectious and parasitic diseases of mammals, birds and bees,
was first published in 1989. Each successive edition has extended and updated the information
provided. This seventh edition includes over 50 updated chapters and guidelines (including a new
guideline on the application of biotechnology to the development of veterinary vaccines, and the
addition of epizootic haemorrhagic disease to the relevant chapter). This edition has a slightly
different structure from former editions: Part 1 contains ten introductory chapters that set general
standards for the management of veterinary diagnostic laboratories and vaccine production
facilities; Part 2 comprises chapters on OIE listed diseases and other diseases of importance to
international trade; Part 3 comprises four guidelines that have been developed on topics such as
biotechnology and antimicrobial susceptibility testing that are intended to give a brief introduction to
their subjects (they are to be regarded as background information rather than strict standards); and
Part 4 is the list of OIE Reference Centres at the time of publication (the list of OIE Reference
Centres is updated by the World Assembly of Delegates (of OIE Member Countries) each year; the
revised list is available on the OIE Web site).
As a companion volume to the Terrestrial Animal Health Code, the Terrestrial Manual sets
laboratory standards for all OIE listed diseases as well as several other diseases of global
importance. In particular it specifies (in blue font) those Prescribed Tests that are recommended
for use in health screening for international trade or movement of animals. The Terrestrial Manual
has become widely adopted as a key reference book for veterinary laboratories around the world.
Aquatic animal diseases are included in a separate Aquatic Manual.
The task of commissioning chapters and compiling the Terrestrial Manual was assigned to the OIE
Biological Standards Commission by the World Assembly of national Delegates. Manuscripts were
requested from specialists (usually the OIE designated experts at OIE Reference Laboratories) in
each of the diseases or the other topics covered. Occasionally, an ad hoc Group of experts was
convened tasked with updating or developing a chapter. After initial scrutiny by the Consultant
Technical Editor, the chapters were sent to scientific reviewers and to experts at OIE Reference
Laboratories. They were also circulated to all OIE Member Countries for review and comment. The
Biological Standards Commission, elected every 3 years by the World Assembly, and the
OIE Terrestrial Manual 2012
iii
Foreword
Consultant Technical Editor took all the resulting comments into consideration, often referring back
to the contributors for further help, before finalising the chapters. The final text has the approval of
the World Assembly.
A procedure for the official recognition of commercialised diagnostic tests, under the authority of the
Assembly, was finalised in September 2004. Data are submitted using a validation template that
was developed by the Biological Standards Commission. Submissions are evaluated by appointed
experts, who advise the Biological Standards Commission before the final opinion of the OIE World
Assembly is sought. All information on the submission of applications can be found on the OIE Web
site.
The Terrestrial Manual continues to expand and to extend its range of topics covered. It is our
sincere hope that it will grow in usefulness to veterinary diagnosticians and vaccine manufacturers
in all the OIE Member Countries. A new paper edition of the Terrestrial Manual is published every
4 years. It is important to note that annual updates to the Terrestrial Manual will be published on the
OIE website once approved by the World Assembly, so readers are advised to check there for the
latest information. This new version of the Terrestrial Manual is published in English and Spanish.
iv
Doctor Bernard Vallat

Director General, OIE
Professor Vincenzo Caporale

President, OIE Biological Standards Commission
September 2012
September 2012
ACKNOWLEDGEMENTS
I am most grateful to the many people whose combined efforts have gone into the preparation of
this Terrestrial Manual. In particular, I would like to express my thanks to:
Dr Bernard Vallat, Director General of the OIE from 2001 to the present, who gave his
encouragement and support to the project of preparing the new edition of this Terrestrial
Manual,
The Members of the OIE Standards Commission, Prof. Vincenzo Caporale, Dr Beverly
Schmitt, Dr Mehdi El Harrak, Dr Paul Townsend, Dr Alejandro Schudel and Dr Hualan Chen
who were responsible for commissioning chapters and, with the Consultant Technical Editor,
for editing all the contributions so as to finalise this edition of the Terrestrial Manual,
The contributors listed on pages xxii to xxxv who contributed their invaluable time and
expertise to write the chapters,
The expert advisers to the Biological Standards Commissions meeting, Dr Adama Diallo and
Dr Peter Wright, the OIE Reference Laboratory experts and other reviewers who also gave
their time and expertise to scrutinising the chapters,
Those OIE Member Countries that submitted comments on the draft chapters that were
circulated to them. These were essential in making the Terrestrial Manual internationally
acceptable,
Ms Sara Linnane who, as Scientific Editor, organised this complex project and made major
contributions to the quality of the text,
Prof. Steven Edwards, Consultant Technical Editor of the Terrestrial Manual, who contributed
hugely to editing and harmonising the contents, but also in collating and incorporating Member
Country comments,
Members of both the OIE Scientific and Technical Department and the Publications
Department, for their assistance.
Dr Karin Schwabenbauer
President of the OIE World Assembly
September 2012
CONTENTS
VOLUME 2
Introduction (How to use this Terrestrial Manual) .....................................................
List of tests for International trade ............................................................................
Common abbreviations used in this Terrestrial Manual ............................................
Glossary of terms .....................................................................................................
Contributors ..............................................................................................................
PART 2
CONTINUED
OIE LISTED DISEASES AND OTHER DISEASES OF IMPORTANCE TO

INTERNATIONAL TRADE
SECTION 2.5.
EQUIDAE
Chapter 2.5.1.
Chapter 2.5.2.
Chapter 2.5.3.
Chapter 2.5.4.
Chapter 2.5.5.
Chapter 2.5.6.
Chapter 2.5.7.
Chapter 2.5.8.
Chapter 2.5.9.
Chapter 2.5.10.
Chapter 2.5.11.
Chapter 2.5.12.
Chapter 2.5.13.
African horse sickness .............................................................................................

Contagious equine metritis .......................................................................................
Dourine .....................................................................................................................
Epizootic lymphangitis ..............................................................................................
Equine encephalomyelitis (Eastern and Western) ....................................................
Equine infectious anaemia .......................................................................................
Equine influenza .......................................................................................................
Equine piroplasmosis ...............................................................................................
Equine rhinopneumonitis ..........................................................................................
Equine viral arteritis ..................................................................................................
Glanders ...................................................................................................................
Horse mange ............................................................................................................
Venezuelan equine encephalomyelitis .....................................................................
SECTION 2.6.
LEPORIDAE
Chapter 2.6.1.
Chapter 2.6.2.
Myxomatosis ............................................................................................................
Rabbit haemorrhagic disease ...................................................................................
SECTION 2.7.
CAPRINAE
Chapter 2.7.1.
Chapter 2.7.2.
Chapter 2.7.3/4.
Chapter 2.7.5.
Chapter 2.7.6.
Chapter 2.7.7.
Chapter 2.7.8.
Chapter 2.7.9.
Chapter 2.7.10
Chapter 2.7.11.
Chapter 2.7.12.
Chapter 2.7.13.
Chapter 2.7.14.
Border disease .........................................................................................................

Caprine and ovine brucellosis (excluding Brucella ovis) ...........................................
Caprine arthritis/encephalitis and Maedi-visna .........................................................
Contagious agalactia ................................................................................................
Contagious caprine pleuropneumonia ......................................................................
Enzootic abortion of ewes (ovine chlamydiosis) .......................................................
Nairobi sheep disease ..............................................................................................
Ovine epididymitis (Brucella ovis).............................................................................
Ovine pulmonary adenocarcinoma (adenomatosis) .................................................
Peste des petits ruminants .......................................................................................
Salmonellosis (S. abortusovis) .................................................................................
Scrapie .....................................................................................................................
Sheep pox and goat pox ..........................................................................................
ix
xi
xv
xvii
xxii
819
831
838
846
852
860
865
879
889
899
913
923
924
931
941
957
968
978
987
995
1008
1017
1018
1027
1032
1044
1045
1055
vii
Contents
SECTION 2.8.
SUIDAE
Chapter 2.8.1.
Chapter 2.8.2.
Chapter 2.8.3.
Chapter 2.8.4.
Chapter 2.8.5.
Chapter 2.8.6.
Chapter 2.8.7.
Chapter 2.8.8.
Chapter 2.8.9.
Chapter 2.8.10.
Chapter 2.8.11.
African swine fever ...................................................................................................

Atrophic rhinitis of swine ...........................................................................................
Classical swine fever (hog cholera) ..........................................................................
Nipah virus encephalitis ...........................................................................................
Porcine brucellosis ...................................................................................................
Porcine cysticercosis ................................................................................................
Porcine reproductive and respiratory syndrome .......................................................
Swine influenza ........................................................................................................
Swine vesicular disease ...........................................................................................
Teschovirus encephalomyelitis (previously enterovirus encephalomyelitis or
Teschen/Talfan disease) ..........................................................................................
Transmissible gastroenteritis ....................................................................................
SECTION 2.9.
OTHER DISEASES1
Chapter 2.9.1.
Chapter 2.9.2.
Chapter 2.9.3.
Chapter 2.9.4.
Chapter 2.9.5.
Chapter 2.9.6.
Chapter 2.9.7.
Chapter 2.9.8.
Chapter 2.9.9.
Chapter 2.9.10.
Chapter 2.9.11.
Chapter 2.9.12.
Bunyaviral diseases of animals (excluding Rift Valley fever)* ..................................

Camelpox .................................................................................................................
Campylobacter jejuni and Campylobacter coli..........................................................
Cryptosporidiosis ......................................................................................................
Cysticercosis* ...........................................................................................................
Hendra and Nipah virus diseases.............................................................................
Listeria monocytogenes. ..........................................................................................
Mange* .....................................................................................................................
Salmonellosis* ..........................................................................................................
Toxoplasmosis .........................................................................................................
Verocytotoxigenic Escherichia coli ...........................................................................
Zoonoses transmissible from non-human primates ..................................................
PART 3
GENERAL GUIDELINES
Guideline 3.1.
Guideline 3.2.
Guideline 3.3.
Guideline 3.4.
Laboratory methodologies for bacterial antimicrobial susceptibility testing ..............

Biotechnology in the diagnosis of infectious diseases ..............................................
The application of biotechnology to the development of veterinary vaccines ...........
The role of official bodies in the international regulation of veterinary biologicals ....
PART 4
OIE REFERENCE EXPERTS AND DISEASE INDEX
List of OIE Reference Laboratories (as of May 2012) ...............................................................................

Alphabetical list of diseases ......................................................................................................................
viii
1067
1080
1089
1105
1106
1113
1114
1127
1138
1145
1152
1163
1175
1183
1190
1214
1225
1239
1256
1268
1287
1297
1308
1311
1322
1338
1348
1363
1403
The diseases on this list that are marked with an asterisk are included in some individual species sections of the OIE List,
but these Terrestrial Manual chapters cover several species and thus give a broader description.
INTRODUCTION
(How to use this Terrestrial Manual)
Arrangement of the Terrestrial Manual
Part 1, the beginning of this Terrestrial Manual, contains ten introductory chapters that set general
standards for the management of veterinary diagnostic laboratories and vaccine facilities.
The main part of the Terrestrial Manual (Part 2) covers standards for diagnostic tests and vaccines
for specific diseases listed in the OIE Terrestrial Animal Health Code. The diseases are in
alphabetical order, subdivided by animal host group. OIE listed diseases are transmissible diseases
that have the potential for very serious and rapid spread, irrespective of national borders. They
have particularly serious socio-economic or public health consequences and are of major
importance in the international trade of animals and animal products.
Four of the diseases in Section 2.9 are included in some individual species sections, but these
chapters cover several host species and thus give a broader description. Some additional diseases
that may also be of importance to trade but that do not have a chapter in the Terrestrial Code are
also included in Section 2.9. This section also includes some important zoonotic infections
The contributors of all the chapters are listed on pages xxiixxxv, but the final responsibility for the
content of the Terrestrial Manual lies with the World Assembly of the OIE.
There is an alphabetical index of the diseases at the end of Volume 2.
Format of chapters
Each disease chapter includes a summary intended to provide information for veterinary officials
and other readers who need a general overview of the tests and vaccines available for the disease.
This is followed by a text giving greater detail for laboratory workers. In each disease chapter,
Part A gives a general introduction to the disease, Part B deals with laboratory diagnosis of the
disease, and Part C (where appropriate) with the requirements for vaccines or in vivo diagnostic
biologicals. The information concerning production and control of vaccines or diagnostics is given
as an example; it is not always necessary to follow these when there are scientifically justifiable
reasons for using alternative approaches. Bibliographic references that provide further information
are listed at the end of each chapter.
Explanation of the tests described and of the table on pages xixiv

The table on pages xixiv lists diagnostic tests in two categories: prescribed and alternative.
Prescribed tests are those that are required by the Terrestrial Animal Health Code for the testing of
animals before they are moved internationally. In the Terrestrial Manual these tests are printed in
blue. At present it is not possible to have prescribed tests for every listed disease. Alternative tests
are those that are suitable for the diagnosis of disease within a local setting, and can also be used
in the import/export of animals after bilateral agreement. There are often other tests described in
the chapters, which may be useful for specific purposes such as diagnosis or surveillance as
indicated in the chapters.
ix
Introduction (How to use this Terrestrial Manual)
General guidelines
Four guidelines that have been developed on topics such as biotechnology and antimicrobial
susceptibility testing are included in Part 3 of this Terrestrial Manual. These are intended to give a
brief introduction to their subjects. They are to be regarded as background information rather than
standards.
List of OIE Reference Laboratories

A list of OIE Reference Laboratories is given in Part 4 of this Terrestrial Manual. These laboratories
have been designated by the OIE as centres of excellence with expertise in their particular field.
They are able to provide advice to other laboratories on methodology. In some cases standard
strains of micro-organisms or reference reagents (e.g. antisera, antigens) can also be obtained
from the OIE Reference Laboratories.
The list of OIE Reference Laboratories will be updated by the World Assembly each year. The
revised list is available on the OIE Web site.
*
* *
LIST OF TESTS FOR INTERNATIONAL TRADE
The table below lists diagnostic tests in two categories: prescribed and alternative. Prescribed tests
are required by the OIE Terrestrial Animal Health Code for the international movement of animals
and animal products and are considered optimal for determining the health status of animals. In the
Terrestrial Manual these tests are printed in blue. At present it is not possible to have prescribed
tests for every listed disease. Alternative tests are those that are suitable for the diagnosis of disease
within a local setting, and can also be used in the import/export of animals after bilateral agreement.
There are often other tests described in the chapters that may also be of some practical value in
local situations or that may still be under development.
Chapter No.
Disease name
2.1.1.
Anthrax
2.1.2.
Aujeszkys disease
2.1.3.
Bluetongue
2.1.4.
Echinococcosis/Hydatidosis
2.1.5.
Foot and mouth disease
2.1.6.
Prescribed tests
Alternative tests
ELISA, VN
Agent id.,
ELISA, PCR
AGID, VN
ELISA*, VN
CF
Heartwater
ELISA, IFA
2.1.7.
Japanese encephalitis
2.1.8.
Leishmaniosis
Agent id.
2.1.9.
Leptospirosis
MAT
2.1.10.
New World screwworm (Cochliomyia

hominivorax) and Old World screwworm
(Chrysomya bezziana)
Agent id.
2.1.11.
Paratuberculosis (Johnes disease)
DTH, ELISA
2.1.12.
Q fever
CF
2.1.13.
Rabies
ELISA, VN
2.1.14.
Rift Valley fever
VN
ELISA, HI
2.1.15.
Rinderpest
ELISA
VN
2.1.16.
Trichinellosis
Agent id.
ELISA
2.1.17.
Trypanosoma evansi infections

(including surra)
2.1.18.
Tularemia
Agent id.
2.1.19.
Vesicular stomatitis
CF, ELISA, VN
2.1.20.
West Nile fever
2.2.1.
Acarapisosis of honey bees
2.2.2.
American foulbrood of honey bees
Please refer to Terrestrial Manual chapters to verify which method is prescribed.
xi
List of tests for international trade
Chapter No.
xii
Disease name
Prescribed tests
Alternative tests
2.2.3.
European foulbrood of honey bees
2.2.4.
Nosemosis of bees
2.2.5.
Small hive beetle infestation

(Aethina tumida)
2.2.6.
Tropilaelaps infestation of honey bees

(Tropilaelaps spp.)
2.2.7
Varroosis of honey bees
2.3.1.
Avian chlamydiosis
2.3.2.
Avian infectious bronchitis
ELISA, HI, VN
2.3.3.
Avian infectious laryngotracheitis
AGID, ELISA, VN
2.3.4.
Avian influenza
Virus isolation
with pathogenicity
testing
AGID, HI
2.3.5.
Avian mycoplasmosis
(Mycoplasma gallisepticum, M. synoviae)
Agg., HI
2.3.6.
Avian tuberculosis
Agent id.,
Tuberculin test
2.3.7.
Duck virus enteritis
2.3.8.
Duck virus hepatitis
2.3.9.
Fowl cholera
2.3.10.
Fowl pox
2.3.11.
Fowl typhoid and Pullorum disease
Agent id., Agg.
2.3.12.
Infectious bursal disease

(Gumboro disease)
AGID, ELISA
2.3.13.
Mareks disease
AGID
2.3.14.
Newcastle disease
Virus isolation
HI
2.3.15.
Turkey rhinotracheitis (avian

metapneumovirus)
2.4.1.
Bovine anaplasmosis
CAT, CF
2.4.2.
Bovine babesiosis
CF, ELISA, IFA
2.4.3.
Bovine brucellosis
BBAT, CF,
ELISA, FPA
2.4.4.
Bovine cysticercosis
Agent id.
2.4.5.
Bovine genital campylobacteriosis
Agent id.
2.4.6.
Bovine spongiform encephalopathy
2.4.7.
Bovine tuberculosis
Tuberculin test
Gamma interferon test
2.4.8.
Bovine viral diarrhoea
Agent id.
2.4.9.
Contagious bovine pleuropneumonia
CF, ELISA
2.4.10.
Dermatophilosis
2.4.11.
Enzootic bovine leukosis
AGID, ELISA
PCR
2.4.12.
Haemorrhagic septicaemia
Agent id.
2.4.13.
Infectious bovine rhinotracheitis/

infectious pustular vulvovaginitis
Agent id.
(semen only),
ELISA, PCR, VN
Chapter No.
Disease name
Prescribed tests
Alternative tests
2.4.14.
Lumpy skin disease
VN
2.4.15.
Malignant catarrhal fever
IFA, PCR, VN
2.4.16.
Theileriosis
Agent id., IFA
2.4.17.
Trichomonosis
Agent id.
Mucus agg.
2.4.18.
Trypanosomosis (Tsetse-transmitted)
IFA
2.5.1.
African horse sickness
CF, ELISA
Agent id. (real-time

PCR), VN
2.5.2.
Contagious equine metritis
Agent id.
2.5.3.
Dourine
CF
ELISA, IFA
2.5.4.
Epizootic lymphangitis
2.5.5.
Equine encephalomyelitis
(Eastern and Western)
CF, HI, PRN
2.5.6.
Equine infectious anaemia
AGID
ELISA
2.5.7.
Equine influenza
HI
2.5.8.
Equine piroplasmosis
ELISA, IFA
CF
2.5.9.
Equine rhinopneumonitis
VN
2.5.10.
Equine viral arteritis
Agent id.
(semen only), VN
2.5.11.
Glanders
CF
2.5.12.
Horse mange
Agent id.
2.5.13.
Venezuelan equine encephalomyelitis
CF, HI, PRN
2.6.1.
Myxomatosis
AGID, CF, IFA
2.6.2.
Rabbit haemorrhagic disease
ELISA, HI
2.7.1.
Border disease
Agent id.
2.7.2.
Caprine and ovine brucellosis

(excluding Brucella ovis)
BBAT, CF,
ELISA, FPA
Brucellin test
Caprine arthritis/encephalitis & Maedi-visna
AGID, ELISA
CF
2.7.3/4.
2.7.5.
Contagious agalactia
2.7.6.
Contagious caprine pleuropneumonia
2.7.7.
Enzootic abortion of ewes

(ovine chlamydiosis)
CF
2.7.8.
Nairobi sheep disease
2.7.9.
Ovine epididymitis (Brucella ovis)
CF
ELISA
2.7.10.
Ovine pulmonary adenocarcinoma

(adenomatosis)
2.7.11.
Peste des petits ruminants
VN
ELISA
2.7.12.
Salmonellosis (S. abortusovis)
2.7.13.
Scrapie
2.7.14.
Sheep pox and goat pox
VN
2.8.1.
African swine fever
ELISA
IFA
2.8.2.
Atrophic rhinitis of swine
xiii
Chapter No.
Disease name
Prescribed tests
2.8.3.
Classical swine fever (hog cholera)
2.8.4.
Nipah virus encephalitis
2.8.5.
Porcine brucellosis
2.8.6.
Alternative tests
ELISA,
FAVN, NPLA
BBAT, CFT,
ELISA, FPA
Porcine cysticercosis
2.8.7.
Porcine reproductive and

respiratory syndrome
ELISA, IFA, IPMA
2.8.8.
Swine influenza
2.8.9.
Swine vesicular disease
VN
ELISA
2.8.10.
Teschovirus encephalomyelitis (previously

enterovirus encephalomyelitis or
Teschen/Talfan disease)
VN
2.8.11.
Transmissible gastroenteritis
VN, ELISA
2.9.1.
Bunyaviral diseases of animals (excluding

Rift Valley fever)
2.9.2.
Camelpox
2.9.3.
Campylobacter jejuni and C. coli
2.9.4.
Cryptosporidiosis
2.9.5.
Cysticercosis
Agent id.
2.9.6.
Hendra and Nipah virus diseases
2.9.8.
Listeria monocytogenes
2.9.8.
Mange
Agent id.
2.9.9.
Salmonellosis
Agent id.
2.9.10.
Toxoplasmosis
2.9.11.
Verocytotoxigenic Escherichia coli
2.9.12.
Zoonoses transmissible from non-human

primates
Note: The tests prescribed by the Terrestrial Animal Health Code for the purposes of international trade are printed
in blue in this Terrestrial Manual.
Abbreviations
Agent id.
Agent identification
HI
Haemagglutination inhibition
Agg.
Agglutination test
IFA
Indirect fluorescent antibody
AGID
Agar gel immunodiffusion
IPMA
Immunoperoxidase monolayer assay
BBAT
Buffered Brucella antigen test
MAT
Microscopic agglutination test
CAT
Card agglutination test
NPLA
Neutralising peroxidase-linked assay
CF
Complement fixation
PCR
Polymerase chain reaction
DTH
Delayed-type hypersensitivity
PRN
Plaque reduction neutralisation
ELISA
Enzyme-linked immunosorbent assay
VN
Virus neutralisation
FAVN
Fluorescent antibody virus neutralisation
No test designated yet
FPA
Fluorescence polarisation assay
xiv
COMMON ABBREVIATIONS USED

IN THIS TERRESTRIAL MANUAL
ABTS
FBS
Fetal bovine serum
FITC
FLK
Fluorescein isothiocyanate
Fetal lamb kidney (cells)
FPA
g
GIT
Fluorescence polarisation assay

Relative centrifugal force
Growth inhibition test
BHK
BLP
BPAT
BSA
BSF
Buffered Brucella antigen test

Bovine fetal kidney (cells)
Beef extract-glucose-peptone-serum
(medium)
Baby hamster kidney (cell line)
Buffered lactose peptone
Buffered plate antigen test
Bovine serum albumin
Bovine serum factors
HA
HAD
HBSS
HEP
HEPES
CAM
CEF
CF
CFU
CIEP
CK
Chorioallantoic membrane
Chicken embryo fibroblast
Complement fixation (test)
Colony-forming unit
Counter immunoelectrophoresis
Calf kidney (cells)
HI
HRPO
IB
ICFTU
ICPI
ID50
Haemagglutination
Haemadsorption
Hanks balanced salt solution
High-egg-passage (virus)
N-2-hydroxyethylpiperazine, N-2ethanesulphonic acid (buffer)
Horseradish peroxidase
Immunoblot test
International complement fixation test unit
Intracerebral pathogenicity index
Median infectious dose
CNS
CPE
CPLM
Central nervous system

Cytopathic effect
Cysteine-peptone-liver infusion maltose
(medium)
Casein-sucrose-yeast (agar)
Diethyl pyrocarbonate
Diethylaminoethyl
Diethylpyrocarbonate
Dulbeccos modified Eagles medium
Dimethyl sulphide
Delayed-type hypersensitivity
Ethylene diamine tetra-acetic acid
Ethylene glycol tetra-acetic acid
Egg-infective dose
Evans modified Tobies medium
Earles yeast lactalbumin (balanced salt
solution)
Fluorescent antibody test
Fluorescent antibody virus neutralisation
IFA
IHA
IPMA
Indirect fluorescent antibody (test)

Indirect haemagglutination
Immunoperoxidase monolayer assay
IU
IVPI
LA
LD
LEP
LPS
MAb
MAT
MCS
MDBK
MDT
MEM
MHC
International units
Intravenous pathogenicity index
Latex agglutination
Lethal dose
Low egg passage (virus)
Lipopolysaccharide
Monoclonal antibody
Microscopic agglutination test
Master cell stock
Madin-Darby bovine kidney (cell line)
Mean death time
Minimal essential medium
Major histocompatibility complex
MLV
m.o.i.
Modified live virus (vaccine)

multiplicity of infection
AGID
ATCC1
BBAT
BFK
BGPS
CSY
DEPC
DEAE
DEPC
DMEM
DMSO
DTH
EDTA
EGTA
EID
ELISA
EMTM
EYL
FAT
FAVN
2,2-azino-di-(3-ethyl-benzthiazoline)-6sulphonic acid
American type culture collection
American Type Culture Collection, P.O. Box 1549, Manassas, Virginia 20108, United States of America.
xv
Common abbreviations used in this Terrestrial Manual
MSV
NI
OGP
OPD
OPG
Master seed virus

Neutralisation index
1-octyl-beta-D-glucopyranoside (buffer)
Orthophenyldiamine (chromogen)
Oxalase-phenol-glycerin (preservative
solution)
Open reading frame
RBC
RFLP
RK
RPM
RSA
Red blood cell

Restriction fragment length polymorphism
Rabbit kidney
Revolutions per minute
Rapid serum agglutination
RT-PCR
SAT
SDS
PAS
PBS
PCR
PD
Polyacrylamide gel electrophoresis

Peroxidaseantiperoxidase (staining
procedure)
Periodic acid-Schiff (reaction)
Phosphate buffered saline
Protective dose
Reverse-transcription polymerase chain

reaction
Serum agglutination test
Sodium dodecyl sulphate
SPF
SPG
SRBC
TCID50
Specific pathogen free

Sucrose phosphate glutamic acid
Sheep red blood cells
Median tissue culture infective dose
PFGE
PFU
PHA
PPD
PPLO
PRN
PSG
Pulsed field gel electrophoresis

Plaque-forming unit
Passive haemagglutination (test)
Purified protein derivative
Pleuropneumonia-like organisms
Phosphate buffered saline glucose
TMB
TSI
VB
VBS
Vero
VN
Tetramethyl benzidine
Triple sugar iron (medium)
Veronal buffer
Veronal buffered saline
African green monkey kidney (cells)
ORF
PAGE
PAP
*
* *
xvi
GLOSSARY OF TERMS
The definitions given below have been selected and restricted to those that are likely to be useful to
users of this OIE Terrestrial Manual.
Absorbance/optical density
Absorbance and optical density are terms used to indicate the strength of reaction. A spectrophotometer is used
to measure the amount of light of a specific wave length that a sample absorbs and the absorbance is
proportional to the amount of a particular analyte present.
Accuracy
Nearness of a test value to the expected value for a reference standard reagent of known activity or titre.
Assay
Synonymous with test or test method, e.g. enzyme immunoassay, complement fixation test or polymerase chain
reaction tests.
Batch
All vaccine or other reagent, such as antigen or antisera, derived from the same homogeneous bulk and identified
by a unique code number.
Cell line
A stably transformed line of cells that has a high capacity for multiplication in vitro.
Centrifugation
Throughout the text, the rate of centrifugation has been expressed as the Relative Centrifugal Force, denoted by
g. The formula is:
(RPM 0.10472)2
Radius (cm) = g
980
where RPM is the rotor speed in revolutions per minute, and where Radius (cm) is the radius of the rotor arm, to
the bottom of the tube, in centimetres.
It may be necessary to calculate the RPM required to achieve a given value of g, with a particular rotor. The
formula is:
RPM =
g 980 /Radius (cm)

0.10472
Cross-reaction
See False-positive reaction.
Cut-off/threshold
Test result value selected for distinguishing between negative and positive results; may include indeterminate or
suspicious zone.
xvii
Glossary of terms
Dilutions
Where dilutions are given for making up liquid reagents, they are expressed as, for example, 1 in 4 or 1/4,
meaning one part added to three parts, i.e. a 25% solution of A in B.
v/v This is volume to volume (two liquids).
w/v This is weight to volume (solid added to a liquid).
Dilutions used in virus neutralisation tests
There are two different conventions used in expressing the dilution used in virus neutralisation (VN) tests. In
Europe, it is customary to express the dilution before the addition of the antigen, but in the United States of
America and elsewhere, it is usual to express dilutions after the addition of antigen.
These alternative conventions are expressed in the Terrestrial Manual as initial dilution or final dilution,
respectively.
Efficacy
Specific ability of the biological product to produce the result for which it is offered when used under the
conditions recommended by the manufacturer.
Equivalency testing
Determination of certain assay performance characteristics of new and/or different test methods by means of an
interlaboratory comparison to a standard test method; implied in this definition is that participating laboratories are
using their own test methods, reagents and controls and that results are expressed qualitatively.
False-negative reaction
Negative reactivity in an assay of a test sample obtained from an animal exposed to or infected with the organism
in question, may be due to lack of analytical sensitivity, restricted analytical specificity or analyte degradation,
decreases diagnostic sensitivity.
False-positive reaction
Positive reactivity in an assay that is not attributable to exposure to or infection with the organism in question,
maybe due to immunological cross-reactivity, cross-contamination of the test sample or non-specific reactions,
decreases diagnostic specificity.
Final product (lot)
All sealed final containers that have been filled from the same homogenous batch of vaccine in one working
session, freeze-dried together in one continuous operation (if applicable), sealed in one working session, and
identified by a unique code number.
Harmonisation
The result of an agreement between laboratories to calibrate similar test methods, adjust diagnostic thresholds
and express test data in such a manner as to allow uniform interpretation of results between laboratories.
Incidence
Estimate of the rate of new infections in a susceptible population over a defined period of time; not to be confused
with prevalence.
In-house checks
All quality assurance activities within a laboratory directly related to the monitoring, validation, and maintenance of
assay performance and technical proficiency.
In-process control
Test procedures carried out during manufacture of a biological product to ensure that the product will comply with
the agreed quality standards.
xviii
Glossary of terms
Inter-laboratory comparison (ring test)
Any evaluation of assay performance and/or laboratory competence in the testing of defined samples by two or
more laboratories; one laboratory may act as the reference in defining test sample attributes.
Master cell (line, seed, stock)
Collection of aliquots of cells of defined passage level, for use in the preparation or testing of a biological product,
distributed into containers in a single operation, processed together and stored in such a manner as to ensure
uniformity and stability and to prevent contamination.
Master seed (agent, strain)
Collection of aliquots of an organism at a specific passage level, from which all other seed passages are derived,
which are obtained from a single bulk, distributed into containers in a single operation and processed together
and stored in such a manner as to ensure uniformity and stability and to prevent contamination.
Performance characteristic
An attribute of a test method that may include analytical sensitivity and specificity, accuracy and precision,
diagnostic sensitivity and specificity and/or repeatability and reproducibility.
Potency
Relative strength of a biological product as determined by appropriate test methods. (Initially the potency is
measured using an efficacy test in animals. Later this may be correlated with tests of antigen content, or antibody
response, for routine batch potency tests.)
Precision
The degree of dispersion of results for a repeatedly tested sample expressed by statistical methods such as
standard deviation or confidence limits.
Predictive value (negative)
The probability that an animal is free from exposure or infection given that it tests negative; predictive values are a
function of the DSe (diagnostic sensitivity) and DSp (diagnostic specificity) of the diagnostic assay and the
prevalence of infection.
Predictive value (positive)
The probability that an animal has been exposed or infected given that it tests positive; predictive values are a
function of the DSe and DSp of the diagnostic assay and the prevalence of infection.
Prevalence
Estimate of the proportion of infected animals in a population at one given point in time; not to be confused with
incidence.
Primary cells
A pool of original cells derived from normal tissue up to and including the tenth subculture.
Production seed
An organism at a specified passage level that is used without further propagation for initiating preparation of a
production bulk.
Proficiency testing
One measure of laboratory competence derived by means of an interlaboratory comparison; implied in this
definition is that participating laboratories are using the same test methods, reagents and controls and that results
are expressed qualitatively.
xix
Glossary of terms
Purity
Quality of a biological product prepared to a final form and:

a)
Relatively free from any extraneous microorganisms and extraneous material (organic or inorganic) as
determined by test methods appropriate to the product; and
b)
Free from extraneous microorganisms or material which could adversely affect the safety, potency or
efficacy of the product.
Reference animal
Any animal for which the infection status can be defined in unequivocal terms; may include diseased, infected,
vaccinated, immunised or nave animals.
Reference Laboratory
Laboratory of recognised scientific and diagnostic expertise for a particular animal disease and/or testing
methodology; includes capability for characterising and assigning values to reference reagents and samples.
Repeatability
Level of agreement between replicates of a sample both within and between runs of the same test method in a
given laboratory.
Reproducibility
Ability of a test method to provide consistent results when applied to aliquots of the same sample tested by the
same method in different laboratories.
Room temperature
The term room temperature is intended to imply the temperature of a comfortable working environment. Precise
limits for this cannot be set, but guiding figures are 1825C. Where a test specifies room temperature, this
should be achieved, with air conditioning if necessary; otherwise the test parameters may be affected.
Safety
Freedom from properties causing undue local or systemic reactions when used as recommended or suggested by
the manufacturer and without known hazard to in-contact animals, humans and the environment.
Sample
Material that is derived from a specimen and used for testing purposes.
Sensitivity (analytical)
Synonymous with Limit of Detection, smallest detectable amount of analyte that can be measured with a defined
certainty; analyte may include antibodies, antigens, nucleic acids or live organisms.
Sensitivity (diagnostic)
Proportion of known infected reference animals that test positive in the assay; infected animals that test negative
are considered to have false-negative results.
Sensitivity (relative)
Proportion of reference animals defined as positive by one or a combination of test methods that also test positive
in the assay being compared.
Specific pathogen free (SPF)
Animals that have been shown by the use of appropriate tests to be free from specified pathogenic
microorganisms, and also refers to eggs derived from SPF birds.
xx
Glossary of terms
Specificity (analytical)
Degree to which the assay distinguishes between the target analyte and other components in the sample matrix;
the higher the analytical specificity, the lower the level of false-positives.
Specificity (diagnostic)
Proportion of known uninfected reference animals that test negative in the assay; uninfected reference animals
that test positive are considered to have false-positive results.
Specificity (relative)
Proportion of reference animals defined as negative by one or a combination of test methods that also test
negative in the assay being compared.
Specimen
Material submitted for testing.
Standard Reagents
International Standard Reagents

Standard reagents by which all other reagents and assays are calibrated; prepared and distributed by an
International Reference Laboratory.
National Standard Reagents

Standard reagents calibrated by comparison with International Standard Reagents; prepared and distributed
by a National Reference Laboratory.
Working Standards (reagents)

Standard reagents calibrated by comparison with the National Standard Reagent, or, in the absence of a
National Standard Reagent, calibrated against a well-characterised in-house standard reagent; included in
routine diagnostic tests as a control and/or for normalisation of test results.
Sterility
Freedom from viable contaminating microorganisms, as demonstrated by approved and appropriate tests.
Test method
Specified technical procedure for detection of an analyte (synonymous with assay).
Tests
Prescribed
Test methods that are required by the OIE Terrestrial Animal Health Code for the international movement of
animals and animal products and that are considered optimal for determining the health status of animals.
Alternative
Test methods considered in this Terrestrial Manual to be suitable for the diagnosis of disease in a local
situation, and that can also be used for import/ export by bilateral agreement.
Screening
Tests of high diagnostic sensitivity suitable for large-scale application.
Confirmatory
Test methods of high diagnostic specificity that are used to confirm results, usually positive results, derived
from other test methods
Working seed
Organism at a passage level between master seed and production seed.
xxi
CONTRIBUTORS
C O N T R IB U T O R S A N D P R O F E S S I O N A L A D D R E S S A T T I ME O F W R I T IN G
The chapters in the Terrestrial Manual are prepared by invited contributors (OIE Reference Experts,
where possible). In accordance with OIE standard procedure, all chapters are circulated to OIE
Member Countries and to other experts in the disease for comment. The OIE Biological Standards
Commission and the Consultant Editor then modify the text to take account of comments received,
and the text is circulated a second time as the final version that will be presented for adoption by
the World Assembly of Delegates to the OIE at the General Session in May of each year. The
Terrestrial Manual is thus deemed to be an OIE Standard Text that has come into being by
international agreement. For this reason, the names of the contributors are not shown on individual
chapters but are listed below. The Biological Standards Commission greatly appreciates the work of
the following contributors (address at the time of writing):
1.1.1. Collection and shipment of
diagnostic specimens
Dr J.E. Pearson (retired)

4016 Phoenix St., Ames, Iowa 50014, USA.
1.1.2. Sampling of diagnostic specimens
Chapter under study
1.1.3. Biosafety and biosecurity in the veterinary

microbiology laboratory and animal facilities
Dr B. Schmitt
National Veterinary Services Laboratories,
Diagnostic Virology Laboratory, P.O. Box 844,
Ames, IA 50010, USA.
Dr P. Le Blanc Smith
CSIRO Livestock Industries, Australian Animal
Health Laboratory (AAHL), Private Bag 24,
Geelong, Victoria 3220,
Australia.
1.1.4. Quality management in veterinary testing

laboratories
Dr R.A. Williams,
AHVLA-Penrith, Merrythought, Calthwaite,
Penrith, Cumbria CA11 9RR, UK.
1.1.5. Principles of validation of diagnostic assays for

infectious diseases1
Dr R.H. Jacobson (retired)

27801 Skyridge Drive, Eugene, Oregon 97405,
USA.
Dr P. Wright (retired)
Aquatic Animal Health, Fisheries and Oceans
Canada, 343 University Avenue, Moncton,
New Brunswick, E1C 9B6, Canada.
xxii
This chapter was updated by consensus of the OIE ad hoc Group on Validation of Diagnostic Assays.
Contributors
1.1.6. Principles of veterinary vaccine production
Dr B. Rippke
Center for Veterinary Biologics, USDA, Animal
and Plant Health Inspection Service, Veterinary
Services, Suite 104, 510 South 17th Street, Ames,
IA 50010, USA.
Dr D.J.K. Mackay
European Medicines Agency, Veterinary
Medicines and Inspections, 7 Westferry Circus,
Canary Wharf, London E14 4HB, UK.
Dr M. Lombard
International Association for Biologicals (IABs),
22 Rue Crillon, 69006 Lyon, France.
1.1.7. Tests for sterility and freedom from

contamination of biological materials
Dr L. Elsken
USDA, APHIS, Center for Veterinary Biologics,
Suite 104, 510 South 17th Street, Ames, Iowa
50010, USA.
1.1.8. Minimum requirements for vaccine production

facilities
Chapter under study
1.1.9. Quality control of vaccines
Chapter under study
1.1.10. International standards for vaccine banks
OIE ad hoc Group on Antigen and Vaccine Banks

for Foot and Mouth Disease
2.1.1. Anthrax
Dr E. Golsteyn-Thomas
Canadian Food Inspection Agency, Lethbridge
Laboratory, P.O. Box 640, Township Road 9-1
Lethbridge, Alberta T1J 3Z4, Canada.
Dr G. Harvey
USDA, APHIS, National Veterinary Services
Laboratories, P.O. Box 844, Ames, Iowa 50010,
USA.
2.1.2. Aujeszkys disease
Dr A. Jestin & Dr M.F. Le Potier

ANSES Ploufragan, Laboratoire dtudes et de
recherches avicoles et porcines, Zoople des
Ctes dArmor-Les Croix, BP 53, 22440
Ploufragan, France.
2.1.3. Bluetongue
Dr P. Daniels
Australian Animal Health Laboratory, CSIRO
Livestock Industries, 5 Portarlington Road,
Geelong, Victoria 3220, Australia.
Dr C.A.L. Oura (formerly)
Institute for Animal Health, Pirbright Laboratory,
Ash Road, Pirbright, Surrey GU24 0NF, UK.
2.1.4. Echinococcosis/Hydatidosis
Dr M. Kamiya
Laboratory of Environmental Zoology, Department
of Biosphere and Environmental Sciences,
Faculty of Environmental Systems, Rakuno
Gakuen University, Midori-machi 582,Ebetsu 0698501, Hokkaido, Japan.
2.1.5. Foot and mouth disease2
Dr D.J. Paton, Dr P.V. Barnett & Dr N.P. Ferris

This chapter was updated by consensus of the OIE ad hoc Group on Vaccine Quality related to Foot and Mouth Disease.
xxiii
Contributors
2.1.6. Heartwater
Dr D. Martinez
CIRAD-EMVT, Campus International de
Baillarguet - TA30/G, 34398 Montpellier Cedex 5,
France.
Dr N. Vachiry
CIRAD-EMVT, Domaine de Duclos, Prise dEau,
97170 Petit-Bourg, Guadeloupe.
Prof. F. Jongejan
Department of Parasitology & Tropical Veterinary
Medicine, Faculty of Veterinary Medicine, Utrecht
University, P.O. Box 80.165, 3508 TD Utrecht,
The Netherlands
AND
Department of Veterinary Tropical Diseases,
Faculty of Veterinary Science, University of
Pretoria, Onderstepoort 0110, South Africa.
2.1.7. Japanese encephalitis
Dr T. Kondo
Epizootic Research Center, Equine Research
Institute, Japan Racing Association, 1400-4
Shiba, Shimotsuke, Tochigi 329-0412, Japan.
2.1.8. Leishmaniosis
Dr L. Gradoni & Dr M. Gramiccia

Dipartimento di Malattie Infettive, Parassitarie ed
Immunomediate, Istituto Superiore di Sanit, Viale
Regina Elena 299, I-00161 Rome, Italy.
2.1.9. Leptospirosis
Prof. C.A. Bolin

Diagnostic Center for Population & Animal Health,
College of Veterinary Medicine, Michigan State
University, 4125 Beaumont Rd, Lansing, Michigan
48910, USA.
2.1.10. New World screwworm (Cochliomyia

hominivorax) and Old World screwworm
(Chrysomya bezziana)
Dr M.J.R. Hall
Department of Entomology, The Natural History
Museum, Cromwell Road, London SW7 5BD, UK.
2.1.11. Paratuberculosis (Johnes disease)
Dr J. Gwozdz
Department of Primary Industries, Victoria,
475 Mickleham Road, Attwood, VIC 3049,
Australia.
2.1.12. Q fever
Dr E. Rousset, Dr K. Sidi-Boumedine &

Dr R. Thiery
Anses Sophia Antipolis, Laboratoire dtudes et
de Recherches sur les Petits Ruminants et les
Abeilles (LERPRA), Les Templiers, 105 route des
Chappes, BP 111, 06902 Sophia Antipolis Cedex,
France.
xxiv
Contributors
2.1.13. Rabies
Dr A. Fooks & Dr D. Horton

AHVLA Weybridge, New Haw, Addlestone, Surrey
KT15 3NB, UK.
Dr T. Mller & Dr C. Freuling
Institute for Epidemiology, Friedrich-Loeffler
Institut, Federal Research Institute for Animal
Health, Seestr. 55, D-16868 Wusterhausen/Dosse
Germany.
Dr Charles Rupprecht
Centers for Disease Control and Prevention,
National Center for Emerging and Zoonotic
Infectious Diseases, Division of High
Consequence Pathogens and Pathology, Poxvirus
and Rabies Branch, 1600 Clifton Rd., MS G33
Atlanta, GA, 30333, USA.
2.1.14. Rift Valley fever
Dr G.H. Gerdes (retired)

Onderstepoort Veterinary Institute, Private Bag
X05, Onderstepoort 0110, South Africa.
2.1.15. Rinderpest
Dr W.P. Taylor
16 Mill Road, Angmering, Littlehampton, West
Sussex BN16 4HT, UK.
Dr P. Roeder
Hollyhedge Cottage, Spats Lane, Headley Down,
Bordon, Hampshire GU35 8SY, UK.
2.1.16. Trichinellosis
Dr A. Gajadhar & Dr L. Forbes

Canadian Food Inspection Agency, Centre for
Foodborne & Animal Parasitology, 116 Veterinary
Road, Saskatoon, Saskatchewan S7N 2R3
Canada.
2.1.17. Trypanosoma evansi infection (surra)3
Dr M. Desquesnes
UMR177-Intertryp (CIRAD-IRD), CIRAD-bios,
Campus international de Baillarguet, TA A-17 / G,
34398 Montpellier Cedex 5, France.
2.1.18. Tularemia
Dr T. Mrner (retired)
Department of Pathology and Wildlife Diseases,
Swedish National Veterinary Institute, Sweden.
2.1.19. Vesicular stomatitis
Dr S.L. Swenson
USA.
2.1.20. West Nile fever
Dr E.N. Ostlund
United States of America.
2.2.1. Acarapisosis of honey bees
Dr W. Ritter
CVUA-Freiberg, FB: Bienen (bee team), Am
Moosweiher 2, D79018 Freiberg, Germany.
2.2.2. American foulbrood of honey bees

2.2.3. European foulbrood of honey bees
Dr D.C. de Graaf
Laboratory of Zoophysiology, University of Ghent,
K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
This chapter was updated by consensus of the OIE ad hoc Group on Diagnostic Tests for Trypanosomoses.
xxv
Contributors
2.2.4. Nosemosis of honey bees
Dr W. Ritter
2.2.5. Small hive beetle infestation (Aethina tumida)
Dr W. Ritter
Dr P. Neumann
Swiss Bee Research Centre, Agroscope
Liebefeld-Posieux, Research Station ALP,
Schwarzenburgstrasse 161, CH-3003 Bern,
Switzerland.
Dr J.D. Ellis
Department of Entomology, The University of
Georgia, Athens, GA 30602, USA.
2.2.6. Tropilaelaps infestation of honey bees

(Tropilaelaps spp.)
2.2.7. Varroosis of honey bees
Dr W. Ritter
2.3.1. Avian chlamydiosis
Dr K. Sachse
Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, Institute of Molecular
Pathogenesis, Naumburger Str. 96a, 07743 Jena,
Germany.
2.3.2. Avian infectious bronchitis
Dr J. Gelb
Dept of Animal and Food Sciences and the Avian
Biosciences Center, University of Delaware,
531 South College Avenue, Newark, Delaware
19716-2150, USA.
2.3.3. Avian infectious laryngotracheitis
Dr R.C. Jones
Department of Veterinary Pathology, University of
Liverpool, Jordan Building, Veterinary Field
Station, Leahurst, Neston, South Wirral
CH64 7TE, UK.
2.3.4. Avian influenza
Dr D. Swayne
Southeast Poultry Research Laboratory,
934 College Station Road, Athens, Georgia 30605
USA.
2.3.5. Avian mycoplasmosis

(Mycoplasma gallisepticum, M. synoviae)
Dr S.H. Kleven
University of Georgia, Poultry Diagnostic and
Research Center, 953 College Stantion Road,
Athens, Georgia 30602-4875, USA.
Dr J.M. Bradbury
University of Liverpool, Department of Veterinary
Pathology, Veterinary Teaching Hospital,
Leahurst, Neston H64 7TE, UK.
2.3.6. Avian tuberculosis
xxvi
Dr D.V. Cousins (formerly)

Australian Reference Laboratory for Bovine
Tuberculosis, Western Australia Dept of
Agriculture and Food, Locked Bag N 4, Bentley
Delivery Centre, Bentley WA 6983, Australia.
Contributors
2.3.7. Duck virus enteritis

2.3.8. Duck virus hepatitis
Dr P.R. Woolcock
California Animal Health and Food Safety
Laboratory System, University of California, Davis
West Health Sciences Drive, P.O. Box 1770,
Davis, California 95617, USA.
2.3.9. Fowl cholera
Dr R. Kunkle
National Animal Disease Center, P.O. Box 70,
Ames, Iowa 50010, USA.
Dr M.A. Wilson
National Animal Disease Center, 1800 N. Dayton
Avenue, Ames, Iowa 50010, USA.
2.3.10. Fowl pox
Dr D.N. Tripathy
University of Illinois at Urbana-Champaign,
College of Veterinary Medicine, Department of
Veterinary Pathbiology, 2001 South Lincoln
Avenue, Urbana, Illinois 61802, USA.
2.3.11. Fowl typhoid and Pullorum disease
Dr R. Davies
KT15 3NB, UK.
2.3.12. Infectious bursal disease (Gumboro disease)
Dr N. Eterradossi
Anses, Laboratoire de Ploufragan-Plouzan,
Laboratoire d'tudes et de recherches avicoles,
porcines et piscicoles, B.P. 53, 22440 PloufraganPlouzan, France.
2.3.13. Mareks disease
Dr Venugopal Nair
Institute for Animal Health, Compton, Berkshire
RG20 7NN, UK.
2.3.14. Newcastle disease
Dr C.L. Afonso & Dr P.J. Miller

SEPRL, USDA, ARS, SAA, 934 College Station
Road, Athens, GA 30605, USA.
Dr Ch. Grund
Friedrich-Loeffler-Institute, Federal Research
Institute for Animal Health, Institute of Diagnostic
Virology, Sdufer 10, D-17493 Greifswald, Insel
Riems, Germany.
Dr G. Koch & Dr B. Peeters
Central Veterinary Institute of Wageningen
University and Research Centre, Department of
Virology, P.O. Box 65, NL-8200 AB Lelystad, The
Netherlands.
Dr Paul W. Selleck
CSIRO, Australian Animal Health Laboratory
(AAHL), 5 Portarlington Road, Private Bag 24,
Geelong 3220, Victoria, Australia.
Dr G.B. Srinivas
Bacteriology Policy, Evaluation, and Licensing,
USDA/APHIS/VS/CVB, 1920 Dayton Avenue,
P.O. Box 844 Ames, Iowa 50010, USA.
xxvii
Contributors
2.3.15. Turkey rhinotracheitis (avian

metapneumovirus)
Dr J. Pedersen
Avian Section, Diagnostic Virology Laboratory,
National Veterinary Services Laboratories,
1920 Dayton Avenue, Ames, IA 50010, USA.
Dr N. Eterradossi
Anses, Laboratoire de Ploufragan-Plouzan,
Laboratoire d'tudes et de recherches avicoles,
porcines et piscicoles, B.P. 53, 22440 PloufraganPlouzan, France.
2.4.1. Bovine anaplasmosis
2.4.2. Bovine babesiosis
Prof. T.F. McElwain

Animal Health Research Center, College of
Veterinary Medicine, 155N Bustad Hall, P.O. Box
647034, Pullman, WA 99164-7034, USA.
Dr P.J. Rolls, Dr R.E. Bock, Dr A.J. de Vos &
Dr S.J. Waldron
Tick Fever Centre, Biosecurity Queensland
280 Grindle Road, Wacol, Queensland 4054,
Australia.
Dr I.E. Echaide
Instituto Nacional de Tecnologa Agropecuaria,
Estacin Experimental Agropecuaria Rafaela,
CC22, CP 2300 Rafaela (Santa Fe), Argentina.
2.4.3. Bovine brucellosis4
Dr K. Nielsen (retired)
Canadian Food Inspection Agency, Animal
Diseases Research Institute, P.O. Box 11300,
Station H, Nepean, Ontario K2H 8P9, Canada.
Dr D.R. Ewalt
Pathobiology Laboratory, National Veterinary
Services Laboratories, 1800 Dayton Road, Ames,
Iowa 50010, USA.
2.4.4. Bovine cysticercosis
See chapter 2.9.5.
2.4.5. Bovine genital campylobacteriosis
Prof. J.A. Wagenaar

Utrecht University, Faculty of Veterinary Medicine,
P.O. Box 80.165, 3508 TD Utrecht, The
Netherlands.
Dr M.A.P. Van Bergen
Central Veterinary Institute of Wageningen UR,
P.O. Box 65, 8200 AB Lelystad, The Netherlands.
2.4.6. Bovine spongiform encephalopathy
Dr M.M. Simmons, Mr M.J. Stack, Dr T. Konold &

Dr K. Webster
AHVLA Weybridge, New Haw, Addlestone,
Surrey KT15 3NB, UK.
2.4.7. Bovine tuberculosis
Dr D.V. Cousins (formerly)

Australian Reference Laboratory for Bovine
Tuberculosis, Western Australia Dept of
Agriculture and Food, Locked Bag N 4, Bentley
Delivery Centre, Bentley WA 6983, Australia.
2.4.8. Bovine viral diarrhoea
Dr T. Drew
KT15 3NB, UK.
All four brucellosis chapters were updated by consensus of the OIE ad hoc Group on Brucellosis.
xxviii
Contributors
2.4.9. Contagious bovine pleuropneumonia
Dr F. Thiaucourt
CIRAD-EMVT, Campus international de
Baillarguet, Montferriez-sur-Lez, B.P. 5035, 34032
Montpellier Cedex 1, France.
2.4.10. Dermatophilosis
Dr D. Martinez
CIRAD, Campus International de Baillarguet TAA15 / G, 34398 Montpellier Cedex 5, France.
2.4.11. Enzootic bovine leukosis
Dr T.W. Vahlenkamp
Institute of Virology, Centre for Infectious
Diseases, Faculty of Veterinary Medicine, Leipzig
University, An den Tierkliniken 29, 04103 Leipzig,
Germany.
2.4.12. Haemorrhagic septicaemia
Dr V.P. Singh
Indian Veterinary Research Institute, Izatnagar
243122 U.P., India.
2.4.13. Infectious bovine rhinotracheitis/

infectious pustular vulvovaginitis
Dr M. Beer
Institute of Diagnostic Virology, Friedrich-LoefflerInstitut, Sdufer 10, D-17493 Greifswald-Insel
Riems, Germany.
2.4.14. Lumpy skin disease
Dr E. Tuppurainen
Ash Road, Pirbright, Woking, Surrey GU24 0NF,
UK.
2.4.15. Malignant catarrhal fever
Dr H.W. Reid
Moredun Research Institute, International
Research Centre, Pentlands Science Park, Bush
Loan, Penicuik EH26 0PZ, Scotland, UK.
2.4.16. Theileriosis
Prof. E. Pipano
Koret School of Veterinary Medicine, The Hebrew
University of Jerusalem, P.O. Box 12 Rehovot,
Israel.
Dr S. Morzaria
FAO Regional Office for Asia and the Pacific,
39 Phra Athit Road, Bangkok 10200, Thailand.
Dr P. Spooner
International Livestock Research Institute,
Naivasha Road, Nairobi 00100, Kenya.
2.4.17. Trichomonosis
Dr A.A. Gajadhar
Centre for Food-borne and Animal Parasitology,
Canadian Food Inspection Agency,
116 Veterinary Road, Saskatoon,
Saskatchewan S7N 2R3, Canada.
Dr S. Parker
Large Animal Clinical Sciences, Western College
of Veterinary Medicine,52 Campus Drive,
Saskatoon, Saskatchewan S7N 5B4, Canada.
2.4.18. Trypanosomosis (Tsetse-transmitted)
Dr M. Desquesnes
UMR177-Intertryp (CIRAD-IRD), CIRAD-bios,
Campus international de Baillarguet, TA A-17 / G,
34398 Montpellier Cedex 5, France.
xxix
Contributors
2.5.1. African horse sickness
Prof. J.M. Snchez-Vizcano

Centro de Vigilancia Sanitaria Veterinaria
(VISAVET), Facultad de Veterinaria, HCV Planta
stano, Universidad Complutense de Madrid
(UCM), Avda Puerta de Hierro s/n, 28040 Madrid
Spain.
Dr C. Gmez-Tejedor Ortiz
Ministerio de Agricultura, Pesca y Alimentacin
Laboratorio Central de Veterinaria, LCV-Algete,
Ctra. Algete Km 8, 28110 Algete (Madrid), Spain.
2.5.2. Contagious equine metritis
Mr P. Todd
AHVLA Bury St Edmunds, Rougham Hill, Bury St
Edmunds, Suffolk IP44, 2RX, UK
Dr M.M. Erdman
USA.
H.J. Roest
Central Veterinary Institute of Wageningen UR,
Department of Bacteriology, P.O. Box 65, 8200
AB Lelystad, The Netherlands.
2.5.3. Dourine
Dr J.B. Katz
USA.
2.5.4. Epizootic lymphangitis
Dr J. Picard
Department of Veterinary Tropical Diseases,
Faculty of Veterinary Science, University of
Pretoria, Private Bag X04, Onderstepoort 0110,
South Africa.
2.5.5. Equine encephalomyelitis

2.5.6. Equine infectious anaemia
Dr E.N. Ostlund
USA.
2.5.7. Equine influenza
Prof. Ann Cullinane

Irish Equine Centre, Johnstown, Naas,
Co. Kildare, Ireland.
2.5.8. Equine piroplasmosis
Dr T. de Waal
University College Dublin, School of Agriculture,
Food Science and Veterinary Medicine, Veterinary
Sciences Centre, Belfield, Dublin 4, Ireland.
2.5.9. Equine rhinopneumonitis
Dr G.P. Allen (deceased)

Department of Veterinary Science,
College of Agriculture, University of Kentucky, 108
M.H. Gluck Equine Research Center, Lexington,
Kentucky 40546-0099, USA.
2.5.10. Equine viral arteritis
Dr P.J. Timoney
University of Kentucky, Department of Veterinary
Science, 108 Gluck Equine Research Center,
Lexington, Kentucky 40546-0099, USA.
2.5.11. Glanders
Dr H. Neubauer
Friedrich-Loeffler Institut, Institut fr Bakterielle
Infektionen und Zoonosen, Naumburger Strasse
96a, 07743 Jena, Germany.
xxx
Contributors
2.5.12. Horse mange
See chapter 2.9.8.
2.5.13. Venezuelan equine encephalomyelitis
Dr E.N. Ostlund
USA.
2.6.1. Myxomatosis
Prof. S. Bertagnoli
cole Nationale Vtrinaire, 23 Chemin de
Capelles, BP 87614, 31076 Toulouse Cedex 03,
France.
2.6.2. Rabbit haemorrhagic disease
Dr A. Lavazza & Dr L. Capucci

Istituto Zooprofilattico Sperimentale della
Lombardia e dellEmilia Romagna, Via Bianchi
7/9, 25124 Brescia, Italy.
2.7.1. Border disease
Dr P.F. Nettleton & Dr K. Willoughby

Research Centre, Pentlands Science Park, Bush
Loan, Penicuik EH26 0PZ, Scotland, UK.
2.7.2. Caprine and ovine brucellosis (excluding

Brucella ovis)5
Dr B. Garin-Bastuji
EU Community/OIE & FAO Reference Laboratory
for Brucellosis, Unit Zoonoses Bactriennes,
Anses, 94706 Maisons-Alfort Cedex, France.
Dr J.M. Blasco
Centro de Investigacin y Tecnologa
Agroalimentaria de Aragn (CITAA), Apartado
727, 50080 Zaragoza, Spain.
2.7.3/4. Caprine arthritis/encephalitis & Maedi-visna
Dr D. Knowles & Dr L.M. Herrmann

USDA- ARS, Animal Disease Research Unit,
3003 ADBF, Washington State University,
Pullman, Washington 99164-6630, USA.
2.7.5. Contagious agalactia
Dr R. Nicholas
AHVLA Weybridge, New Haw, Addlestone,
Surrey KT15 3NB, UK.
Dr G.R. Loria
Istituto Zooprofilattico Sperimentale della Sicilia,
Palermo, Italy.
2.7.6. Contagious caprine pleuropneumonia
Dr F. Thiaucourt
CIRAD-EMVT, Campus international de
Baillarguet, Montferriez-sur-Lez, B.P. 5035, 34032
Montpellier Cedex 1, France.
2.7.7. Enzootic abortion of ewes (ovine chlamydiosis)
Dr K. Sachse
Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, Institute of Molecular
Pathogenesis, Naumburger Str. 96a, 07743 Jena,
Germany.
Dr D. Longbottom
Research Centre, Pentlands Science Park
Bush Loan, Penicuik EH26 0PZ, UK.
2.7.8. Nairobi sheep disease
See chapter 2.9.1.
xxxi
Contributors
2.7.9. Ovine epididymitis (Brucella ovis)6
Dr B. Garin-Bastuji
EU Community/OIE & FAO Reference Laboratory
for Brucellosis, Unit Zoonoses Bactriennes,
Anses, 94706 Maisons-Alfort Cedex, France.
Dr J.M. Blasco
Centro de Investigacin y Tecnologa
Agroalimentaria de Aragn (CITAA), Apartado
727, 50080 Zaragoza, Spain.
2.7.10. Ovine pulmonary adenocarcinoma

(adenomatosis)
Dr M.J. Sharp
AHVLA, Lasswade Laboratory, Pentlands Science
Park, Bush Loan, Penicuik EH26 0PZ, Scotland,
UK.
2.7.11. Peste des petits ruminants7
Dr A. Diallo
FAO/IAEA Agriculture and Biotechnology
Laboratory, International Atomic Energy Agency,
A-2444 Seibersdorf, Austria.
2.7.12. Salmonellosis (S. abortusovis)
See chapter 2.9.9.
2.7.13. Scrapie
Dr M.M. Simmons, Mr M. Stack, Dr T. Konold &

Dr D. Matthews
KT15 3NB, UK.
2.7.14. Sheep pox and goat pox
Dr E. Tuppurainen
UK.
2.8.1. African swine fever
Dr C.A.L. Oura (formerly)

Dr M. Arias
Centro de Investigacin en Sanidad Animal
(CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
6
7
8
2.8.2. Atrophic rhinitis of swine
Dr K.B. Register
USDA, ARS, National Animal Disease Center,
2300 Dayton Avenue, Ames, Iowa 50010, USA.
2.8.3. Classical swine fever (hog cholera)
Dr T. Drew
KT15 3NB, UK.
2.8.4. Nipah virus encephalitis
See chapter 2.9.6.
2.8.5. Porcine brucellosis8
Dr S. Olsen,
USDA, ARS, National Animal Disease Center,
2300 Dayton Avenue, Ames, Iowa 50010, USA.
2.8.6. Porcine cysticercosis
See chapter 2.9.5.
This chapter was updated by consensus of the OIE ad hoc Group on Peste des Petits Ruminants.
xxxii
Contributors
2.8.7. Porcine reproductive and respiratory syndrome
Dr L.R. Ludemann
USDA, APHIS, Center for Veterinary Biologics,
Laboratory, P.O. Box 844, Ames, Iowa 50010,
USA.
Dr K.M. Lager
Virus and Prion Diseases of Livestock Research
Unit, National Animal Disease Center, USDAARS, Ames, Iowa 50010, USA.
2.8.8. Swine influenza
Dr S.L. Swenson
National Veterinary Services Laboratories, P.O.
Box 844, Ames, Iowa 50010, USA.
2.8.9. Swine vesicular disease
Dr D. Paton
Dr E. Brocchi,
Istituto Zooprofilattico Sperimentale della e
dell'Emilia Romagna (IZSLER), Via A. Bianchi
No. 9, 25124 Brescia, Italy.
Mr N. Knowles
2.8.10. Teschovirus encephalomyelitis (previously
enterovirus encephalomyelitis or Teschen/Talfan
disease)
UK.
2.8.11. Transmissible gastroenteritis
Dr L.J. Saif
The Ohio State Universtiy, Ohio Agricultural
Research and Development Center, Food Animal
Health Research Program, 1680 Madison
Avenue, Wooster, Ohio 44691-4096, USA.
2.9.1. Bunyaviral diseases of animals (excluding

Rift Valley fever)
Dr G.H. Gerdes (retired)

Onderstepoort Veterinary Institute, Private Bag
X05, Onderstepoort 0110, South Africa.
2.9.2. Camelpox
Dr H. Elliott
International Animal Health Division , DEFRA,
1A Page Street, London SW1P 4PQ, UK.
Dr E. Tuppurainen
2.9.3. Campylobacter jejuni and C. coli
Prof. J.A. Wagenaar

Utrecht University, Faculty of Veterinary Medicine,
P.O. Box 80.165, 3508 TD Utrecht, The
Netherlands.
Dr W.F. Jacobs-Reitsma
RIKILT Institute of Food Safety, Wageningen-UR
P.O. Box 230, 6700 AE Wageningen, The
Netherlands.
2.9.4. Cryptosporidiosis
Prof. H. Smith
Scottish Parasite Diagnostic Laboratory, Stobhill
General Hospital, Glasgow G21 3UW, UK.
2.9.5. Cysticercosis
Dr S. Lloyd
Department of Clinical Veterinary Medicine,
University of Cambridge, Madingley Road,
Cambridge CB3 0ES, UK.
xxxiii
Contributors
2.9.6. Hendra and Nipah virus diseases
Dr P. Daniels
Australian Animal Health Laboratory, CSIRO
Livestock Industries, 5 Portarlington Road,
Geelong, Victoria 3220, Australia.
Dr M. Narasiman
Veterinary Research Institute, 59, Jalan Sultan
Azlan Shah, 31400 Ipoh, Perak, Malaysia.
Dr C.G. Gay
ARS, Research, Education, and Economics,
USDA, 5601 Sunnyside Avenue, Beltsville,
Maryland 20705-5148, USA.
Dr J.A. Roth
Center for Food Security and Public Health,
Institute for International Cooperation in Animal
Biologics
College of Veterinary Medicine, Iowa State
University, Ames, Iowa 50011, USA.
Dr H.M. Weingartl
Special Pathogens Unit, National Centre for
Foreign Animal Disease, Canadian Food
Inspection Agency, 1015 Arlington St., Winnipeg,
MB, R3E 3M4, Canada
2.9.7. Listeria monocytogenes
Dr J. Lopez (retired)
Canadian Food Inspection Agency,
National Centre for Foreign Animal Disease,
1015 Arlington Street, Winnipeg, Manitoba
R3E 3M4, Canada.
2.9.8. Mange
Dr J.L. Schlater & Dr J.W. Mertins

Parasitology and Clinical Pathology Section,
Pathobiology Laboratory, National Veterinary
Services Laboratories, USDA, APHIS, VS, P.O.
Box 844, Ames, Iowa 50010, USA.
2.9.9. Salmonellosis
Dr R. Davies
KT15 3NB, UK.
2.9.10. Toxoplasmosis
Dr D. Buxton & Dr S.W. Maley

Moredun Research Institute, Pentlands Science
Park, Bush Loan, by Edinburgh EH26 0PZ,
Scotland, UK.
2.9.11. Verocytotoxigenic Escherichia coli
Dr F.A. Clifton-Hadley
KT15 3NB, UK.
2.9.12. Zoonoses transmissible from non-human

primates
FELASA Working Group on Non-Human Primate

Health: H. Weber (Convenor), E. Berge, J. Finch,
P. Heidt, F.-J. Kaup, G. Perretta, B. .Verschuere &
S. Wolfensohn
FELASA, BCM Box 2989, London WC1N 3XX,
UK.
Guideline 3.1. Laboratory methodologies for bacterial

antimicrobial susceptibility testing
Dr D. White
US Food and Drug Administration, Center for
Veterinary Medicine, Office of Research, 8401
Muirkirk Road, Laurel, MD 20708, USA.
xxxiv
Contributors
Guideline 3.2. Biotechnology in the diagnosis of

infectious diseases
Dr G. Viljoen, Dr I. Naletoski & Dr A. Diallo

Joint FAO/IAEA Division of Nuclear Techniques in
Food and Agriculture, Department of Nuclear
Sciences and Applications, International Atomic
Energy Agency, A-2444 Seibersdorf, Austria.
Guideline 3.3. The Application of Biotechnology to the

Development of Veterinary Vaccines
Dr A.A. Potter, Dr V. Gerdts, Dr G. Mutwiri,

Dr S. Tikoo & De S. van Drunen Littelvan den Hurk
Vaccine and Infectious Disease Organization,
120 Veterinary Road, Saskatoon S7N 5E3,
Canada.
Guideline 3.4. The role of official bodies in the

international regulation of veterinary biologicals
Dr Ph. Vannier (retired)

Anses Ploufragan, Laboratoire dtudes et de
recherches avicoles et porcines, Zoople des
Ctes dArmor-Les Croix, BP 53, 22440
Ploufragan, France.
Dr R. Hill
Center for Veterinary Biologics, USDA, APHIS,
Veterinary Services, P.O. Box 844, Ames
Iowa 50010, USA.
Dr O. Itoh
National Veterinary Assay Laboratory, JMAFF,
1-15-1 Tokura, Kokubunji, Tokyo 185-8511,
Japan.
Dr C. Lambert
Anses Fougres, Agence nationale du
mdicament vtrinaire, B.P. 203, 35302
Fougres Cedex, France.
xxxv
NB: Ve rsion a dopted by the Worl d A ssembly of De legates of the OIE in May 2012
SECTION 2.5.
EQUIDAE
CHAPTER 2.5.1.
AFRICAN HORSE SICKNESS
SUMMARY
Description of the disease: African horse sickness (AHS) is an infectious but noncontagious viral
disease affecting all species of equidae caused by an Orbivirus of the family Reoviridae and
characterised by alterations in the respiratory and circulatory functions. AHS is transmitted by at
least two species of Culicoides. Nine different serotypes have been described.
All serotypes of AHS occur in eastern and southern Africa. AHS serotypes 9, 4 and 2 have been
found in North and West Africa from where they occasionally spread into countries surrounding the
Mediterranean. Examples of outbreaks that have occurred outside Africa are: in the Middle East
(19591963), in Spain (serotype 9, 1966, serotype 4, 19871990), and in Portugal (serotype 4,
1989).
Laboratory diagnosis of AHS is essential. Although the clinical signs and lesions are characteristic,
they can be confused with those of other equine diseases.
As a viral disease, the laboratory diagnosis of AHS can be based on the identification of infectious
virus, virus nucleic acid, viral antigens or specific antibodies. Over the past few years, a wide
variety of laboratory tests have been adapted for the detection of both AHS virus (AHSV) and
specific antibodies.
Identification of the agent: it is particularly important to perform virus isolation and serotyping
whenever outbreaks occur outside the enzootic regions in order to choose a homologous serotype
for the vaccine.
AHSV can be isolated from blood collected during the early febrile stage. For virus isolation, the
other tissues of choice for diagnosis are spleen, lung, and lymph nodes, collected at necropsy.
Sample preparations can be inoculated in cell cultures, such as baby hamster kidney-21 (BHK-21),
monkey stable (MS), African green monkey kidney (Vero) or insect cells (KC), intravenously in
embryonated eggs, and intracerebrally in newborn mice. Several enzyme-linked immunosorbent
assays (ELISAs) for the rapid detection of AHSV antigen in blood, spleen tissues and supernatant
from infected cells have been developed. Identification of AHSV RNA has also been achieved using
a reverse-transcription polymerase chain reaction method. Virus isolates can be serotyped by a
type-specific serological test such as virus neutralisation (VN) and by reverse-transcription
polymerase chain reaction and sequencing.
Serological tests: Horses that survive natural infection develop antibodies against the infecting
serotype of AHSV within 812 days post-infection. This may be demonstrated by several
serological methods, such as complement fixation test, ELISA, immunoblotting and VN. The latter
test is used for serotyping.
Requirements for vaccines: Attenuated (monovalent and polyvalent) live vaccines for use in
horses, mules and donkeys, are currently commercially available. Subunit vaccines have been
evaluated experimentally.
819
Chapter 2.5.1. African horse sickness
A. INTRODUCTION
African horse sickness (AHS) (Peste equina africana, Peste equine) is an infectious, non-contagious arthopodborne disease of equidae, caused by a double-stranded RNA Orbivirus belonging to the family Reoviridae. The
genus Orbivirus also includes bluetongue virus and epizootic haemorrhagic disease virus, which have similar
morphological and biochemical properties with distinctive pathological and antigenic properties as well as host
ranges. Nine antigenically distinct serotypes of AHSV have been identified by virus neutralisation; some crossreaction has been observed between 1 and 2, 3 and 7, 5 and 8, and 6 and 9, but no cross-reactions with other
known orbiviruses occur.
The virion is an unenveloped particle of a size around 70 nm. The genome of AHS virus (AHSV) is composed of
ten double-stranded RNA segments, which encode seven structural proteins (VP1-7), most of which have been
completely sequenced for AHSV serotypes 4, 6 and 9 (Roy et al., 1991; Venter et al., 2000; Williams et al., 1998)
and four nonstructural proteins (NS1, NS2, NS3, NS3A) (Grubman & Lewis, 1992; Laviada et al., 1993). Proteins
VP2 and VP5 form the outer capsid of the virion, and proteins VP3 and VP7 are the major inner capsid proteins.
Proteins VP1, VP4 and VP6 constitute minor inner capsid proteins. The NS3 proteins are the second most
variable AHSV proteins (Van Niekert et al., 2001), the most variable being the major outer capsid protein, VP2.
This protein, VP2, is the determinant of AHSV serotypes and, together with VP5, the target for virus neutralisation
activity (Martinez-Torrecuadrada et al., 2001). At least two field vectors are involved in the transmission of the
virus: Culicoides imicola and C. bolitinos.
AHS is enzootic in sub-Saharan Africa, although occasional outbreaks have occurred in northern Africa (1965,
19891990), the Middle East (19591961), and in Europe (Spain, 1966, 19871990 and Portugal, 1989)
(Sanchez-Vizcano, 2004).
There are four classical clinical forms of AHS: pulmonary, cardiac, mixed, and horse sickness fever. The
peracute, pulmonary form occurs in fully susceptible animals and has a short course, often only a few hours, and
a high mortality rate. The animal exhibits respiratory distress, an extended head and neck, and profuse sweating.
Terminally, froth exudes from the nostrils. The cardiac, oedematous form has a more subacute course with
mortality reaching 50%. The head and neck may show severe swelling that can extend down to the chest.
Swelling of the supraorbital fossae is characteristic and may include conjunctival swelling with petechiae.
Paralysis of the oesophagus may result in aspiration pneumonia and sublingual haemorrhages are always a poor
prognostic sign. The mixed, acute form is most commonly seen and has feature of both the cardiac and
pulmonary forms. Mortality can reach 70%. Horsesickness fever is an often overlooked, mild form of the disease
and is seen in resistant equidae such as zebra and donkeys (Coetzer & Guthrie, 2005).
The disease has both a seasonal (late summer/autumn) and a cyclical incidence with major epizootics in southern
Africa during warm-phase events (Baylis et al., 1999). Mortality due to AHS is related to the species of equidae
affected and to the strain or serotype of the virus. Among equidae, horses are the most susceptible to AHS with a
mortality rate of 5095%, followed by mules with mortality around 50%. In enzootic regions of Africa, donkeys are
very resistant to AHS and experience only subclinical infections. In European and Asian countries, however,
donkeys are moderately susceptible and have a mortality rate of 10%. Zebras are also markedly resistant with no
clinical signs, except fever, and may have extended viraemia (up to 40 days).
A laboratory diagnosis is essential to establish a correct and confirmatory diagnosis. Although some clinical signs
and lesions are characteristic, AHS can be confused with other diseases. For example, the supraorbital swelling,
which is often present in horses with subacute AHS, is, in combination with an appropriate history, sufficient for a
tentative diagnosis. Other signs and lesions are less specific for AHS, and other diseases such as equine
encephalosis, equine infectious anaemia, equine morbillivirus pneumonia, equine viral arteritis, babesiosis and
purpura haemorrhagica should be excluded (OIE Technical Disease Cards: http://www.oie.int/en/animal-health-inthe-world/technical-disease-cards/).
Attenuated (monovalent and polyvalent) live vaccines for use in horses, mules and donkeys, are currently
commercially available.
There is no evidence that humans can become infected with any field strain of AHSV, either through contact with
naturally or experimentally infected animals or by virus manipulation in laboratories.
B. DIAGNOSTIC TECHNIQUES
Several techniques are already available for AHS viral identification ranging from the rapid capture (indirect
sandwich) enzyme-linked immunosorbent assay (ELISA), using either polyclonal antibodies (PAbs) or monoclonal
antibodies (MAbs), to the polymerase chain reaction (PCR) test, including a new reverse-transcription (RT) PCR
for discrimination of the nine AHSV serotypes or cell culture and inoculation of newborn mice. If possible more
than one test should be performed to diagnose an outbreak of AHS, especially the index case. The initial test can
820
be a quick test such as ELlSA or PCR, followed by virus isolation in tissue culture. Virus neutralisation (VN) for
serotype identification or RT-PCR with sequencing should be performed as early in the outbreak as possible so
that the serotype can be identified and the correct vaccine selected.
At present, there are no international standards for viruses or diagnostic reagents, and there is no standard
methodology for the identification of AHSV. However, a viral and antibody panel has been evaluated, and
comparative studies between different ELISAs for AHSV antigen and antibody determination have been carried
out in different laboratories, including in the EU Reference Laboratory for AHS. The results have demonstrated a
high level of correlation for both antigen and antibody determination with an in-house test and commercial kits
(Rubio et al., 1998; Villalba, 2009). Similar studies have been conducted with several RT-PCR assays (Aquero,
2009) also providing a high level of correlation.
A very important aspect of the diagnosis is the selection of samples and their safe transportation to the laboratory.
1.
Identification of the agent
a)
Virus isolation
Unclotted whole blood collected during the early febrile stage of the disease from sick animals, as well as
small pieces (24 g) of spleen, lung and lymph nodes from animals that have died, are the samples of
choice for diagnosis. Samples should be kept at 4C during transportation and short-term storage prior to
processing.
Cell culture
Successful direct isolation of AHSV has been performed on baby hamster kidney (BHK-21), monkey stable
(MS) and African green monkey kidney (Vero) mammalian cell lines and on Culicoides and mosquito insect
cell lines. Blood samples collected in an appropriate anticoagulant can be used undiluted as the inoculum.
After 1560 minutes of adsorption at ambient temperature or at 37C, the cell cultures are washed and
maintenance medium is added. Alternatively and more commonly, the blood is washed, lysed and diluted
1/10. This procedure removes unwanted antibody, which could neutralise free virus, and promotes release
of virus associated with the red blood cell membranes. When tissue samples, such as spleen, lung, etc., are
used, a 10% tissue suspension is prepared in phosphate buffered saline (PBS) or cell culture medium,
containing antibiotics.
A cytopathic effect (CPE) may appear between 2 and 10 days post-infection with mammalian cells. Three
blind passages should be performed before considering the samples to be negative. No CPE is observed in
insect cells but the presence of the virus can be detected in the supernatant after 57 days by real-time RTPCR. Supernatant from infected insect cells can then be passed onto mammalian cells, which will show CPE
after one or two passages.
Newborn mice
This method of isolation of AHSV involves the intracerebral inoculation of two families of 13-day-old mice.
In positive cases, animals develop nervous signs between 3 and 15 days post-inoculation. The brains from
sick animals may be collected, homogenised and re-inoculated intracerebrally into at least six 13-day-old
mice. This second passage should present a shortened incubation period (25 days) and 100% mortality.
Virus may be typed directly from mouse brain by conventional neutralisation (VN) or by RNA extraction and
sequencing.
b)
Nucleic acid methods
Reverse-transcription polymerase chain reaction (an alternative test for international trade)
The reverse-transcription polymerase chain reaction (RT-PCR) is a highly sensitive technique that allows the
detection of a very low number of copies of RNA molecules, but this does not necessarily indicate the
presence of infectious virus. The RNA strand is first reverse transcribed into its complementary DNA
(cDNA), followed by amplification of the resulting DNA using polymerase chain reaction. This technique has
greatly improved the detection of AHSV-RNA by improving the sensitivity of detection and shortening the
time required for the diagnosis.
Several agarose gel-based RT-PCR assays for the specific detection of AHSV RNA have been described
targeted at viral segments 3, 7 or 8 (Aradaib, 2009; Bremer et al., 1998; Laviada et al., 1997; Sakamoto et
al., 1994; Stone-Marschat et al., 1994; Zientara et al., 1994). The most widely used method employs primers
corresponding to the 5 end (nucleotides 121) and 3 end (nucleotides 11601179) of RNA segment 7
amplifying the complete viral segment.
821
Real-time RT-PCR (rRT-PCR) methods for the highly sensitive and specific detection of AHSV RNA have
been recently developed based on the use of a pair of primers and a Taqman probe from conserved
sequences of viral segments 5 or 7 (Agero et al.. 2008; Fernndez-Pinero et al., 2009; Rodriguez-Sanchez
et al., 2008). A duplex RRT-PCR has also been described that targets segments 8 and 9 of the genome and
uses Taqman probes (Quan et al., 2010). Although both gel-based and rRT-PCR procedures can detect
reference strains from the nine virus serotypes, rRT-PCR provides advantages over agarose gel-based RTPCR methods, with its faster analysis time, higher sensitivity, and suitability for high-throughput automation.
Nevertheless gel-based RT-PCR methods, particularly those amplifying long RNA fragments (Laviada et al.,
1997; Zientara et al., 1994), can be very useful in the further genetic characterisation of the virus by
sequencing of the amplicons.
Details of AHSV agarose gel-based RT-PCR and real-time RT-PCR are given below.
Extraction of viral RNA
To assure a good reaction it is necessary to extract from the sample an AHSV RNA of high quality. The
extraction of nucleic acids from clinical samples can be performed by a variety of in-house and commercially
available methods.
An example of an in-house RNA extraction is given below:
i)
1 g of tissue sample is homogenised in 1 ml of denaturing solution (4 M guanidium thiocyanate, 25 mM

sodium citrate, 0.1 M 2-mercaptoethanol, 0.5% sarcosyl).
ii)
After centrifugation, 1 g of yeast RNA, 0.1 ml of 2 M sodium acetate pH 4, 1 ml of phenol and 0.2 ml
of chloroform/isoamyl alcohol mixture (49/1) are added to the supernatant.
iii)
The suspension is vigorously shaken and cooled on ice for 15 minutes.
iv)
After centrifugation, the RNA present in the aqueous phase is phenol extracted, ethanol precipitated
and resuspended in sterile water
Commercial kits use different approaches for RNA isolation. Most are based on one of the following
procedures:
Phenolchloroform extraction of nucleic acids;
Adsorption of nucleic acids to filter system;
Adsorption of nucleic acids to magnetic beads system.
Agarose gel-based RT-PCR procedure (Laviada et al., 1997; Zientara et al., 1994)
Denaturation of extracted RNA has to be performed prior to the RT-PCR procedure. The sequences of the
PCR primers used are 5-GTT-AAA-ATT-CGG-TTA-GGA-TG-3, which corresponds to the messenger RNA
polarity, and 5-GTA-AGT-GTA-TTC-GGT-ATT-G-3, which is complementary to the messenger RNA
polarity. All the components required for the reverse transcription and PCR are included in the reaction tube
containing the denatured RNA. A one-step RT-PCR is carried out by incubating in a thermocycler as follows:
45 minutes to 1 hour at 3755C, 510 minutes at 95C, then 40 cycles of: 9495C for 1 minute, 55C for
11.5 minutes, 7072C for 22.5 minutes, followed by a final extension step of 78 minutes at 7072C.
Analysis of the PCR products is carried out by agarose gel electrophoresis. AHS-positive samples will
resolve in a 1179 base-pair band that can be used as template in the sequencing reaction, using
independently the PCR primers, for obtaining the nucleotide sequence of viral segment 7.
Real-time RT-PCR Procedure (Agero et al., 2008)

This group-specific real-time RT-PCR has been employed with good results by the participating national
reference laboratories of the European Union (EU) Member States in a proficiency test organised by the EU
reference laboratory for AHS (Agero, 2009). The rRT-PCR is carried out as follows:
822
i)
2 l of isolated RNA is mixed with forward (5-CCA-GTA-GGC-CAG-ATC-AAC-AG-3) and reverse (5CTA-ATG-AAA-GCG-GTG-ACC-GT-3) primers (2.5 l of each primer at 8 M) and RNAse-free water
up to 7 l.
ii)
This mixture is denatured by heating at 95C for 5 minutes, followed by rapid cooling on ice.
iii)
cDNA synthesis and hot-start PCR amplification are carried out in one-step, in a volume of 20 l
containing the denatured RNA-primers mixture, 0.1 l of the fluorogenic MGB-TaqMan probe (5-FAMGCT-AGC-AGC-CTA-CCA-CTA-MGB-3, probe was labelled in 5 with 6-carboxyfluorescein, FAM, and
in 3 with a non-fluorescent quencher bound to an MGB group) at 50 M (final concentration: 0.25 M),
adequate buffer and enzymes (RTase and DNA polymerase at a concentration recommended by the
manufacturer).
iv)
Amplification conditions consist of a first reverse-transcription step at 48C for 25 minutes, followed by
10 minutes at 95C (hot start) and 40 cycles of 15 seconds at 95C, 35 seconds at 55C and
30 seconds at 72C (or 40 cycles at 97C for 2 seconds and 55C for 30 seconds if reagents and
thermocycler allowing fast reactions are used). Fluorescence data are acquired at the end of the 55C
step.
v)
Samples are considered positives if the fluorescence increases significantly over the base level. If no
fluorescence is detected during the whole real-time RT-PCR, samples are considered negative.
Inactivated virus of serotypes 1-9 reference strains can be obtained from the OIE Reference Laboratory in
Spain in order to set up the RT-PCR detection method.
c)
AHSV typing
Until recently, the VN test has been the method of choice for typing as well as the gold standard test for
identifying AHSV isolated from the field using type-specific antisera (Verwoerd, 1979). This technique takes
5 or more days before results are obtained. The development of type-specific gel-based RT-PCR (Sailleau
et al., 2000), and real-time RT-PCR using hybridisation probes (Koekemoer, 2008) for identification and
differentiation of AHSV genotypes, provides a rapid typing method for AHSV in tissue samples and blood.
There is a good correlation between the results obtained with the type-specific RT-PCR and the VN test,
however, the sensitivity of these assays is lower than that obtained with the diagnostic group-specific realtime RT-PCR (Agero et al., 2008). Type-specific rRT-PCR assays based on the use of Taqman-MGB
probes have been developed more recently (Tena, 2009) and have a similar sensitivity to the group-specific
rRT-PCR. However, the genetic variation that may appear over time in the AHSV genome, in particular in
the VP2 coding region, where specific primers/probes for typing assays have to be designed, makes the
detection of all genetic variants within each serotype by this type of technique difficult (Koekemoer, 2008).
Therefore, although molecular methods are able to rapidly type AHSV in many positive field samples, VN
should be kept as the gold standard for serotyping AHSV isolates.
Typing of the nine AHSV types has also been carried out with probes developed from a set of cloned fulllength VP2 genes (Koekemoer et al., 2000). This technique can be used as an alternative to PCR
amplification of genome segment 2.
2.
Serological tests
Indirect and competitive blocking ELISAs using either soluble AHSV antigen or a recombinant protein VP7
(Hamblin et al., 1990; Laviada et al., 1992b; Maree & Paweska, 2005) have proved to be good methods for the
detection of anti-AHSV group-reactive antibodies, especially for large-scale investigations (Rubio et al., 1998).
Both of these tests have been recognised by the European Commission (2002). The competitive blocking ELISA
can also be used for testing wildlife as species-specific anti-globulin is not required with this method. An
immunoblotting test has also been adapted for anti-AHS antibody determination (Laviada et al., 1992b), which is
especially suitable for small numbers of sera. The complement fixation (CF) test has been widely used, but some
sera are anti-complementary, particularly donkey and zebra sera.
a)
Blocking enzyme-linked immunosorbent assay (a prescribed test for international trade)

The competitive blocking ELISA technique aims at detecting specific antibodies against AHSV, present in
any equine species. VP7 is the main antigenic protein within the molecular structure of AHSV and it is
conserved across the nine AHSV serotypes. An MAb directed against VP7 is used in this test, allowing high
sensitivity and specificity. Moreover, other species apart from horses (e.g. donkeys, zebra, etc.) can be
tested thus preventing the problem of specificity experienced occasionally using the indirect ELISAs. VP7
recombinant antigen is non-reactive, which provides a high level of security (European Commission, 2002).
The principle of this test is to block the specific reaction between the recombinant VP7 protein absorbed on
an ELISA plate and a conjugated MAb against VP7. AHSV antibodies present in a suspect serum sample
will block this reaction. A decrease in the amount of colour is evidence of the presence of AHSV antibodies
in the serum sample.
Test procedure
i)
Solid phase: coat ELISA plates (e.g. high adsorption capacity Nunc Maxisorb) with 50100 ng of
recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9.6. Incubate overnight at 4C.
823
ii)
Wash the plates three times with PBS 0.1 containing 0.135 M NaCl and 0.05% (v/v) Tween 20
(washing solution). Gently tap the plates on to absorbent material to remove any residual wash.
iii)
Test samples: serum samples to be tested, and positive and negative control sera (if not ready to use
by kit manufacturer), are diluted 1/5 in diluent containing 0.35 M NaCl, 0.05% Tween 20; and
0.1% Kathon, 100 l per well. Incubate for 1 hour at 37C.
iv)
Wash the plates five times with PBS 0.1 containing 0.135 M NaCl and 0.05% (v/v) Tween 20
(washing solution). Gently tap the plates on to absorbent material to remove any residual wash.
v)
Conjugate: dispense 100 l/well of horseradish peroxidise-conjugated MAb anti-VP7. This MAb should
be previously diluted 1/50001/15000 in a 1/1 solution of StabiliZyme Select Stabilizer (SurModics.
Reference: SZ03) in distilled water. Incubate for 30 minutes at 37C.
vi)
Wash the plates as described in step iv.
vii)
Substrate/chromogen: add 100 l/well of 1/10 diluted ABTS substrate solution, 5 mg/ml substrate
buffer (0.1 M phosphate/citrate buffer, pH 4, containing 0.03% H2O2) and incubate for 10 minutes at
room temperature. Colour development is stopped by adding 100 l/well of 2% (w/v) of SDS.
viii) Read the plates at 405 nm.

ix)
Interpretation of results: determine the blocking percentage (BP) of each sample by applying the
following formula:
BP=
Abs(control) Abs(sample)
Abs(control) Abs(control+)
100
Samples showing BP value lower than 45% are considered negative for antibodies to AHSV. Samples
showing BP value higher than 50% are considered positive for antibodies to AHSV. Samples with BP value
between 45% and 50% are considered doubtful and must be retested. If the result is the same, resample
and test 2 weeks later.
b)
Indirect enzyme-linked immunosorbent assay (a prescribed test for international trade)

The recombinant VP7 protein has been used as antigen for AHSV antibody determination with a high degree
of sensitivity and specificity (Laviada et al., 1992b; Wade-Evans et al., 1993). Other advantages of this
antigen are its stability and its lack of infectivity. The conjugate used in this method is a horseradish
peroxidase anti-horse gamma-globulin reacting with horse, mules and donkeys. The method described by
Maree & Paweska (2005) uses protein G as conjugate that also reacts with zebra serum.
Test procedure
i)
Solid phase: Coat ELISA plates (e.g. high adsorption capacity Nunc Maxisorb) with recombinant
AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9.6. Incubate overnight at 4C.
ii)
Wash the plates five times with distilled water containing 0.01% (v/v) Tween 20 (washing solution).
Gently tap the plates on to absorbent material to remove any residual wash.
iii)
Block the plates with PBS, pH 7.2 + 5% (w/v) skimmed milk, 200 l/well, for 1 hour at 37C.
iv)
Remove the blocking solution and gently tap the plates on to absorbent material.
v)
Test samples: Serum samples to be tested, and positive and negative control sera, are diluted 1/25 in
PBS + 5% (w/v) skimmed milk + 0.05% (v/v) Tween 20, 100 l per well. Incubate for 1 hour at 37C.
For titration, add twofold dilution series from 1/25 (100 l/well), one serum per plate column, and do the
same with positive and negative controls. Incubate for 1 hour at 37C.
vi)
Wash the plates as described in step ii.
vii)
Conjugate: Dispense 100 l/well of horseradish peroxidase conjugated anti-horse gamma-globulin

diluted in PBS + 5% milk + 0.05% Tween 20, pH 7.2. Incubate for 1 hour at 37C or protein A
peroxidase (Maree & Paweska, 2005).
viii) Wash the plates as described in step ii.

ix)
824
Substrate: Add 200 l/well of substrate solution (10 ml DMAB + 10 ml of MBTH + 5 l H2O2). Colour
development is stopped by adding 50 l of 3 N H2SO4 after approximately 510 minutes (before the
negative control begins to be coloured). Other substrates such as ABTS (2,2-azino-di-[3-ethylbenzothiazoline]-6-sulphonic acid), TMB (tetramethyl benzidine), or OPD (orthophenyldiamine) can
also be used.
c)
x)
Read the plates at 600 nm (or 620 nm).
xi)
Interpretation of results: Calculate the cut-off value by adding 0.6 to the value of the negative control.
(0.06 is the standard deviation derived with a group of 30 negative sera) Test samples giving
absorbance values lower than the cut-off are regarded as negative. Test samples giving absorbance
values greater than the cut-off + 0.15 are regarded as positive. Test samples giving intermediate
absorbance values are doubtful and a second technique must be employed to confirm the result.
Complement fixation (a prescribed test for international trade)

The CF test has been used extensively in the past, but currently its use is decreasing and has been replaced
in many laboratories by ELISA as a screening technique. This progressive replacement is because of the
higher sensitivity and degree of standardisation of ELISA as well as a significant number of sera with anticomplementary activity. Nevertheless the CF test is a useful tool in endemic areas for the demonstration and
titration of group-specific IgM antibodies against AHSV notably following a recent infection or vaccination.
Reagents
i)
Veronal buffered saline containing 1% gelatin (VBSG).
ii)
Serum samples, free from erythrocytes, must be heat inactivated: horse serum at 56C, zebra serum at
60C and donkey serum at 62C, for 30 minutes.
iii)
The antigen is a sucrose/acetone extract of AHSV-infected mouse brain. The control antigen is
uninfected mouse brain, extracted in the same way. In the absence of an international standard serum,
the antigen should be titrated against a locally prepared positive control serum. In the test, four to eight
units are used. The antigen may also be obtained by inoculation of the virus in suitable cell culture (see
Section B.1 above).
iv)
The complement is a normal guinea-pig serum.
v)
The haemolysin is a hyperimmne rabbit serum against sheep red blood cells (SRBCs).
vi)
The SRBCs are obtained by aseptic puncture of the jugular vein and preserved in Alsevers solution1 or
sodium citrate.
vii)
The haemolytic system (HS) is prepared by diluting the haemolysin to contain two haemolytic doses
and using this to sensitise washed SRBCs. The SRBCs are standardised to a 3% concentration.
viii) Control sera: A positive control serum is obtained locally and validated. Serum from a healthy antibodynegative horse is used as the negative control serum.
Test procedure
i)
The reaction is performed in 96-well round-bottom microtitre plates in a final volume of 100 l/well or in
tubes if the macro-technique is used, at 4C for 18 hours.
ii)
All the sera, samples and controls are diluted 1/5 in VBSG and 25 l of each serum is added in
duplicate. A twofold dilution series of each serum is done from 1/5 to 1/180.
iii)
Add 25 l of the antigen diluted according to the previous titration.
iv)
Add 25 l of the complement diluted according to a previous titration.
v)
Incubate at 4C for 18 hours.
vi)
25 l of HS is added to all wells on the microtitre plate.
vii)
The plate is incubated for 30 minutes at 37C.
viii) Plates are then centrifuged at 200 g, and the wells are scored for the presence of haemolysis. Control
of sera, complement, antigen and HS are used
ix)
Results are read using 50% haemolysis as the end point. The inverse of the highest dilution of serum
specifically fixing complement with the CF antigen is called the titre.
x)
A titre of 1/10 or more is positive, under 1/10 is negative.
20.5 g dextrose (114 mM), 7.9 g sodium citrate 2H2O (27 mM), 4.2 g NaCl (71 mM), H2O to 1 litre. Adjust to pH with 1 M
citric acid.
825
d)
Virus neutralisation (VN)

Serotype-specific antibody can be detected using the VN test (House et al., 1990). The VN test may have
additional value in epidemiological surveillance and transmission studies, mainly in endemic areas where
multiple serotypes are likely to be present.
VN Test procedure
i)
Stock virus is diluted to yield 100 TCID50 (50% tissue culture infective dose), with a range of 30
300 TCID50, per 25 l, and 25 l is added to each of four microtitre wells containing 25 l serum
dilutions. For screening, a final serum dilution of 1/10 is used. Doubling dilutions are used for titrations.
ii)
Serum/virus mixtures are incubated for 60 minutes at 37C 5% CO2 and 95% humidity prior to the
addition of 0.1 ml of Vero cell suspension (200,000 cells/ml) to each test well.
iii)
A back titration of virus stock is prepared for each test using four wells per tenfold dilution, 25 l per
well. Test plates are incubated at 37C, 5% CO2, 95% humidity for 45 days, until the back titration
indicates that the stock virus contains 30300 TCID50.
iv)
The plates are then fixed and stained in a solution of 0.15% (w/v) crystal violet in 2% (v/v)
glutaraldehyde and rinsed. Alternatively, they may be fixed with 70% ethanol and stained with 1% basic
fuschsin.
v)
The 50% end-point titre of the serum is calculated by the SpearmanKrber method and expressed as
the negative log10.
C. REQUIREMENTS FOR VACCINES

1.
Background
a)
Rationale and intended use of the product

Polyvalent or monovalent live attenuated AHS vaccines, based on the selection in Vero cell culture of
genetically stable macroplaques, have been used for the control of AHSV in and out of Africa (Erasmus,
1976; Sanchez-Vizcano, 2004). Polyvalent vaccines are commercially available.
An inactivated monovalent (serotype 4) AHSV vaccine based on virus purification and inactivation with
formalin was produced commercially in the early 1990s (House et al., 1992), but is not available at the
present time. Subunit AHSV vaccines based on serotype 4 outer capsid protein VP2 and VP5 plus inner
capsid protein VP7, derived from single and dual recombinant baculovirus expression vectors have been
used experimentally in different combinations to immunise horses (Martinez et al., 1996). The protective
efficacy of VP2 in a subunit vaccine was also evaluated (Scanlen et al., 2002). However, these vaccines are
not commercially available.
2.
Outline of production and minimum requirements for conventional vaccines
At present only the live attenuated AHS vaccines (polyvalent or monovalent) are commercially available.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.6 Principles of veterinary vaccine
production. The guidelines given here and in chapter 1.1.6 are intended to be general in nature and may be
supplemented by national and regional requirements.
Live attenuated African horse sickness vaccine
a)
Characteristics of the seed

i)
Biological characteristics
The seed virus is prepared by selection in Vero cells of genetically stable large plaques from low
passage levels of AHSV. The plaque mutants are then further multiplied by three passages in Vero
cells. A large quantity of this antigen is lyophilised and stored at 20C as seed stock antigen.
ii)
Quality criteria
The seed virus must be shown to be free of contaminating viruses, bacteria and mycoplasmas by the
appropriate techniques. The serotype identity of the seed virus is confirmed.
826
b)
Method of manufacture
i)
Procedure
At the onset of a production run, working antigens are produced from the seed stock antigen in either
BHK-21 or Vero cell cultures. The working antigens are tested for sterility, purity and identity and
should contain at least 1 106 plaque-forming units (PFU)/ml of infectious virus.
ii)
Requirements for substrates and media

Roller bottle cultures of Vero or BHK-21 cells are grown using gamma-irradiated bovine serum in the
growth medium. Once the cultures are confluent, the medium is poured off and the cells are seeded
with the working antigens. After 1 hour, maintenance medium is added to the cultures. Incubation is
continued at 37C for 23 days. When the CPE is advanced, both cells and supernatant medium are
harvested. The products from the same serotype are pooled and stored at 4C.
iii)
In-process control
The pooled harvests of the individual serotypes are tested for sterility and assayed for infectivity by
plaque titration on Vero cell cultures. The minimum acceptable titre is 1 106 PFU/ml.
Finally, two quadrivalent vaccines are constituted by mixing equal volumes of serotypes 1, 3, 4, 5 and
2, 6, 7, 8 respectively. Subsequently, AHSV serotype 5 was withdrawn from this vaccine. A monovalent
type can also be prepared. After addition of suitable stabiliser, the vaccine is distributed in 1.0 ml
volumes into glass vials and freeze-dried.
iv)
Final product batch test

Sterility
Following lyophilisation, five bottles of vaccine are selected at random and tested for sterility by
internationally accepted methods. Tests for sterility and freedom from contamination of biological
products are given in chapter 1.1.7.
Safety
Innocuity of a vaccine is determined by the inoculation of reconstituted vaccine into mice (0.25 ml
intraperitoneally), guinea-pig (1.0 ml intraperitoneally), and a horse (5.0 ml subcutaneously). All the
animals are observed daily for 14 days. The rectal temperature of the horse is taken twice daily for
14 days and should never exceed 39C.
Batch potency
Potency is largely based on virus concentration in the vaccine.
The minimum immunising dose for each serotype is about 1 103 PFU/dose. The infectivity titre of the
final product is assayed by plaque titration in Vero cell cultures and should contain at least 1
105 PFU/dose. The horse used for safety testing is also used for determining the immunogenicity of a
vaccine.
Serum samples are collected on the day of vaccination and 21 days later, and are tested for
neutralising antibodies against each serotype by the plaque-reduction test using twofold serum
dilutions and about 100 PFU of virus. The horse should develop a neutralising antibody titre of at least
20 against at least three of the four serotypes in the quadrivalent vaccine.
c)
Requirements for authorisation

No specific guideline is described for AHS vaccine. However a guideline is described in the EU for
Bluetongue virus under exceptional circumstances that could probably be used for AHS virus. This guideline
includes the minimum date requirements for the authorisation under exceptional circumstances for vaccine
production for emergency use against bluetongue virus (Regulation EC N726/2004, in particular Articles 38,
39 and 43 thereof and Article 26 of Direction 2001/82/EC), including guidance measures to facilitate the
rapid inclusion of new or different virus serotypes.
3.
Vaccines based on biotechnology
a)
Vaccines available and their advantages

None is available commercially. Experimental subunit vaccines have been described (Section C.1.a above).
827
b)
Special requirements for biotechnological vaccines, if any

None.
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horse sickness virus. J. Virol. Methods, 167, 4552.
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(2008). Novel gel-based and real-time PCR assays for the improved detection of African horse sickness virus. J. Virol.
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HAMBLIN C. (1998). Validation of ELISA for the detection of African horse sickness virus antigens and antibodies. Arch.
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sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. J. Gen. Virol., 81, 831
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horsesickness subunit vaccine candidate is determined by adjuvant. Vaccine, 20, 10791088.
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African Horse Sickness (AHS) Reference Laboratories. Brussels, Belgium, 1 December 2009.
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H., Wagner R.R., eds. Plenum Press, London, UK, Vol. 14, 285345.
829
VILLALBA R. (2009). Results of the AHS Interlaboratory comparison test 2009: RT-PCR. Annual Meeting of EC
National African Horse Sickness (AHS) Reference Laboratories. Brussels, Belgium, 1 December 2009.
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antigen, synthesised in bacteria, and anti-VP7 monoclonal antibodies in a competitive ELISA. J. Virol. Methods,
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WILLIAMS C.F., INOUE T., LUCUS A.M., ZANOTTO P.M. & ROY Y.P. (1998). The complete sequence of four major
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*
* *
NB: There are OIE Reference Laboratories for African horse sickness
(see Table in Part 4 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list:
http://www.oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories/ ).
Please contact the OIE Reference Laboratories for any further information on
diagnostic tests, reagents and vaccines for African horse sickness
830
CHAPTER 2.5.2.
CONTAGIOUS EQUINE METRITIS
SUMMARY
Definition of the disease: Contagious equine metritis is an inflammatory disease of the proximal
and distal reproductive tract of the mare caused by Taylorella equigenitalis, which usually results in
temporary infertility. It is a nonsystemic infection, the effects of which are restricted to the
reproductive tract of the mare.
When present, clinical signs include endometritis, cervicitis and vaginitis of variable severity and a
slight to copious mucopurulent vaginal discharge. Recovery is uneventful, but prolonged
asymptomatic or symptomatic carriage is established in a proportion of infected mares. Direct
venereal contact during natural mating presents the highest risk for the transmission of
T. equigenitalis from a contaminated stallion or an infected mare. Direct venereal transmission can
also take place by artificial insemination using infective raw, chilled and possibly frozen semen.
Indirectly, infection may be acquired through fomite transmission, manual contamination,
inadequate observance of appropriate biosecurity measures at the time of breeding and at semencollection centres. Stallions can become asymptomatic carriers of T. equigenitalis. The principal
sites of colonisation by the bacterium are the urogenital membranes (urethral fossa, urethral sinus,
terminal urethra and penile sheath). The sites of persistence of T. equigenitalis in the majority of
carrier mares are the clitoral sinuses and fossa and infrequently the uterus. Foals born of carrier
mares may also become carriers. The organism can infect equid species other than horses, e.g.
donkeys.
Identification of the agent: To avoid loss of viability, individual swabs should be fully submerged
in Amies charcoal medium and transported to the testing laboratory under temperature-controlled
conditions for plating out within 48 hours of collection. Growth of T. equigenitalis is likely to take 3
6 days and may take up to 14 days, but usually does not take longer than 6 days at 37C on
specialised media in an atmosphere of 510% CO2. An incubation time of at least 7 days is
advisable before certifying cultures negative for T. equigenitalis. Identification should include
biochemical characterisation, antigenic testing using specific antibodies and molecular genotyping.
The fastidious nature of T. equigenitalis makes it difficult to isolate and test-breeding of stallions for
detection of the carrier state has been used as a valuable adjunct to cultural examination.
Another species of Taylorella, T. asinigenitalis, has been isolated from male donkeys and horse
mares and stallions in the United States of America and a number of European countries. This
bacterium has not been associated with naturally occurring disease; it has been detected in the
genital tract of male donkeys and would appear to be passed to other donkeys and horses during
mating.
Serological tests: Serology is of value in detecting recent, but not chronic, infection in the mare.
Serum antibody to T. equigenitalis can be detected in mares for 37 weeks after infection. It may
also be demonstrated in the occasional carrier mare, but never in the stallion. No individual
serological test described to date has been shown reliably to detect infection. Serological tests can
be used as an adjunct to culture for T. equigenitalis in screening mares recently bred to a carrier
stallion, but must not be used as a substitute for culture.
Requirements for vaccines: Effective vaccines are not yet available.
A. INTRODUCTION
Contagious equine metritis was first described in the United Kingdom (UK) in 1977 (Crowhurst, 1977), after which
it was diagnosed in a number of countries world-wide. It first presented as disease outbreaks characterised by a
831
Chapter 2.5.2. Contagious equine metritis
mucopurulent vaginal discharge originating from inflammation of the endometrium and cervix, resulting in
temporary infertility. The fastidious growth characteristics and slow growth of the causative bacterium, Taylorella
equigenitalis, caused difficulties in initial attempts at culture (Platt et al., 1977), but the disease was reproduced by
experimental challenge with isolated laboratory-grown bacteria (Platt et al., 1978; Swaney & Sahu 1978). Using
appropriate culture conditions, T. equigenitalis can be isolated from infective vaginal discharge. Mares may
experience more than one episode of the disease in a short period of time (Timoney et al., 1977; 1979). Serum
antibody persists for 37 weeks after infection, but often it is not detectable for up to 1521 days after recovery
from acute infection in the mare (Dawson et al., 1978). Most mares recover uneventfully, but some may become
carriers of T. equigenitalis for many months (Platt et al., 1978). Colonisation by T. equigenitalis in the mare is
most consistently demonstrated by swabbing the clitoral fossa and recesses of the clitoral sinuses; it may be
recovered from the cervix and endometrium in pure culture (Platt et al., 1978). Sampling of the clitoral sinuses
should be done with mini-tipped swabs, not the standard type of bacteriological swab. Carriage does not always
adversely affect conception (Timoney et al., 1978), and in such cases pregnancy may proceed to term. The
offspring of such pregnancies may become contaminated during passage through the birth canal, and such foals
may become long-term subclinical carriers (Timoney & Powell, 1982). Many primary cases of infection with
T. equigenitalis in the mare are subclinical, and a frequent indicator of infection is a mare returning in oestrus
prematurely after being bred to a putative carrier stallion.
Carrier mares and stallions act as reservoirs of T. equigenitalis, but stallions, because they mate with numerous
mares, play a much more important role in dissemination of the bacterium. The urogenital membranes of stallions
become contaminated at coitus, leading to a carrier state that may persist for many months or years (Schluter et
al., 1991). Failure to observe appropriate hygienic measures when breeding mares and stallions may also spread
the organism. Other sites of the horses body are not known to harbour T. equigenitalis. Most carrier mares are
clitoral carriers of T. equigenitalis. Long-term persistence of the organism in the uterus, though uncommon, can
occur. To detect such carriers of T. equigenitalis, an endometrial or deep cervical swab sample should be taken
routinely in addition to sampling the clitoral area of all mares. Abortion in the mare due to T. equigenitalis is a very
rare occurrence.
Another species of Taylorella, T. asinigenitalis (Jang et al., 2001) has been isolated from male donkeys and horse
mares and stallions in the United States of America (Katz et al., 2000; Meade et al., 2010), and a number of
European countries (Baverud et al., 2006; Breuil et al., 2011; Franco et al., 2009). This bacterium, which has not
been associated with naturally occurring disease, resides in the genital tract of male donkeys and can be passed
to donkeys and horses during mating.
Taylorella equigenitalis is not known to infect humans and it should be handled in the laboratory at biosafety level
2.
Disease control
Prior infection or vaccination are not fully protective (Fernie et al., 1980; Timoney et al., 1979), and failure of
antibody to persist has meant that control of infection has relied entirely on prevention of transmission through the
detection of T. equigenitalis on swabs of the reproductive tract of stallions and mares. In spite of difficulties in
culturing T. equigenitalis, screening mares and stallions prior to and while on the stud farm has successfully
eliminated the disease from thoroughbred horses in countries using a voluntary code of practice. These have
been based on the widely adopted UKs Horserace Betting Levy Boards Code of Practice
(www.hblb.org.uk/codes.htm), which is reviewed and updated annually and will reflect current information. The
key recommendations of that Code of Practice are summarised below.
At the start of the breeding season, swabs are taken from all stallions, including those in their first breeding
season, from the urethra, urethral fossa and sinus, penile sheath and pre-ejaculatory fluid, on two occasions no
fewer than 7 days apart. Mares are classified according to the degree of risk that they represent, and the
frequency of sampling is adjusted accordingly. High-risk mares are defined in the Code of Practice as: (a) those
from which T. equigenitalis has been isolated (the high risk status will remain until three sets of negative swabs
have been taken at three different oestrous periods in each of 2 years); (b) mares that have visited any premises
on which T. equigenitalis has been isolated within the previous 12 months; (c) mares arriving from countries
currently listed in the Code of Practice as higher risk; d) all mares that have been in countries that are currently
listed in the Code of Practice as higher risk within the last 12 months.
High risk stallions are defined as: (a) stallions that have not previously been used for breeding purposes;
(b) stallions from which T equigenitalis has been isolated (the high risk status will remain until treatment has
been undertaken and required swab results are negative); (c) stallions that have been , on any premises on which
T equigenitalis has been isolated within the last 12 months; (d) stallions that have mated a mare which has not
been swabbed negative in accordance with the Code of Practice. Low risk stallions are any stallions not defined
as high risk. There is a potentially serious problem with contagious equine metritis in this group of horses,
especially those belonging to non-thoroughbred breeds.
832
Results of laboratory tests for T. equigenitalis should be entered on an officially approved certificate, which is sent
to the veterinarians and stallion stud farm managers who supervise the breeding. The certificate should record the
animals name, the sites and date of swabbing, the name of the veterinarian taking the swabs, identity of the
testing laboratory, the date the swabs were received and cultured by the laboratory, and whether the swabs were
negative or positive, or whether the culture was overgrown by other bacteria to an extent that the laboratory could
not be confident that small numbers of T. equigenitalis would be detected and, therefore, requiring another set of
swabs to be collected.
Difficulties with the culture of T. equigenitalis due to its fastidious nature necessitate the use of a quality control
system that should be approved before a laboratory is permitted to undertake official testing for contagious equine
metritis and to issue certificates of the test results. The task of quality control should be undertaken by an
experienced, reliable, and impartial microbiology laboratory authorised for the purpose.
Any mares with an abnormal vaginal exudate, or returning to oestrus prematurely, should be investigated and
managed as though infected with T. equigenitalis until results of laboratory testing prove otherwise. Other causes
of endometritis include Pseudomonas aeruginosa, Streptococcus zooepidemicus and certain capsule types of
Klebsiella pneumoniae. Swabs should be examined for these bacteria, and an attempt made to culture and
identify K. pneumoniae and P. aeruginosa so as to establish a differential diagnosis.
If carriers of T. equigenitalis are detected, the organism can be eliminated by treatment with systemic and/or local
antibiotics combined with antiseptic washing of the sites of persistence in the mare and the stallion. Particular
attention should be paid to the clitoral fossa and cleansing the recesses of the clitoral sinuses of mares, where
colonisation by T. equigenitalis is frequently found in carrier animals. A course of treatment may take several
weeks and may need to be repeated before swabbing consistently fails to recover T. equigenitalis from the
treated stallion or mares (Crowhurst et al., 1979). A variable number of carrier mares can be refractive to several
courses of treatment. These may require surgery and ablation of the clitoral sinuses for permanent elimination of
the carrier state in such animals.
Control measures for countries regarded as free from T. equigenitalis infection should be based on the screening
of animals prior to importation and/or during a post-importation quarantine period using swabbing and testing
regimes broadly based on those described above for breeding populations.
1.
Identification of the agent (the prescribed test for international trade)
Various bacteria may be present on the urogenital membranes of horses as harmless commensals and may
interfere with the culture of T. equigenitalis. Some may initially be present in small numbers, but multiply on the
swab before it is cultured depending on the ambient temperature the swab is exposed to in transit to the testing
laboratory. Overgrowth of these organisms on the culture plates may obscure the presence of T. equigenitalis.
Swabs must be placed in a transport medium with activated charcoal, such as Amies medium, to absorb inhibitory
by-products of bacterial metabolism (Swerczek, 1978). The numbers of viable T. equigenitalis decline on swabs
over time, and this effect is more pronounced at higher temperatures (Sahu et al., 1979). Swabs must be kept
cool during transportation and should arrive and be plated out at the laboratory no later than 48 hours after they
were taken. Negative culture results from swabs plated out more than 48 hours after they were taken are
unreliable. Antibiotic treatment for whatever cause should cease at least 7 days (systemic treatment) or 21 days
(local treatment) before swabbing. The presence of antibiotics may sublethally damage T. equigenitalis, which
nonetheless persists on the urogenital membranes but cannot be grown on laboratory media.
Each swab must be inoculated on to 5% (v/v) heated blood, (chocolate), agar plates, produced by heating the
liquid medium, containing blood, at 7080C for 12 minutes. When cooled to 4550C, trimethoprim (1 g/ml),
clindamycin (5 g/ml), and amphotericin B (515 g/ml), is added to the medium (Timoney et al., 1982).
Thymidine, which will inactivate trimethoprim, is present in bacteriological media containing peptone, so it is
important to add 5% lysed horse blood at this stage. Lysed horse blood contains thymidine phosphorylase, which
will inactivate thymidine, thus allowing the trimethoprim to exert its selective effect. This is the preferred medium
for isolating T. equigenitalis; this medium has been used successfully to isolate equally well both streptomycin
resistant and sensitive biotypes of the pathogen and to suppress the growth of many commensal bacteria and
inhibit fungal growth. As inhibiters may prevent the isolation of some strains of T. equigenitalis, swabs should also
be inoculated on to plates of 5% chocolate blood agar with a rich peptone agar base containing additional
cysteine (0.83 mM), sodium sulphite (1.59 mM) and a fungicide (515 g/ml amphotericin B). While
T. equigenitalis can be cultivated on blood agar, it will grow better on chocolate blood agar as described above.
833
Some manufacturers1 produce a peptone agar base that is quality controlled for its ability to support the growth of
T. equigenitalis. The quality of the commercial agar should be confirmed by the testing laboratory. An important
feature of all good T. equigenitalis media is the absence of fermentable carbohydrates. These do not enhance the
growth of T. equigenitalis, but their fermentation by other bacteria inhibits T. equigenitalis growth (Atherton, 1983;
Fernie et al., 1980). A third medium containing streptomycin sulphate (200 g/ml) is sometimes used as some
isolates of T. equigenitalis are resistant to this concentration of antibiotic, which serves to reduce the extent of
growth of other bacteria that might otherwise obscure the presence of small numbers of T. equigenitalis
(Swerczek, 1978). However, a streptomycin-sensitive biotype is now the most common strain isolated and will not
be detected on this medium; consequently, it should only be used in conjunction with medium without
streptomycin. Growth by other bacteria, for example Proteus mirabilis, however, may be so extensive that the
laboratory should report that they cannot issue a negative result. In this event, further swabs should be requested
in the hope that the problem will not recur.
Occasionally, the urogenital membranes of stallions or mares will be persistently colonised by another bacterium
that interferes with detection of T. equigenitalis. It may be necessary to attempt to eliminate this by washing and
antibiotic treatment. Swabbing for T. equigenitalis shall not recommence until at least 7 days (systemic treatment)
or 21 days (local treatment) after treatment has stopped. The use of Timoneys medium (Timoney et al., 1982),
described above, should overcome this difficulty in most cases
All culture media should be subjected to quality control and must support growth of a small inoculum of the
suspect organism before their use on suspect samples. The reference strain of T. equigenitalis must also be
cultured in parallel with the test samples to ensure that the culture conditions are optimal for isolation of this
organism.
The fastidious nature of T. equigenitalis makes it difficult to isolate. Test breeding of stallions has been used to
increase the sensitivity of detection of the carrier state and it has been a valuable adjunct to cultural examination.
The numbers of Taylorella present on the external genitalia of stallions can be very low and may be missed by
culturing alone, but can be detected after multiplication in the mare that has been test bred. The use of test
breeding as an additional diagnostic tool can be especially important in countries that are considered free from
contagious equine metritis.
Plates must be incubated at 3537C in 510% (v/v) CO2 in air or by use of a candle jar. At least 72 hours is
normally required before colonies of T. equigenitalis become visible, after which time daily inspection is needed.
Rarely, visual detection of colonies may take up to 14 days (Ward et al., 1984). A standard incubation time of at
least 7 days is advisable before certifying cultures negative for T. equigenitalis. Plates should be examined for
contaminants after the first 24 hours incubation. Colonies of T. equigenitalis may be up to 23 mm in diameter,
smooth with an entire edge, glossy and yellowish grey. Laboratories should be aware that certain countries
require the prolonged incubation period as a standard procedure and should therefore ascertain the particular
import requirements of those countries and/or indicate the incubation period on which their cultural findings are
based.
Taylorella equigenitalis is a Gram-negative, nonmotile, bacillus or cocco-bacillus that is often pleomorphic (up to
6 m long) and may exhibit bipolar staining. It is catalase positive, phosphatase positive, and strongly oxidase
positive. It is otherwise inert in tests for biochemical activity. If a slow-growing organism is isolated that fits the
description for cellular morphology and that is strongly oxidase positive, it should be tested for reactivity with T.equigenitalis-specific antiserum.
A variety of serotyping tests have been developed ranging in complexity from slide agglutination to direct or
indirect immunofluorescence. Each method has its advantages and disadvantages. The disadvantage of the slide
agglutination test is that occasionally autoagglutination of isolates occurs: culturing in bottled CO2 in air, as
opposed to in a candle jar, may reduce autoagglutination (Ter Laak & Wagenaars, 1990). It has been suggested
that immunofluorescence can be used to identify autoagglutinating isolates, some workers have reported crossreaction with Mannheimia haemolytica but this is very rare. If a cross-reaction is suspected, it may be necessary
to repeat the test using adsorbed antisera (Ter Laak & Wagenaars, 1990). The specificity of the
immunofluorescence test can be improved by the use of monoclonal antibodies that are now available.
Nevertheless polyclonal and monoclonal antibodies can be used in the differentiation of T. equigenitalis and
T. asinigenitalis (Breuil et al., 2010) 2.
Antiserum is produced by vaccinating rabbits with killed T. equigenitalis. A number of different immunisation
regimes can be employed, ranging from those used for producing Escherichia-coli-typing antisera to immunisation
together with an adjuvant, such as Freunds incomplete. Monoclonal antibodies are available commercially that
1
2
834
For example, Mast Diagnostics, Mast House, Derby Road, Bootle, Merseyside L20 1EA, United Kingdom (UK), and
Lab M, Tomley House, Wash Lane, Bury BL9 6AU, UK.
Institut Pourquier, 326 rue de la Galera, Parc Euromedicine, 34090 Montpelier, France. E-mail: info@institut-pourquier.fr
provide a highly specific means of identifying T. equigenitalis. A standard strain, such as NCTC 111843, should be
used for immunisation. However, the most important consideration is the specificity of the antiserum produced. It
should agglutinate T. equigenitalis, but fail to agglutinate other bacteria that might be cultured from horse
urogenital membranes, even if rarely. In particular, it should not agglutinate any oxidase-positive and Gramnegative rods, such as Mannheimia haemolytica, Actinobacillus equuli, Bordetella bronchiseptica (to which
T. equigenitalis is closely related, see Bleumink-Pluym et al. (1993), Oligella urethralis and Pseudomonas
aeruginosa. Taylorella asinigenitalis has similar, though not identical, colonial appearance and cultural
characteristics and gives identical biochemical test results to those used to confirm the identity of T. equigenitalis.
There is even serological cross-reactivity between the two organisms. Differentiation of T. asinigenitalis from
T. equigenitalis is possible using the polymerase chain reaction (PCR) or 16S rDNA sequencing and biochemical
reactivity (Baverud et al., 2006; Breuil et al., 2011; Duquesne et al., 2007; Wakeley et al., 2006).
A latex agglutination kit is available commercially for the antigenic identification of T. equigenitalis. It is based on
polyclonal antibodies produced using methods similar to those described above. This is widely used by routine
testing laboratories for the confirmation of the identity of colonies growing on selective medium that give a
biochemical reaction consistent with T. equigenitalis. As T. equigenitalis is antigenically relatively distinct, and
small amounts of cross-reactive antibody are easily absorbed during production of the reagent, the test has
proved to be highly specific and sensitive. It should be emphasised that it will not necessarily distinguish strains of
T. equigenitalis from T. asinigenitalis.
Molecular testing methods such as PCR and real-time PCR have been applied to the detection of
T.equigenitalis both directly (using swabs taken from sampling sites) and indirectly (from cultures grown from
swabs) (Bleumink-Pluym et al., 1994). In studies carried out in the UK the PCR was shown to be highly specific
and was able to detect very small numbers of T. equigenitalis in the presence of very large numbers of bacteria
comprising the background flora harvested from culture plates inoculated with samples from the equine urogenital
tract. In Japan the field application of the PCR was evaluated for the eradication of contagious equine metritis. It
was demonstrated that the PCR was more sensitive than culture for the detection of T. equigenitalis from genital
swabs of horses (Anzai et al., 2002; Moore et al., 2001). A real-time PCR was developed in the UK for use
directly on genital swabs and compared with culture (Wakeley et al., 2006) and this has been subsequently used
for pre-breeding screening studies (Ousey et al., 2009). There was no significant difference in the performance of
the direct PCR and culture, but the PCR had the added advantage of speed of result and also differentiated
T. equigenitalis from T. asinigenitalis. Commercial PCR kits are available for the detection of T. equigenitalis and
these may be used to enhance the testing capabilities of authorised laboratories.
2.
Serological tests
No serological test described to date will, by itself, reliably detect infection for diagnosis and control. However, the
complement fixation test has been used successfully as an adjunct to culture for T. equigenitalis in screening
mares between 21 and 45 days after being bred to a suspect carrier stallion.

Effective vaccines that protect against contagious equine metritis or prevent colonisation by T. equigenitalis are
not yet available.
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contagious equine metritis in equine genital swabs by direct polyermase chain reaction. Res. Vet. Sci., 82, 4749.
FERNIE, BATTY I., WALKER P.D., PLATT H., MACKINTOSH M.E. & SIMPSON D.J. (1980). Observations on vaccine and
post-infection immunity in contagious equine metritis. Res. Vet. Sci., 28, 362367.
FRANCO A., DONATI V., TROIANO P., LORENZETTI R., ZINI M., AUTORINO G.L., PETRELLA A., MAGGI A. & BATTISTI A.
(2009). Detection of Taylorella asinigenitalis in donkey jacks in Italy. Vet. Rec., 165, 540541.
JANG S.S., DONAHUE J.M., ARATA A.B., GORIS J., HANSEN L.M., EARLEY D.L., VANDAMME P.A., TIMONEY P.J. & HIRSH
D.C. (2001). Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus
asinus). Int. J. Syst. Evol. Microbiol., 51, 971976.
KATZ J.B., EVANS L.E., HUTTO D.L., SCHROEDER-TUCKER L.C., CAREW A.M., DONAHUE J.M. & HIRSH D.C. (2000).
Clinical, bacteriologic, serologic and pathologic features of infections with atypical Taylorella equigenitalis in
mares. J. Am. Vet. Med. Assoc., 216 (12), 19451948.
MEADE B.J., TIMONEY P.J., DONAHUE J.M., BRANSCUM A.J., FORD R. & ROWE R. (2010). Initial occurrence of
Taylorella asinigenitalis and its detection in nurse mares, a stallion and donkeys in Kentucky. Prev. Vet. Med., 95,
292296.
MOORE J.E., BUCKLEY T.C., MILLAR B.C., GIBSON P., CANNON G., EGAN C., COSGROVE H., STANBRIDGE S., ANZAI T.,
MATSUDA M. & MURPHY P.G. (2001). Molecular surveillance of the incidence of Taylorella equigenitalis and
Pseudomonas aeruginosa from horses in Ireland by sequence-specific PCR. Equine Vet. J., 33, 319322.
OUSEY J.C., PALMER L., CASH R.S.G., GRIMES K.J., FLETCHER A.P., BARRELET A., FOOTE A.K., MANNING F.M. &
RICKETTS S.W. (2009) An investigation into the suitability of a commercial real-time PCR assay to screen for
Taylorella equigenitalis in routine prebreeding equine genital swabs. Equine Vet. J., 41 (9), 878882.
PLATT H., ATHERTON J.G., DAWSON F.L.M. & DURRANT D.S. (1978). Developments in contagious equine metritis.
Vet. Rec., 102, 19.
PLATT H., ATHERTON J.G., SIMPSON D.J., TAYLOR C.E.D., ROSENTHAL R.O., BROWN D.F.J. & WREGHITT T.G. (1977).
Genital infection in mares. Vet. Rec., 101, 20
SAHU S.P., DARDIRI A.H., ROMMEL F.A. & PIERSON R.E. (1979). Survival of contagious equine metritis bacteria in
transport media. Am. J. Vet. Res., 40, 10401042
SCHLUTER H., KULLER H., FREIDREICH U., SELBITZ H., MARWITZ T., BEYER C. & ULLRICH E. (1991). Epizootiology and
treatment of contagious equine metritis (CEM), with particular reference to the treatment of infected stallions.
Prakt. Tierarzt., 72, 503511.
SWANEY L.M. & SAHU S.P. (1978). CEM: bacteriological methods. Vet. Rec., 102, 43.
SWERCZEK T.W. (1978). Inhibition of the CEM organism by the normal flora of the reproductive tract. Vet. Rec.,
103, 125.
836
TER LAAK E.A. & WAGENAARS C.M.F. (1990). Autoagglutination and the specificity of the indirect fluorescent
antibody test applied to the identification of Taylorella equigenitalis. Res. Vet. Sci., 49, 117119
TIMONEY P.J., OREILLY P.J., MCARDLE J.F., WARD J. & HARRINGTON A.M. (1979). Responses of mares to
rechallenge with the organism of contagious equine metritis 1977. Vet. Rec., 104, 264.
TIMONEY P.J. & POWELL D.G. (1982). Isolation of the contagious equine metritis organism from colts and fillies in
the United Kingdom and Ireland. Vet. Rec., 111, 478482.
TIMONEY P.J., SHIN S.J. & JACOBSON R.H. (1982). Improved selective medium for isolation of the contagious equine
metritis organism. Vet. Rec., 111,107108.
TIMONEY P.J., WARD J. & KELLY P. (1977). A contagious genital infection of mares. Vet. Rec., 101, 103
TIMONEY P.J., WARD J. & MCARDLE J.F. (1978). CEM and the foaling mare. Vet. Rec., 102, 246247
WAKELEY P.R., ERRINGTON J., HANNON S., ROEST H.I.J., CARSON T. HUNT B. & HEATH P. (2006). Development of a
real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from
T. asinigenitalis. Vet. Microbiol., 118, 247254.
WARD J., HOURIGAN M., MCGUIRK J. & GOGARTY A. (1984). Incubation times for primary isolation of contagious
equine metritis organism. Vet. Rec., 114, 298.
*
* *
NB: There are OIE Reference Laboratories for Contagious equine metritis
diagnostic tests, reagents and vaccines for Contagious equine metritis
837
CHAPTER 2.5.3.
DOURINE
SUMMARY
Dourine is a chronic or acute contagious disease of breeding solipeds that is transmitted directly
from animal to animal during coitus. The causal organism is Trypanosoma equiperdum (Doflein,
1901).
Dourine is the only trypanosomosis that is not transmitted by an invertebrate vector. Trypanosoma
equiperdum differs from other trypanosomes in that it is primarily a tissue parasite that rarely
invades the blood. There is no known natural reservoir of the parasite other than infected equids. It
is present in the genital secretions of both infected males and females. The incubation period,
severity, and duration of the disease vary considerably; it is often fatal, but spontaneous recoveries
do occur but latent carriers do exist. Subclinical infections occur, and donkeys and mules are more
resistant than horses and may remain inapparent carriers. Infection is not always transmitted by an
infected animal at every copulation. Rats can be infected experimentally, and can be used to
maintain strains of the parasite indefinitely. Trypanosoma equiperdum strains are best stored in
liquid nitrogen.
The clinical signs are marked by periodic exacerbation and relapse, ending in death, sometimes
after paraplegia or, possibly, recovery. Fever, local oedema of the genitalia and mammary glands,
cutaneous eruptions, incoordination, facial paralysis, ocular lesions, anaemia, and emaciation may
all be observed. Oedematous cutaneous plaques, 58 cm in diameter and 1 cm thick, are
pathognomonic.
Identification of the agent: Definitive diagnosis depends on the recognition of clinical signs and
identification of the parasite. As this is rarely possible, diagnosis is usually based on clinical signs,
together with serological evidence from complement fixation (CF) tests.
Serological tests: Humoral antibodies are present in infected animals whether or not they display
clinical signs. The CF test is used to confirm infection in clinical cases or in latent carriers.
Noninfected animals, especially donkeys, often yield unclear results. The indirect fluorescent
antibody test can be used to confirm infection or resolve inconclusive CF test results. Enzymelinked immunosorbent assays are also used.
Requirements for vaccines and diagnostic biologicals: There are no vaccines available. The
only effective control is through the slaughter of infected animals. Good hygiene is essential during
assisted matings because infection may be transmitted through contaminated fomites.
A. INTRODUCTION
Dourine is a chronic or acute contagious disease of breeding solipeds that is transmitted directly from animal to
animal during coitus. The causal organism is Trypanosoma equiperdum (Doflein, 1901). Dourine is also known
under other names: mal de cot, el dourin, morbo coitale maligno, Beschlseuche, slapsiekte, sluchnaya bolyezn,
and covering disease (Barner, 1963; Hoare, 1972).
Although the disease has been known since ancient times, its nature was established only in 1896 when Rouget
discovered trypanosomes in infected Algerian horses. Dourine is the only trypanosomosis that is not transmitted
by an invertebrate vector. Trypanosoma equiperdum differs from other trypanosomes in that it is primarily a tissue
parasite that is rarely detected in the blood. There is no known natural reservoir of the parasite other than infected
equids.
838
Chapter 2.5.3. Dourine
Infection is transmitted during copulation, more commonly from stallion to mare, but also from mare to stallion,
due to the presence of the parasite in the seminal fluid and mucous exudate of the penis and sheath of the
infected male, and in the vaginal mucus of the infected female. Initially, parasites are found free on the surface of
the mucosa or between the epithelial cells of a newly infected animal. Invasion of the tissues takes place, and
oedematous patches appear in the genital tract. Parasites then may pass into the blood, where they are carried to
other parts of the body. In typical cases, this metastatic invasion gives rise to characteristic cutaneous plaques.
The incubation period, severity and duration of the disease vary considerably. In South Africa, the disease is
typically chronic, usually mild, and may persist for several years (Henning, 1955). In other areas, such as northern
Africa and South America, the disease tends to be more acute, often lasting only 12 months or, exceptionally,
1 week. Although dourine is a fatal disease with an average mortality of 50% (especially in stallions), spontaneous
recovery can occur. Subclinical infections are recognised. Donkeys and mules are more resistant than horses.
As trypanosomes are not continually present in the genital tract throughout the course of the disease,
transmission of the infection does not necessarily take place at every copulation involving an infected animal.
Transmission of infection from mare to foal can occur via the mucosa, such as the conjunctiva. Mares milk has
been shown to be infectious. Animals other than equids can be infected experimentally. Rat-adapted strains can
be maintained indefinitely; infected rat blood can be satisfactorily cryopreserved. Antigens for serological tests are
commonly produced from infected laboratory rats.
The disease is marked by stages of exacerbation, tolerance or relapse, which vary in duration and which may
occur once or several times before death or recovery. The signs most frequently noted are: pyrexia, tumefaction
and local oedema of the genitalia and mammary glands, oedematous cutaneous eruptions, knuckling of the joints,
incoordination, facial paralysis, ocular lesions, anaemia, and emaciation. A pathognomonic sign is the
oedematous plaque consisting of an elevated lesion in the skin, up to 58 cm in diameter and 1 cm thick. The
plaques usually appear over the ribs, although they may occur anywhere on the body, and usually persist for
between 3 and 7 days. They are not a constant feature.
Generally, the oedema disappears and returns at irregular intervals. During each recess, an increasing extent of
permanently thickened and indurated tissue can be seen. The vaginal mucosa may show raised and thickened
semitransparent patches. Folds of swollen membrane may protrude through the vulva. It is not uncommon to find
oedema of the mammary glands and adjacent tissues. Depigmentation of the genital area, perineum, and udder
may occur. In the stallion, the first clinical sign is a variable swelling involving the glans penis and prepuce. The
oedema extends posteriorly to the scrotum, inguinal lymph nodes, and perineum, with an anterior extension along
the inferior abdomen. In stallions of heavy breeds, the oedema may extend over the whole floor of the abdomen.
Pyrexia is intermittent; nervous signs include incoordination, mainly of the hind limbs, lips, nostrils, ears, and
throat. Facial paralysis is usually unilateral. In fatal cases, the disease is usually slow and progressive, with
increasing anaemia and emaciation, although the appetite remains good almost throughout.
At post-mortem examination, gelatinous exudates are present under the skin. In the stallion, the scrotum, sheath,
and testicular tunica are thickened and infiltrated. In some cases the testes are embedded in a tough mass of
sclerotic tissue and may be unrecognisable. In the mare, the vulva, vaginal mucosa, uterus, bladder, and
mammary glands may be thickened with gelatinous infiltration. The lymph nodes, particularly in the abdominal
cavity, are hypertrophied, softened and, in some cases, haemorrhagic. The spinal cord of animals with paraplegia
is often soft, pulpy and discoloured, particularly in the lumbar and sacral regions.
1.
A definitive diagnosis depends on the recognition of the clinical signs and the demonstration of the parasite. This
is rarely possible because: (a) although the clinical signs and gross lesions in the developed disease may be
pathognomonic, they cannot always be identified with certainty, especially in the early stages or in latent cases;
they can be confused with other conditions, such as coital exanthema (moreover, in some countries [e.g. in South
America], T. evansi infections give rise to similar clinical signs); (b) the trypanosomes are only sparsely present
and are extremely difficult to find, even in oedematous areas; and (c) the trypanosomes are only fleetingly present
in the blood, and in small numbers that defy detection. For unknown reasons, no parasite strain of T. equiperdum
has been isolated in any country of the world since 1982 and most of the strains currently available in national
veterinary diagnostic laboratories are related to T. evansi (Claes et al., 2003).
In practice, diagnosis is based on clinical evidence supported by serology. Recently, other approaches have been
studied and reported on (Claes et al., 2003).
839
In infected animals, trypanosomes are present, in low numbers only, in lymph and oedematous fluids of the
external genitalia, in the vaginal mucus, and fluid contents of plaques. They are usually undetectable in the blood,
but may be found in the urethral or vaginal mucus collected from preputial or vaginal washings or scrapings 4
5 days after infection. Later, parasites may be found in the fluid contents of oedemas and plaques, especially
shortly after their eruption. The skin of the area over the plaque should be washed, shaved and dried, and the
fluid contents aspirated by syringe. Blood vessels should be avoided. The fresh aspirate is examined
microscopically for motile trypanosomes. These are present for a few days only, so that lesions should be
examined at intervals. The parasite is rarely found in thick blood films, but is sometimes detectable after
centrifuging blood and examining the recentrifuged plasma.
As dourine is the only trypanosome to affect horses in temperate climates, the observation of trypanosomes in
thick blood films is sufficient for a positive diagnosis. In countries where nagana or surra occur, it is difficult to
distinguish T. equiperdum microscopically (morphology, motility) from other members of the subgenus
Trypanozoon (T. evansi, T. brucei). In particular, T. equiperdum and T. evansi cannot be differentiated on the
basis of morphological criteria. Both are monomorphic, slender trypomastigotes with a free flagellum, although
pleomorphic, stumpy, proteonuclear forms are recognised. However, in kinetopastic strains, the presence of maxicircles in T. equperdum and the absence in T. evansi provides a possible differentiation (Wassal et al., 1991).
Typical strains of the parasite range in length from 15.6 to 31.3 m.
2.
Serological tests
Humoral antibodies are present in infected animals, whether they display clinical signs or not. The complement
fixation (CF) test (UK Ministry of Agriculture, Fisheries and Food [MAFF], 1986) is used to confirm clinical
evidence and to detect latent infections. Uninfected equids, particularly donkeys and mules, often give
inconsistent or nonspecific reactions because of the anticomplementary effects of their sera. In the case of
anticomplementary sera, the indirect fluorescent antibody (IFA) test is of advantage. There is no internationally
adopted protocol. Cross-reactions are possible due to the presence in some countries of other trypanosomes, for
example, T. cruzi and T. evansi. Enzyme-linked immunosorbent assays (ELISAs) are also used. Trypanosoma
equiperdum is closely related to other Old World trypanosomes, including T. brucei and T. evansi. Members of
this genus all share conserved cytoskeletal elements that provoke a strong and cross-reactive serological
response. All diagnostic antigens and antisera (monoclonal and polyclonal) currently available for use in
serodiagnostic testing contain these conserved elements or antibodies to them, and therefore none of the
serological procedures described below is specific for dourine. The diagnosis of dourine must include history,
clinical, and pathological findings as well as serology. Significant improvements in dourine serodiagnosis will
require development of more trypanosome-specific subunit antigens and antibodies to them.
a)
Complement fixation test (the prescribed test for international trade)

Standard or microplate techniques may be used (Herr et al., 1985). Guinea-pig serum (available
commercially) is used as a source of complement. Other reagents are sheep red blood cells (RBCs) washed
in veronal buffer, and rabbit haemolytic serum (i.e. rabbit anti-sheep RBC) (commercial) as well as known
negative and positive control sera.
840
Antigen production
i)
A rat is inoculated with T. equiperdum cryopreserved stock. The rat must be free from T. lewisi, which
could be achieved by injection with neoarsphenamine, but is better accomplished by using specificpathogen free rats. Adult rats receive 0.51.0 ml of rapidly thawed frozen stabilate, intramuscularly or
intraperitoneally. At maximum parasitaemia, blood is collected into an anticoagulant, such as heparin,
which will serve as a stock culture for the inoculation of additional rats.
ii)
Twenty large rats are inoculated intramuscularly or intraperitoneally with 0.51.0 ml of this stock
culture. All rats are to have a heavy infection concurrently. If necessary, the dose is adjusted and
additional rats are inoculated to reach maximum parasitaemia at the desired time of 7296 hours. Rats
usually die within 35 days; prior to this, blood is taken from the tail for thick smears and examined
microscopically. When parasitaemia is maximal, the rat is killed and blood is collected in Alsevers or
acidcitratedextrose (ACD) saline solution. If parasitaemia is not synchronous, blood can be collected
and held in Alsevers or ACD saline at 4C until blood has been collected from all the rats.
iii)
The blood is filtered through muslin gauze and centrifuged at 800 g for 4 minutes. The RBCs are
mostly deposited while the trypanosomes remain in suspension.
iv)
The supernatant fluid is transferred to a fresh tube; the upper layer of RBCs is mixed with
trypanosomes to a second tube, and the next layer to a third. Alsevers or ACD saline is added to tubes
2 and 3 to prevent clotting of cells. All tubes are mixed and centrifuged at 1500 g for 5 minutes.
v)
The supernatant fluid is discarded and the upper white layer of trypanosomes is transferred from all
tubes into a clean tube. The next pink layer is transferred into a second tube, and the lower layer to a
third tube.
vi)
Physiological saline is added and mixed and the tubes are centrifuged again at 1500 g for 5 minutes to
separate the trypanosomes. The washing step is repeated until all the trypanosomes are collected as a
pure white mass. Ten rats should produce 35 g of antigen. This purification procedure can also be
carried out using a column of DEAE (diethylaminoethyl) cellulose in a solution of phosphate buffered
saline (PBS) containing glucose, pH 8.0 (Lanham & Godfrey, 1970).
vii)
The concentrated trypanosomes are diluted with two volumes of veronal buffer and 5%
polyvinylpyrrolidone as a cryopreservative. Before use in CF tests, the antigen must be dispersed to a
fine suspension with a hand-held or motorised ground glass homogeniser chilled in ice (Watson, 1920).
This antigen may be divided into aliquots, frozen and lyophilised.
The antigen is standardised by titration against a 1/5 dilution of a standard low-titre antiserum.
Sera: Positive and negative sera should be inactivated at 58C for 30 minutes before being used in the tests.
Mule and donkey sera are normally inactivated at 62C for 30 minutes. The USDA complement fixation
protocol calls for inactivation of sera for 35 minutes (United States Department of Agriculture [USDA], 2006).
Dilutions of sera that are positive in the screening test are titrated against two units of antigen. Test sera are
screened at a dilution of 1/5. Sera showing more than 50% complement fixation at this dilution are usually
deemed to be positive.
Anticomplementary sera: If the anticomplementary control shows only a trace, this may be ignored. For all
other anticomplementary sera, the activity must be titrated. A duplicate series of dilutions is made and the
sample is retested using T. equiperdum antigen in the first row and veronal buffer only in the second. The
second row gives the titre of the anticomplementary reaction. Provided the first row shows an end-point that
is at least three dilutions greater than the second, the anticomplementary effect may be ignored and the
sample rated as positive. If the results are any closer, a fresh sample of serum must be requested. Dilution
of the serum 1/2 and heat inactivation at 6063C for 30 minutes may result in reduction or removal of the
anticomplementary effect.
Buffers and reagents: 0.15 M veronal buffered saline, pH 7.4, is used for diluting reagents and for washing
sheep RBCs. Antigen is pretested by checkerboard titration, and two units are used in the test. Guinea-pig
complement (C) is tested for its haemolytic activity, and diluted to provide two units for the test. Sheep RBCs
in Alsevers or ACD saline solution are washed three times. A 3% solution is used for the haemolytic system.
The USDA protocol calls for 2% solution in the microtitration procedure with confirmation in a tube test with
3% RBC (USDA, 2006). Titrated rabbit-anti-sheep RBCs the rabbit haemolytic serum is taken at double
the concentration of its haemolytic titre (two units). All test sera, including positive and negative control sera,
are inactivated at a 1/5 dilution before testing.
Primary dilutions
i)
100 l of test serum is diluted with 400 l of veronal buffer (1/5).
ii)
100 l of both positive and negative control sera is diluted with 400 l of veronal buffer (1/5).
iii)
The solutions are incubated in a water bath at 58C for 30 minutes to inactivate complement and
destroy anticomplementary factors.
Screening test procedure
i)
25 l of inactivated test serum is placed in each of three wells.
ii)
25 l of inactivated control serum is placed in each of three wells.
iii)
25 l of T. equiperdum antigen diluted to contain two units is placed in the first well only for each
serum.
iv)
25 l of complement diluted to contain two units is added to the first two wells only for each serum.
v)
25 l veronal buffer, pH 7.4, is added to the second well for each serum (anticomplementary well).
vi)
50 l veronal buffer, pH 7.4, is added to the third well for each serum (lysis activity well).
vii)
The complement control is prepared.
viii) The plate is shaken on a microshaker sufficiently to mix the reagents.

ix)
The plate is incubated for 1 hour in a water bath, incubator or in a humid chamber at 37C.
841
x)
The haemolytic system is prepared. After the first 50 minutes of incubation, the sheep RBCs are
sensitised by mixing equal volumes of rabbit haemolytic serum, diluted to contain two units per 50 l,
and a 3% suspension of washed RBCs; the solution is mixed well and incubated for 10 minutes at
37C.
xi)
After incubation, 50 l of haemolytic system is added to each well.
xii)
The plate is shaken on a microshaker sufficiently to mix the reagents.
xiii) The plate is incubated for 30 minutes at 37C. To aid in reading the results, the plates can be
centrifuged after incubation.
xiv) Reading the results: the plate is viewed from above with a light source beneath it. The fixation in every
well is assessed by estimating the proportion of cells not lysed. The degree of fixation is expressed as
0, 1+, 2+, 3+, 4+ (0%, 25%, 50%, 75% or 100% cells not lysed). Reactions are interpreted as follows:
4+, 3+, 2+ = positive, 1+ = suspicious, trace = negative, complete haemolysis = negative.
xv)
b)
End-point titration: All sera with positive reactions at 1/5 are serially double diluted and tested
according to the above procedure for end-point titration.
Indirect fluorescent antibody test

An IFA test for dourine can also be used (MAFF, 1986) as a confirmatory test or to resolve inconclusive
results obtained by the CF test. The test is performed as follows:
Antigen: (For method, see preparation of CF test antigen in Section B.2.a) Blood is collected into
heparinised vacutainers or into a solution of acidcitratedextrose from an animal in which the number of
trypanosomes is still increasing (about ten parasites per 10 microscope field should be present).
i)
The blood is centrifuged for 10 minutes at 800 g.
ii)
One to two volumes of PBS is added to the packed RBCs, the mixture is agitated, and smears are
made that evenly cover the whole slide.
iii)
The smears are air-dried and then wrapped in bundles of four, with paper separating each slide. The
bundles of slides are wrapped in aluminium foil, sealed in an airtight container over silica gel, and
stored at 20C or 76C.
iv)
Slides stored at 20C should retain their activity for about 1 year, at 76C they should remain
useable for longer.
Acidcitratedextrose solution: Use 15 ml per 100 ml of blood.

Conjugate: Fluorescein-labelled sheep anti-horse immunoglobulins.
Test procedure
i)
The antigen slides are allowed to reach room temperature in a desiccator. An alternative method is to
remove slides directly from the freezer and fix them in acetone for 15 minutes.
ii)
The slides are marked out.
iii)
Separate spots of test sera diluted in PBS are applied, and the slides are incubated in a humid
chamber in a water bath at 37C for 30 minutes.
iv)
The slides are washed in PBS, pH 7.2, three times for 5 minutes each, and air-dried.
v)
Fluorescein-labelled conjugate is added at the correct dilution. Individual batches of antigen and
conjugate should be titrated against each other using control sera to optimise the conjugate dilution.
The slides are incubated in a humid chamber in a water bath at 37C for 30 minutes.
vi)
The slides are washed in PBS, three times for 5 minutes each, and air-dried. An alternative method, to
reduce background fluorescence, is to counter-stain, using Evans Blue (0.01% in distilled water) for
1 minute, rinse in PBS and then air dry
vii)
The slides are mounted in glycerol/PBS (50/50) or immersion oil (commercially available, nonfluorescing grade).
viii) The slides are then examined under UV illumination. Incident light illumination is used with barrier filter
K 530 and exciter filter BG 12. Slides may be stored at 4C for 45 days. Sera diluted at 1/80 and
above showing strong fluorescence of the parasites are usually considered to be positive. Estimating
the intensity of fluorescence demands experience on the part of the observer.
842
Standard positive and negative control sera should be included in each batch of tests, and due consideration
should be given to the pattern of fluorescence in these controls when assessing the results of test sera.
c)

The ELISA has been developed and compared with other serological tests for dourine (Bundesinstitut fr
gesundheitlichen Verbraucherschutz und Veterinrmedizin, 1995; Wassall et al., 1991).
Carbonate buffer, pH 9.6, for antigen coating on to microtitre plates: Na2CO3 (1.59 g); NaHCO3 (2.93g); and
distilled water (1 litre).
Blocking buffer: Carbonate buffer + 3% fetal calf serum (FCS).
PBS, pH 7.4, with Tween 20 (PBST) for washing: KH2PO4 (0.2 g); Na2HPO4 12 H2O (2.94 g); NaCl
(8.0 g); KCl (0.2 g in 1 litre distilled water), and Tween 20 (0.5 ml).
Sample and conjugate buffer: PBST + 6% FCS.
Citric phosphate buffer: Citric acid monohydrate (4.2 g in 200 ml distilled water); Na2HPO4 12 H2O (in
200 ml distilled water). Both components are mixed at equal volumes.
Substrate indicator system: ABTS (2,2-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]) (40 mg) is
dissolved in citric phosphate buffer (100 ml), and stored at 4C in the dark. Just before use, 100 l of
1/40 H2O2 is added to 10 ml of ABTS.
Conjugate: Rabbit anti-horse IgG (H+L) PO or IgY anti-horse Ig-PO.
Antigen: Lyophilised T. equiperdum antigen (0.5 ml) is reconstituted with coating buffer (5 ml), sonicated
twice for 10 seconds each at 12 m peak to peak, and centrifuged at 10,000 g for 4 minutes. The
supernatant is further diluted to a pretested working dilution (e.g. 1/500).
Test procedure
i)
Wells in columns 2, 4, 6, etc., are charged with 50 l of antigen, columns 1, 3, 5, etc., are charged with
the same amount of carbonate buffer. The plate is incubated for 40 minutes at 37C in a humid
chamber, washed in tap water, and 50 l of blocking buffer is added to each well. The plate is
incubated for 20 minutes, washed in tap water followed by three wash cycles with PBST, with soaking
times of 3 minutes/cycle.
ii)
50 l of test samples and equine control sera prediluted 1/100 in sample/conjugate buffer is added in
parallel to wells with and without antigen. The plate is incubated for 30 minutes, washed in tap water,
followed by three wash cycles with PBST.
iii)
Properly diluted conjugate in sample/conjugate buffer is added in volumes of 50 l to all wells. The
plate is incubated for 30 minutes with subsequent washing as above.
iv)
100 l of substrate indicator system is added to all wells.
v)
The reaction is stopped after 15 minutes at room temperature by the addition of 25 l of 37 mM NaCN.
Alternatively, commercially available detergents can be used after pretesting. The results are read
photometrically at a wavelength of 405 nm.
vi)
Calculation of results: absorbance (with antigen) minus absorbance (without antigen) = net extinction.
A reaction exceeding a net extinction of 0.3 is regarded as a positive result.
A competitive ELISA has also been described for detecting antibody against Trypanosoma equiperdum
(Katz et al., 2000).
d)
Other serological tests

Other serological tests have been used, including radioimmunoassay, counter immunoelectrophoresis and
agar gel immunodiffusion (AGID) tests (Caporale et al., 1981; Hagebock et al., 20199303). The AGID has
been used to confirm positive tests and to test anticomplementary sera. A seven-well pattern in 0.8%
agarose in Tris buffer is used, with the CF test antigen in the centre well and positive control sera and
843
unknown sera in alternate peripheral wells. A method has been published for diagnosing equine
piroplasmosis, glanders and dourine at the same time, using immunoblotting (Katz et al., 1999). A card
agglutination test has been developed that compares favourably with the CF test (Claes et al., 2005).
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

There are no biological products available. Control of the disease depends on compulsory notification and
slaughter of infected animals. Good hygiene at assisted matings is also essential.
REFERENCES
BARNER R.D. (1963). Protozoal diseases. In: Equine Medicine and Surgery, Bone J.F. et al., eds. American
Veterinary Publications, Santa Barbara, California, USA, 205210.
BUNDESINSTITUT FR GESUNDHEITLICHEN VERBRAUCHERSCHUTZ UND VETERINRMEDIZIN (BgVV) (Federal Institute for
Consumer Health Protection and Veterinary Medicine) (1995). Working Protocols: ELISA on Dourine. BgVV. P.O.
Box 33 00 13, D-14191 Berlin, Germany.
CAPORALE V.P., BIANCIFIORI F., DI MATTEO A., NANNINI D. & URBANI G. (1981). Comparison of various tests for the
serological diagnosis of Trypanosoma equiperdum infection in the horse. Comp. Immunol. Microbiol. Infect. Dis.,
4, 243246.
CLAES F., AGBO E.C., RADWANSKA M., TE PAS M.F., BALTZ T., DE WAAL D.T., GODDEERIS B.M., CLAASSEN E. &
BUSCHER P. (2003). How does T. equiperdum fit into the Trypanozooan genus? A cluster analysis and multiplex
genotyping approach. Parasitol., 126, 425431.
CLAES F., ILGEKBAYEVA G.D., VERLOO D., SAIDOULDIN T.S., GEERTS S., BUSCHER P. & GODDEERIS B.M. (2005).
Comparison of serological tests for equine trypanosomosis in naturally infected horses from Kazakhstan. Vet.
Parasitol., 131 (34), 221225.
HAGEBOCK J.M., CHIEVES L., FRERICHS W.M. & MILLER C.D. (1993). Evaluation of agar gel immunodiffusion and
indirect fluorescent antibody assays as supplemental tests for dourine in equids. Am. J. Vet. Res., 54, 1201
1208.
HENNING M.W. (1955). Animal Diseases in South Africa, Third Edition. Central News Agency, South Africa.
HERR S., HUCHZERMAYER H.F.K.A., TE BRUGGE L.A., WILLIAMSON C.C., ROOS J.A. & SCHIELE G.J. (1985). The use of
a single complement fixation technique in bovine brucellosis, Johnes disease, dourine, equine piroplasmosis and
Q fever serology. Onderstepoort J. Vet. Res., 52, 279282.
HOARE C.A. (1972). The Trypanosomes of Mammals. A Zoological Monograph. Blackwell Scientific Publications,
Oxford & Edinburgh, UK.
KATZ J.B., CHIEVES L.P., HENNAGER S.G., NICHOLSON J.M., FISHER T.A. & BYERS P.E. (1999). Serodiagnosis of
equine piroplasmosis, dourine and glanders using an arrayed immunoblotting method. J. Vet. Diagn. Invest., 11,
292294.
KATZ J.B., DEWALD R. & NICHOLSON J. (2000). Procedurally similar competitive immunoassay systems for the
serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum and Burkholderia mallei infection in
horses. J. Vet. Diagn. Invest., 12, 4650.
LANHAM S.M. & GODFREY D.G. (1970). Isolation of salivarian trypanosomes from man and other mammals using
DEAE-cellulose. Exp. Parasitol., 28, 521534.
MINISTRY OF AGRICULTURE, FISHERIES AND FOOD (1986). A Manual of Veterinary Parasitological Laboratory
Techniques, Third Edition. Her Majestys Stationery Office, London, UK.
UNITED STATES DEPARTMENT OF AGRICULTURE (2006). Complement Fixation Test for Detection of Antibodies to
Trypanosoma equiperdum Microtitration Test. United States Department of Agriculture, Animal and Plant Health
Inspection Service, National Veterinary Services Laboratories, Ames, Iowa, USA.
844
WASSALL D.A., GREGORY R.J.F. & PHIPPS L.P. (1991). Comparative evaluation of enzyme-linked immunosorbent
assay (ELISA) for the serodiagnosis of dourine. Vet. Parasitol., 39, 233239.
WATSON A.E. (1920). Dourine in Canada 19041920. History, Research and Suppression. Dominion of Canada
Department of Agriculture, Health of Animals Branch, Ottawa, Canada.
*
* *
NB: There is an OIE Reference Laboratory for Dourine
diagnostic tests and reagents for dourine
845
CHAPTER 2.5.4.
EPIZOOTIC LYMPHANGITIS
SUMMARY
Epizootic lymphangitis is a contagious, chronic disease of horses and other Equidae characterised
clinically by a spreading, suppurative, ulcerating pyogranulomatous dermatitis and lymphangitis.
This is seen particularly in the neck, legs and chest. It can also present as an ulcerating
conjunctivitis, or rarely a multifocal pneumonia. Transmission is by contact of infected material with
traumatised skin, by biting flies, ticks or inhalation. The causative agent, Histoplasma capsulatum
var. farciminosum, is a thermally dimorphic, fungal soil saprophyte. Differential diagnoses include
glanders (farcy), caused by Burkholderia mallei, ulcerative lymphangitis due to Corynebacterium
pseudotuberculosis, sporotrichosis caused by Sporothrix schenckii, and the skin lesions of
histoplasmosis caused by H. capsulatum var. capsulatum. Amphotericin B injection with local
wound drainage and inorganic iodides are used to treat early cases.
Identification of the agent: Identification of the agent is made by its appearance in smears of the
exudate or in histological sections of the lesion material. The yeast form of the organism is present
in large numbers in well established lesions, and appears as pleomorphic ovoid to globose
structures, approximately 25 m in diameter, located both extracellularly and intracellularly in
macrophages and giant cells. Organisms are usually surrounded by a halo when stained with
Gram stain, haematoxylin and eosin, Periodic acidSchiff reaction or Gomori methenaminesilver
stain. The mycelial form of the organism grows slowly under aerobic conditions at 2530C on a
variety of media, including Mycobiotic agar, enriched Sabourauds dextrose agar, brainheart
infusion agar, and pleuropneumonia-like organism nutrient agar. Conversion to the yeast phase at
37C must be demonstrated.
Serological and other tests: Antibodies to H. capsulatum var. farciminosum develop at or before
the onset of clinical signs. Assays reported for detection of antibody include fluorescent antibody,
enzyme-linked immunosorbent assay, and passive haemagglutination tests. In addition, a skin
hypersensitivity test has been described.
Requirements for vaccines and diagnostic biologicals: Killed and live vaccines have been used
on a limited scale in endemic areas, but they are not readily available.
A. INTRODUCTION
Epizootic lymphangitis is a contagious, chronic disease of horses, mules and donkeys. The disease is
characterised clinically by a suppurative, ulcerating, and spreading pyogranulomatous, multifocal dermatitis and
lymphangitis. It is seen most commonly in the extremities, chest wall and the neck, but it can also be present as
an ulcerating conjunctivitis of the palpebral conjunctiva, or rarely as a multifocal pneumonia. The organism may
also invade open lesions including ruptured strangles abscesses and castration wounds. It has also been called
pseudofarcy or pseudoglanders. Another synonym is equine histoplasmosis, which may be a more accurate
name for the disease, as not all clinical cases present obvious lymphangitis. The form that the disease takes
seems to depend primarily on the route of entry (Singh, 1965). The traumatised skin is either infected directly by
infected pus, nasal or ocular excretions or indirectly by soil or contaminated harnesses, grooming equipment,
feeding and watering utensils, wound dressings or flies. It is also believed that ticks may play a role in the
transmission of this agent (Ameni & Terefe, 2004). The conjunctival form of the disease is believed to be spread
by flies of the Musca or Stomoxys genera (Singh, 1965). The pulmonary form of the disease is infrequent and is
presumed to occur after inhalation of the organism. The incubation period is from about 3 weeks to 2 months
(Ameni, 2006). In all cases, the lesions are nodular and granulomatous in character, and the organism, once
established, spreads locally by invasion and then via the lymphatics. There is often thickening, or cording, of
lymphatics, with the formation of pyogranulomatous nodules. Regional lymph nodes may by enlarged and
inflamed. Lesions usually heal spontaneously after 23 months, resulting in stellate scar formation. However,
846
Chapter 2.5.4. Epizootic lymphangitis
extensive lesions with high mortality rates can occur in areas where there is poor veterinary care and nutrition
(Ameni, 2006).
The causative agent, Histoplasma capsulatum var. farciminosum, is a thermally dimorphic fungus. The mycelial
form is present in soil; the yeast form is usually found in lesions. Histoplasma farciminosum was formerly
described as an independent species, but this assessment has been changed and it is now considered to be a
variety of H. capsulatum due to the close morphological similarities of both the mycelial and yeast forms (Ueda et
al, 2003). Antigenically, H. capsulatum var. farciminosum and H. capsulatum var. capsulatum are
indistinguishable, however the latter is the cause of disseminated histoplasmosis, is endemic in North America
and has a wide host range (Robinson & Maxie, 1993). DNA sequences of four protein-coding genes have been
analysed to elucidate the evolutionary relationships of H. capsulatum varieties. This indicated that H. capsulatum
var. farciminosum is deeply buried in the branch of SAm Hcc group A, (H60 to -64, -67, -71, -74 and -76), looking
as if it were an isolate of South American H. capsulatum var. capsulatum (Kasuga et al, 1999).
The cutaneous form of the disease may be confused with farcy (the skin form of glanders), which is caused by
Burkholderia mallei, ulcerative lymphangitis, which is caused by Corynebacterium pseudotuberculosis, indolent
ulcers acaused by Rhodococcus equi, sporotrichosis caused by Sporothrix schenckii, and histoplasmosis caused
by H. capsulatum var. capsulatum, cryptococcosis, strangles. sarcoids and cutaneous lymphosarcomas
(Jungerman & Schwartzman, 1972; Lehmann et al, 1996).
The disease is more common in the tropics and subtropics and is endemic in north, east and north-east Africa,
and some parts of Asia, including some countries bordering the Mediterranean Sea, India, Pakistan and Japan.
The disease is common in Ethiopia, especially in cart horses, affecting an average of 18.8% of horses in warm,
humid areas between 1500 and 2300 metres above sea level (Ameni, 2006; Ameni & Terefe, 2004). Reports from
other parts of the world are sporadic and all cases must be verified by laboratory testing. The prevalence of the
disease increases with assembling of animals; it was much more common, historically, when large numbers of
horses were stabled together for cavalry and other transportation needs. Mainly, it is horses, mules, and donkeys
that are affected by the disease, although infection may occur in camels, cattle and dogs (Ueda et al, 2003).
Experimentally, other animals are refractory to infection subsequent to inoculation, with the exception of certain
laboratory animal species such as mice, guinea-pigs and rabbits (Herve et al, 1994; Singh, 1965). Infection in
humans has also been reported (Al-Ani et al, 1998; Chandler et al, 1980; Guerin et al, 1992).
The disease is eradicated by the humane slaughter of infected horses, disinfection of infected premises and
restricting the movement of equids from infected premises. In endemic areas where eradication is not possible,
inorganic iodides can be used for therapy in early cases (Al-Ani, 1999). Localised nodules can also be lanced, the
pus drained and the nodules packed with a 7% tincture of iodine. If affordable, amphotericin B can be used.
As the clinical signs of epizootic lymphangitis can be confused with those of other diseases in the field, definitive
diagnosis rests on laboratory confirmation.
1.
Material should be collected directly from unruptured nodules. For microbiological isolation, the material should be
placed in a liquid nutrient medium with antibacterials and kept refrigerated until culturing, which should be
attempted as soon as possible. For direct examination, swabs of lesion material can be smeared on glass slides
and fixed immediately. For histopathology, sections of lesion material, including both viable and nonviable tissue,
should be placed in 10% neutral buffered formalin. Confirmation of the disease is dependent on the
demonstration of H. capsulatum var. farciminosum.
a)
Direct microscopic examination
Gram-stained smears
Smears can be stained directly with Grams stain and examined for the typical yeast form of the organism,
which will appear as Gram-positive, pleomorphic, ovoid to globose structures, approximately 25 m in
diameter (Al-Ani et al., 1998). They may occur singly or in groups, and may be found either extracellularly or
within macrophages. A halo around the organisms (unstained capsule) is frequently observed.
Histopathology
In haematoxylin and eosin (H&E)-stained histological sections, the appearance of the lesion is quite
characteristic and consists of pyogranulomatous inflammation with fibroplasia. Langhans giant cells are
common. The presence of numerous organisms, both extracellularly and intracellularly within macrophages
847
or multinucleated giant cells in tissue sections stained with H&E, Periodic acidSchiff reaction and Gomori
methenaminesilver stain are observed (Robinson & Maxie, 1993). There is some indication that the number
of organisms increases with chronicity. The organisms are pleomorphic, often described as slightly lemonshaped basophilic masses, varying from 2 to 5 m in diameter, that are surrounded by a halo when stained
with H&E or Grams stain (Al-Ani, 1999).
Electron microscopy
Electron microscopy has been applied to skin biopsy samples of 1.52.0 mm immediately prefixed in
phosphate buffered 2% glutaraldehyde solution at 4C and post-fixed in 1% osmium tetroxide. Ultra-thin
sections were cut and stained with uranyl acetate and lead citrate. Examination demonstrated the fine
internal structure of the organism, H. capsulatum var. farciminosum, including the cell envelope, plasma
membrane, cell wall, capsule and inner cell structures (Al-Ani, 1999).
b)
Culture
The mycelial form of H. capsulatum var. farciminosum grows slowly on laboratory media (28 weeks at
26C). Media that can be used include Mycobiotic agar (Al-Ani et al, 1998), Sabourauds dextrose agar agar
enriched with 2.5% glycerol, brainheart infusion agar supplemented with 10% horse blood, and
pleuropneumonia-like organism (PPLO) nutrient agar enriched with 2% dextrose and 2.5% glycerol, pH 7.8
(Guerin et al, 1992; Robinson & Maxie, 1993). The addition of antibiotics to the media is recommended:
cycloheximide (0.5 g/litre) and chloramphenicol (0.5 g/litre). Broad-spectrum antibacterial activity is obtained
if gentamicin (50 mg/litre) and penicillin G (6 106 units/litre) are used instead of chloramphenicol. Colonies
appear in 28 weeks as dry, grey-white, granular, wrinkled mycelia. The colonies become brown with aging.
Aerial forms occur, but are rare. The mycelial form produces a variety of conidia, including chlamydospores,
arthroconidia and some blastoconidia. However, the large round double-walled macroconidia that are often
observed in H. capsulatum var. capsulatum are lacking.
As a confirmatory test the yeast form of H. capsulatum var. farciminosum can be induced by subculturing
some of the mycelium into brainheart infusion agar containing 5% horse blood or by using Pines medium
alone at 3537C in 5% CO2. Yeast colonies are flat, raised, wrinkled, white to greyish brown, and pasty in
consistency (Robinson & Maxie, 1993). However, complete conversion to the yeast phase may only be
achieved after four to five repeated serial transfers on to fresh media every 8 days.
c)
Animal inoculation
Experimental transmission of H. capsulatum var. farciminosum has been attempted in mice, guinea-pigs and
rabbits. Immunosuppressed mice are highly susceptible to experimental infection and can be used for
diagnostic purposes (Al-Ani, 1999).
2.
Serological tests
There are published reports of various tests to detect antibodies as well as a skin hypersensitivity test for
detection of cell-mediated immunity. Antibodies usually develop at or just after the onset of clinical signs.
a)
Fluorescent antibody tests
The following non-quantitative procedure is as described by Fawi (1969).

i)
Slides containing the organisms are made by smearing the lesion contents on to a glass slide or by
emulsifying the cultured yeast phase of the organism in a saline solution and creating a thin film on a
glass slide.
ii)
The slides are heat-fixed by passing the slide through a flame.
iii)
The slides are then washed in phosphate buffered saline (PBS) for 1 minute.
iv)
Undiluted test sera are placed on the slides, which are then incubated for 30 minutes at 37C.
v)
The slides are washed in PBS three times for 10 minutes each.
vi)
Fluorescein isothiocyanate (FITC)-conjugated anti-horse antibody at an appropriate dilution is flooded

over the slides, which are then incubated for 30 minutes at 37C.
vii)
Washing in PBS is repeated three times for 10 minutes each.
viii) The slides are examined using fluorescence microscopy.
848
Direct fluorescent antibody test
The following procedure is as described by Gabal et al (1983).
b)
i)
The globulin fraction of the test serum is precipitated, and then re-suspended to its original serum
volume in saline. The serum is then conjugated to FITC.
ii)
Small colony particles of the cultured mycelial form of the organism are suspended in 12 drops of
saline on a glass slide. With a second slide, the colony particles are crushed and the solution is
dragged across the slide to create a thin film.
iii)
The smears are heat-fixed.
iv)
The slides are washed in PBS for 1 minute.
v)
The slides are incubated with dilutions of conjugated serum for 60 minutes at 37C.
vi)
The slides are washed in PBS three times for 5 minutes each.
vii)
The slides are examined using fluorescence microscopy.
Indirect Enzyme-linked immunosorbent assay

The following procedure is as described by Gabal & Mohammed (1985).
i)
The mycelial form of the organism is produced on Sabourauds dextrose agar in tubes, and incubated
for 4 weeks at 26C. Three colonies are ground in 50 ml of sterile PBS. The suspension is diluted
1/100 and the 96-well microtitre plates are coated with 100 l/well.
ii)
The plates are incubated at 4C overnight.
iii)
The plates are washed with PBS containing Tween 20 (0.5 ml/litre) (PBS-T) three times for 3 minutes
each.
iv)
The plates are incubated with 5% bovine serum albumin, 100 l/well, at 2325C for 30 minutes, with
shaking.
v)
The plates are washed with PBS-T three times for 3 minutes each.
vi)
The sera are serially diluted using twofold dilution in duplicate in PBS-T, starting with a 1/50 dilution
and incubated for 30 minutes at 2325C.
vii)
viii) Peroxidase-labelled goat anti-horse IgG is diluted 1/800 and used at 100 l/well, with incubation for
30 minutes at 2325C, with shaking.
c)
ix)
x)
Finally, 100 l/well of hydrogen peroxide and ABTS (2,2-Azino-di-[3-ethyl-benzthiazoline]-6-sulphonic

acid) in a citric acid buffer, pH 4, is added.
xi)
The plates are read at 60 minutes in a spectrophotometer at wavelength 405 nm.
xii)
The absorbance values are obtained twice from each serum dilution and the standard deviation and
average percentage of the absorbance values of the different serum samples are considered in the
interpretation of the results.
Passive haemagglutination test

The following procedure is as described by Gabal & Khalifa (1983).
i)
The organism is propagated for 8 weeks on Sabourauds dextrose agar. Five colonies are scraped,
ground, suspended in 200 ml of saline, and sonicated for 20 minutes. The remaining mycelial elements
are filtered out, and the filtrate is diluted 1/160.
ii)
Normal sheep red blood cells (RBCs) are washed, treated with tannic acid, washed, and re-suspended
as a 1% cell suspension.
iii)
Different dilutions of the antigen preparation are mixed with the tanned RBCs and incubated in a water
bath at 37C for 1 hour. The RBCs are collected by centrifugation, washed three times in buffered
saline and re-suspended to make a 1% cell suspension.
iv)
Test sera are inactivated by heating at 56C for 30 minutes and then absorbed with an equal volume of
washed RBCs.
849
d)
v)
Dilutions of serum (0.5 ml) are placed in test tubes with 0.05 ml of antigen-coated tanned RBCs.
vi)
Agglutination is recorded at 2 and 12 hours.
vii)
Agglutination is detected when the RBCs form a uniform mat on the bottom of the tube. A negative test
is indicated by the formation of a button of RBCs at the bottom of the tube.
Skin hypersensitivity tests

Two skin hypersensitivity tests for the diagnosis of epizootic lymphangitis have been described. The first test
was described by Gabal & Khalifa (1983) and adapted by Armeni et al (2006).
i)
A pure culture of H. farcimonsum is propagated for 8 weeks on Sabourauds dextrose agar containing
2.5% glycerol. Five colonies are scraped, ground, suspended in 200 ml of saline, undergo five freeze
thaw cycles and are sonicated at an amplitude of 40 for 20 minutes. The remaining mycelial elements
are removed by centrifugation at 1006 g at 4C for 11 minutes. Sterility of the preparation is verified by
incubating an aliquot on Sabourauds dextrose agar at 26C for 4 weeks.
ii)
Animals are inoculated intradermally with 0.1 ml containing 0.2 mg/ml protein in the neck.
iii)
The inoculation site is examined for the presence of a local indurated and elevated area at 24
48 hours post-injection. An increase in skin thickness of > 4 mm is considered to be positive.
Alternatively, a histofarcin test has been described by Soliman et al (1985).

i)
The mycelial form of the organism is grown on polystyrene discs floating on 250 ml of PPLO media
containing 2% glucose and 2.5% glycerine at 2325C for 4 months.
ii)
The fungus-free culture filtrate is mixed with acetone (2/1) and held at 4C for 48 hours.
iii)
The supernatant is decanted and the acetone is allowed to evaporate.
iv)
Precipitate is suspended to 1/10 original volume in distilled water.
v)
Animals are inoculated intradermally with 0.1 ml of antigen in the neck.
vi)
The inoculation site is examined for the presence of a local indurated and elevated area at 24, 48 and
72 hours post-injection.

Control of the disease is usually through elimination of the infection. This is achieved by culling infected horses
and application of strict hygiene practices to prevent spread of the organism. There are published reports on the
use of killed (Al-Ani et al, 1998) and live attenuated vaccines (Zhang et al, 1986) in areas where epizootic
lymphangitis is endemic, apparently with relatively good results.
The antigens used for skin hypersensitivity testing are described in the previous section.
REFERENCES
AL-ANI F.K. (1999). Epizootic lymphangitis in horses: a review of the literature. Rev. sci. tech. Off. int. Epiz., 18,
691699.
AL-ANI F.K., ALI A.H. & BANNA H.B. (1998). Histoplasma farciminosum infection of horses in Iraq. Veterinarski
Arhiv., 68, 101107.
AMENI G. (2006). Preliminary trial on the reproducibility of epizootic lymphangitis through experimental infection of
two horses. Short Communication. Veterinary J., 172 (3), 553555.
AMENI G. & TEREFE W. (2004). A cross-sectional study of epizootic lymphangitis in cart-mules in western Ethiopia.
Preventive Vet. Med., 66, 9399.
AMENI G. TEREFE W. & HAILU A. (2006). Histofarcin test for the diagnosis of epizootic lymphanigitis in Ethiopia:
development, optimisation and validation in the field. Veterinary J., 171, 358362.
850
CHANDLER F.W., KAPLAN W. & AJELLO L. (1980). Histopathology of Mycotic Diseases. Year Book Medical
Publishers, Chicago, USA, 7072 and 216217.
FAWI M.T. (1969). Fluorescent antibody test for the serodiagnosis of Histoplasma farciminosum infections in
Equidae. Br. Vet. J., 125, 231234.
GABAL M.A., BANA A.A. & GENDI M.E. (1983). The fluorescent antibody technique for diagnosis of equine
histoplasmosis (epizootic lymphangitis). Zentralbl. Veterinarmed. [B], 30, 283287.
GABAL M.A. & KHALIFA K. (1983). Study on the immune response and serological diagnosis of equine
histoplasmosis (epizootic lymphangitis). Zentralbl. Veterinarmed. [B], 30, 317321.
GABAL M.A. & MOHAMMED K.A. (1985). Use of enzyme-linked immunosorbent assay for the diagnosis of equine
Histoplasma farciminosi (epizootic lymphangitis). Mycopathologia, 91, 3537.
GUERIN C., ABEBE S. & TOUATI F. (1992). Epizootic lymphangitis in horses in Ethiopia. J. Mycol. Med., 2, 15.
HERVE V., LE GALL-CAMPODONICO P., BLANC F., IMPROVISI, L., DUPONT, B, MATHIOT C. & LE GALL F. (1994).
Histoplasmose a Histoplasma farciminosum chez un cheval africain. J. Mycologie Med., 4, 54.
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KASUGA T., TAYLOR T.W. & WHITE T.J. (1999). Phylogenetic relationships of varieties and geographical groups of
the human pathogenic fungus Histoplasma capsulatum darling. J. Clin. Microbiol., 37, 653663.
LEHMANN P.F. HOWARD D.H. & MILLER J.D. (1996). Veterinary Mycology. Springer-Verlag, Berlin, Germany, 96,
251263.
ROBINSON W.F. & MAXIE M.G. (1993). The cardiovascular system. In: Pathology of Domestic Animals, Vol. 3.
Academic Press, New York, USA, 8284.
SINGH T. (1965). Studies on epizootic lymphangitis. I. Modes of infection and transmission of equine
histoplasmosis (epizootic lymphangitis). Indian J. Vet. Sci., 35, 102110.
SOLIMAN R., SAAD M.A. & REFAI M. (1985). Studies on histoplasmosis farciminosii (epizootic lymphangitis) in Egypt.
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461.
UEDA Y., SANO A. TAMURA M., INOMATA T., KAMEI K., YOKOYAMA K., KISHI F., ITO J., Y., MIYAJI M. & NISHIMURA K.
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from a paraffin-embedded skin sample from a dog in Japan. Vet. Microbiol., 94, 219224.
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vaccine against epizootic lymphangitis in horses. Chinese J. Vet. Sci. Tech., 7, 35.
*
* *
851
CHAPTER 2.5.5.
EQUINE ENCEPHALOMYELITIS
SUMMARY
Eastern and Western equine encephalomyelitis viruses belong to the genus Alphavirus of the family
Togaviridae. Alternate infection of birds and mosquitoes maintain these viruses in nature. The
disease occurs sporadically in horses and humans from mid-summer to late autumn. Horses and
humans are tangential dead-end hosts. The disease in horses is characterised by fever, anorexia,
and severe depression. In severe cases, the disease in horses progresses to hyperexcitability,
blindness, ataxia, severe mental depression, recumbency, convulsions, and death. Eastern equine
encephalomyelitis (EEE) virus infection in horses is often fatal, while Western equine
encephalomyelitis (WEE) virus can cause a subclinical or mild disease with less than 30% mortality.
EEE and WEE have been reported to cause disease in poultry, game birds and ratites. Sporadic
cases of EEE have been reported in cows, sheep, pigs, deer, and dogs.
Identification of the agent: A presumptive diagnosis of EEE or WEE can be made when
susceptible horses display the characteristic somnolence and other signs of neurological disease in
areas where haematophagous insects are active. There are no characteristic gross lesions.
Histopathological lesions can provide a presumptive diagnosis. EEE virus can usually be isolated
from the brain and sometimes other tissues of dead horses, however WEE virus is rarely isolated.
EEE and WEE viruses can be isolated from field specimens by inoculating newborn mice,
embryonating chicken eggs, cell cultures, or newly hatched chickens. The virus is identified by
complement fixation (CF), immunofluorescence, or plaque reduction neutralisation (PRN) tests.
EEE and WEE viral RNA may also be detected by reverse-transcription polymerase chain reaction
methods.
Serological tests: Antibody can be identified by PRN, haemagglutination inhibition (HI), CF tests,
or IgM capture enzyme-linked immunosorbent assay.
Requirements for vaccines and diagnostic biologicals: EEE and WEE vaccines are safe and
immunogenic. They are produced in cell culture and inactivated with formalin.
A. INTRODUCTION
Eastern equine encephalomyelitis (EEE) and Western equine encephalomyelitis (WEE) viruses are members of
the genus Alphavirus of the family Togaviridae. The natural ecology for virus maintenance occurs via alternate
infection of birds and ornithophilic mosquitoes. Clinical disease may be observed in humans and horses, both of
which are dead-end hosts for these agents. EEE has been diagnosed in Quebec and Ontario in Canada, central
and eastern regions of the United States of America (USA), the Caribbean Islands, Mexico, and Central and
South America. Disease caused by the WEE virus has been reported in the western USA and Canada, Mexico,
and Central and South America (Morris, 1989; Reisen & Monath, 1989; Walton et al., 1981). Highlands J virus,
antigenically related to WEE virus, has been isolated in eastern USA. Although Highlands J virus is generally
believed not to cause disease in mammals, it has been isolated from the brain of a horse dying of encephalitis in
Florida (Karabatsos et al., 1988).
Even though the mortality is lower for WEE, the clinical signs of EEE and WEE can be identical. The disease
caused by either virus is also known as sleeping sickness. Following an incubation period of 514 days, clinical
signs include fever, anorexia, and depression. A presumptive diagnosis of EEE or WEE virus infection in
unvaccinated horses can be made if the characteristic somnolence is observed during the summer in temperate
climates or the wet season in tropical and subtropical climates, when the mosquito vector is plentiful. However, a
number of other diseases, such as West Nile virus and Venezuelan equine encephalomyelitis (chapters 2.1.20
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OIE Manual Terrestrial 2012
Chapter 2.5.5. Equine encephalomyelitis (Eastern and Western)
and 2.5.14, respectively), produce similar clinical signs and the diagnosis must be confirmed by the described
diagnostic test methods. WEE virus infection in horses is often observed over a wide geographical area, e.g.
sporadic cases over 1000 square miles. EEE virus infections are usually observed in limited geographical areas.
Isolated events of high mortality in captive-raised game birds, primarily pheasants, chukars, aquarium penguins,
and quail have been traced to WEE, EEE, or Highlands J virus infection (Morris, 1989; Reisen & Monath, 1989;
Tuttle et al., 2005). Most encephalomyelitis infections in domestic fowl are caused by EEE virus and occur on the
east coast states of the USA. The virus is introduced by mosquitoes, but transmission within the flocks is primarily
by feather picking and cannibalism. Both EEE and WEE viruses have caused a fatal disease in ratites.
Haemorrhagic enteritis has been observed in emus infected with EEE and WEE viruses, and morbidity and
mortality rates may be greater than 85%. Highlands J and EEE viruses have been found to produce depression,
somnolence, decreased egg production, and increased mortality in turkeys (Guy, 1997). EEE virus has been
reported to cause disease in cows (McGee et al., 1992; Pursell et al., 1976), sheep (Bauer et al., 2005), pigs
(Elvinger et al., 1996), white-tailed deer (Tate et al., 2005), and dogs (Farrar et al., 2005).
EEE virus causes severe disease in humans with a mortality rate of 3070% and a high frequency of permanent
sequelae in patients who survive. WEE is usually mild in adult humans, but can be a severe disease in children.
The fatality rate is between 3 and 14%. Severe infection and death caused by EEE and WEE viruses have been
reported in laboratory workers; therefore, any work with these viruses must be performed at containment level 3
(see Chapter 1.1.3 Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities). It is
recommended that personnel be immunised against EEE and WEE viruses (United States Department of Health
and Human Services, 1999). Precautions should also be taken to prevent human infection when performing postmortem examinations on horses suspected of being infected with the equine encephalomyelitis viruses.
1.
The most definitive method for diagnosis of EEE or WEE is the isolation of the viruses. EEE virus can usually be
isolated from the brains of horses, unless more than 5 days have elapsed between the appearance of clinical
signs and the death of the horse. EEE virus can frequently be isolated from brain tissue even in the presence of a
high serum antibody titre. WEE virus is rarely isolated from tissues of infected horses. Brain is the tissue of choice
for virus isolation, but the virus has been isolated from other tissues, such as the liver and spleen. It is
recommended that a complete set of these tissues be collected in duplicate, one set for virus isolation and the
other set in formalin for histopathological examination. Specimens for virus isolation should be sent refrigerated if
they can be received in the laboratory within 48 hours of collection; otherwise, they should be frozen and sent with
dry ice. A complete set of tissues will allow the performance of diagnostic techniques for other diseases. For
isolation, a 10% suspension of tissue is prepared in phosphate buffered saline (PBS), pH 7.8, containing bovine
serum albumin (BSA) (fraction V; 0.75%), penicillin (100 units/ml), and streptomycin (100 g/ml). The suspension
is clarified by centrifugation at 1500 g for 30 minutes.
EEE and WEE viruses can be isolated in a number of cell culture systems. The most commonly used cell cultures
are primary chicken or duck embryo fibroblasts, continuous cell lines of African green monkey kidney (Vero),
rabbit kidney (RK-13), or baby hamster kidney (BHK-21). Isolation is usually attempted in 25 cm2 cell culture
flasks. Confluent cells are inoculated with 1.0 ml of tissue suspension. Following a 12-hour absorption period,
maintenance medium is added. Cultures are incubated for 68 days, and one blind passage is made. EEE and
WEE viruses will produce a cytopathic change in cell culture. Cultures that appear to be infected are frozen. The
fluid from the thawed cultures is used for virus identification.
The newborn mouse is also considered to be a sensitive host system. Inoculate intracranially one or two litters of
14-day-old mice with 0.02 ml of inoculum using a 26-gauge 3/8 inch (9.3 mm) needle attached to a 1 ml
tuberculin syringe. The inoculation site is just lateral to the midline into the midportion of one lateral hemisphere.
Mice are observed for 10 days. Mice that die within 24 hours of inoculation are discarded. From 2 to 10 days
postinoculation, dead mice are collected daily and frozen at 70C. Mouse brains are harvested for virus
identification by aspiration using a 20-gauge 1 inch (2.5 cm) needle attached to a 1 ml tuberculin syringe. A
second passage is made only if virus cannot be identified from mice that die following inoculation.
The chicken embryo is considered to be less sensitive than newborn mice when used for primary isolation of EEE
and WEE viruses. Tissue suspensions can be inoculated by the yolk-sac route into 68-day-old embryonating
chicken eggs. There are no diagnostic signs or lesions in the embryos infected with these viruses. Inoculated
embryos should be incubated for 7 days, but deaths usually occur between 2 and 4 days post-inoculation. Usually
only one passage is made unless there are dead embryos from which virus cannot be isolated. Newly hatched
chickens are susceptible and have been used for virus isolation. If this method is used, precautions must be taken
to prevent aerosol exposure of laboratory personnel, as infected birds can shed highly infectious virus.
853
EEE or WEE viruses can be identified in infected mouse or chicken brains, cell culture fluid, or amnionic-allantoic
fluid by complement fixation. A 10% brain suspension is prepared in veronal (barbitone) buffer; egg and cell
culture fluids are used undiluted or diluted 1/10 in veronal buffer. The fluid or suspension is centrifuged at 9000 g
for 30 minutes, and the supernatant fluid is tested against hyperimmune serum or mouse ascitic fluid prepared
against EEE and WEE viruses using a standard CF procedure (United States Department Of Health, Education
and Welfare, 1974). The CF test requires the overnight incubation at 4C of serum-antigen with 7 units of
complement. Virus can be identified in cell culture by direct immunofluorescent staining. The less commonly used
method of virus identification is the neutralisation test, as outlined below.
Reverse-transcription polymerase chain reaction (RT-PCR) methods to detect EEE, WEE and VEE viral nucleic
acid in mosquitoes and vertebrate tissues have been described, although few have been extensively validated for
mammalian samples (Lambert et al., 2003; Linssen et al., 2000; Monroy et al., 1996; Vodkin et al., 1993). A
multiplex PCR method was developed to expedite differential diagnosis in cases of suspected EEE or West Nile
arboviral encephalomyelitis in horses (Johnson et al., 2003). The assay has enhanced speed and sensitivity
compared with cell culture virus isolation and has been used effectively in the USA during several recent
arbovirus seasons. Recently, a combination of an RT-PCR with an enzyme-linked immunosorbent assay (ELISA:
RT-PCR-ELISA) was reported as a method to identify alpha-viruses that are pathogenic to humans (Wang et al.,
2006).
Antigen-capture ELISA has been developed for EEE surveillance in mosquitoes. This can be used in countries
that do not have facilities for virus isolation or PCR (Brown et al., 2001). Immunohistochemical procedures for
diagnosis of EEE have also been described (Patterson et al., 1996).
2.
Serological tests
Serological confirmation of EEE or WEE virus infection requires a four-fold or greater increase or decrease in
antibody titre in paired serum samples collected 1014 days apart. Most horses infected with EEE or WEE virus
have a high antibody titre when clinical disease is observed. Consequently, a presumptive diagnosis can be made
if an unvaccinated horse with appropriate clinical signs has antibody against only EEE or WEE virus. The
detection of IgM antibody by the ELISA can also provide a presumptive diagnosis of acute infection (Sahu et al.,
1994). The plaque reduction neutralisation (PRN) test or, preferably, a combination of PRN and
haemagglutination inhibition (HI) tests is the procedure most commonly used for the detection of antibody against
EEE and WEE viruses. There may be cross-reactions between antibody against EEE and WEE virus in the CF
and HI tests. CF antibody against both EEE and WEE viruses appears later and does not persist; consequently, it
is less useful for the serological diagnosis of disease.
a)
Complement fixation
The CF test is frequently used for the demonstration of antibodies, although the antibodies detected by the
CF test may not persist for as long as those detected by the HI or PRN tests. A sucrose/acetone mouse
brain extract is commonly used as antigen. The positive antigen is inactivated by treatment with 0.1% betapropiolactone.
In the absence of an international standard serum, the antigen should be titrated against a locally prepared
positive control serum. The normal antigen, or control antigen, is mouse brain from uninoculated mice
similarly extracted and diluted.
Sera are diluted 1/4 in veronal buffered saline containing 1% gelatin (VBSG), and inactivated at 56C for
30 minutes. Titrations of positive sera may be performed using additional twofold dilutions. The CF antigens
and control antigen (normal mouse brain) are diluted in VBSG to their optimal amount of fixation as
determined by titration against the positive sera; guinea-pig complement is diluted in VBSG to contain
5 complement haemolytic units-50% (CH50). Sera, antigen, and complement are reacted in 96-well roundbottom microtitre plates at 4C for 18 hours. The sheep red blood cells (SRBCs) are standardised to 2.8%
concentration. Haemolysin is titrated to determine the optimal dilution for the lot of complement used.
Haemolysin is used to sensitise 2.8% SRBCs and the sensitised cells are added to all wells on the microtitre
plate. The test is incubated for 30 minutes at 37C. The plates are then centrifuged (200 g), and the wells
are scored for the presence of haemolysis. The following controls are used: (a) serum and control serum
each with 5 CH50 and 2.5 CH50 of complement; (b) CF antigen and control antigen each with 5 CH50, and
2.5 CH50 of complement; (c) complement dilutions of 5 CH50, 2.5 CH50, and 1.25 CH50; and (d) cell control
wells with only SRBCs and VBSG diluent. These controls test for anticomplementary serum and
anticomplementary antigen, activity of complement used in the test, and integrity of the SRBC indicator
system in the absence of complement, respectively.
To avoid anticomplementary effects, sera should be separated from the blood as soon as possible. Positive
and negative control sera should be used in the test.
854
b)
The antigen for the HI test is the same as described above for the CF test. The antigen is diluted so that the
amount used in each haemagglutinating unit (HAU) is from four to eight times that which agglutinates 50% of
the RBCs in the test system. The haemagglutination titre and optimum pH for each antigen are determined
with goose RBCs diluted in pH solutions ranging from pH 5.8 to pH 6.6, at 0.2 intervals.
Sera are diluted 1/10 in borate saline, pH 9.0, and then inactivated at 56C for 30 minutes. Kaolin treatment is
used to remove nonspecific serum inhibitors. Alternatively, nonspecific inhibitors may be removed by acetone
treatment of serum diluted 1/10 in PBS followed by reconstitution in borate saline. Sera should be absorbed
before use by incubation with a 0.05 ml volume of washed packed goose RBCs for 20 minutes at 4C.
Following heat inactivation, kaolin treatment and absorption, twofold dilutions of the treated serum are
prepared in borate saline, pH 9.0 with 0.4% bovalbumin. Serum dilutions (0.025 ml/well) are prepared in a
96-well round-bottom microtitre plate in twofold dilutions in borate saline, pH 9.0, with 0.4% bovalbumin.
Antigen (0.025 ml/well) is added to the serum. Plates are incubated at 4C overnight. RBCs are derived from
normal white male geese1 and washed three times in dextrose/gelatin/veronal (DGV), and a 7.0%
suspension is prepared in DGV. The 7.0% suspension is then diluted 1/24 in the appropriate pH solution,
and 0.05 ml per well is added immediately to the plates. Plates are incubated for 30 minutes at 37C.
Positive and negative control sera are incorporated into each test. A test is considered to be valid only if the
control sera give the expected results. Titres of 1/10 and 1/20 are suspect, and titres of 1/40 and above are
positive.
c)

The ELISA is performed by coating flat-bottomed plates with anti-equine IgM capture antibody (Sahu et al.,
1994). The antibody is diluted according to the manufactures recommendations in 0.5 M carbonate buffer,
pH 9.6, and 50 l is added to each well. The plates are incubated at 37C for 1 hour, and then at 4C
overnight. Prior to use, the coated plates are washed three times with 200300 l/well of 0.01 M PBS
containing 0.05% Tween 20. After the second wash, 200 l/well of PBS/Tween/5% nonfat dried milk is
added and the plates are incubated at room temperature for 1 hour. Following incubation, the plates are
washed again three times with PBS/Tween. Test and control sera are diluted 1/400 in 0.01 M PBS, pH 7.2,
containing 0.05% Tween 20, and 50 l is added to each well. The plates are incubated at 37C for
90 minutes and then washed three times. Next, 50 l of viral antigen is added to all wells. (The dilution of the
antigen will depend on the source and should be empirically determined.) The plates are incubated overnight
at 4C, and washed three times. Then, 50 l of horseradish-peroxidase-conjugated monoclonal antibody
(MAb) to encephalitis virus2 is added. The plates are incubated for 90 minutes at 37C and then washed six
times. Finally, 50 l of freshly prepared ABTS (2,2-Azino-bis-[3-ethylbenzo-thiazoline-6-sulphonic acid])
substrate + hydrogen peroxidase is added, and the plates are incubated at room temperature for 15
40 minutes The absorbance of the test serum is measured at 405 nm. A test sample is considered to be
positive if the absorbance of the test sample in wells containing virus antigen is at least twice the
absorbance of negative control serum in wells containing virus antigen and at least twice the absorbance of
the sample tested in parallel in wells containing normal antigen.
d)

The PRN test is very specific and can be used to differentiate between EEE and WEE virus infections. The
PRN test is performed in duck embryo fibroblast, Vero, or BHK-21 cell cultures in 25 cm2 flasks or six-well
plates. Volumes listed are for flasks; the volume should be halved for wells in six-well plates. The sera can
be screened at a 1/10 and 1/100 final dilution. Endpoints can be established using the PRN or HI test.
Serum used in the PRN assay is tested against 100 plaque-forming units (PFU) of virus (50 PFU for six-well
plates). The virus/serum mixture is incubated at 37C for 75 minutes before inoculation on to confluent cell
culture monolayers in 25 cm2 flasks. The inoculum is adsorbed for 1 hour, followed by the addition of 6 ml of
overlay medium. The overlay medium consists of two solutions that are prepared separately. Solution I
contains 2 Earles Basic Salts Solution without phenol red, 4% fetal bovine serum, 100 g/ml gentamicin,
200 g/ml nystatin, 0.45% solution of sodium bicarbonate, and 0.002% neutral red. When duck embryo
fibroblasts are used, Solution 1 also contains 6.6% yeast extract lactalbumin hydrolysate. Solution II consists
of 2% Noble agar that is sterilised and maintained at 47C. Equal volumes of solutions I and II are adjusted
to 47C and mixed together just before use. The test is incubated for 4872 hours, and endpoints are based
on a 90% reduction in the number of plaques compared with the virus control flasks, which should have
about 100 plaques.
1
2
RBCs from adult domestic white male geese are preferred, but RBCs from other male geese can be used. If cells from
female geese are used, there may be more test variability. It has been reported that rooster RBCs cause a decrease in
the sensitivity of the test.
Available from: Centers for Disease Control and Prevention, Biological Reference Reagents, 1600 Clifton Road NE, Mail
Stop C21, Atlanta, Georgia 30333, United States of America.
855

Inactivated vaccines against EEE and WEE viruses are available commercially. Attenuated EEE and WEE virus
vaccines have not proven satisfactory. The vaccines licensed for use in the USA are prepared using the following
combinations: EEE and WEE; EEE, WEE, and Venezuelan equine encephalomyelitis (VEE); and EEE and VEE.
In addition, tetanus toxoid, inactivated influenza virus, and inactivated West Nile virus have been combined with
EEE and WEE or EEE, WEE, and VEE. Early vaccines were produced from virus propagated in embryonating
chicken eggs and inactivated with formalin. Current vaccines are prepared from virus propagated in cell culture,
and inactivated with formalin (Maire et al., 1970) or monoethylamine.
1.
Seed management
a)

Standard strains of EEE and WEE viruses that were isolated over 20 years ago have been used for vaccine
production and have been proven to produce a protective immunity. Strains of EEE virus that differ
antigenically and in molecular structure have been identified from different geographical regions. However,
the North American and Caribbean isolates appear to be similar (Weaver et al., 1994). Strains of WEE virus
isolated from different countries have been found to be similar both by MAb testing and RNA oligonucleotide
fingerprinting analysis (Reisen & Monath, 1989). A recent well-characterised isolate from the country where
the vaccine is to be used would be advantageous. Viruses that are selected must be immunogenic and
replicate to high titres in cell culture.
b)
Method of culture
Primary chicken embryo fibroblasts and Vero cells have been used for propagation of viruses used for
vaccine production. The fibroblasts should be prepared from specific pathogen free embryos. Other
susceptible cell lines could also be used.
c)
Validation as a vaccine
If a cell line is used, the master cell stock is tested to confirm the identity of the cell line, species of origin,
and freedom from extraneous agents. If primary cell cultures are used, a monolayer from each batch of each
subculture should be tested for extraneous agents including bacteria, fungi, mycoplasma, and viruses. The
master seed virus should also be tested to ensure freedom from bacteria, fungi, mycoplasma, and
extraneous viruses (see Chapter 1.1.7 Tests for sterility and freedom from contamination of biological
materials for a description of the method).
The vaccines are administered by the intramuscular (in most cases) or intradermal route in the cervical
region in two doses given 24 weeks apart. Annual revaccination is recommended. All foals vaccinated
before 1 year of age should be revaccinated before the next vector season.
2.
Details of the manufacture of vaccines currently on the market are not available. Consequently, the information
provided here is intended only as background reference material on the vaccines and not as a method of
manufacture. The virus and cell culture system should be selected so that a high virus titre, 106 TCID50 (50%
tissue culture infective dose) per ml, is obtained in under 48 hours. Virus for vaccine production can be prepared
from the supernatant fluid from infected cell cultures. The fluid is harvested when 70100% of the monolayers
have the characteristic cytopathic changes. The virus titre is determined by titration in cell culture or mice. The
fluid is clarified by low speed centrifugation and filtered through gauze. The virus is inactivated by adding formalin
to a final concentration of 1/2000 (0.05%) and holding at 37C for 24 hours. Residual formaldehyde is neutralised
by sodium bisulphite (Maire et al., 1970). The residual free formalin content in the inactivated vaccine should not
exceed 0.2% formaldehyde.
3.
In-process control
Cultures should be examined daily for virus-induced cytopathic effect. The harvested virus should be tested for
microbial contamination. The efficacy of the inactivation process should be checked by testing for viable virus.
4.
Batch control
a)
Sterility
Tests for sterility and freedom from contamination of biological materials may be found in chapter 1.1.7.
856
b)
Safety
The inactivated vaccine is safety tested by inoculating subcutaneously at least ten 6- to 12-hour-old chickens
with 0.5 ml of the vaccine. The chickens are observed each day for 10 days for unfavourable reactions that
are attributable to the vaccine (United States Code of Federal Regulations, 2000). Safety testing can also be
carried out by inoculating intracerebrally at least eight 14-day-old mice with 0.02 ml of the vaccine, and
observing for 7 days. It is critical that safety tests be conducted on each lot of vaccine to insure that there is
no residual virulent virus present.
c)
Potency
Potency testing is performed by inoculating each of ten guinea pigs with either EEE or WEE virus, using
one-half the horse dose on two occasions, 1421 days apart, by the route recommended for the horse.
Serum samples from each vaccinate and each control are tested 1421 days after the second dose using
the PRN test. The EEE titres should be 1/40, and the WEE titres should be 1/40 (US Code of Federal
Regulations), using Vero cells. If duck embryo fibroblasts are used in the PRN test, the titres will be lower.
An alternative potency test is to use intracerebral challenge, 1421 days after the second vaccination. Each
guinea pig is inoculated with 0.1 ml of virus containing 100 LD50 (50% lethal dose). Simultaneous titration is
carried out. In order for the vaccine to be approved, 80% of the guinea pigs must survive both viruses.
d)
Duration of immunity
Comprehensive studies on duration of immunity are not available. An annual revaccination is recommended.
Foals that are vaccinated before 1 year of age should be revaccinated before the next vector season.
e)
Stability
The lyophilised vaccine is stable and immunogenic for 3 years if kept refrigerated at 27C. After 3 years,
vaccine should be discarded. The vaccines should be used immediately after reconstitution.
f)
Preservatives
The preservatives used are thimerosal at a 1/10,000 dilution and antibiotics (neomycin, polymyxin
amphotercin B and gentamicin).
g)
Precautions (hazards)
Severe infection and death caused by EEE and WEE viruses have been reported in laboratory workers;
therefore, any work with these viruses must be carried out at least in a containment level 3 laboratory (see
chapter 1.1.3) using biological safety cabinets, and it is recommended that personnel be immunised against
EEE and WEE viruses (United States Department of Health and Human Services, 1999).
Pregnant mares and foals under 2 weeks old should not be vaccinated.
5.
Tests on the final product
a)
Safety
See Section C.4.b.
b)
Potency
See Section C.4.c.
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*
* *
NB: There is an OIE Reference Laboratory for Equine encephalomyelitis (Eastern and Western):
diagnostic tests, reagents and vaccines for equine encephalomyelitis (Eastern and Western)
859
CHAPTER 2.5.6.
EQUINE INFECTIOUS ANAEMIA
SUMMARY
Equine infectious anaemia (EIA) is a persistent viral infection of equids. The causative agent, EIA
virus (EIAV) is a lentivirus in the family Retroviridae, subfamily Orthoretrovirinae. Other members of
the lentivirus genus include: bovine immunodeficiency virus; caprine arthritis encephalitis virus;
feline immunodeficiency virus; human immunodeficiency virus 1; human immunodeficiency virus 2;
and maedi/visna virus. EIA can be diagnosed on the basis of clinical signs, pathological lesions,
serology and molecular methods. Infected horses remain viraemic carriers for life and, with very
rare exceptions, yield a positive serological test result. Antibody response usually persists and
antibody-positive animals, older than 68 months, are identified as virus carriers (below 68 months
of age, serological reactions can be due to maternal antibodies; status can be confirmed by
molecular techniques). Infected equids are potential virus reservoirs. Biting flies are mechanical
vectors for the virus in nature.
Identification of the agent: Virus from a horse can be isolated by inoculating suspect blood into a
susceptible horse or on to leukocyte cultures prepared from susceptible horses. Recognition of
infection in horses that have been inoculated experimentally may be made on the basis of clinical
signs, haematological changes and a positive antibody response determined by an
immunodiffusion test or enzyme-linked immunosorbent assay (ELISA) or by molecular techniques.
Successful virus isolation in horse leukocyte cultures is confirmed by the detection of specific EIA
antigen, by immunofluorescence assay, polymerase chain reaction, reverse-transcriptase assay, or
by the inoculation of culture fluids into susceptible horses. Virus isolation is rarely attempted
because of the time, difficulty and expense involved.
Serological tests: Agar gel immunodiffusion (AGID) tests and ELISAs are simple, reliable tests for
the demonstration of EIAV infection. When ELISAs are positive they should be confirmed using the
AGID test. EIA antigens can be prepared from infected tissue cultures or by using recombinant
DNA technology.
Requirements for vaccines and diagnostic biologicals: There are no biological products
currently available.
A. INTRODUCTION
Equine infectious anaemia (EIA) occurs world-wide. The infection, formerly known as swamp fever, is limited to
equids. The disease is characterised by recurrent febrile episodes, thromboctopenia, anaemia, rapid loss of
weight and oedema of the lower parts of the body. If death does not result from one of the acute clinical attacks, a
chronic stage develops and the infection tends to become inapparent. The incubation period is normally 1
3 weeks, but may be as long as 3 months. In acute cases, lymph nodes, spleen and liver are hyperaemic and
enlarged. Histologically these organs are infiltrated with nests of immature lymphocytes and plasma cells. Kupffer
cells in the liver often contain haemosiderin or erythrocytes. The enlarged spleen may be felt on rectal
examination. Differential diagnoses include equine viral arteritis (Chapter 2.5.10), and other causes of oedema,
fever, or anemia.
Once a horse is infected with EIA virus (EIAV), its blood remains infectious for the remainder of its life. This
means that the horse is a viraemic carrier and can potentially transmit the infection to other horses (Cheevers &
McGuire, 1985). Transmission occurs by transfer of blood from an infected horse. In nature, spread of the virus is
most likely via interrupted feeding of bloodsucking horseflies on a clinically ill horse and then on susceptible
horses. Transmission can also occur by the iatrogenic transfer of blood through the use of contaminated needles.
In utero infection of the fetus may occur (Kemen & Coggins, 1972). The virus titre is higher in horses with clinical
signs and the risk of transmission is higher from these animals than the carrier animals with a lower virus titre.
860
Chapter 2.5.6. Equine infectious anaemia
Agar gel immunodiffusion (AGID) tests (Coggins et al., 1972) and enzyme-linked immunosorbent assays
(ELISAs) (Suzuki et al., 1982) are accurate, reliable tests for the detection of EIA in horses, except for animals in
the early stages of infection and foals of infected dams. In other rare circumstances, misleading results may occur
when the level of virus circulating in the blood during an acute episode of the disease is sufficient to bind available
antibody, and if initial antibody levels never rise high enough to be detectable (Toma, 1980). Although the ELISA
will detect antibodies somewhat earlier and at lower concentrations than the AGID test, positive ELISAs are
confirmed using the AGID test. This is because false-positive results have been noted with ELISAs. The AGID
test also has the advantage of distinguishing between EIA and non-EIA antigenantibody reactions by lines of
identity.
The EIAV is a lentivirus in the family Retroviridae, subfamily Orthoretrovirinae. Other members of the lentivirus
genus include: bovine immunodeficiency virus; caprine arthritis encephalitis virus; feline immunodeficiency virus;
human immunodeficiency virus 1; human immunodeficiency virus 2; and maedi/visna virus. Nucleic acid
sequence comparisons have demonstrated a marked relatedness among these viruses.
1.
a)
Virus isolation and identification

Virus isolation is usually not necessary to make a diagnosis.
Isolation of the virus from suspect horses may be made by inoculating their blood on to leukocyte cultures
prepared from horses free of infection. Virus production in cultures can be confirmed by detection of specific
EIA antigen by ELISA (Shane et al., 1984), by imunofluorescence assay (Weiland et al., 1982), by molecular
tests or by subinoculation into susceptible horses. Virus isolation is rarely attempted because of the difficulty
of growing horse leukocyte cultures.
When the exact status of infection of a horse cannot be ascertained, the inoculation of a susceptible horse
with suspect blood should be employed. In this case a horse that has previously been tested for antibody
and shown to be negative is given an immediate blood transfusion from the suspect horse, and its antibody
status and clinical condition are monitored for at least 45 days. Usually, 125 ml of whole blood given
intravenously is sufficient to demonstrate infection, but in rare cases it may be necessary to use a larger
volume of blood (250 ml) or washed leukocytes from such a volume (Coggins & Kemen, 1976).
b)

A nested polymerase chain reaction (PCR) assay to detect EIA proviral DNA from the peripheral blood of
horses has been described (Nagarajan & Simard, 2001). The nested PCR method is based on primer
sequences from the gag region of the proviral genome. It has proven to be a sensitive technique to detect
field strains of EAV in white blood cells of EIA infected horses; the lower limit of detection is typically around
10 genomic copies of the target DNA (Nagarajan & Simard, 2001; 2007). A real-time reverse-transcriptase
PCR assay has also been described (Cook et al., 2002). To confirm the results of these very sensitive
assays, it is recommended that duplicate samples of each diagnostic specimen be processed. Because of
the risk of cross contamination, it is also important that proper procedures are followed. Methods to insure
the validity of PCR testing are discussed in detail in Chapter 1.1.5 Principles and methods of validation of
diagnostic assays for infectious diseases.
The following are some of the circumstances where the PCR assay maybe used for the detection of EIAV
infection in horses:
Conflicting results on serologic tests;
Suspected infection but negative or questionable serologic results;
Complementary test to serology for the confirmation of positive results;
Confirmation of early infection, before serum antibodies to EIAV develop;
Ensuring that horses that are to be used for antiserum or vaccine production or as blood donors are
free of EIAV;
Confirmation of the status of a foal from an infected mare.
861
2.
Serological tests
Due to the persistence of EIAV in infected equids, detection of serum antibody to EIAV confirms the diagnosis of
EIAV infection.
a)
Agar gel immunodiffusion test (the prescribed test for international trade)
Precipitating antibody is rapidly produced as a result of EIA infection, and can be detected by the AGID test.
Specific reactions are indicated by precipitin lines between the EIA antigen and the test serum and
confirmed by their identity with the reaction between the antigen and the positive standard serum. Horses in
the first 23 weeks after infection will usually give negative serological reactions. In rare cases the postinfection time prior to the appearance of detectable antibody may extend up to 60 days.
Reagents for AGID are available commercially from several companies. Alternately, AGID antigen and
reference serum may be prepared as described below.
Preparation of antigen
Specific EIA antigen may be prepared from the spleen of acutely infected horses (Coggins et al., 1973), from
infected equine tissue culture (Malmquist et al., 1973), from a persistently infected canine thymus cell line
(Bouillant et al., 1986), or from proteins expressed in bacteria or baculovirus using the recombinant DNA
technique (Archambault et al., 1989; Kong et al., 1997). Preparation from infected cultures or from
recombinant DNA techniques gives a more uniform result than the use of spleen cells and allows for better
standardisation of reagents.
To obtain a satisfactory antigen from spleen, a horse must be infected with a highly virulent strain of EIAV.
The resulting incubation period should be 57 days, and the spleen should be collected 9 days after
inoculation, when the virus titre is at its peak and before any detectable amount of precipitating antibody is
produced. Undiluted spleen pulp is used in the immunodiffusion test as antigen (Coggins et al., 1973).
Extraction of antigen from the spleen with a saline solution and concentration with ammonium sulphate does
not give as satisfactory an antigen as selection of a spleen with a very high titre of EIA antigen.
Alternatively, equine fetal kidney or dermal cells or canine thymus cells are infected with a strain of EIAV
adapted to grow in tissue culture (American Type Culture Collection). Virus is collected from cultures by
precipitation with 8% polyethylene glycol or by pelleting by ultracentrifugation. The diagnostic antigen, p26,
is released from the virus by treatment with detergent or ether (Malmquist et al., 1973). EIAV core proteins,
expressed in bacteria or baculovirus, are commercially available and find practical use as high quality
antigens for serological diagnosis.
The p26 is an internal structural protein of the virus that is coded for by the gag gene. The p26 is more
antigenically stable among EIAV strains than the virion glycoproteins gp45 and gp90 (Montelaro et al.,
1984). There is evidence of strain variation in the p26 amino acid sequence; however there is no evidence to
indicate that this variation influences any of the serological diagnostic tests (Zhang et al., 1999).
Preparation of standard antiserum
A known positive antiserum may be collected from a horse previously infected with EIAV. This serum should
yield a single dense precipitation line that is specific for EIA, as demonstrated by a reaction of identity with a
known standard serum. It is essential to balance the antigen and antibody concentrations in order to ensure
the optimal sensitivity of the test. Reagent concentrations should be adjusted to form a narrow precipitation
line approximately equidistant between the two wells containing antigen and serum.
862
Test procedure (Association Franaise de Normalisation [AFNOR], 2000; Coggins et al., 1973; PEARSON
& COGGINS, 1979)
i)
Immunodiffusion reactions are carried out in a layer of agar in Petri dishes. For Petri dishes that are
100 mm in diameter, 1517 ml of 1% Noble agar in 0.145 M borate buffer (9 g H3BO3, plus 2 g NaOH
per litre), pH 8.6 ( 0.2) is used. Six wells are punched out of the agar surrounding a centre well of the
same diameter. The wells are 5.3 mm in diameter and 2.4 mm apart. Each well must contain the same
volume of reagent.
ii)
The antigen is placed in the central well and the standard antiserum is placed in alternate exterior
wells. Serum samples for testing are placed in the remaining three wells.
iii)
The dishes are maintained at room temperature in a humid environment.
iv)
After 2448 hours the precipitation reactions are examined over a narrow beam of intense, oblique light
and against a black background. The reference lines should be clearly visible at 24 hours, and at that
time any test sera that are strongly positive may also have formed lines of identity with those between
the standard reagents. A weakly positive reaction may take 48 hours to form and is indicated by a
slight bending of the standard serum precipitation line between the antigen well and the test serum
well. Sera with high precipitating antibody titres may form broader precipitin bands that tend to be
diffuse. Such reactions can be confirmed as specific for EIA by dilution at 1/2 or 1/4 prior to retesting;
these then give a more distinct line of identity. Sera devoid of EIA antibody will not form precipitation
lines and will have no effect on the reaction lines of the standard reagents.
v)
b)
Interpretation of the results: Horses that are in the early stages of an infection may not give a positive
serological reaction in an AGID test. Such animals should be bled again after 34 weeks. In order to
make a diagnosis in a young foal, it may be necessary to determine the antibody status of the dam. If
the mare passes EIA antibody to the foal through colostrum, then a period of 6 months or longer after
birth must be allowed for the maternal antibody to wane. Sequential testing of the foal at monthly
intervals may be useful to observe the decline in maternal antibody. To conclude that the foal is not
infected, a negative result must be obtained (following an initial positive result) at least 2 months after
separating the foal from contact with the EIA positive mare or any other positive horse. Alternatively
PCR could be performed on the blood of the foal to determine the presence/absence of EIA proivirus.

There are four ELISAs that are approved by the United States Department of Agriculture for the diagnosis of
equine infectious anaemia and are available internationally; a competitive ELISA and three non-competitive
ELISAs. The competitive ELISA and two non-competitive ELISAs detect antibody produced against the p26
core protein antigen. The third non-competitive ELISA incorporates both p26 core protein and gp45 (viral
transmembrane protein) antigens. Typical ELISA protocols are used in all tests. If commercial ELISA
materials are not available, a non-competitive ELISA using p26 antigen purified from cell culture material
may be employed (Shane et al., 1984).
A positive test result by ELISA should be retested using the AGID test to confirm the diagnosis because
some false-positive results have been noted with the ELISA. The results can also be confirmed by the
immunoblot technique. A standard antiserum for immunodiffusion, which contains the minimum amount of
antibody that should be detected by laboratories, is available from the OIE Reference Laboratories (see
Table given in Part 4 of this Terrestrial Manual). Uniform methods for EIA control have been published
(United States Department of Agriculture [USDA], 2002).

No biological products are available currently.
REFERENCES
ASSOCIATION FRANAISE DE NORMALISATION (AFNOR) (2000). Animal Health Analysis Methods. Detection of
Antibodies against Equine Infectious Anaemia by the Agar Gel Immunodiffusion Test. NF U 47-002. AFNOR, 11
avenue Francis de Pressens, 93571 Saint-Denis La Plaine Cedex, France.
ARCHAMBAULT D., WANG Z., LACAL J.C., GAZIT A., YANIV A., DAHLBERG J.E. & TRONICK S.R. (1989). Development of
an enzyme-linked immunosorbent assay for equine infectious anaemia virus detection using recombinant
Pr55gag. J. Clin. Microbiol., 27, 11671173.
BOUILLANT A.M.P., NELSON K., RUCKERBAUER C.M., SAMAGH B.S. & HARE W.C.D. (1986). The persistent infection of
a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral
antigens. J. Virol. Methods, 13, 309321.
CHEEVERS W.M. & MCGUIRE T.C. (1985). Equine infectious anaemia virus; immunopathogenesis and persistence.
Rev. Infect. Dis., 7, 8388.
COGGINS L. & KEMEN M.J. (1976). Inapparent carriers of equine infectious anaemia (EIA) virus. In: Proceedings of
the IVth International Conference on Equine Infectious Diseases. University of Kentucky, Lexington, Kentucky,
USA, 1422.
COGGINS L., NORCROSS N.L. & KEMEN M.J. (1973). The technique and application of the immunodiffusion test for
equine infectious anaemia. Equine Infect. Dis., III, 177186.
863
COGGINS L., NORCROSS N.L. & NUSBAUM S.R. (1972). Diagnosis of equine infectious anaemia by immunodiffusion
test. Am. J. Vet. Res., 33, 1118.
COOK R.F., COOK S.J., LI F.L., MONTELARO R.C., & ISSEL C.J. (2002). Development of a multiplex real-time reverse
transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). Virol Methods, 105, 171179.
KEMEN M.J. & COGGINS L. (1972). Equine infectious anaemia: transmission from infected mares to foals. J. Am.
Vet. Med. Assoc., 161, 496499.
KONG X. K., PANG H., SUGIURA T., SENTSUI H., ONODERA T., MATSUMOTO Y. & AKASHI H. (1997). Application of equine
infectious anaemia virus core proteins produced in a Baculovirus expression system, to serological diagnosis.
Microbiol. Immunol., 41, 975980.
MALMQUIST W.A., BARNETT D. & BECVAR C.S. (1973). Production of equine infectious anaemia antigen in a
persistently infected cell line. Arch. Gesamte Virusforsch., 42, 361370.
MONTELARO R.C., PAREKH B., ORREGO A. & ISSEL C.J. (1984). Antigenic variation during persistent infection by
equine infectious anaemia, a retrovirus. J. Biol. Chem., 16, 1053910544.
NAGARAJAN M.M. & SIMARD C. (2001). Detection of horses infected naturally with equine infectious anemia virus by
nested polymerase chain reaction. J. Virol. Methods, 94, 97109.
NAGARAJAN M.M. & SIMARD C. (2007). Gag genomic heterogeneity of equine infectious anemia virus (EIAV) in
naturally infected horses in Canada: implication on EIA diagnosis and peptide-based vaccine development. Virus
Res., 129, 228235.
PEARSON J.E. & COGGINS L. (1979). Protocol for the immunodiffusion (Coggins) test for equine infectious anaemia.
Proc. Am. Assoc. Vet. Lab. Diagnosticians, 22, 449462.
SHANE B.S., ISSEL C.J. & MONTELARO R.C. (1984). Enzyme-linked immunosorbent assay for detection of equine
infectious anemia virus p26 antigen and antibody. J. Clin. Microbiol., 19, 351355.
SUZUKI T., UEDA S. & SAMEJINA T. (1982). Enzyme-linked immunosorbent assay for diagnosis of equine infectious
anaemia. Vet. Microbiol., 7, 307316.
TOMA B. (1980). Rponse srologique ngative persistante chez une jument infecte. Rec. Med. Vet., 156, 5563.
UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) (2002). Equine Infectious Anemia Uniform Methods and
Rules. Animal and Plant Health Inspection Service, USDA:
http://www.aphis.usda.gov/animal_health/animal_diseases/eia/
WEILAND F., MATHEKA H.D. & BOHM H.O. (1982). Equine infectious anaemia: detection of antibodies using an
immunofluorescence test. Res. Vet. Sci., 33, 347350.
ZHANG W., AUYONG D.B., OAKS J.L. & MCGUIRE T.C. (1999). Natural variation of equine infectious anemia virus
Gag-protein cytotoxic T lymphocyte epitopes. Virology, 261, 242252.
*
* *
NB: There are OIE Reference Laboratories for Equine infectious anaemia:
diagnostic tests and reagents for equine infectious anaemia
864
CHAPTER 2.5.7.
EQUINE INFLUENZA
SUMMARY
Equine influenza is an acute respiratory infection of horses, donkeys and mules caused by two distinct
subtypes (H7N7, formerly equi-1, and H3N8, formerly equi-2) of influenza A virus within the genus
Influenzavirus A of the family Orthomyxoviridae. Viruses of the H7N7 subtype have not been isolated
since the late 1970s. Equine influenza viruses of both subtypes are considered to be of avian ancestry
and highly pathogenic avian H5N1 has recently been associated with an outbreak of respiratory
disease in donkeys in Egypt. In fully susceptible equidae, clinical signs include pyrexia and a harsh
dry cough followed by a mucopurulent nasal discharge. In partially immune vaccinated animals, one
or more of these signs may be absent. Vaccinated infected horses can still shed the virus and serve
as a source of virus to their cohorts. Characteristically, influenza spreads rapidly in a susceptible
population. The disease is endemic in many countries with substantial equine populations. In recent
years, infection has been introduced into Australia and re-introduced into South Africa and Japan; to
date New Zealand and Iceland are reported to be free of equine influenza virus.
While normally confined to equidae, equine H3N8 influenza has crossed the species barrier to
dogs. Extensive infection of dogs has been reported in North America where it normally produces
mild fever and coughing but can cause fatal pneumonia. While equine influenza has not been
shown to cause disease in humans, serological evidence of infection has been described primarily
in individuals with an occupational exposure to the virus. During 20042006 influenza surveillance
in central China (Peoples Rep. of) two equine H3N8 influenza viruses were also isolated from pigs.
Identification of the agent: Embryonated hens eggs and/or cell cultures can be used for virus
isolation from nasopharyngeal swabs or nasal and tracheal washes. Isolates should always be sent
immediately to an OIE Reference Laboratory. Infection may also be demonstrated by detection of
viral nucleic acid or antigen in respiratory secretions using the reverse-transcription polymerase
chain reaction (RT-PCR) or an antigen-capture enzyme-linked immunosorbent assay (ELISA),
respectively.
Serological tests: Diagnosis of influenza virus infections is usually only accomplished by tests on
paired sera; the first sample should be taken as soon as possible after the onset of clinical signs
and the second approximately 2 weeks later. Antibody levels are determined by haemagglutination
inhibition (HI) or single radial haemolysis (SRH).
Requirements for vaccines and diagnostic biologicals: Spread of infection and severity of
disease may be reduced by the use of potent inactivated equine influenza vaccines containing
epidemiologically relevant virus strains. Inactivated equine influenza vaccines contain whole viruses
or their subunits. The vaccine viruses are propagated in embryonated hens eggs or tissue culture,
concentrated, and purified before inactivation with agents such as formalin or beta-propiolactone.
Inactivated vaccines provide protection by inducing humoral antibody to the haemagglutinin protein.
Responses are generally short-lived and multiple doses are required to maintain protective levels of
antibody. An adjuvant is usually required to stimulate durable protective levels of antibody. Live
attenuated virus and viral vectored vaccines have been licensed in some countries.
Vaccine breakdown has been attributed to inadequate vaccine potency, inappropriate vaccination
schedules, and outdated vaccine viruses that are compromised as a result of antigenic drift. An invitro potency test (single radial diffusion) can be used for in-process testing of the antigenic content
of inactivated products before addition of an adjuvant. In process testing of live and vectored
vaccines relies on titration of infectious virus. A surveillance programme is underway to monitor
antigenic drift among equine influenza viruses and to provide information on strain selection for
vaccines.
865
Chapter 2.5.7. Equine influenza
A. INTRODUCTION
Equine influenza is caused by two subtypes: H7N7 (formerly subtype 1) and H3N8 (formerly subtype 2) of
influenza A viruses (genus Influenzavirus A of the family Orthomyxoviridae); however there have been very few
reports of H7N7 subtype virus infections in the last 30 years (Webster, 1993).
In fully susceptible equidae, clinical signs include pyrexia, nasal discharge and a harsh dry cough; pneumonia in
young foals and donkeys and encephalitis in horses have been described as rare events (Daly et al., 2006;
Gerber, 1970). Clinical signs associated with infection in dogs also include fever and a cough; occasionally
infection results in suppurative bronchopneumonia and peracute death (Crawford et al., 2005). Characteristically,
influenza spreads rapidly in a susceptible population. The virus is spread by the respiratory route, and indirectly
by contaminated personnel, vehicles and fomites. The incubation period in susceptible horses may be less than
24 hours. In partially immune vaccinated animals the incubation period may be extended, one or more clinical
signs may be absent and spread of the disease may be limited. This makes clinical diagnosis of equine influenza
more difficult as other viral diseases, such as equine herpesvirus-associated respiratory disease, may clinically
resemble a mild form of influenza. Horses infected with equine influenza virus become susceptible to secondary
bacterial infection and may develop mucopurulent nasal discharge, which can lead to diagnosis of bacterial
disease with the underlying cause being overlooked.
Equine influenza viruses are believed to be of avian ancestry, and more recent transmission of avian viruses to
horses and donkeys has been recorded. The sequence analysis of an H3N8 virus isolated in 1989 from horses
during a limited influenza epidemic in North Eastern China (Peoples Rep. of) established that the virus was more
closely related to avian influenza viruses than to equine influenza viruses (Guo et al., 1992). Recently, avian
H5N1 was associated with respiratory disease of donkeys in Egypt (Abdel-Moneim et al., 2010).
Equine influenza viruses have the potential to cross species barriers and have been associated with respiratory
disease in dogs primarily in North America (Crawford et al., 2005). Isolated outbreaks of equine influenza have
also occurred in dogs within the UK but the virus has not become established in the canine population. Close
contact with infected horses was thought to be involved in each outbreak in the UK. Equine influenza viruses have
also been isolated from pigs in central China (Peoples Rep. of) (Tu et al., 2009). Despite the occasional
identification of seropositive persons with occupational exposure there is currently little evidence of zoonotic
infection of people with equine influenza (Alexander & Brown, 2000).
In endemic countries the economic losses due to equine influenza can be minimised by vaccination and many
racing authorities and equestrian bodies have mandatory vaccination policies. Vaccination does not produce
sterile immunity; vaccinated horses may shed virus and contribute silently to the spread of EI. Appropriate risk
management strategies to deal with this possibility should be developed.
Laboratory diagnosis of acute equine influenza virus infections is based on virus detection in nasal swabs
collected from horses with acute respiratory illness. Alternatively, the demonstration of a serological response to
infection may be attempted with paired serum samples. Ideally, both methods are used. Equine influenza virus
may be isolated in embryonated hens eggs or cell culture. Infection may also be demonstrated by detection of
viral antigen in respiratory secretions using an antigen capture enzyme-linked immunosorbent assay (ELISA) or of
viral genome using reverse-transcription polymerase chain reaction (RT-PCR) assays. All influenza viruses are
highly contagious for susceptible hosts and care must therefore be taken during the handling of infected eggs or
cultures to avoid accidental cross-contamination. Standard strains should not be propagated in the diagnostic
laboratory, at least never at the same time or in the same place where diagnostic samples are being processed.
All working areas must be efficiently disinfected before and after virus manipulations, which should preferably be
conducted within biohazard containment level 2 and class II safety cabinets.
It is important to obtain samples as soon as possible after the onset of clinical signs, preferably within 35 days.
These samples include nasopharyngeal swabs and nasal or tracheal washings, the latter taken by endoscopy.
Swabs may consist of absorbent cotton wool sponge/gauze on wire, and should be long enough to be passed via
the ventral meatus into the nasopharynx. Swabs should be transferred to a tube containing transport medium
immediately after use. This medium consists of phosphate buffered saline (PBS) containing either 40% glycerol or
2% tryptose phosphate broth with 2% antibiotic solution (penicillin [10,000 units], streptomycin [10,000 units] in
sterile distilled water [100 ml]), and 2% fungizone (250 mg/ml stock). If the samples are to be inoculated within 1
2 days they may be held at 4C, but, if kept for longer, they should be stored at 70C or below. Samples should
be kept cool during transport to the laboratory.
Sample processing should follow the quality procedures outlined in Chapter 1.1.4 Quality management in veterinary
testing laboratories, taking measures to prevent cross contamination. The liquid is expelled from the swab by squeezing
with forceps and the swab is then disposed of suitably. Further antibiotics may be added if samples appear to be heavily
866
contaminated with bacteria. The remainder of the fluid is stored at 70C. Samples treated with antibiotics are allowed
to stand on ice for 3060 minutes and are then centrifuged at 1500 g for 15 minutes to remove bacteria and debris; the
supernatant fluids are used for inoculation. The remainder of the fluid is stored at 70C. Filtration of samples is not
advised as influenza virus may adsorb on to the filter and be lost from the sample.
1.
Isolation of infectious virus may be carried out in embryonated hens eggs or cell cultures. Traditionally, eggs
have been preferred for isolation of equine influenza as many clinical isolates do not grow well in cells without
serial passage. Comparison of H3N8 viruses isolated in eggs and MadinDarby canine kidney (MDCK) cells
indicated that MDCK cells are capable of selecting variant viruses that are not representative of the predominant
virus in clinical specimens (Ilobi et al., 1994). However, some viruses have been successfully isolated in MDCK
cells but not in eggs and selection of variants has also occurred as a result of culture in eggs (Oxburgh &
Klingborn, 1999). Ideally, isolation should be attempted using both substrates.
RT-PCR and real-time RT-PCR assays are being widely used in diagnostic laboratories as a more sensitive
alternative to virus isolation (Quinlivan et al., 2005). Influenza virus antigen in nasal secretions may also be
detected directly by a sensitive antigen-capture ELISA for the H3N8 virus using a monoclonal antibody (MAb)
against the equine influenza virus nucleoprotein (Livesay et al., 1993).This assay is not commercially available,
other than as a diagnostic service, but commercial self-contained kits for detecting human influenza are available
and have been shown to detect equine influenza antigen (Chambers et al., 1994; Yamanaka et al., 2008). This
approach is less sensitive than RT-PCR but provides a rapid result on which management decisions may be
based. It should not be used to the exclusion of virus isolation. It is essential that new viruses be isolated and sent
to reference laboratories for characterisation as part of the surveillance programme to monitor antigenic drift and
emergence of new viruses and to provide isolates for inclusion in updated vaccines. Positive RT-PCR and ELISA
results are useful in the selection of samples for virus isolation attempts if resources are limited, or for the
selection of specimens to be sent to a reference laboratory for virus isolation and characterisation.
a)
Virus isolation in embryonated hens eggs

Fertile eggs are set in a humid incubator at 3738C and turned twice daily; after 1011 days, they are
examined by candling and live embryonated eggs are selected for use. The area above the air sac is
cleansed with alcohol and a small hole is made through the shell. Inoculum may be introduced into the
amniotic or the allantoic cavity. Several eggs/sample are inoculated (0.1 ml) in the amniotic cavity with no
additional dilution of the sample (sample may also be diluted 1/10 and 1/100 in PBS containing antibiotics).
The syringe is withdrawn approximately 1 cm and a further 0.1 ml is inoculated into the allantoic cavity.
Alternatively, many laboratories opt to inoculate into the allantoic cavity alone through a second hole drilled
just below the line of the air sac. The hole(s) is/are sealed with wax or Sellotape, and the eggs are incubated
at 3435C for 3 days. The embryos that die within 24 hours following inoculation should be discarded. The
eggs that contain embryos that die more than 24 hours after inoculation or contain live embryos after 3 days
are examined for the presence of EI virus.
The eggs are transferred to 4C for 4 hours or overnight to kill the embryos and to reduce bleeding at
harvest. The shells are disinfected, and the amniotic and/or allantoic fluid is harvested by pipette, each
harvest being kept separate. These are tested for haemagglutination (HA) activity by mixing twofold dilutions
of the harvested fluid in equal volumes (0.025 ml) with chicken red blood cells (RBCs) (0.5% [v/v] packed
cells in PBS) in V- or U-bottomed microtitre plates or 0.4% guinea-pig RBCs (0.4% [v/v] packed cells in PBS)
in V- or U-bottomed plates. Plates are incubated for approximately 30 minutes preferably at 4C to prevent
neuraminidase activity. If chicken RBCs are used, the plates may be read by tilting to 70 so that nonagglutinated cells stream to the bottom of the well. Non-agglutinated guinea-pig cells appear as a button at
the bottom of the well and may take longer to settle. If there is no HA activity, aliquots of each harvest are
pooled and passaged into further eggs. All HA positive samples are divided into aliquots and stored at
70C; one aliquot is titrated for HA immediately. The HA titre is the reciprocal of the greatest dilution to
show agglutination. If the HA titre is 1/16 or more, the isolate is characterised immediately. If titres are low,
positive samples should be passaged. Care should be taken to avoid generation of defective interfering
particles by prediluting the inoculum 1/10, 1/100, 1/1,000. Positive samples arising from the highest dilution
should be selected as stocks for storage. It may be necessary to undertake as many as five passages to
isolate the virus, particularly from vaccinated horses. If virus has not been recovered by the fifth passage,
further passages are unlikely to be successful.
b)
Virus isolation in cell cultures

Cultures of the MDCK cell line (MDCK, ATCC CCL34) may be used to isolate equine influenza viruses. The
cells are grown to confluence in tubes and then infected in triplicate with 0.250.5 ml of each sample,
processed as described above. Prior to inoculation, the cell monolayer is washed at least once with tissue
culture medium containing trypsin (2 g/ml) without serum. The cultures are maintained with serum-free
medium containing 0.52 g/ml trypsin (treated with TPCK [L-1-tosylamine-2-phenylethyl chloromethyl
867
ketone] to remove chymotrypsin, available pretreated, e.g. from Sigma), and examined daily for evidence of
cytopathic effects (CPE). If positive, or after 7 days in any case, the supernatant fluids are tested for HA.
Fluids with titres of 1/16 are characterised immediately. Negative fluids and those with titres <1/16 are
repassaged up to five passages.
Alternatively, the cells are screened for evidence of haemadsorption (HAD). This procedure detects
expression of viral antigens at the cell surface. The medium is removed from the cultures and the tubes are
washed with PBS. One or two drops of a 50% suspension of chicken or guinea-pig RBCs are added, the
tubes are rotated carefully, and kept at room temperature (23C 2C) for 30 minutes. Unbound RBCs are
washed off with PBS, and the cultures are examined microscopically for evidence of HAD.
c)
Haemagglutinin subtyping
The HA subtype of new isolates of equine influenza viruses is best determined by haemagglutination
inhibition (HI; Section B.2.a) using H7N7- and H3N8-specific antisera. Isolates may first be treated with
Tween 80/ether, which destroys viral infectivity and reduces the risk of cross-contamination. In the case of
H3N8 viruses particularly, this treatment enhances the HA activity (John & Fulginiti, 1966). However,
treatment with Tween 80/ether also decreases specificity and may increase the variability of the results
obtained. Standard antigens must be titrated in parallel with tests to identify viruses and should include
H7N7 strains (e.g. A/eq/Prague/56, A/eq/Newmarket/77) and H3N8 strains (e.g. A/eq/Newmarket/2/93, and
A/eq/South Africa/4/03 and A/eq/Richmond/1/07). Virus strains may be obtained from OIE Reference
Laboratories (see Table given in Part 4 of this Terrestrial Manual). Additionally, recent isolates from the
same geographical area should be included if available. The standard antigens should be treated with
Tween 80/ether to avoid cross-contamination. Test antigens and standard antigens are always back-titrated
to confirm their antigen content.
New isolates of equine influenza viruses may be further characterised by HI using strain-specific antisera.
The species in which antibodies are raised will influence the cross-reactivity of the antiserum, with ferrets
providing the most strain-specific antibody (Mumford, 1992). The specificity and cross reactivity of the sera
are also influenced by the immunisation schedule. Sera obtained 3 weeks after a single antigen application
are considered to be most discriminative.
All isolates should be sent immediately to an International Reference Laboratory designated by OIE or the
World Health Organization (WHO) for inclusion in the strain surveillance programme to monitor antigenic
drift and emergence of new viruses.
d)
Neuraminidase subtyping
Subtyping of neuraminidase requires specific antisera and no routine technique is available. Subtyping can
also be done using specific PCR primers.
e)

RT-PCR assays both conventional and real-time, are increasingly used for the detection of equine influenza
genome in nasal secretions. Real-time Light Cycler RT-PCR using primers M52C (5-CTT-CTA-ACC-GAGGTC-GAA-ACG-3) and M253R (5-AGG-GCA-TTT-TGG-ACA-AAG-CGT-CTA-3) designed to amplify a
244 base pair amplicon from nucleotides 32 to 276 of the matrix gene, which is highly conserved across
influenza A viruses, has been shown to be more sensitive for the detection of positive samples than virus
culture in eggs or detection of nucleoprotein using a kit for the detection of human influenza (Tu et al., 2009).
For several years this assay has been used effectively for diagnosis and surveillance by one of the OIE
reference laboratories. However it has not been validated by interlaboratory comparisons at international
level. A probe-based real-time RT-PCR assay based on the matrix gene and developed for the detection of
a wide range of influenza type A strains including avian H5N1, was combined with an automated DNA
extraction system to establish a high throughput assay used in the mass screening of horses during the
eradication programme in Australia in 2007 (Foord et al., 2009).This type of technology is constantly
evolving. TaqMan real-time RT-PCR assays specific for equine-2 influenza (H3N8) and equine-1 (H7N7)
viruses have recently been described and a commercial RT-PCR kit for the detection of equine influenza has
become available (Lu et al., 2009). These RT-PCR assays have not yet been validated in line with the OIE
Validation Template.
Although the genetic sequence of isolates can also be derived from PCR assays it remains essential to
isolate infectious virus in order to examine the antigenic properties of new isolates and evaluate antigenic
drift in the field.
2.
Serological tests
Influenza infections can be detected by performing serological tests on paired sera to show a rise in specific
antibody. These tests should be carried out whether virus isolation has been attempted or not. They are robust
868
and may prove positive in the absence of virus isolation. Two simple methods exist, HI and single radial
haemolysis (SRH), each equally efficient and widely used. The complement fixation (CF) test can also be applied,
but is not in general use. The paired serum samples, i.e. the acute and convalescent samples, should be tested
together to minimise the impact of inter-assay variability. The standard antigens are described above (Section
B.1.c). If available, isolates from recent cases should be included. Freeze-dried post-infection equine antisera to
A/eq/Newmarket/77 (H7N7), A/eq/Newmarket/1/93 (American lineage H3N8), A/eq/Newmarket/2/93 (European
lineage H3N8), A/eq/South Africa/4/03 (Florida Clade 1, sublineage of American lineage) and an influenzanegative equine serum, are available from the European Directorate for the Quality of Medicines (EDQM)1. These
sera have been assigned SRH values through an international collaborative study and can be used as primary
reference sera for the assay (Daly et al., 2007; Mumford, 2000).
a)
Haemagglutination inhibition test

The antigen is first treated with Tween 80/ether in order to increase the sensitivity of the test, particularly for
H3N8 viruses. The test is best done in microtitre plates using the appropriate dilution equipment. A
macrotest may be used, for which antigen is diluted to a final HA titre of 1/8 per well and the volumes for
PBS, sera and antigen are 0.5 ml. Sera are pretreated to remove nonspecific haemagglutinins, then
inactivated at 56C for 30 minutes. Pretreatments include the use of one of the following: (a) kaolin and
RBCs absorption, (b) potassium periodate, or (c) Vibrio cholerae receptor-destroying enzyme. Potassium
periodate or V. cholerae receptor-destroying enzyme is the treatment of choice. The treated sera are diluted
in PBS, a standard dose of antigen is added (HA titre of 1/4 per well for microtitration assay), and these are
kept at room temperature (23C 2C) for 30 minutes. After gentle mixing, RBCs are added and the test is
read 30 minutes later. The HI titres are read as the highest dilution of serum giving complete inhibition of
agglutination. Either chicken RBCs (1% [v/v] packed cells) in V-bottomed microtitre plates or guinea-pig
RBCs (0.5% [v/v] packed cells) in V- or U-bottomed plates may be used. If chicken RBCs are used, the
plates may read by tilting to 70 so that non-agglutinated cells stream to the bottom of the well. Nonagglutinated guinea-pig cells appear as a button in the bottom of the well and may take longer to settle. The
HI titre is the reciprocal of the greatest dilution showing complete inhibition of agglutination. Currently, a cutoff point for positive samples has not been determined for the HI test and thus, low titres should be
investigated further. Titre increases of fourfold or more between paired sera indicate recent infection.
Tween 80/ether treatment of the virus
i)
To 39.5 ml of infective allantoic fluid, add 0.5 ml of a 10% (v/v) suspension of Tween 80 in PBS to give
a 0.125% (v/v) concentration of Tween 80.
ii)
After mixing gently at room temperature for 5 minutes, add 20 ml of diethyl ether to give a final
concentration of 33.3% by volume, and mix the suspension well at 4C for 15 minutes.
iii)
After allowing the layers to separate by standing, remove the aqueous layer containing the disrupted
virus particles to a glass bottle with a loose lid and allow the excess ether to evaporate off overnight
(John & Fulginiti, 1966). Safety precautions while handling ether must be strictly observed and work
should be confined to a fume hood.
iv)
Store treated virus in aliquots at 70C.
Titration of haemagglutination
i)
Add 25 l of PBS to all wells in a row of a microtitre plate.
ii)
Add 25 l of virus to first well then generate a twofold dilution series across the plate leaving the last
well as a negative control.
iii)
Add an extra 25 l of PBS to all wells.
iv)
Add 50 l of RBCs to all wells. Leave at room temperature or at 4C (particularly if ambient

temperatures are high), for 30 minutes. The HA titre is taken as the last virus dilution giving partial HA.
Potassium periodate pretreatment of sera
i)
Mix one volume (150 l) of serum with two volumes (300 l) of freshly prepared 0.016 M potassium
periodate (0.38 g in 100 ml PBS), and leave at 22C ( 2C) for 15 minutes.
ii)
Add a further one volume of 3% glycerol in PBS to neutralise any excess periodate solution, mix and
leave at room temperature (23C 2C) for 15 minutes.
iii)
Inactivate in a 56C water bath for 30 minutes.
Headquarters: EDQM - Council of Europe, 7 alle Kastner, CS 30026, F-67081 Strasbourg, France.
869
b)
Test procedure
i)
Dispense 25 l of PBS to all wells of a microtitre plate.
ii)
Add serum (25 l) to the first well of a row of 12, then generate twofold serial dilutions (1/8 to at least
1/512, allowing for dilution of 1/4 from treatment of serum), leaving the last well as a control.
iii)
Dilute the antigen to give a dose of 4 HA units (4 minimum agglutinating dose, i.e. titre/4).
iv)
Add 25 l to each well, and incubate at room temperature (22C 2C) for 30 minutes.
v)
Add 50 l of RBCs to each well. Leave at room temperature or at 4C (particularly if ambient

temperatures are high), for 30 minutes.
vi)
The plates may be read by tilting to 70 so that non-agglutinated cells stream to the bottom of the well.
No agglutination is recorded as a positive result.
Single radial haemolysis

In this test, viral antigens are coupled to fixed RBCs that are suspended in agarose containing guinea-pig
complement (C). Wells are punched in the agarose and filled with test sera. Influenza antibodies and C lyse
the antigen-coated RBCs, resulting in a clear, haemolytic zone around the well; the size of this zone is
directly proportional to the level of strain-specific antibody in the serum sample (Morley et al., 1995).
Special immunodiffusion plates (MP Biomedical) may be used for the assay, but simple Petri dishes are also
suitable. Sheep RBCs collected into Alsevers solution are washed three times. The C can be obtained
commercially, or normal guinea-pig serum can be used. The viral antigens are egg-grown stocks or purified
preparations; the strains used are the same as for the HI tests. The viruses are coupled to RBCs by
potassium periodate or by chromic chloride. The coupled antigen/RBCs preparations are mixed with C,
together with a 1% solution of agarose (low melting grade) in PBS. Care must be taken to ensure that the
temperature is not allowed to rise above 42C at any time. The mixture is poured into plates and left to set.
Wells of 3 mm in diameter and 12 mm apart are punched in the solidified agarose, at least 6 mm from the
edge of the plates. Such plates may be stored at 4C for 12 weeks. Plates are prepared for each antigen.
Sera are inactivated at 56C for 30 minutes, but no further treatment is necessary. Paired sera should be
assayed on the same plate. As a minimum, a subtype-specific antiserum should be included as a control
serum in one well on each plate. All sera are tested in a control plate containing all components except virus
to check for nonspecific lysis. Alternatively, an unrelated virus, such as A/PR/8/34 (H1N1), may be used in
the control plate. Sera that show haemolytic activity for sheep RBCs must be pre-absorbed with sheep
RBCs. Zones of lysis should be clear and not hazy or translucent. All clear zones should be measured and
the area of haemolysis calculated.
Preparation of reagents
i)
Saline/HEPES: 0.85% NaCl (4.25 g/500 ml); 0.05 M HEPES (N-2-hydroxyethylpiperazine, N-2ethanesulphonic acid; 5.95 g/500 ml); and 0.02% sodium azide. Make to pH 6.5 with NaOH.
ii)
Saline/HEPES/BSA: as saline/HEPES with 0.2% (w/v) bovine serum albumin (BSA).
iii)
CrCl3 stock solution (2.25 M) 6 g/10 ml: Make fresh 1/400 dilution in 0.85% NaCl for each assay.
iv)
PBS (London)/PBS A: NaCl (10.00 g); KCl (0.25 g); Na2PO4 (1.45 g); KH2PO4 (0.25 g); and Na azide
(0.20 g). Make up to 1 litre with distilled water.
v)
Agarose in PBS: Place flask containing PBS A on a stirrer. Slowly add 10 g agarose to the stirring
solution. Liquefy in a pressure cooker. Dispense into glass bottles for storage at 22C ( 2C).
vi)
Virus antigen: Allantoic fluid containing infectious virus is harvested and stored at 70C. A short
titration curve determines the optimum ratio of virus antigen to RBCs to be used when preparing
sensitised sheep RBCs. The H7N7 influenza strains always produce clear zones; the H3N8 strains
sometimes produce hazy zones, in which case it is necessary to concentrate the virus by
centrifugation.
vii)
Sheep blood: Collect blood into an equal volume of Alsevers solution and store at 4C. It may be
necessary to test bleed several sheep, as characteristics of RBCs from individual sheep vary. Keep the
blood for 2 days before use, it may then be usable for up to 3 weeks, providing sterility is maintained.
viii) Complement: Use commercially available guinea-pig complement or collect serum from young guineapigs of 300350 g body weight and store in small volumes at 70C. For use, thaw in cold water and
hold at 4C prior to mixing.
ix)
870
Treatment of sera: Use undiluted sera heat inactivated at 56C for 30 minutes. Avoid repeated freeze
thaw cycles.
Test procedure
i)
Wash sheep RBCs at least three times in saline/HEPES.
ii)
Prepare an appropriate volume of 8% RBCs (v/v packed cells) in saline/HEPES, having first calculated
the number of plates required and allowing 1 ml per 6 11 cm immunoplate and 12 ml extra.
iii)
Add a predetermined volume2 of virus antigen to the 8% RBCs solution. Hold the mixture at 4C for
10 minutes. Haemagglutination may be observed.
iv)
SLOWLY add CrCl3 (1/400 in 0.85% NaCl) at half the total volume of virus/RBCs suspension. Hold at
22C (2C) for 5 minutes with occasional mixing.
v)
Sediment the sensitised RBCs by centrifugation at 1500 g for 5 minutes.
vi)
Gently resuspend in saline/HEPES/BSA and centrifuge at 1500 g for 5 minutes.
vii)
Resuspend RBCs to an 8% suspension in PBS A.
During the sensitisation process, melt the agarose in a pressure cooker. Shortly before use, pipette 7.8 ml
volumes to Universal bottles and retain at 42C. Check that the agar has cooled to 42C before use.
Preparation of plates
i)
Add 0.9 ml of virus-sensitised sheep RBCs to 7.8 ml of agarose (42C). Mix quickly, but gently.
ii)
Add 0.3 ml of undiluted guinea-pig serum. Mix again and pour into immunoplates on a levelling table.
Allow to set and air dry without a lid for 5 minutes.
iii)
Place lids on plates and store at 4C in a humid box until used.
iv)
Prepare control plates with unsensitised cells or cells sensitised with an unrelated virus. Batches of
prepared plates can be stored for several weeks.
v)
Punch 3 mm holes in the set gels to a prepared template, allow for 16 test sera and a positive control
serum. On antigen control plates, prepare five rows of eight wells.
vi)
Pipette 10 l of heat-inactivated (56C for 30 minutes) test sera and a positive control serum to
appropriate wells. Incubate at 34C for 20 hours in a humid box.
vii)
Measure zone diameters, and calculate areas of haemolysis after the area of the well has been
deducted.
Interpretation of the results
For results to be valid, positive and negative control sera should give results assigned through the
international collaborative study (Daly et al., 2007; Mumford, 2000) or if not using these international
standards, results consistent with those expected on the basis of prior experience. Areas of haemolysis for
the control sera should be clear and intra-laboratory variation should be no more than 5% for the control
serum. Results may be expressed as mm2 or as a ratio of the control serum value. Sera giving positive
results in the control plate should be adsorbed with sheep RBCs. For diagnostic purposes, acute and
convalescent sera should be tested in duplicate on the same plate. Increase in zone areas produced by
convalescent serum compared with acute serum is evidence of infection. The increase in area deemed to be
significant depends on the reproducibility of the test within the laboratory, but should be equivalent to a
twofold or more increase in antibody concentration. This area can be calculated from a standard curve
generated from a dilution series of a standard antiserum.

1.
Background
a)

Equine influenza is a self-limiting disease and its economic significance is primarily due to the contagious
nature of the virus and the disruption to equestrian activities. Rapid diagnosis, movement restrictions and
vaccination are the key control measures for equine influenza. Vaccination decreases clinical signs and virus
Prepare three plates by adding 0.6, 1.2 or 1.8 ml of virus antigen to 2 ml RBCs. Add 1.3, 1.6 and 1.9 ml CrCl3 respectively
and resuspend to 2 ml in PBS A. Optimum volume of virus antigen is that which results in the largest and clearest zones
with appropriate reference serum.
871
shedding. Influenza vaccines are widely available and are routinely used in competition horses in Europe,
the Americas, and Asia. In some countries, vaccination is mandatory for sport horses and racehorses that
are competing under rules of equestrian organisations. Following a primary course of three doses at
intervals of around 0, 1 and 6 months, an annual booster is the usual minimum requirement. More frequent
vaccination is recommended for young horses and some equestrian organisations require biannual
boosters.
Equine influenza virus vaccines typically consist of inactivated whole viruses or their glycoprotein subunits,
with or without adjuvant. Live attenuated virus and canary pox vectored vaccines have become available
commercially in some countries. Immunity generated by inactivated vaccines administered via the
intramuscular route is reliant on stimulation of circulating antibody to the HA, which neutralises virus; some
products have been shown to stimulate antibody in respiratory secretions. Critically the integrity and
conformation of the HA should be maintained during inactivation procedures to ensure that the vaccine
stimulates appropriate neutralising antibody. This can be tested by use of an immunological assay such as
SRD (single radial diffusion), which measures immunologically active HA capable of reacting with specific
anti-HA antibodies. The immunogenicity of the vaccine can be confirmed by measurement of HA antibody
stimulated in small animal models or the target species. An immune stimulating complex (ISCOM) based
vaccine against equine influenza has also been shown to stimulate systemic IFN gamma synthesis.
Canary pox-vectored vaccines, also administered via the intramuscular route, have the potential for cellmediated immunity (CMI) priming, as indicated, by increased equine influenza virus-specific IFN gamma and
IL-2 mRNA expression in vitro. Infectious titre of the recombinant canary pox virus carrying equine influenza
HA genes is used as an in vitro measure of potency and immunogenicity is assessed by measurement of
antibody stimulated in the target species.
Antibody to HA as measured by SRH, stimulated by inactivated whole virus, subunit or canary pox-vectored
vaccine correlates well with protection against infection in an experimental challenge model system (Edlund
Toulemonde et al., 2005; Mumford, 1983; 1994). In contrast, a cold-adapted temperature-sensitive mutant
used as a live attenuated vaccine replicates in the upper respiratory tract and does not stimulate high levels
of circulating antibody to HA but nevertheless provides protection against challenge infection. Immunity is
presumed to be mediated through mucosal or cellular responses rather than circulating antibody. As with the
vectored vaccine, in-process control testing is reliant on measurement of the infectious virus titre.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.6 Principles of veterinary
vaccine production. The guidelines given here and in chapter 1.1.6 are intended to be general in nature as
manufacturers are obliged to meet European Pharmacopeia, USDA or other national and regional
requirements.
All equine influenza vaccines should contain epidemiologically relevant strains

A formal equine influenza global surveillance programme has been in place since 1995.
The OIE Reference Laboratories and other collaborating laboratories collect data on
outbreaks of equine influenza and strain variation that is reviewed annually by an Expert
Surveillance Panel (ESP) including representatives from OIE and WHO. This panel
makes recommendations on the need to update vaccines, and these are published
annually in the OIE Bulletin (Expert Surveillance Panel, 2011). The criteria for updating
equine influenza vaccines are similar to those for human influenza vaccines and based
on analysis of evidence of disease in well vaccinated horses, antigenic changes, genetic
changes and, when possible, supporting experimental challenge data.
H7N7: Many vaccines still contain an H7N7 strain. However, the Expert Surveillance
Panel has recommended that the H7N7 component should be omitted as there have
been few reports of infections with this subtype have been substantiated during the past
30 years.
H3N8: Antigenic variants of H3N8 viruses co-circulate (Bryant et al., 2009) and it is
important to include a strain or strains that are epidemiologically relevant as
recommended by the OIE Expert Surveillance Panel and published in the OIE Bulletin
(Expert Surveillance Panel, 2010). OIE Reference Laboratories can provide help in
selecting suitable vaccine strains.
872
2.
a)

i)
For each vaccine strain, a prototype batch should be prepared to establish its suitability as a vaccine
strain, i.e. purity and safety should be confirmed by standard techniques. The ability of seed-lot viruses
to grow to high titre and generate sufficient antigenic mass to stimulate adequate antibody responses
in the target species, should be confirmed. Additionally, vaccine virus derived in MDCK cells should be
fully characterised to ensure that antigenic variants have not arisen during the culture process, such
that the vaccine virus is no longer representative of the original isolate.
ii)
Quality criteria (sterility, purity, freedom from extraneous agents)

Virus strains may be obtained from OIE Reference Laboratories (see Table given in Part 4 of this
Terrestrial Manual). Viruses selected as vaccine strains should be described in terms of origin and
passage history. The strains are propagated in the allantoic cavity of 10-day-old embryonated hens
eggs or cell cultures, such as MDCK. All manipulations must be conducted separately for each strain.
Viral growth is monitored by HA tests. Passaged virus is identified by serological tests, such as HI or
SRH or by PCR using specific primers. If vaccine virus is grown in cell culture, antigenic studies with
ferret sera and MAbs should be undertaken to ensure that variant viruses have not been selected
during passage to prepare master and working seed viruses. Master and working seed viruses are
divided into aliquots and stored in freeze-dried form at 20C or at 70C following testing for
extraneous agents. Records of storage conditions should be maintained.
The master seed lot of each vaccine strain selected should be processed at one time to assure a
uniform composition, tested for extraneous agents, and fully characterised. Antisera or MAbs for use in
HI tests to characterise vaccine strains may be obtained from OIE and WHO Reference Laboratories.
Working seed lots are derived from a master seed lot and should be of uniform composition, free from
extraneous agents, and fully characterised. Aliquots of the working seed are used for production of
vaccine.
Master and working seed lots should be prepared in specific pathogen free eggs or, as a minimum, in
eggs derived from a healthy flock.
If MDCK cells are used to propagate vaccine virus, master cell lines should be established and stored
in liquid nitrogen, and should be tested for freedom from extraneous agents according to National
Control Authority Guidelines.
Examination of seed viruses for extraneous agents including mycoplasmas and other equine viruses
should be performed by appropriate techniques, including inoculation of susceptible tissue cultures and
examination for cytopathic effect or application of fluorescent antibodies for antigen detection.
The presence of other common equine respiratory pathogens, e.g. equine herpesviruses 1, 2, 4,
equine picornaviruses, equine viral arteritis, and equine adenoviruses, should be specifically excluded.
The absence of bacteria should be confirmed by standard sterility tests and toxicity tests in small
animals.
b)
i)
Procedure
Production is based on a seed-lot system that has been validated with respect to the characteristics of
the vaccine strains. Where eggs are used, each strain of virus is inoculated separately into the allantoic
cavity of 911-day-old embryonated hens eggs from a healthy flock. The eggs are incubated at a
suitable temperature for 23 days, and the allantoic fluid is collected. Alternatively, each strain is
inoculated separately into MDCk cell cultures. The viral suspensions of each strain are collected
separately and inactivated. If necessary, they may be purified. Suitable adjuvants and antimicrobial
preservatives may be added.
Monovalent virus pools should be inactivated as soon as possible after their preparation, by a method
approved by the National Control Authority. If formalin (37% formaldehyde) or beta-propiolactone (2oxetanone) is used, the concentration by volume should not exceed 0.2%. Ideally, pools should be held
at 4C and should be inactivated within 5 days of harvest. Inactivation of the vaccine must be
demonstrated. A suitable method consists of inoculating 0.2 ml of undiluted monovalent pool and 1/10
and 1/100 dilutions of the monovalent pool into the allantoic cavities of groups of fertile eggs (10 eggs
in each group), and incubating the eggs at 3337C for 3 days. At least 8 of the 10 eggs should survive
at each dosage level. A volume of 0.5 ml of allantoic fluid is harvested from each surviving egg. The
fluid harvested from each group is pooled, and 0.2 ml of each of the three pools is inoculated,
undiluted, into a further group of 10 fertile eggs. Haemagglutinin activity should not be detected in
873
these new groups of eggs. In some countries, the requirement that 80% of the eggs should survive
during incubation may be impossible to satisfy, in which case the National Control Authority should
then specify a modified requirement to be satisfied. Before inactivation, samples should be collected for
bacterial and fungal sterility tests.
Monovalent material may be concentrated and purified by high-speed centrifugation or other suitable
methods approved by the National Control Authority, either before or after the inactivation procedure. It
is important to concentrate and purify the virus under optimum conditions, e.g. temperatures that
preserve its antigenic properties.
The monovalent virus pool shall be shown not to contain viable influenza virus when tested by
inoculation of embryonated hens eggs, by a method approved by the National Control Authority.
Alternatively, the satisfactory inactivation can also be demonstrated by inoculating monolayers of
MDCK cells.
ii)

Vaccines should be produced in eggs or in a cell line that meet the requirements of the National
Control Authority. Wherever practicable the use of substances of animal origin for example, serum and
trypsin should be kept to a minimum. Substances of animal origin used during production should be
subjected to a suitable, validated sterilisation or inactivation procedure or be tested for the absence of
extraneous organisms.
iii)
In-process controls
Relevant in-process controls should be applied before and after inactivation and before and after
concentration and purification.
In-process controls include: (a) identity of virus strains (tested by HI); (b) sterility; (c) virus titre;
(d) haemagglutinin content (tested by chicken RBCs agglutinating units, CCA [chick cell agglutination]);
and (e) immunologically active HA (tested by SRD or another suitable immunochemical method).
Single radial diffusion test

SRD is a reliable method for measuring immunologically active HA in terms of g HA, and is used
routinely for potency testing of human influenza vaccines (Wood et al., 1983b).
The potency of inactivated equine influenza vaccine depends on the concentration of immunologically
active haemagglutinin (Wood et al., 1983a).
Assessment of the antigenic content of the vaccine by CCA alone may be misleading, as the sensitivity
of this assay is a reflection of the ability of virus strains to agglutinate RBCs. Disruption of virus may
lead to an apparent increase in HA as measured by CCA. The CCA assay does not provide a measure
of the antigenic properties of the HA (HA may retain its properties to bind to RBCs while losing its
ability to stimulate antibody).
Most equine influenza vaccines contain more than one variant of the H3N8 subtype. In this situation, it
is not possible to judge the potency of individual H3N8 components from serological tests performed
on sera collected from horses or small animals vaccinated with the final product, because of crossreactivity between the two isolates of the same subtype. Thus, it is important that a reliable method,
such as SRD, be used to measure the potency of individual components before and after inactivation
and prior to mixing and formulation with adjuvant.
In the SRD test, virus preparations are compared with a calibrated reference preparation of known HA
content. Antigens are allowed to diffuse through a gel containing an antiserum specific for a particular
HA. The distance diffused by the antigen before precipitation by the antibody incorporated in the gel is
directly related to the concentration of haemagglutinin in the antigen preparations.
Standard reagents for SRD testing are available from the WHO International Laboratory for Biological
Standards3. Reagents for A/eq/Prague/56 (H7N7), and the H3N8 strains A/eq/Miami/63,
A/eq/Kentucky/81, A/eq/Newmarket/1/93 (American lineage) and A/eq/Newmarket/2/93 (European
lineage) are currently available.
iv)
Final product batch tests

Sterility and purity
874
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG,
UK.
Safety
a)
For inactivated or subunit vaccines absence of viral infectivity should be demonstrated by two
passages in 10 embryonated hen eggs. The allantoic fluid should have no haemagglutinating
activity.
b)
Using no fewer than three horses, each horse is inoculated intramuscularly (at two different sites)
with the dose of vaccine specified by the manufacturer; these inoculations are repeated 2
4 weeks later. The animals are kept under observation for 10 days after the second set of
injections. No abnormal local or systemic reaction should ensue.
c)
If vaccine is to be used in mares, safety should be demonstrated by giving two doses of vaccine
to no fewer than two pregnant mares at the prescribed interval within the trimester for which the
vaccine is recommended. Once safety has been demonstrated on a prototype batch, safety
testing in pregnant mares may be omitted for routine testing of subsequent batches of the final
product.
Batch potency
Following mixing of viral antigens and adjuvants, aliquots should be potency tested in vivo using horses
and guinea-pigs or a suitable alternative immunochemical assay. Adjuvants cause interference in
quantitative in-vitro tests, such as CCA and SRD, although SRD may be used on the final product as a
qualitative assay to demonstrate the presence of antigen for each vaccine strain. For repeated batch
tests, only guinea-pigs or a suitable alternative immunochemical assay are used, subject to agreement
of the National Control Authority.
a)
Serological responses in horses

For a valid in-vivo potency test, naive seronegative horses must be selected for vaccination.
Young horses or ponies (not less than 6 months old) should be screened for the presence of
antibody using H7N7 and H3N8 viruses including recently isolated viruses relevant to the area in
which the horses were reared. If HI tests are used for screening, H3N8 viruses should be treated
with Tween 80/ether to maximise the sensitivity of the test. Alternatively, SRH may be used to
establish the seronegative status of animals.
To test a vaccine for efficacy in horses, inject a volume corresponding to one vaccine dose by the
recommended route into each of five susceptible seronegative horses. After the period
recommended between the first and second doses, as stated on the label, a volume of vaccine
corresponding to the second dose of vaccine is injected into each horse.
Three blood samples are collected from each animal, the first at the time of the first vaccination,
the second 1 week after the first vaccination to check for anamnestic response, and the third
2 weeks after the second vaccination.
The serological assay used to measure the antibody response to the viruses contained in the
vaccine must be standardised for a valid in-vivo potency test. The SRH assay (see Section B.2.b)
is preferred as standard reference sera are available for quality control purposes from the
European Pharmacopoeia4. These sera should be tested in parallel with the test sera to ensure
that the test is valid with respect to sensitivity; the values obtained should not vary by more than
20% from the SRH values assigned in an international collaborative study (Daly et al., 2007;
Mumford, 2000). Due to poor repeatability and reproducibility of the HI test between different
laboratories, no HI titre could be assigned to these sera.
Following vaccination the antibody value measured by SRH should not be less than 150 mm2.
This is higher than the value required in the European Pharmacopoeia Monograph for inactivated
equine influenza vaccines (85 mm2) as this value is not considered to be protective. If the value
found for any horse after the first vaccination indicates that there has been an anamnestic
response, the result is not taken into account. A supplementary test is carried out, as described
above, replacing the horses that showed an anamnestic response with an equal number of new
animals.
If the HI test is used, the antibody titre of each serum taken after the second vaccination in each
test should not be less than 1/64 (calculated for the original serum, taking into account the
predilution of 1/8). Alternatively, the antibody levels stimulated by the vaccine under test should
be shown to be at least equal to the antibody levels stimulated by a standard vaccine tested in
parallel that has been shown previously to protect horses against challenge infection.
Serum to A/eq/Newmarket/1/77
A/eq/Newmarket/2/93 (E0850022).
(Catalogue
number
E0850010),
A/eq/Newmarket/1/93
(E0850021)
and
875
b)
Challenge studies in horses

It may be desirable in certain cases to undertake challenge studies in horses to demonstrate
potency, particularly if vaccines are being assessed for their ability to protect against antigenically
dissimilar viruses. Challenge studies may be carried out by exposing vaccinated horses/ponies to
an aerosol of virulent influenza virus no fewer than 2 weeks after the second dose of vaccine.
Comparisons of clinical signs, virus excretion and serological responses are made with a group of
unvaccinated control animals challenged at the same time. The timing of the challenge procedure
will reflect the claims to be made on the data sheet regarding duration of immunity. Protection at
2 weeks post-vaccination when antibody levels are at their peak level does not necessarily
indicate good duration of immunity under field conditions.
If tests for potency in horses have been carried out with satisfactory results on a representative
batch of vaccine, these tests may be omitted as a routine control on other batches of vaccine
prepared using the same seed-lot system, subject to agreement by the National Control Authority.
c)
Serological responses of guinea-pigs

Inject each of no fewer than five guinea-pigs free from specific antibodies with one vaccine dose.
Collect blood samples 21 days later, and test the serum by SRH or HI (see Sections B.2.a and
B.2.b). Perform the tests of each serum using, respectively, the antigen(s) prepared from the
strain(s) used in the production of the vaccine. The antibody titre of each serum in each test
should not be less than the titre stimulated by a standard vaccine that has been shown to
stimulate protective levels of antibody in horses.
c)

i)
Safety requirements
The safety requirements may vary with the National Authority but usually include the assessment of the
administration of an overdose and of the repeated administration of one dose to young horses and
pregnant mares, if the vaccine is intended for use in pregnant mares. For live vaccines the
dissemination by the vaccinated horse and the spread of the vaccine strain from vaccinated to
unvaccinated horses along with the possible consequences must be investigated. Reversion to
virulence studies should be performed by serial passaging of the live vaccine.
ii)
Efficacy requirements
The efficacy requirements may vary with the National Authority but usually include the assessment of
the serological response in horses and virus challenge studies in susceptible horses (see Section
C.2.b.iv batch potency b).
Where claims for duration of immunity are made on the data sheet, these should be supported with
data on the duration of protective levels of antibody maintained in horses vaccinated according to the
recommended schedule. Antibody levels quoted as protective should be validated in challenge studies
or by comparison with published reports.
iii)
Stability
Vaccines should be stored at 53C and protected from light. The shelf life quoted on the data sheet
should be demonstrated by testing the potency of aliquots over time using the guinea-pig potency test
(see Section C.2.b.iv batch potency c).
iv)
Maintaining epidemiologically relevant strains in vaccines

To enable vaccine manufacturers to respond quickly to recommendations from the Expert Surveillance
Panel to update vaccine strains, the Committee for Veterinary Medicinal Products (CVMP) for the
European Agency for the Evaluation of Medicinal Products (EMEA) has developed a fast-track
licensing system to be used when vaccine strains are updated (EMEA, 1998).
3.
a)

A canary pox recombinant vaccine is available in some countries and was used in the equine influenza
control and eradication programme in Australia in 2007. A nucleoprotein ELISA was used to differentiate
horses vaccinated with the recombinant vaccine from horses that had been exposed to virus by natural
infection (DIVA) This DIVA was possible because the canary pox recombinant expresses only the
haemagglutinin gene of equine influenza. There is some evidence that this vaccine may be used to prime
the immune system in the face of maternally derived antibodies (Minke et al., 2008).
876
b)
Special requirements
National Authorities frequently require an environmental risk assessment. This may include an assessment
of the potential direct and indirect, immediate or delayed adverse effects of the genetically modified vaccine
on human and animal health and on the environment. The phenotypic and genetic stability of the vaccine
and its potential interactions with other organisms may need to be evaluated.
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PRODUCTS (CVMP) (1998). Note for Guidance: Harmonisation of Requirements for Equine Influenza Vaccines
Specific Requirements for Substitution or Addition of a Strain or Strains. Document EMEA/CVMP/112/98-FINAL,
EMEA, London, UK.
EXPERT SURVEILLANCE PANEL (2011). ESP on Equine Influenza Vaccine Composition, OIE Headquarters, Paris, 24
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FOORD A.J., SELLECK P., COLLING A., KLIPPEL J., MIDDLETON D. & HEINE H.G. (2009). Real-time RT-PCR for
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GERBER H. (1970). Clinical features, sequelae and epidemiology of equine influenza. In: Equine Infectious
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LIVESAY G.J., ONEILL T., HANNANT D, YADAV M.P. & MUMFORD J.A. (1993). The outbreak of equine influenza (H3N8)
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MUMFORD J. (2000). Collaborative study for the establishment of three European Pharmacopoeia biological
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immunogenicity of experimental equine influenza ISCOM vaccines. Vaccine, 12, 857863.
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vaccine 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8). J. Hyg.
(Camb.), 90, 385395.
OXBURGH L. & KLINGBORN B. (1999). Cocirculation of two distinct lineages of equine influenza virus subtype H3N8.
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QUINLIVAN M., DEMPSEY E., RYAN F., ARKINS S. & CULLINANE A. (2005). Real-time reverse transcription PCR for
detection and quantitative analysis of equine influenza virus. J. Clin. Microbiol., 43, 50555057.
TU J., ZHOU H., JIANG T., LI C., ZHANG A., GUO X., ZOU W., CHEN H. & JIN M. (2009). Isolation and molecular
characterization of equine H3N8 influenza virus from pigs in China. Arch. Virol., 154, 887890.
WEBSTER R.G. (1993). Are equine 1 influenza viruses still present in horses? Equine Vet. J., 25, 537538.
WOOD J.M., MUMFORD J., FOLKERS C., SCOTT A.M. & SCHILD G.C. (1983b). Studies with inactivated equine influenza
vaccine 1. Serological responses of ponies to graded doses of vaccine. J. Hyg. (Camb.), 90, 371384.
WOOD J.M., SCHILD G.C., FOLKERS C., MUMFORD J. & NEWMAN R.W. (1983a). The standardisation of inactivated
equine influenza vaccines by single-radial immunodiffusion. J. Biol. Stand., 11, 133136.
YAMANAKA T., TSUJIMURA K., KONDO T. & MATSUMURA T. (2008). Evaluation of antigen detection kits for diagnosis of
equine influenza. J. Vet. Med. Sci., 70, 189192.
*
* *
NB: There are OIE Reference Laboratories for Equine influenza
diagnostic tests, reagents and vaccines for equine influenza
878
CHAPTER 2.5.8.
EQUINE PIROPLASMOSIS
SUMMARY
Equine piroplasmosis is a tick-borne protozoal disease of horses, mules, donkeys and zebra. The
aetiological agents are blood parasites named Theileria equi and Babesia caballi. Theileria equi
was previously designated as Babesia equi. Infected animals may remain carriers of these
parasites for long periods and act as sources of infection for ticks, which act as vectors. The
parasites are found inside the red blood cells of the infected animals.
The introduction of carrier animals into areas where tick vectors are prevalent can lead to an
epizootic spread of the disease.
Identification of the agent: Infected horses can be identified by demonstrating the parasites in
stained blood or organ smears during the acute phase of the disease. Romanovsky-type staining
methods, such as Giemsa, give the best results. In carrier animals, low parasitaemias make it
extremely difficult to detect parasites, especially in the case of B. caballi infections, although they
may sometimes be demonstrated by using a thick blood smear technique.
Paired merozoites joined at their posterior ends are a diagnostic feature of B. caballi infection. The
parasites in the erythrocytes measure 2 5 m. The merozoites of T. equi are less than 23 m
long, and are pyriform, round or ovoid. A characteristic of T. equi is the arrangement of four pearshaped merozoites forming a tetrad known as a Maltese cross.
Molecular techniques for the detection of T. equi and B. caballi based on species-specific
polymerase chain reaction (PCR) assays, targeting the 18S rRNA gene, have been developed and
continue to expand. These tests have been shown to be highly specific and sensitive and promise
to play an increasing role in the diagnosis of infections.
Serological tests: Infections in carrier animals are best demonstrated by testing their sera for the
presence of specific antibodies.
Currently, the indirect fluorescent antibody (IFA) test and the competitive enzyme-linked
immunosorbent assay (C-ELISA) are the primary tests used for qualifying horses for importation.
The complement fixation (CF) test, for many years the primary test, has been replaced by the IFA
and C-ELISA. These tests have proven to be more effective at detecting long-term infected animals
and animals treated with antiparasitic drugs; these animals may be CF negative but still be infected.
The IFA test and C-ELISA have been shown to be highly specific for each of the two species of
piroplasmosis agents involved. Indirect ELISAs may also be used to detect antibodies to both
species in infected horses, although cross-reactions between T. equi and B. caballi occur.
Application of recombinant T. equi and B. caballi merozoite proteins in diagnostic assays appear to
be very promising in the accurate determination of equine piroplasmosis infection.
available.
A. INTRODUCTION
Equine piroplasmosis is a tick-borne protozoal disease of horses, mules, donkeys and zebra. The aetiological
agents of equine piroplasmosis are Theileria equi and Babesia caballi. Twelve species of Ixodid ticks in the
genera Dermacentor, Rhipicephalus and Hyalomma have been identified as transstadial vectors of B. caballi and
T. equi, while eight of these species were also able to transmit B. caballi infections transovarially (De Waal &
879
Chapter 2.5.8. Equine piroplasmosis

Potgieter, 1987; Neitz, 1956; Stiller & Frerich, 1979; Stiller et al., 1980). Infected animals may remain carriers of
these blood parasites for long periods and act as sources of infection for tick vectors.
The parasites occur in southern Europe, Asia, countries of the Commonwealth of Independent States, Africa,
Cuba, South and Central America, and certain parts of the southern United States of America. Theileria equi has
also been reported from Australia (but, apparently never established itself in this region), and is now believed to
have a wider general distribution than B. caballi.
During the life cycle of Babesia, initially the sporozoites invade red blood cells (RBCs) where they transform into
trophozoites (Friedhoff, 1970; Potgieter & Els, 1977). In this situation the trophozoites grow and divide into two
round, oval or pear-shaped merozoites. The mature merozoites are now capable of infecting new RBCs and the
division process is then repeated.
Babesia caballi: the merozoites in the RBCs are pear-shaped, 25 m long and 1.33.0 m in diameter (Levine,
1985). The paired merozoites joined at their posterior ends are considered to be a diagnostic feature of B. caballi
infection (Mehlhorn & Schein, 1984).
Theileria equi: the merozoites of this organism are relatively small, less than 23 m long (Levine, 1985), and are
pyriform, round or ovoid. A characteristic of T. equi is the arrangement of four pear-shaped merozoites,
measuring about 2 m in length, forming a tetrad known as the Maltese cross arrangement (Holbrook et al.,
1968).
In T. equi infection it has been shown that sporozoites inoculated into horses via a tick bite invade the
lymphocytes (Schein et al., 1981). The sporozoites undergo development in the cytoplasm of these lymphocytes
and eventually form Theileria-like schizonts. Merozoites released from these schizonts enter RBCs. The
taxonomic position of T. equi has been controversial and only relatively recently has it been redescribed as a
Theileria (Mehlhorn & Schein, 1998). Further support for the close relation with Theileria spp. also comes from the
homology found between 30 and 34 kDa T. equi surface proteins and similar sized proteins of various Theileria
spp. (Kappmeyer et al., 1993; Knowles et al., 1997). However, the position of T. equi in phylogenetic trees based
on the small subunit ribosomal RNA genes is variable and mostly appear as a sister clade of the Theilerids
(Criado-Fornelio et al., 2003; Nagore et al., 2004) leading some to suggest that T. equi is ancestral to the
Theilerids (Criado-Fornelio et al., 2003) or a different group altogether (Allsopp et al., 1994).The clinical signs of
equine piroplasmosis are often nonspecific, and the disease can easily be confused with other conditions.
Piroplasmosis can occur in peracute, acute and chronic forms. The acute cases are more common, and are
characterised by fever that usually exceeds 40C, reduced appetite and malaise, elevated respiratory and pulse
rates, congestion of mucous membranes, and faecal balls that are smaller and drier than normal.
Clinical signs in subacute cases are similar. In addition, affected animals show loss of weight, and fever is
sometimes intermittent. The mucous membranes vary from pale pink to pink, or pale yellow to bright yellow.
Petechiae and/or ecchymoses may also be visible on the mucous membranes. Normal bowel movements may be
slightly depressed and the animals may show signs of mild colic. Mild oedematous swelling of the distal part of
the limbs sometimes occurs.
Chronic cases usually present nonspecific clinical signs such as mild inappetence, poor performance and a drop
in body mass. The spleen is usually found to be enlarged on rectal examination.
A rare peracute form where horses are found either dead or moribund has been reported (Littlejohn, 1963).
1.
Infected horses may be identified by demonstrating the parasites in stained blood, optimally collected from
superficial skin capillaries, or organ smears during the acute phase of the disease. Romanovsky-type staining
methods, such as the Giemsa method, usually give the best results (Shute, 1966). However, even in acute clinical
cases of B. caballi infection, the parasitaemia is very low and difficult to detect. Experienced workers sometimes
use a thick blood smear technique (Mahoney & Saal, 1961) to detect very low parasitaemia. Thick films are made
by placing a small drop (approximately 50 l) of blood on to a clean glass slide which is then air-dried, heat fixed
at 80C for 5 minutes, and stained in 5% Giemsa for 2030 minutes.
An accurate identification of the species of the parasite is sometimes desirable, as mixed infections of T. equi and
B. caballi probably occur frequently.
880

Identification of equine piroplasmosis in carrier animals by means of blood smear examination is not only very
difficult but also inaccurate and therefore serological methods are preferred for this (see below). However, falsenegative or false-positive reactions may be encountered in the course of serological tests (Donnelly et al., 1980;
Frerichs et al., 1969; Tenter & Freidhoff, 1986). In such cases, the passage of whole blood, although a
cumbersome and expensive exercise, is a very useful technique to determine the true position. Large quantities of
whole blood (500 ml) are transfused into a susceptible, preferably splenectomised, horse. This animal is then kept
under close observation for clinical signs of disease. Diagnosis is confirmed by the presence of parasites in its
RBCs.
In an additional technique, a specific uninfected tick vector is fed on a suspect animal, and the organism can then
either be identified in the tick itself or through the transmission of the organism by the tick vector to another
susceptible animal.
Success in the establishment of in-vitro cultures of T. equi and B. caballi may be one alternative to supplement
the methods described above, in order to identify carriers of the parasites (Holman et al., 1993; 1994; Zweygarth
et al., 1995; 1997). Babesia caballi parasites were successfully cultured from the blood of two horses that tested
negative by the complement fixation (CF) test (Holman et al., 1993). Similarly, T. equi could be cultured from
horses that did not show any patent parasitaemia at the time of the initiation of the cultures (Zweygarth et al.,
1995; 1997).
Molecular techniques for the detection of T. equi and B. caballi have been described. These methods are based
on species-specific polymerase chain reaction (PCR) assays, which mainly target the 18S rRNA gene
(Bashiruddin et al., 1999; Criado-Fornelio et al., 2003; Sahagun-Ruiz et al., 1997). Further refinements to the
technique includes nested PCR (Rampersad et al., 2003), loop-mediated isothermal amplification (LAMP)
(Alhassan et al., 2007) with reported increased sensitivity; a highly sensitive reverse line blot assay (RLB) and
multiplex PCR for simultaneous detection and identification of Theileria and Babesia species in horses (Alhassan
et al., 2005; Nagore et al., 2004).
2.
Serological tests
It is extremely difficult to diagnose the organisms in carrier animals by means of the microscopic examination of
blood smears. Furthermore, it is by no means practical on a large scale. The serological testing of animals is
therefore recommended as a preferred method of diagnosis, especially when horses are destined to be imported
into countries where the disease does not occur, but the vector is present.
Sera should be collected and dispatched to diagnostic laboratories in accordance with the specifications of that
laboratory. Horses for export that have been subjected to serological tests and shown to be free from infection,
should be kept free of ticks to prevent accidental infections.
A number of serological techniques have been used in the diagnosis of piroplasmosis, such as the CF test, the
indirect fluorescent antibody (IFA) test and the enzyme-linked immunosorbent assay (ELISA). In addition, a
simple and rapid immunochromatographic test for T. equi has also recently been described and might be a very
useful test for the mass screening of serum samples (Huang et al., 2004).
a)
Indirect fluorescent antibody test (a prescribed test for international trade)

The IFA test has been successfully applied to the differential diagnosis of T. equi and B. caballi infections
(Madden & Holbrook, 1968). The recognition of a strong positive reaction is relatively simple, but any
differentiation between weak positive and negative reactions requires considerable experience in
interpretation. A detailed description of the protocol of the IFA test has been given (Madden & Holbrook,
1968; Morzaria et al., 1977). An example of an IFA protocol is given below.
Antigen production
Blood for antigen is obtained from horses with a rising parasitaemia, ideally 25%. Carrier animals that have
already produced antibodies are not suitable for antigen production. Blood (about 15 ml) is collected into
235 ml of phosphate buffered saline (PBS), pH 7.2. The RBCs are washed three times in cold PBS (1000 g
for 10 minutes at 4C). The supernatant fluid and the white cell layer are removed after each wash. After the
last wash, the packed RBCs are reconstituted to the initial volume with 4% bovine serum albumin fraction V
made up in PBS, i.e. the original packed cell volume = 30%, so that one-third consists of RBCs. If the
original RBC volume is 15 ml, then 5 ml of packed RBCs + 10 ml of 4% bovine albumin in PBS constitutes
the antigen. After thorough mixing, the antigen is placed on to prepared wells on a glass slide using a
template or a syringe (Morzaria et al., 1977). Alternatively, the cells can be spread smoothly on to
microscope slides, covering the entire slide with an even, moderately thick film. These slides are allowed to
dry, wrapped in soft paper and sealed in plastic bags or wrapped in aluminium foil, and stored at 20C for
up to 1 year.
881
Test procedure
i)
Each sample of serum is tested against an antigen of B. caballi and of T. equi.
ii)
Prior to use, the frozen antigen slides are removed from storage at 20C and incubated at 37C for
10 minutes.
iii)
The antigen smears are then removed from their protective covering and fixed in cold dry acetone
(20C) for 1 minute. Commercially produced slides are available that are pre-fixed.
iv)
If smears were prepared on the whole slide surface, squares (1421 in number, i.e. 23 rows of
7 each) are formed on the antigen smears with nail varnish or rapidly drying mounting medium (i.e.
Cystoseal).
v)
Test, positive and negative control sera are diluted from 1/80 to 1/1280 in PBS. Negative and positive
control sera are included in each test.
vi)
Sera are applied (10 l each) at appropriate dilutions to the different wells or squares on the antigen
smear, incubated at 37C for 30 minutes, and washed several times in PBS and once in water.
vii)
An anti-horse immunoglobulin prepared in rabbits and conjugated with fluorescein isothiocyanate (this
conjugate is available commercially) is diluted in PBS and applied to the smear, which is then
incubated and washed as before.
viii) After the final wash, two drops of a solution containing equal parts of glycerin and PBS are placed on
each smears and mounted with a cover-slip.
ix)
b)
The smear is then examined under the microscope for the fluorescing parasites. Sera diluted 1/80 or
more that show strong fluorescence are usually considered to be positive, although due consideration
is also given to the patterns of fluorescence of the positive and negative controls.
Enzyme-linked immunosorbent assay (a prescribed test for international trade)

In recent years a number of recombinant antigens for the use in ELISAs have been described. Recombinant
T. equi (EMA-1; EMA-2; Be82 and Be158) and B. caballi proteins (RAP-1; Bc48; Bc134) have been
produced in Escherichia coli (Hirata et al., 2002; 2005; Huang et al., 2003; Ikadai et al., 2000; Kappmeyer et
al., 1999; Knowles et al., 1992; Tamaki et al., 2004) or in insect cells by baculovirus (Xuan et al., 2001;
Tanaka et al., 1999). Recombinant antigens produced in E. coli or by baculovirus have the obvious
advantage of avoiding the need to infect horses for antigen production, and of eliminating the crossreactions that have been experienced in the past with the crude ELISA antigens. They also provide a
consistent source of antigen for international distribution and standardisation.
Indirect ELISAs using EMA-2 and BC48 have shown high sensitivity and specificity in detecting antibodies in
infected horses (Huang et al., 2003; Ikadai et al., 1999; Tanaka et al., 1999). Initial results from these tests
are promising and further validation of the assays is underway.
A competitive inhibition ELISA (C-ELISA) using EMA-1 protein and a specific monoclonal antibody (MAb)
that defines this merozoite surface protein epitope, have been used in a C-ELISA for T. equi (Knowles et al.,
1992). This C-ELISA overcomes the problem of antigen purity, as the specificity of this assay depends only
on the specificity defined by the MAb T. equi epitope. A 94% correlation was shown between the C-ELISA
and the CF test in detecting antibodies to T. equi. Sera that gave discrepant results were evaluated for their
ability to immunoprecipitate 35S-methionine-labelled in-vitro translated products of T. equi merozoite mRNA.
Samples that were C-ELISA positive and CF test negative clearly precipitated multiple T. equi proteins.
However, immunoprecipitation results with serum samples that were C-ELISA negative and CF test positive
were inconclusive (Knowles et al., 1991). This C-ELISA for T. equi was also recently validated in Morocco
and Israel, giving a concordance of 91% and 95.7% with the IFA test, respectively (Rhalem et al., 2001;
Shkap et al., 1998). A similar C-ELISA has been developed using the recombinant B. caballi rhoptryassociated protein 1 (RAP-1) and an MAb reactive with a peptide epitope of a 60 kDa B. caballi antigen
(Kappmeyer et al., 1999). The results of 302 serum samples tested with this C-ELISA and the CF test
showed a 73% concordance. Of the 72 samples that were CF test negative and C-ELISA positive, 48 (67%)
were shown to be positive on the IFA test, while four of the five samples that tested CF test positive and CELISA negative were positive on the IFA test (Kappmeyer et al., 1999).
A test protocol for an equine piroplasmosis C-ELISA has been described and used for additional validation
studies (Katz et al., 2000; United States Department of Agriculture [USDA], 2005). The apparent specificity
of the T. equi and B. caballi C-ELISAs lay between 99.2% and 99.5% using sera from 1000 horses
presumed to be piroplasmosis-free. One thousand foreign-origin horses of unknown infection status were
tested by the C-ELISA and the CF test with an apparent greater sensitivity of the C-ELISA. The results were
1.1% (T. equi) and 1.3% (B. caballi) more seropositive animals detected by C-ELISA than by the CF test; the
additional positive results were confirmed by IFA testing. A similar study of 645 foreign-origin horses tested
882

for import and pre-import purposes used heat-treated sera (58C for 30 minutes), and resulted in 3.6%
(T. equi) and 2.1% (B. caballi) more seropositive animals detected by the C-ELISA than by the CF test. Both
C-ELISAs were highly reproducible well-to-well, plate-to-plate, and day-to-day, with overall variances of
1.2% and 1.6% for the T. equi and B. caballi tests, respectively.
The C-ELISA protocol is given below.
A detailed description of antigen production and a test protocol has been given by the National Veterinary
Services Laboratories of the USDA (2005). A commercial kit is now available that is based on the same
antigens and monoclonal antibodies).
Solutions
Antigen coating buffer: prepare the volume of antigen coating buffer required using the following amounts of
ingredients per litre: 2.93 g sodium bicarbonate; 1.59 g sodium carbonate; sufficient ultra-pure water to
dissolve, and make up to 1 litre with ultra-pure water. Adjust to pH 9.6.
C-ELISA wash solution (high salt diluent): prepare the volume of C-ELISA wash solution required by using
the following amounts of ingredients per litre: 29.5 g sodium chloride; 0.22 g monobasic sodium phosphate;
1.19 g dibasic sodium phosphate; 2.0 ml Tween 20; sufficient ultra-pure water to dissolve, and make up to
1 litre with ultra-pure water. Mix well. Adjust pH to 7.4. Sterilise by autoclaving at 121C.
Antigen production
Frozen transformed E. coli culture is inoculated at a 1/10,000 dilution into any standard non-selective
bacterial growth broth (e.g. Luria broth) containing added carbenicillin (100 g/ml) and isopropylthiogalactoside (IPTG, 1 mM). Cultures are incubated on an orbital shaker set at 200 rpm at 37C overnight.
Cells grown overnight are harvested by centrifugation (5000 g for 10 minutes), washed in 50 mM Tris/HCl
and 5 mM ethylene diamine tetra-acetic acid (EDTA) buffer, pH 8.0, and harvested again as before. (Antigen
is available from the National Veterinary Services Laboratories, P.O. Box 844, Ames, Iowa 50010, USA.)
Cells are resuspended to 10% of the original volume in the Tris/EDTA buffer to which 1 mg/ml of lysozyme
has been added, and incubated on ice for 20 minutes. Nonidet P-40 detergent (NP-40) is then added to a
final 1% concentration (v/v), vortexed, and the mixture is incubated on ice for 10 minutes. The material is
next sonicated four times for 30 seconds each time at 100 watts, on ice, allowing 2 minutes between
sonications for the material to remain cool. The sonicate is centrifuged at 10,000 g for 20 minutes. The
resulting supernatant is dispensed in 0.5 ml aliquots in microcentrifuge tubes and may then be stored at
70C for several years. The presence of heterologous host bacterial antigens does not interfere with the
binding of specific equine anti-piroplasma antibodies or the binding of the paired MAbs to their respective
expressed recombinant antigen epitopes and is confirmed by the following procedures. The antigencontaining supernatants are quality controlled by titrating them with their paired MAbs and with reference
monospecific equine antisera to verify both an adequate level of expression and complete specificity for the
homologous species of piroplasmosis agent. Normal serum (negative serum) controls must not interfere with
binding of the MAbs or positive equine reference sera to the expressed antigen preparation.
Test procedure
i)
Microtitration plates are prepared by coating the wells with 50 l of either T. equi antigen or B. caballi
antigen diluted in antigen-coating buffer. The dilution used is determined by standard serological
titration techniques. The plate is sealed with sealing tape, stored overnight at 4C, and frozen at
70C. Plates can be stored at 70 C for up to 6 months.
ii)
The primary anti-T. equi or anti-B. caballi MAb and secondary antibody-peroxidase conjugate is diluted
as directed by the manufacturer at the time of use in the C-ELISA, with antibody-diluting buffer
(supplied with the test kit).
iii)
Plates are thawed at room temperature, the coating solution is decanted, and the plates are washed
twice with C-ELISA wash solution.
iv)
The serum controls and test serum samples are diluted 1/2 with serum-diluting buffer before 50 l of
sera is added to wells. Each unknown serum sample is tested in single or duplicate wells. Positive
control sera and blanks are tested in duplicate while negative controls are tested in triplicate on
different parts of the plate. Plates are incubated covered, at room temperature (2125C) for
30 minutes in a humid chamber, and then washed three times in C-ELISA wash solution.
v)
All wells then receive 50 l/well of diluted primary anti-T. equi or anti-B. caballi MAb. (The MAb is
produced in a cell culture bioreactor and is available from the National Veterinary Services
883

Laboratories, P.O. Box 844, Ames, Iowa 50010, USA.) Plates are incubated covered for 30 minutes at
room temperature (2125C) in a humid chamber, and then washed three times in C-ELISA wash
solution.
vi)
Diluted secondary peroxidase anti-murine IgG (50 l/well) conjugate is added to each well. Plates are
incubated covered for 30 minutes at room temperature (2125C) in a humid chamber, and then
washed three times in C-ELISA wash solution.
vii)
Chromogenic enzyme substrate (50 l/well) is added to all wells, and plates are incubated for
15 minutes at room temperature (2125C) during colour development.
viii) The colour development is stopped by adding 50 l of stop solution to all wells and the plates are read
immediately on a plate reader.
c)
ix)
The plates are read at 620, 630 or 650 nm wavelength (OD). The average OD is calculated for the
duplicate wells for all control sera and blank wells. For a valid test, the mean of the negative controls
must produce an OD >0.300 and <2.000. The mean positive control sera must produce an inhibition of
40%.
x)
Per cent inhibition [%I] is calculated as follows: %I = 100 [(Sample OD 100) (Mean negative
control OD)].
xi)
If a test samples produces 40% inhibition it is considered positive. If the test sample produces <40%
inhibition it is considered negative.
Complement fixation
The CF test has been used in the past by some countries to qualify horses for importation (Taylor et al.,
1969). A detailed description of antigen production and a test protocol has been given (for example: USDA,
1997). The CF test may not identify all infected animals, especially those that have been drug-treated or that
produce anti-complementary reactions, or because of the inability of IgG(T) (the major immunoglobulin
isotype of equids) to fix guinea-pig complement (McGuire et al., 1971). Therefore the IFA test and C-ELISA
have replaced the CF as the prescribed tests for international trade.
An example of a CF test protocol is given below.
Solutions
Alsevers solution: prepare 1 litre of Alsevers solution by dissolving 20.5 g glucose; 8.0 g sodium citrate;
4.2 g sodium chloride in sufficient distilled water. Adjust to pH 6.1 using citric acid, and make up the volume
to 1 litre with distilled water. Sterilise by filtration.
Stock veronal buffer (5): dissolve the following in 1 litre of distilled water: 85.0 g sodium chloride; 3.75 g
sodium 5,5 diethyl barbituric; 1.68 g magnesium chloride (MgCl2.6H2O); 0.28 g calcium chloride. Dissolve
5.75 g of 5,5 diethyl barbituric acid in 0.5 litre hot (near boiling) distilled water. Cool this acid solution and
add to the salt solution. Make up to 2 litres with distilled water and store at 4C. To prepare a working
dilution, add one part stock solution to four parts distilled water. The final pH should be from 7.4 to 7.6.
Antigen production
Blood is obtained from horses with a high parasitaemia (e.g. 37% parasitaemia for B. caballi and 6085%
for T. equi), and mixed with equal volumes of Alsevers solution as an anticoagulant. The plasma/Alsevers
supernatant and buffy coat are removed when the RBCs have settled to the bottom of the flask. The RBCs
are washed several times with cold veronal buffer and then disrupted. The antigen is recovered from the
lysate by centrifugation at 30,900 g for 30 minutes.
The recovered antigen is washed several times in cold veronal buffer by centrifugation at 20,000 g for
15 minutes. Polyvinyl pyrrolidone 40,000 (15%[ w/v]) is added as a stabiliser and the preparation is mixed
on a magnetic stirrer for 30 minutes, strained through two thicknesses of sterile gauze, dispensed into 2 ml
volumes and freeze-dried. The antigen can then be stored at below 50C for several years.
884
Test procedure
i)
The specificity and potency of each batch of antigen should be checked against standard antisera of
known specificity and potency. Optimal antigen dilutions are also determined in a preliminary
checkerboard titration.
ii)
Test sera are inactivated for 30 minutes at 58C (donkey and mule sera are inactivated at 62.5C for
35 minutes) and tested in dilutions of 1/5 to 1/5120. Veronal buffer is used for all dilutions.

iii)
Complement is prepared and titrated spectrophotometrically to determine the 50% haemolytic dose
(CH50) (Stiller et al., 1980) and used in the test at five times CH50. The haemolytic system consists of
equal parts of a 2% sheep (RBC) suspension and veronal buffer with 5 minimum haemolytic doses
(MHDs) of haemolysin (amboceptor) (USDA, 1997). Some laboratories use twice the 100% haemolytic
dose, which gives equivalent sensitivity.
iv)
The test has been adapted to microtitration plates (Herr et al., 1985). The total volume of the test is
0.125 ml, made up of equal portions (0.025 ml) of antigen, complement (five times CH50) and diluted
serum. Incubation is performed for 1 hour at 37C.
v)
A double portion (0.05 ml) of the haemolytic system is added and the plates are incubated for a further
45 minutes at 37C with shaking after 20 minutes.
vi)
The plates are centrifuged for 1 minute at 200 g before being read over a mirror.
vii)
A lysis of 50% is recorded as positive, with the titre being the greatest serum dilution giving 50% lysis.
A titre of 1/5 is regarded as positive. A full set of controls (positive and negative sera) must be included
in each test as well as control antigen prepared from normal (uninfected) horse RBCs.
Anticomplementary samples are examined by the IFA test. Donkey sera are frequently anticomplementary.

No biological products are available currently.
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(2001). Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation
of its diagnostic potential in an enzyme-linked immunosorbant assay. J. Clin. Microbiol., 39, 705709.
ZWEYGARTH E., JUST M.C. & DE WAAL D.T. (1995). Continuous in vitro cultivation of erythrocytic stages of Babesia
equi. Parasitol. Res., 81, 355-358.
ZWEYGARTH E., JUST M.C. & DE WAAL D.T. (1997). In vitro cultivation of Babesia equi: detection of carrier animals
and isolation of parasites. Onderstepoort J. Vet Res., 64, 5156.
*
* *
NB: There are OIE Reference Laboratories for Equine piroplasmosis
diagnostic tests and reagents for equine piroplasmosis
888
CHAPTER 2.5.9.
EQUINE RHINOPNEUMONITIS
SUMMARY
Equine rhinopneumonitis (ER) is a collective term for any one of several highly contagious, clinical
disease entities of equids that may occur as a result of infection by either of two closely related
herpesviruses, equid herpesvirus-1 and -4 (EHV-1 and EHV-4).
Infection by either EHV-1 or EHV-4 is characterised by a primary respiratory tract disease of
varying severity that is related to the age and immunological status of the infected animal.
Infections by EHV-1 in particular are capable of progression beyond the respiratory mucosa to
cause the more serious disease manifestations of abortion, perinatal foal death, or neurological
dysfunction.
Identification of the agent: The standard method of identification of the herpesviral agents of ER
continues to be laboratory isolation of the virus from appropriate clinical or necropsy material,
followed by seroconfirmation of its identity. The viruses can be isolated in equine cell culture from
nasopharyngeal samples taken from horses during the febrile stage of respiratory tract infection,
from liver, lung, spleen, or thymus of aborted fetuses and early foal deaths, and from the leukocyte
fraction of the blood of animals with acute EHV-1 disease. Positive identification of viral isolates as
EHV-1 or EHV-4 can be achieved by immunofluorescence with type-specific monoclonal
antibodies.
A rapid presumptive diagnosis of rhinopneumonitis abortion can be achieved by direct
immunofluorescent detection of viral antigen in cryostat sections of tissues from aborted fetuses,
using conjugated polyclonal antiserum.
Sensitive and reliable methods for EHV-1/4 detection by polymerase chain reaction or
immunoperoxidase staining have been developed and are useful adjuncts to standard virus
cultivation techniques for diagnosis of ER.
Post-mortem demonstration of histopathological lesions of EHV-1 in tissues from aborted fetuses,
cases or perinatal foal death or in the central nervous system of neurologically affected animals
complements the laboratory diagnosis of ER.
Serological tests: Because most horses will possess some level of antibody to EHV-1/4, the
demonstration of specific antibody in the serum collected from a single blood sample is not
sufficient for a positive diagnosis of recent, active ER. Paired, acute and convalescent sera from
animals suspected of being infected with EHV-1 or EHV-4 can be tested for a four-fold or greater
rise in virus-specific antibody titre by either virus neutralisation, or enzyme-linked immunosorbent
assay, or complement fixation.
Requirements for vaccines and diagnostic biologicals: Both live attenuated and inactivated
viral vaccines of varying composition are commercially available for use in assisting in the control of
ER. While vaccination is helpful in reducing the incidence of abortion in mares, and in ameliorating
the severity of clinical signs of respiratory infection in young horses, it should not be considered to
be a substitute for strict adherence to the well established tenets of sound management practices
known to reduce the risk of rhinopneumonitis. Revaccination at frequent intervals is recommended
with each of the products, as the duration of vaccine-induced immunity is relatively short.
Standards for production and licensing of both attenuated and inactivated EHV vaccines are
established by appropriate veterinary regulatory agencies in the countries of vaccine manufacture
and use. A single set of internationally recognised standards for ER vaccines is not available. In
each case, however, vaccine production is based on the system of a detailed outline of production
889
Chapter 2.5.9. Equine rhinopneumonitis
employing a well characterised cell line and a master seed lot of vaccine virus that has been
validated with respect to virus identity, safety, virological purity, immunogenicity, and the absence of
extraneous microbial agents.
A. INTRODUCTION
Equine rhinopneumonitis (ER) is an historically derived term that describes a constellation of several disease
entities of horses that may include respiratory disease, abortion, neonatal foal pneumonitis, or
myeloencephalopathy (Allen & Bryans, 1986; Allen et al., 1999; Bryans & Allen, 1988; Crabb & Studdert, 1995).
The disease has been recognised for over 60 years as a threat to the international horse industry, and is caused
by either of two members of the Herpesviridae family, equid herpesvirus-1 and -4 (EHV-1 and EHV-4). EHV-1 and
EHV-4 are closely related alphaherpesviruses of horses with nucleotide sequence identity within individual
homologous genes ranging from 55% to 84%, and amino acid sequence identity from 55% to 96% (Telford et al.,
1992; 1998). The two herpesviruses are enzootic in all countries in which large populations of horses are
maintained as part of the cultural tradition or agricultural economy. There is no recorded evidence that the two
herpesviruses of ER pose any health risks to humans working with the agents.
ER is highly contagious among susceptible horses, with viral transmission to cohort animals occurring by
inhalation of aerosols of virus-laden respiratory secretions. Extensive use of vaccines has not eliminated EHV
infections, and the world-wide annual financial burden from these equine pathogens is immense.
In horses under 3 years of age, clinical ER usually takes the form of an acute, febrile respiratory illness that
spreads rapidly through the group of animals. The viruses infect and multiply in epithelial cells of the respiratory
mucosa. Signs of infection become apparent 28 days after exposure to virus, and are characterised by fever,
inappetence, depression, and nasal discharge. The severity of respiratory disease varies with the age of the
horse and the level of immunity resulting from previous vaccination or natural exposure. Subclinical infections with
EHV-1/4 are common, even in young animals. Although mortality from uncomplicated ER is rare and complete
recovery within 12 weeks is the normal pattern, the respiratory infection is a frequent and significant cause of
interrupted schedules among horses assembled for training, racing, or competitive equestrian events. Fully
protective immunity resulting from infection is of short duration, and convalescent animals are susceptible to
reinfection by EHV-1/4 after several months. Although reinfections by the two herpesviruses cause less severe or
clinically inapparent respiratory disease, the risks of subsequent abortion and/or central nervous system (CNS)
disease are not eliminated. The greatest clinical threats to individual breeding, racing, or pleasure horse
operations posed by ER are the potential abortigenic and neurological sequelae of EHV-1 respiratory infection.
1.
Identification of the agent (Allen et al., 2004)
Because ER is a highly contagious disease with the potential for occurring as explosive outbreaks with high
mortality from abortigenic or neurological sequelae, rapid diagnostic methods are important. Although several
rapid and innovative diagnostic techniques based on enzyme-linked immunosorbent assay (ELISA), polymerase
chain reaction (PCR), immunohistochemical staining with peroxidase, or nucleic acid hybridisation probes have
been recently described, their use is often restricted to specialised reference laboratories, and thus the method of
choice for diagnosis of ER by diagnostic virology laboratories handling many routine samples continues to be the
traditional methodology of cell culture isolation followed by sero-identification of the isolated viruses. Successful
laboratory isolation of EHV-1/4 depends on strict adherence to proper methods for both sample collection and
laboratory processing.
a)
Collection of samples
Samples of nasopharyngeal exudate for virus isolation are best obtained from horses during the very early,
febrile stages of the respiratory disease, and are collected via the nares by swabbing the nasopharyngeal
area with a 5 5 cm gauze sponge attached to the end of a 50 cm length of flexible, stainless steel wire
encased in latex rubber tubing. A guarded uterine swab devise can also be used. After collection, the swab
should be removed from the wire and transported immediately to the virology laboratory in 3 ml of cold (not
frozen) fluid transport medium (serum-free MEM [minimal essential medium] with antibiotics). Virus infectivity
can be prolonged by the addition of bovine serum albumin or gelatine to 0.1% (w/v).
Virological examination of fetal tissues from suspect cases of EHV-1 abortion is most successful when
performed on aseptically collected samples of liver, lung, thymus, and spleen. The tissue samples should be
transported to the laboratory and held at 4C until inoculated into tissue culture. Samples that cannot be
890
processed within a few hours should be stored at 70C. In ante-mortem cases of EHV-1 neurological
disease, the virus can often be isolated from the leukocyte fraction of the blood of acutely infected horses or,
less often, from the nasopharynx of the affected animal or cohort animals. For attempts at virus isolation
from blood leukocytes, a 20 ml sample of sterile blood, collected in citrate, or heparin anticoagulant (EDTA
[ethylene diamine tetra-acetic acid] should not be used as it can destroy the cell cultures). The samples
should be transported without delay to the laboratory on ice, but not frozen. Although the virus has, on
occasion, been isolated from post-mortem cases of EHV-1 neurological disease by culture of samples of
brain and spinal cord, such attempts to isolate virus are often unsuccessful; however, they maybe useful for
PCR examination.
b)
Virus isolation
For efficient primary isolation of EHV-4 from horses with respiratory disease, equine-derived cell cultures
must be used. Both EHV-1 and EHV-4 may be isolated from nasopharyngeal samples using primary equine
fetal kidney cells or cell strains of equine fibroblasts derived from dermal (E-Derm) or lung tissue. EHV-1 can
be isolated on other cell types, as will be discussed later. The nasopharyngeal swab and its accompanying
3 ml of transport medium are transferred into the barrel of a sterile 10 ml syringe. Using the syringe plunger,
the fluid is squeezed from the swab into a sterile tube. A portion of the expressed fluid is then filtered
through a sterile, 0.45 m membrane syringe filter unit into a second sterile tube. Filtration will decrease
bacterial contamination, but may also lower virus titre. Recently prepared cell monolayers in 25 cm2 tissue
culture flasks are inoculated with 0.5 ml of the filtered, as well as the unfiltered, nasopharyngeal swab
extract. Cell monolayers in multiwell plates incubated in a 5% CO2 environment may also be used. Virus is
allowed to attach by incubating the inoculated monolayers at 37C on a platform rocker for 1.52 hours.
Monolayers of uninoculated control cells should be incubated in parallel with sterile transport medium only.
At the end of the attachment period, the inocula are removed and the monolayers are rinsed twice with
phosphate buffered saline (PBS) to remove virus-neutralising antibody that may be present in the
nasopharyngeal secretions. After addition of 5 ml of supplemented maintenance medium (MEM containing
2% fetal calf serum [FCS] and twice the standard concentrations of antibiotics [penicillin, streptomycin,
gentamicin, and amphotericin B]), the flasks are incubated at 37C. The use of positive control virus samples
to validate the isolation procedure carries the risk that this may lead to eventual contamination of diagnostic
specimens. This risk can be minimised by using routine precautions and good laboratory technique,
including the use of biosafety cabinets, inoculating positive controls after the diagnostic specimens,
decontaminating the surfaces in the hood while the inoculum is adsorbing and using a positive control of
relatively low titre. Inoculated flasks should be inspected daily by microscopy for the appearance of
characteristic herpesvirus cytopathic effect (CPE) (focal rounding, increase in refractility, and detachment of
cells). Cultures exhibiting no evidence of viral CPE after 1 week of incubation should be blind-passaged into
freshly prepared monolayers of cells, using small aliquots of both media and cells as the inoculum. Further
blind passage is usually not productive.
A number of cell types may be used for isolation of EHV-1 from the tissues of aborted fetuses or from postmortem cases of neurological disease (e.g. rabbit kidney [RK-13], baby hamster kidney [BHK-21], Madin
Darby bovine kidney [MDBK], pig kidney [PK-15], etc.), but equine-derived cell cultures are most sensitive
and must be used if the infrequent cases of EHV-4 abortion are to be detected. Around 10% (w/v) pooled
tissue homogenates of liver, lung, thymus, and spleen (from aborted fetuses) or of CNS tissue (from cases
of neurological disease) are used for virus isolation. These are prepared by first mincing small samples of
tissue into 1 mm cubes in a sterile Petri dish with dissecting scissors, followed by macerating the tissue
cubes further in serum-free culture medium with antibiotics using a mechanical tissue grinder (e.g. TenBroeck or Stomacher). After centrifugation at 1200 g for 10 minutes, the supernatant is removed and 0.5 ml
is inoculated into duplicate cell monolayers in 25 cm2 flasks. Following incubation of the inoculated cells at
37C for 1.52 hours, the inocula are removed and the monolayers are rinsed twice with PBS. After addition
of 5 ml of supplemented maintenance medium, the flasks are incubated at 37C for up to 1 week or until
viral CPE is observed.
Culture of peripheral blood leukocytes for the presence of EHV-1 can be attempted in horses during the
early stages of the myeloencephanlopathy. Buffy coats may be prepared from unclotted blood by
centrifugation at 600 g for 15 minutes, and the buffy coat is taken after the plasma has been carefully
removed. The buffy coat is then layered onto Ficoll 1,090, centrifuged at 400 g for 20 minutes and the
leukocyte-rich interface is then layered onto Ficoll 1.077 and centrifuged in the same way. The PBMC
interface (without most granulocytes) is washed twice in PBS (300 g for 10 minutes) and resuspended in
1 ml of MEM containing 2% FCS. Then, 0.5 ml of the rinsed cell suspension is added to each of the
duplicate monolayers of equine fibroblast, equine fetal or RK-13 cell monolayers in 25 cm2 flasks containing
810 ml freshly added maintenance medium. The flasks are incubated at 37C for 7 days; the inoculum is
not removed. Because CPE may be difficult to detect in the presence of the massive inoculum of leukocytes,
each flask of cells is freezethawed after 7 days of incubation and the contents centrifuged at 300 g for
10 minutes. Finally, 0.5 ml of the cell-free, culture medium supernatant is transferred to freshly made cell
891
monolayers that are just subconfluent. These are incubated and observed for viral CPE for at least 56 days
before discarding as negative.
c)
Seroconfirmation of virus identity

The basis for identification of any herpesvirus isolate recovered from specimens submitted from suspected
cases of ER is its immunoreactivity with specific antisera. Specific identification of an isolate as EHV-1 or
EHV-4 can be quickly and simply accomplished by immunofluorescent (FA) detection of viral antigen in the
infected cell culture using type-specific monoclonal antibodies (MAbs), which are available from OIE
Reference Laboratories for equine rhinopneumonitis. The test, which is type-specific and accurate, can be
performed on a small aliquot of infected cells from the same container inoculated with clinical or postmortem material. An isolate made in a laboratory that lacks MAbs or FA capability can be confirmed as EHV1/4 by virus neutralization using a virus-specific polyclonal antiserum or by the PCR (see section B.1.f).
Cell monolayers infected with the isolate are removed by scraping from the flask when at least 75% CPE is
evident. The cells are pelleted from the culture medium and resuspended in 0.5 ml of PBS. 50 l of the cell
suspension is placed into two wells of a multiwell microscope slide, air-dried, and fixed for 10 minutes with
100% acetone. Control cell suspensions (uninfected, EHV-1 infected, or EHV-4 infected) are also spotted
into each of two wells of the same slide. Control cells may be prepared in advance and stored frozen in
small aliquots. A drop of an appropriate dilution of MAb specific for EHV-1 is added to one well of each cell
pair, and a drop of MAb specific for EHV-4 is added to each of the other wells. After 30 minutes incubation
at 37C in a humid chamber, unreacted antibody is removed by two 10-minute washes with PBS. MAbs
bound to viral antigen can be detected with goat anti-mouse IgG conjugated with fluorescein isothiocyanate
(FITC). A drop of diluted conjugate is added to each well and, after 30 minutes at 37C, the wells are again
washed twice with PBS. Cells are examined with a fluorescence microscope, and positive fluorescence with
the antibody of appropriate specificity indicates the virus type.
d)
Virus detection by direct immunofluorescence

Direct immunofluorescent detection of EHV-1 antigens in samples of post-mortem tissues collected from
aborted equine fetuses provides an indispensable method to the veterinary diagnostic laboratory for making
a rapid preliminary diagnosis of herpesvirus abortion (Gunn, 1992). Side-by-side comparisons of the
immunofluorescent and cell culture isolation techniques on more than 100 cases of equine abortion have
provided evidence that the diagnostic reliability of direct immunofluorescent staining of fetal tissues obtained
at necropsy approaches that of virus isolation attempts from the same tissues. In the United States of
America (USA), specific and potent polyclonal antiserum to EHV-1, prepared in swine and conjugated with
FITC, is provided to veterinary diagnostic laboratories for this purpose by the National Veterinary Services
Laboratories of the United States Department of Agriculture (USDA). The antiserum cross-reacts with EHV-4
and hence is not useful for serotyping. Freshly dissected samples (5 5 mm pieces) of fetal tissue (lung,
liver, thymus, and spleen) are frozen, sectioned on a cryostat at 20C, mounted on to microscope slides,
and fixed with 100% acetone. After air-drying, the sections are incubated at 37C in a humid atmosphere for
30 minutes with an appropriate dilution of the conjugated swine antibody to EHV-1. Unreacted antibody is
removed by two washes in PBS, and the tissue sections are then covered with aqueous mounting media
and a cover-slip, and examined for fluorescent cells indicating the presence of EHV antigen. Each test
should include a positive and negative control consisting of sections from known EHV-1 infected and
uninfected fetal tissue.
e)
Virus detection by immunoperoxidase staining

Enzyme immunohistochemical (IH) staining methods (e.g. immunoperoxidase) have been developed
recently as procedures for detecting EHV-1 antigen in paraffin-embedded tissues of aborted equine fetuses
or neurologically affected horses (Schultheiss et al., 1993; Whitwell et al., 1992). Such ancillary IH
techniques for antigen detection may facilitate identification of the virus in archival tissue samples or in
clinical cases in which traditional laboratory methods for EHV-1 detection have been unsuccessful.
Immunoenzymatic staining for EHV-1 is particularly useful for the simultaneous evaluation of morphological
lesions and the identification of the infectious agent. Immunoperoxidase staining for EHV-1 or EHV-4 may
also be done on infected cell monolayers (van Maanen et al., 2000). Adequate controls must be included
with each immunoperoxidase test run for evaluation of both the method specificity and antibody specificity.
f)
Virus detection by polymerase chain reaction

The PCR can be used for rapid amplification and diagnostic detection of nucleic acids of EHV-1 and -4 in
clinical specimens, paraffin-embedded archival tissue, or inoculated cell cultures (Borchers & Slater, 1993;
Lawrence et al., 1994; OKeefe et al., 1994; Varrasso et al., 2001; Wagner et al., 1992). A variety of typespecific PCR primers have been designed to distinguish between the presence of EHV-1 and EHV-4. The
correlation between PCR and virus isolation techniques for diagnosis of EHV-1 or EHV-4 is high (Varrasso
et al., 2001). Diagnosis of ER by PCR is rapid, sensitive, and does not depend on the presence of infectious
892
virus in the clinical sample. It now forms an integral part of a range of diagnostic tests currently available for
ER, each with its own advantages and limitations.
For diagnosis of active infection by EHV, PCR methods are most reliable with samples from aborted fetuses
or from nasopharyngeal swabs and peripheral blood leukocytes of foals and yearlings; they are most useful
in explosive epizootics of abortion or respiratory tract disease in which a rapid identification of the virus is
critical for guiding management strategies. PCR examinations of spinal cord and brain tissue, as well as
PBMC, are important in seeking a diagnosis on a horse with neurological signs. However, the interpretation
of the amplification by PCR of genomic fragments of EHV-1 or EHV-4 in lymph nodes or trigeminal ganglia
from adult horses is complicated by the high prevalence of latent EHV-1 and EHV-4 DNA in such tissues
(Welch et al., 1992).
A simple multiplex PCR assay for simultaneous detection of both EHV-1 and EHV-4 has been described
(Wagner et al., 1992). A more sensitive protocol for nested PCR detection of EHV-1 or EHV-4 in clinical or
pathological specimens (nasal secretions, blood leukocytes, brain and spinal cord, fetal tissues, etc.) is
described here (Borchers & Slater, 1993). This procedure has been used successfully; however, the
technology in this area is changing rapidly and other simpler more sensitive techniques are becoming
available.
i)
Prepare template DNA from test specimens: Following sample homogenisation and cell (and virion)
lysis in the presence of a chaotropic salt, nucleic acids bind selectively to silica or cationic resin
substrates. Substrate-bound nucleic acids are purified in a series of rapid wash steps followed by
recovery with low-salt elution. The reagents for performing such steps for rapid nucleic acid isolation
are available in kit format from a number of commercial sources (e.g. High Pure PCR Template
Preparation Kit, Roche Molecular Biochemicals, Indianapolis, USA; QIAamp DNA Kit, Qiagen,
Valencia, USA).
ii)
Nested primer sequences specific for EHV-1 (based on those described in Borchers & Slater, 1993):
BS-1-P1 = 5-TCT-ACC-CCT-ACG-ACT-CCT-TC-3 (917936)
gB1-R-2 = 5-ACG-CTG-TCG-ATG-TCG-TAA-AAC-CTG-AGA-G-3 (23902363)
BS-1-P3 = 5-CTT-TAG-CGG-TGA-TGT-GGA-AT-3 (13771396)
gB1-R-a = 5-AAG-TAG-CGC-TTC-TGA-TTG-AGG-3 (21472127)
iii)
Nested primer sequences specific for EHV-4 (Borchers & Slater, 1993):
BS-4-P1 = 5-TCT-ATT-GAG-TTT-GCT-ATG-CT-3 (17051724)
BS-4-P2 = 5-TCC-TGG TTG-TTA-TTG-GGT-AT-3 (26562637)
BS-4-P3 = 5-TGT-TTC-CGC-CAC-TCT-TGA-CG-3 (18571876)
BS-4-P4 = 5-ACT-GCC-TCT-CCC-ACC-TTA-CC-3 (24562437)
iv)
PCR conditions for first stage amplification: Specimen template DNA (12 ug in 2 l) is added to a PCR
mixture (total volume of 50 l) containing 1 PCR buffer (50 mM KCl, 10 mM Tris/HCl, pH 9.0, 0.1%
Triton X-100), 200 M of each deoxynucleotide triphosphate (dNTP), 2.5 mM MgCl2, 2.0 M of each
outer primer (BS-1-P1 and gB1-R-2 for EHV-1 detection and, in a separate reaction mixture, BS-4-P1
and BS-4-P2 for EHV-4 detection) and 0.5 u Taq DNA polymerase. Cycling parameters are: initial
denaturation at 94C for 4 minutes; 40 cycles of 94C for 30 seconds, 60C for 30 seconds, and 72C
for 90 seconds; with a final extension at 72C for 10 minutes. Separate reaction mixtures containing
either known viral DNA or no DNA (water) should be prepared and amplified as positive and negative
controls.
v)
PCR conditions for second stage (nested) amplification: Two l of a 1/10 dilution of the first
amplification product is added to a fresh PCR mixture (total volume of 50 l) containing 1 PCR buffer,
200 M of each dNTP, 2.5 mM MgCl2, 2.0 M of each nested primer (BS-1-P3 and gB1-R-a for EHV-1
detection and, in a separate reaction mixture, BS-4-P3 and BS-4-P4 for EHV-4 detection) and 0.5 u
Taq DNA polymerase. Cycling parameters are: initial denaturation at 94C for 4 minutes; 40 cycles of
94C for 30 seconds, 60C for 30 seconds, and 72C for 1 minute; with a final extension at 72C for
10 minutes.
vi)
Gel analysis of amplified products: 10 l of each final amplified product, including controls, is mixed
with 2 l of 6 loading dye and electrophoresed on a 1.5% agarose gel in Tris/acetate or Tris-Borate
running buffer, along with a 100 base pairs (bp) DNA ladder. The gel is stained with ethidium bromide
893
and viewed by UV transillumination for amplified products of either 770 bp for EHV-1 or 580 bp for
EHV-4.
g)
Histopathology
Histopathological examination of sections of formalin-fixed, paraffin-embedded tissues from aborted fetuses
or from neurologically affected horses is an essential part of the laboratory diagnosis of these two clinical
manifestations of ER. In aborted fetuses, typical herpetic intranuclear inclusion bodies present within
bronchiolar epithelium or in cells at the periphery of areas of hepatic necrosis are pathognomonic lesions for
EHV-1. The characteristic, but not pathognomonic, microscopic lesion associated with EHV-1 neuropathy is
a degenerative thrombotic vasculitis of small blood vessels in the brain or spinal cord (perivascular cuffing
and infiltration by inflammatory cells, endothelial proliferation and necrosis, and thrombus formation).
2.
Serological tests
Because of the ubiquity of the viral agents of ER and the high seroprevalence among horses in most parts of the
world, the demonstration of a negative antibody titre to EHV-1/4 by serological testing of horses designated for
export is not part of present veterinary regulations that seek to prevent international spread of infectious diseases
of horses. Serological testing can, however, be a useful adjunct procedure for assisting in the diagnosis of ER in
horses. Serodiagnosis of ER is based on the demonstration of significant increases in antibody titres in paired
sera taken during the acute and convalescent stages of the disease. The results of tests performed on sera from
a single collection date are, in most cases, impossible to interpret with any degree of confidence. The initial (acute
phase) serum sample should be taken as soon as possible after the onset of clinical signs, and the second
(convalescent phase) serum sample should be taken 24 weeks later. Acute phase sera from mares after
abortion or from horses with EHV-1 neurological disease may already contain maximal titres of EHV-1 antibody,
with no increase in titres detectable in sera collected at later dates. In such cases, serological testing of paired
serum samples from clinically unaffected cohort members of the herd for rising antibody titres against EHV-1/4
may provide information useful for retrospective diagnosis of ER within the herd. Finally, the serological detection
of antibodies to EHV-1 in heart or umbilical cord blood or other fluids of equine fetuses can be of diagnostic value
in rare cases of virologically negative fetuses aborted as a result of EHV-1 infection; in some cases, the EHV 1/4
genome maybe identified from these tissues by PCR.
Serum antibody levels to EHV-1/4 may be determined by ELISA (Dutta et al., 1983), virus neutralisation (VN)
(Thomson et al., 1976), or complement fixation (CF) tests (Thomson et al., 1976). Because there are no
internationally recognised reagents or standardised techniques for performing any of the serological tests for
detection of EHV-1/4 antibody, antibody titre determinations on the same serum may differ from one laboratory to
another. Furthermore, all of the serological tests mentioned detect antibodies that are cross-reactive between
EHV-1 and EHV-4. Nonetheless, the demonstration, by any of the tests, of a four-fold or greater rise in antibody
titre to EHV-1 or EHV-4 during the course of a clinical illness provides serological confirmation of recent infection
with one of the viruses. The ELISA and CF test have the advantage that they provide results faster and do not
require cell culture facilities. Recently, a type-specific ELISA that can distinguish between antibodies to EHV-1
and EHV-4 was developed and made commercially available (Crabb et al., 1995). The microneutralisation test is
a widely used and sensitive serological assay for detecting EHV-1/4 antibody and will thus be described here.
a)
Virus neutralisation test

This serological test is most commonly performed in flat-bottom 96-well microtitre plates (tissue culture
grade) using a constant dose of virus and doubling dilutions of equine test sera. At least two replicate wells
for each serum dilution are required. Serum-free MEM is used throughout as a diluent. Virus stocks of
known titre are diluted just before use to contain 100 TCID50 (50% tissue culture infective dose) in 25 l.
Monolayers of E-Derm or RK-13 cells are monodispersed with EDTA/trypsin and resuspended at a
concentration of 5 105/ml. Note that RK-13 cells can be used with EHV-1 but do not give clear CPE with
EHV-4. Antibody positive and negative control equine sera and controls for cell viability, virus infectivity, and
test serum cytotoxicity, must be included in each assay. End-point VN titres of antibody are calculated by
determining the reciprocal of the highest serum dilution that protects 100% of the cell monolayer from virus
destruction in both of the replicate wells.
A suitable test procedure is as follows:
894
i)
Inactivate test and control sera for 30 minutes in a water bath at 56C.
ii)
Add 25 l of serum-free MEM to all wells of the microtitre assay plates.
iii)
Pipette 25 l of each test serum into duplicate wells of both rows A and B of the plate. The first row
serves as the serum toxicity control and the second row as the first dilution of the test. Make doubling
dilutions of each serum starting with row B and proceeding to the bottom of the plate by sequential
mixing and transfer of 25 l to each subsequent row of wells. Six sera can be assayed in each plate.
iv)
Add 25 l of the appropriately diluted EHV-1 or EHV-4 virus stock to each well (100 TCID50/well)
except those of row A, which are the serum control wells for monitoring serum toxicity for the indicator
cells. Note that the final serum dilutions, after addition of virus, run from 1/4 to1/256.
v)
A separate control plate should include titration of both a negative and positive horse serum of known
titre, cell control (no virus), virus control (no serum), and a virus titration to calculate the actual amount
of virus used in the test.
vi)
Incubate the plates for 1 hour at 37C in 5% CO2 atmosphere.
vii)
Add 50 l of the prepared E-Derm or RK-13 cell suspension (5 105 cells/ml) in MEM/10% FCS to
each well.
viii) Incubate the plates for 45 days at 37C in an atmosphere of 5% CO2 in air.
ix)
Examine the plates microscopically for CPE and record the results on a worksheet. Alternatively, the
cell monolayers can be scored for CPE after fixing and staining as follows: after removal of the culture
fluid, immerse the plates for 15 minutes in a solution containing 2 mg/ml crystal violet, 10% formalin,
45% methanol, and 45% water. Then, rinse the plates vigorously under a stream of running tap water.
x)
Wells containing intact cell monolayers stain blue, while monolayers destroyed by virus do not stain.
Verify that the cell control, positive serum control, and serum cytotoxicity control wells stain blue, that
the virus control and negative serum control wells are not stained, and that the actual amount of virus
added to each well is between 101.5 and 102.5 TCID50. Wells are scored as positive for neutralisation of
virus if 100% of the cell monolayer remains intact. The highest dilution of serum resulting in complete
neutralisation of virus (no CPE) in both duplicate wells is the end-point titre for that serum.
xi)
Calculate the neutralisation titre for each test serum, and compare acute and convalescent phase
serum titres from each animal for a four-fold or greater increase.

Both live attenuated and inactivated vaccines are available as licensed, commercially prepared products for use
as prophylactic aids in reducing the burden of disease in horses caused by EHV-1/4 infection. Clinical experience
has demonstrated that none of the vaccine preparations should be relied on to provide an absolute degree of
protection from ER. Multiple doses, repeated annually, of each of the currently marketed ER vaccines are
recommended by their respective manufacturers, with vaccination schedules that vary with the particular vaccine.
At least sixteen vaccine products for ER, each containing different permutations of EHV-1, EHV-4, and the two
subtypes of equine influenza virus, are currently marketed by five veterinary biologicals manufacturers.
The clinical indications stated on the product label for use of the several available vaccines for ER are either
herpesvirus-associated respiratory disease, abortion, or both. Only four vaccine products have met the regulatory
requirements for claiming efficacy in providing protection from herpesvirus abortion as a result of successful
vaccination and challenge experiments in pregnant mares. None of the vaccine products has been conclusively
demonstrated to prevent the occurrence of neurological disease sometimes associated with EHV-1 infection.
1.
Seed management
a)
Characteristics and culture

The master seed virus (MSV) for ER vaccines must be prepared from strains of EHV-1 and/or EHV-4 that
have been positively and unequivocally identified by both serological and genetic tests. Seed virus must be
propagated in a cell line approved for equine vaccine production by the appropriate regulatory agency. A
complete record of original source, passage history, medium used for propagation, etc., shall be kept for the
master seed preparations of both the virus(es) and cell stock(s) intended for use in vaccine production.
Permanently stored stocks of both MSV and master cell stock (MCS) used for vaccine production must be
demonstrated to be pure, safe and, in the case of MSV, also immunogenic. Generally, the fifth passage from
the MSV and the twentieth passage from the MCS are the highest allowed for vaccine production. Results of
all quality control tests on master seeds must be recorded and made a part of the licensee's permanent
records.
895
b)
i)
Purity
Tests for master seed purity include prescribed procedures that demonstrate the virus and cell seed
stocks to be free from bacteria, fungi, mycoplasmas, and extraneous viruses. Special tests must be
performed to confirm the absence of equine arteritis virus, equine infectious anaemia virus, equine
influenza virus, equine herpesvirus-2, -3, and -5, equine rhinovirus, the alphaviruses of equine
encephalomyelitis, bovine viral diarrhoea virus (BVDV common contaminant of bovine serum), and
porcine parvovirus (PPV potential contaminant of porcine trypsin). The purity check should also
include the exclusion of the presence of EHV-1 from EHV-4 MSV and vice versa.
ii)
Safety
Samples of each lot of MSV to be used for preparation of live attenuated ER vaccines must be tested
for safety in horses determined to be susceptible to the virulent wild-type virus, including pregnant
mares in the last 4 months of gestation. Vaccine safety must be demonstrated in a safety field trial in
horses of various ages from three different geographical areas. The safety trial should be conducted by
independent veterinarians using a prelicensing batch of vaccine. EHV-1 vaccines making a claim for
efficacy in controlling abortion must be tested for safety in a significant number of late gestation
pregnant mares, using the vaccination schedule that will be recommended by the manufacturer for the
final vaccine product.
iii)
Immunogenicity
Tests for immunogenicity of the EHV-1/4 MSV stocks should be performed in horses on an
experimental test vaccine prepared from the highest passage level of the MSV allowed for use in
vaccine production. The test for MSV immunogenicity consists of vaccination of horses with low
antibody titres to EHV-1/4, with doses of the test vaccine that will be recommended on the final product
label. Second serum samples should be obtained and tested for significant increases in neutralising
antibody titre against the virus, 21 days after the final dose.
iv)
Efficacy
An important part of the validation process is the capacity of a prelicensing lot of the ER vaccine to
provide a significant level of clinical protection in horses from the particular disease manifestation of
EHV-1/4 infection for which the vaccine is offered, when used under the conditions recommended by
the manufacturer's product label. Serological data are not acceptable for establishing the efficacy of
vaccines for ER. Efficacy studies must be designed to ensure appropriate randomisation of test
animals to treatment groups, blinding of the recording of clinical observations, and the use of sufficient
numbers of animals to permit statistical evaluation for effectiveness in prevention or reduction of the
specified clinical disease. The studies should be performed on fully formulated experimental vaccine
products (a) produced in accordance with, (b) at or below the minimum antigenic potency specified in,
and, (c) produced with the highest passage of MSV and MCS allowed by the approved Outline of
Production (see Section C.2). Vaccine efficacy is demonstrated by vaccinating a minimum of 20 EHV1/4-susceptible horses possessing serum neutralising antibody titres 32, followed by challenge of the
vaccinates and ten nonvaccinated control horses with virulent virus. A significant difference in the
clinical signs of ER must be demonstrated between vaccinates and nonvaccinated control horses. The
vaccination and challenge study must be performed on an identical number of pregnant mares and
scored for abortion if the vaccine product will make a label usage claim for prevention of or as an aid
in the prevention of abortion caused by EHV-1.
2.
A detailed protocol of the methods of manufacture to be followed in the preparation of vaccines for ER must be
compiled, approved, and filed as an Outline of Production with the appropriate licensing agency. Specifics of the
methods of manufacture for ER vaccines will differ with the type (live or inactivated) and composition (EHV-1 only,
EHV-1 and EHV-4, EHV-4 and equine influenza viruses, etc.) of each individual product, and also with the
manufacturer.
3.
In-process control
Cells, virus, culture medium, and medium supplements of animal origin that are used for the preparation of
production lots of vaccine must be derived from bulk stocks that have passed the prescribed tests for bacterial,
fungal, and mycoplasma sterility; nontumorgenicity; and absence of extraneous viral agents.
4.
Batch control
Each bulk production lot of ER vaccine must pass tests for sterility, safety, and immunogenic potency.
896
a)
Sterility
Samples taken from each batch of completed vaccine are tested for bacteria, fungi, and mycoplasma
contamination. Procedures to establish that the vaccine is free from extraneous viruses are also required;
such tests should include inoculation of cell cultures that allow detection of the common equine viruses, as
well as techniques for the detection of BVDV and PPV in ingredients of animal origin used in the production
of the batch of vaccine.
b)
Safety
Tests to assure safety of each production batch of ER vaccine must demonstrate complete inactivation of
virus (for inactivated vaccines) as well as a level of residual virus-killing agent that does not exceed the
maximal allowable limit (e.g. 0.2% for formaldehyde). Safety testing in laboratory animals is also required.
c)
Potency
Batch control of antigenic potency for EHV-1 vaccines only may be tested by measuring the ability of
dilutions of the vaccine to protect hamsters from challenge with a lethal dose of hamster-adapted EHV-1
virus. Although potency testing on production batches of ER vaccine may also be performed by vaccination
of susceptible horses followed by either viral challenge or assay for seroconversion, the recent availability of
virus type-specific MAbs has permitted development of less costly and more rapid in-vitro immunoassays for
antigenic potency. The basis for such in-vitro assays for ER vaccine potency is the determination, by use of
the specific MAb, of the presence of at least the minimal amount of viral antigen within each batch of vaccine
that correlates with the required level of protection (or seroconversion rate) in a standard animal test for
potency.
d)
Tests to establish the duration of immunity to EHV-1/4 achieved by immunisation with each batch of vaccine
are not required. The results of many reported observations indicate that vaccination-induced immunity to
EHV-1/4 is not more than a few months in duration; these observations are reflected in the frequency of
revaccination recommended on ER vaccine product labels.
e)
Stability
At least three production batches of vaccine should be tested for shelf life before reaching a conclusion on
the vaccines stability. When stored at 4C, inactivated vaccine products generally maintain their original
antigenic potency for at least 1 year. Lyophilised preparations of the live virus vaccine are also stable during
storage for 1 year at 4C. Following reconstitution, live virus vaccine is unstable and cannot be stored
without loss of potency.
5.
Before release for labelling, packaging, and commercial distribution, randomly selected filled vials of the final
vaccine product must be tested by prescribed methods for freedom from contamination and safety in laboratory
test animals.
a)
Safety
See Section C.4.b.
b)
Potency
See Section C.4.c.
REFERENCES
ALLEN G.P. & BRYANS J.T. (1986). Molecular epidemiology, pathogenesis and prophylaxis of equine herpesvirus-1
infections. In: Progress in Veterinary Microbiology and Immunology, Vol. 2, Pandey R., ed. Karger, Basel,
Switzerland & New York, USA, 78144.
ALLEN G.P., KYDD J.H., SLATER J.D. & SMITH K.C. (1999). Recent advances in understanding the pathogenesis,
epidemiology, and immunological control of equid herpesvirus-1 (EHV-1) abortion. Equine Infect. Dis., 8, 129
146.
ALLEN G.P., KYDD J.H., SLATER J.D. & SMITH K.C. (2004). Equid herpesvirus-1 (EHV-1) and -4 (EHV-4) infections.
In: Infectious Diseases of Livestock, Coetzer J.A.W., Thomson G.R. & Tustin R.C., eds. Oxford University Press,
Cape Town, South Africa.
897
BORCHERS K. & SLATER J. (1993). A nested PCR for the detection and differentiation of EHV-1 and EHV-4. J. Virol.
Methods, 45, 331336.
BRYANS J.T. & ALLEN G.P. (1988). Herpesviral diseases of the horse. In: Herpesvirus Diseases of Animals,
Wittman G., ed. Kluwer, Boston, USA, 176229.
CRABB B.S., MACPHERSON C.M., REUBEL G.H., BROWNING G.F., STUDDERT M.J. & DRUMMER H.E. (1995). A typespecific serological test to distinguish antibodies to equine herpesviruses 4 and 1. Arch. Virol., 140, 245258.
CRABB B.S. & STUDDERT M.J. (1995). Equine herpesviruses 4 (equine rhinopneumonitis virus) and 1 (equine
abortion virus). Adv. Virus Res., 45, 153190.
DUTTA S.K., TALBOT N.C. & MYRUP A.C. (1983). Detection of equine herpesvirus-1 antigen and the specific
antibody by enzyme-linked immunosorbent assay. Am. J. Vet. Res., 44, 19301934.
GUNN H.M. (1992). A direct fluorescent antibody technique to diagnose abortion caused by equine herpesvirus.
Irish Vet. J., 44, 3740.
LAWRENCE G.L., GILKERSON J., LOVE D.N., SABINE M. & WHALLEY J.M. (1994). Rapid, single-step differentiation of
equid herpesvirus 1 and 4 from clinical material using the polymerase chain reaction and virus-specific primers. J.
Virol. Methods, 47, 5972.
OKEEFE J.S., JULIAN A., MORIARTY K., MURRAY A. & WILKS C.R. (1994). A comparison of the polymerase chain
reaction with standard laboratory methods for the detection of EHV-1 and EHV-4 in archival tissue samples. N.Z.
Vet. J., 42, 9396.
SCHULTHEISS P.C., COLLINS J.K. & CARMAN J. (1993). Use of an immunoperoxidase technique to detect equine
herpesvirus-1 antigen in formalin-fixed paraffin-embedded equine fetal tissues. J. Vet. Diagn. Invest., 5, 1215.
TELFORD E.A.R., WATSON M.S., MCBRIDE K. & DAVISON A.J. (1992). The DNA sequence of equine herpesvirus-1.
Virology, 189, 304316.
TELFORD E.A.R., WATSON M.S., PERRY J., CULLINANE A.A. & DAVISON A.J. (1998). The DNA sequence of equine
herpesvirus 4. J. Gen. Virol., 79, 11971203.
THOMSON G.R., MUMFORD J.A., CAMPBELL J., GRIFFITHS L. & CLAPHAM P. (1976). Serological detection of equid
herpesvirus 1 infections of the respiratory tract. Equine Vet. J., 8, 5865.
MAANEN C., VREESWIJK J., MOONEN P., BRINKHOF J. DE BOER-LUIJTZE E. & TERPSTRA C. (2000). Differentiation
and genomic and antigenic variation among fetal, respiratory, and neurological isolates from EHV1 and EHV4
infections in The Netherlands. Vet. Q., 22 (2): 8893.
VAN
VARRASSO A., DYNON K., FICORILLI N., HARTLEY C.A., STUDDERT M.J. & DRUMMER H.E. (2001). Identification of
equine herpesviruses 1 and 4 by polymerase chain reaction. Aust. Vet. J., 79, 563569.
WAGNER W.N., BOGDAN J., HAINES D., TOWNSEND H.G.G. & MISRA V. (1992). Detection of equine herpesvirus and
differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction. Can. J. Microbiol., 38,
11931196.
WELCH H.M., BRIDGES C.G., LYON A.M., GRIFFITHS L. & EDINGTON N. (1992). Latent equid herpesviruses 1 and 4:
detection and distinction using the polymerase chain reaction and cocultivation from lymphoid tissues. J. Gen.
Virol., 73, 261268.
WHITWELL K.E., GOWER S.M. & SMITH K.C. (1992). An immunoperosidase method applied to the diagnosis of
equine herpesvirus abortion, using conventional and rapid microwave techniques. Equine Vet. J., 24, 1012.
*
* *
NB: There are OIE Reference Laboratories for Equine rhinopneumonitis
diagnostic tests, reagents and vaccines for equine rhinopneumonitis
898
CHAPTER 2.5.10.
EQUINE VIRAL ARTERITIS
SUMMARY
Equine viral arteritis (EVA) is a contagious viral disease of equids caused by equine arteritis virus
(EAV), an RNA virus classified in the family Arteriviridae. Only one major serotype of the virus has
been identified so far. Equine arteritis virus is found in horse populations in many countries worldwide. Although infrequently reported in the past, confirmed outbreaks of EVA appear to be on the
increase.
The majority of naturally acquired infections with EAV are subclinical. Where present, clinical signs
of EVA can vary in range and severity. The disease is characterised principally by fever,
depression, anorexia, dependent oedema, especially of the limbs, scrotum and prepuce in the
stallion, conjunctivitis, an urticarial-type skin reaction, abortion and, rarely, a fulminating pneumonia,
enteritis or pneumo-enteritis in young foals. Apart from mortality in young foals, the case-fatality
rate in outbreaks of EVA is very low. Affected horses almost invariably make complete clinical
recoveries. A long-term carrier state can occur in a variable percentage of infected stallions, but not
in mares, geldings or sexually immature colts.
Identification of the agent: EVA cannot be differentiated clinically from a number of other
respiratory and systemic equine diseases. Diagnosis of EAV infection is based on virus isolation,
detection of nucleic acid or viral antigen, or demonstration of a specific antibody response. Virus
isolation should be attempted from appropriate clinical or post-mortem specimens in rabbit, equine,
or monkey kidney cell culture. The identity of isolates of EAV should be confirmed by neutralisation
test, reverse-transcription polymerase chain reaction (RT-PCR) assay, or by immunocytochemical
methods, namely indirect immunofluorescence or avidinbiotinperoxidase techniques.
Detection and identification of EAV nucleic acid in suspect cases of the disease can also be
attempted using the RT-PCR assay and appropriate viral-specific RNA primers.
Where mortality is associated with a suspected outbreak of EVA, a wide range of tissues should be
examined for histological evidence of panvasculitis that is especially pronounced in the small
arteries throughout the body. The characteristic vascular lesions present in the mature animal are
not a notable feature in EVA-related abortions. In such cases, equine arteritis viral antigens may be
visualised by immunohistochemical examination of placental and various fetal tissues.
Serological tests: A variety of serological tests, including virus neutralisation (VN), complement
fixation (CF), indirect fluorescent antibody, agar gel immunodiffusion, the enzyme-linked
immunosorbent assay (ELISA), and the fluorescent microsphere immunoassay assay (MIA) have
been used for the detection of antibody to EAV. The tests currently in widest use are the
complement-enhanced VN test and the ELISA. The VN test is a very sensitive and highly specific
assay of proven value in diagnosing acute infection and in seroprevalence studies. Several ELISAs
have been developed, none of which have been as extensively validated as the VN test though
some appear to offer comparable specificity and close to equivalent sensitivity. The CF test is less
sensitive than either procedure, but it can be used for diagnosing recent infection.
Requirements for vaccines and diagnostic biologicals: Two commercial tissue culture vaccines
are currently available against EVA. One is a modified live virus (MLV) vaccine prepared from virus
that has been attenuated for horses by multiple serial transfers in primary equine and rabbit cell
cultures. It has been shown to be safe and protective for stallions and nonpregnant mares.
Vaccination of foals under 6 weeks of age and of pregnant mares in the final 2 months of gestation
is contraindicated. There is no evidence of back reversion to virulence of the vaccine virus following
its use in the field over more than 20 years. The second vaccine is an inactivated, adjuvanted
899
Chapter 2.5.10. Equine viral arteritis
product prepared from virus grown in equine cell culture that can be used in nonbreeding and
breeding horses. In the absence of appropriate safety data, the vaccine is not currently
recommended for use in pregnant mares.
A. INTRODUCTION
Equine viral arteritis (EVA) is a contagious viral disease of equids caused by equine arteritis virus (EAV), a
positive-sense, single-stranded RNA virus, and the prototype member of the genus Arterivirus, family
Arteriviridae, order Nidovirales (Cavanagh, 1997). Epizootic lymphangitis pinkeye, fivre typhoide and
rotlaufseuche are some of the descriptive terms used in the past to refer to a disease that clinically very closely
resembled EVA. The natural host range of EAV would appear to be restricted to equids, although very limited
evidence would suggest it may also include new world camelids, viz. alpacas and llamas (Weber et al., 2006).
The virus does not present a human health hazard (Timoney & McCollum, 1993). EAV is present in the horse
population of many countries world-wide (Timoney & McCollum, 1993). There has been an increase in the
incidence of EVA in recent years that has been linked to the greater frequency of movement of horses and use of
transported semen (Balasuriya et al., 1998).
While the majority of cases of acute infection with EAV are subclinical, certain strains of the virus can cause
disease of varying severity (Timoney & McCollum, 1993). Typical cases of EVA can present with all or any
combination of the following clinical signs: fever, depression, anorexia, leukopenia, dependent oedema, especially
of the limbs, scrotum and prepuce of the stallion, conjunctivitis, ocular discharge, supra or periorbital oedema,
rhinitis, nasal discharge, a local or generalised urticarial skin reaction, abortion, stillbirths and, rarely, a fulminating
pneumonia, enteritis or pneumo-enteritis in young foals. Regardless of the severity of clinical signs, affected
horses almost invariably make complete recoveries. The case-fatality rate in outbreaks of EVA is very low;
mortality is usually only seen in very young foals, particularly those congenitally infected with the virus (Timoney &
McCollum, 1993; Vaala et al., 1992), and very rarely in otherwise healthy adult horses.
EVA cannot be differentiated clinically from a number of other respiratory and systemic equine diseases, the most
common of which are equine influenza, equine herpesvirus 1 and 4 infections, infection with equine rhinitis A and
B viruses, equine adenoviruses and streptococcal infections, with particular reference to purpura haemorrhagica.
The disease also has clinical similarities to equine infectious anaemia, African horse sickness fever, cases of
Hendra virus infection, Getah virus infection and toxicosis caused by hoary alyssum (Berteroa incana) (Timoney &
McCollum, 1993). After infection, EAV replicates in macrophages and circulating monocytes (Del Piero, 2000) and
is shed in various secretions/excretions of acutely infected animals, in especially high concentration from the
respiratory tract (McCollum et al., 1971).
A variable percentage of acutely infected stallions later become long-term carriers in the reproductive tract and
constant semen shedders of the virus (Timoney & McCollum, 1993; 2000). The carrier state, which has been
shown to be androgen dependant, has been found in the stallion, but not in the mare, gelding or sexually
immature colt (Timoney & McCollum, 1993). Unequivocal evidence of the carrier state has only been found in
stallions serologically positive for antibodies to the virus (Timoney & McCollum, 2000). While temporary downregulation of circulating testosterone levels using a GnRH antagonist or by immunisation with GnRH would
appear to have expedited clearance of the carrier state in some stallions, the efficacy of either treatment strategy
has yet to be fully established. Concern has been expressed that such a therapeutic approach could be used to
deliberately mask existence of the carrier state.
1.
a)
Virus isolation
Where an outbreak of EVA is suspected, or when attempting to confirm a case of subclinical EAV infection,
virus isolation should be attempted from nasopharyngeal and conjunctival swabs, unclotted blood samples,
and semen from stallions considered to be possible carriers of the virus (Timoney & McCollum, 1993). To
optimise the chances of virus isolation, the relevant specimens should be obtained as soon as possible after
the onset of fever in affected horses. As heparin can inhibit the growth of EAV in rabbit kidney cells (RK-13
cell line), its use as an anticoagulant is contraindicated as it may interfere with isolation of the virus from
whole blood. Acid citrate dextrose or ethylenediaminetetraacetic acid (EDTA) are the anticoagulants of
choice to use in obtaining unclotted blood samples. Where EVA is suspected in cases of mortality in young
foals or older animals, isolation of EAV can be attempted from a variety of tissues, especially the lymphatic
glands associated with the alimentary tract and related organs, and also the lungs, liver and spleen
(McCollum et al., 1971). In outbreaks of EVA-related abortion and/or cases of stillborn foals, placental and
900
fetal fluids and a wide range of placental, lymphoreticular and other fetal tissues (especially lung) can be
productive sources of virus (Timoney & McCollum, 1993).
Swabs for attempted isolation should be immersed in a suitable viral transport medium and these, together
with any fluids or tissues collected for virus isolation and/or reverse-transcription polymerase chain reaction
(RT-PCR) testing should be shipped either refrigerated or frozen in an insulated container to the laboratory,
preferably using an overnight delivery service. Unclotted blood samples must be transported refrigerated but
not frozen. Where possible, specimens should be submitted to a laboratory with established competency to
test for this infection.
Although reportedly not always successful in natural cases of EAV infection (Timoney & McCollum, 1993),
virus isolation should be attempted from clinical specimens or necropsied tissues using rabbit, equine or
monkey kidney cell culture (Timoney et al., 2004; Timoney & McCollum, 1993). Selected cell lines, e.g. RK13 (ATCC CCL-37), LLC-MK2 (ATCC CCL-7), and primary horse or rabbit kidney cell culture can be used,
with RK-13 cells being the cell system of choice (Timoney et al., 2004). Experience over the years has
shown that primary isolation of EAV from semen can present more difficulty than from other clinical
specimens or from infected tissues unless an appropriate cell culture system is used. Several factors have
been shown to influence primary isolation of EAV from semen in RK-13 cells. Higher isolation rates have
been obtained using 3- to 5-day-old monolayers, a large inoculum size in relation to the cell surface area in
the inoculated flasks or multiwell plates, and most importantly, the incorporation of carboxymethyl cellulose
(medium viscosity, 400800 cps) in the overlay medium. It should be noted that most RK-13 cells, including
ATCC CCL-37, are contaminated with bovine viral diarrhoea virus, the presence of which appears to
enhance sensitivity of this cell system for the primary isolation of EAV, especially from semen. There is
considerable evidence to indicate that primary isolation rates of EAV may be increased by using RK-13 cells
of high passage history1 (Timoney et al., 2004).
Inoculated cultures are examined daily for the appearance of viral cytopathic effect (CPE), which is usually
evident within 26 days. In the absence of visible CPE, culture supernatants should be subinoculated on to
confluent cell monolayers after 47 days. While the vast majority of isolations of EAV are made on the first
passage in cell culture, a small minority only become evident on the second or subsequent passages in vitro
(Timoney & McCollum, 1993; 2000). The identity of isolates of EAV can be confirmed in a one-way
neutralisation test, by standard RT-PCR or real-time RT-PCR assay (Balasuriya et al., 1998) or by an
immunocytochemical method (Little et al., 1995), indirect immunofluorescence (Crawford & Henson, 1973)
or the avidinbiotinperoxidase (ABC) technique (Little et al., 1995). A polyclonal rabbit antiserum has been
used to identify EAV in infected cell cultures. Mouse monoclonal antibodies (MAbs) to the nucleocapsid
protein (N) (Balasuriya et al., 1998) and major envelope glycoprotein (GP5) of EAV (Balasuriya et al., 1998)
and a monospecific rabbit antiserum to the unglycosylated envelope protein (M) (Balasuriya et al., 1998)
have also been developed and these can detect various strains of the virus in RK-13 cells as early as 12
24 hours after infection (Balasuriya et al., 1998; Little et al., 1995).
Virus isolation from semen (a prescribed test for international trade)

There is considerable evidence that short- and long-term carrier stallions shed EAV constantly in the semen,
but not in respiratory secretions or urine; nor has it been demonstrated in the buffy coat (peripheral blood
mononuclear cells) of such animals (Timoney & McCollum, 1993; 2000). Stallions should first be blood
tested using the virus neutralisation (VN) test or an appropriately validated enzyme-linked immunosorbent
assay (ELISA) or other serological test procedure. Virus isolation should be attempted from the semen of
stallions serologically positive (titre 1/4) for antibodies to EAV that do not have a certified history of
vaccination against EVA with confirmation that they were serologically negative (titre <1/4) at time of initial
vaccination. Virus isolation is also indicated in the case of shipped semen where the serological status and
possible vaccination history of the donor stallion is not available. It is recommended that virus isolation from
semen be attempted from two samples, which can be collected on the same day, on consecutive days or
after an interval of several days or weeks. There is no evidence that the outcome of attempted virus isolation
from particular stallions is influenced by the frequency of sampling, the interval between collections or time
of the year. Isolation of EAV should be carried out preferably on portion of an entire ejaculate collected using
an artificial vagina or a condom and a teaser or phantom mare. When it is not possible to obtain semen by
this means, a less preferable alternative is to collect a dismount sample at the time of breeding. Care should
be taken to ensure that no antiseptics/disinfectants are used in the cleansing of the external genitalia of the
stallion prior to collection. Samples should contain the sperm-rich fraction of the ejaculate with which EAV is
associated as the virus is not present in the pre-sperm fraction of semen (Timoney & McCollum, 1993;
2000). Immediately following collection, the semen should be refrigerated on crushed ice or on freezer packs
for transport to the laboratory with a minimum of delay. Where there is likely to be a delay in submitting a
specimen for testing, the semen can be frozen at or below 20C for a short period before being dispatched
Such a line (RK-13-KY) is available from the OIE Reference Laboratory for Equine viral arteritis at the Maxwell H. Gluck
Equine Research Center, University of Kentucky, United States of America.
901
to the laboratory. Freezing a sample has not been found to militate against isolation of EAV from the semen
of a carrier stallion. In situations where it is not feasible to determine the carrier status of a stallion by virus
isolation or RT-PCR procedures, the stallion can be test bred to two seronegative mares, which are checked
for seroconversion to the virus 28 days after breeding (Timoney & McCollum, 1993).
Test procedure
i)
On receipt in the laboratory, it should be noted whether each semen sample is frozen, chilled or at
ambient temperature. Every sample should be checked to ensure that it contains the sperm-rich
fraction of the ejaculate. This can be established by microscopic examination of a wet-mount
preparation of a sample. Additionally, specimens of ejaculate should be visually inspected for colour
and presence of gross particulate contamination. If a semen specimen is contaminated with blood,
which can result from trauma to the external genitalia of the stallion at time of collection, a repeat
sample should be requested as testing such a specimen from a serologically positive stallion may
compromise the reliability of the virus isolation result.
ii)
Although no longer considered an essential step, pretreatment of semen before inoculation into cell
culture by short-term sonication (for three 15-second cycles); facilitates effective mixing and dispersion
of a sample.
iii)
After removal of culture medium, 3- to 5-day-old confluent monolayer cultures of RK-13 cells, either in
25 cm2 tissue culture flasks or multiwell plates, are inoculated with serial decimal dilutions (101 to
103) of seminal plasma in tissue culture maintenance medium containing 2% fetal bovine serum and
antibiotics. An inoculum of 1 ml per 25 cm2 flask is used and no fewer than two flasks per dilution of
seminal plasma are inoculated. Inoculum size and number of wells inoculated per dilution of a
specimen should be pro-rated where multiwell plates are used. Appropriate dilutions of a virus positive
control semen sample or virus control of known titre diluted in culture medium should be included in
each test.
iv)
The flasks are closed, lids replaced on multiwell plates and inoculated cultures gently rotated to
disperse the inoculum over the cell monolayers.
v)
Inoculated cultures are then incubated for 1 hour at 37C either in an aerobic incubator or an incubator
containing a humidified atmosphere of 5% CO2 in air, depending on whether flasks or multiwell plates
are used.
vi)
Without removing any of the inoculum or washing the cell monolayers, the latter are overlaid with
0.75% carboxymethyl cellulose containing medium with antibiotics.
vii)
The flasks or plates are reincubated at 37C and checked microscopically for viral CPE, which is
usually evident within 26 days.
viii) In the absence of visible CPE, culture supernatants are subinoculated onto 3- to 5-day-old confluent
cell monolayer cultures of RK-13 cells after 57 days. After removal of the overlay medium,
monolayers are stained with 0.1% formalin-buffered crystal violet solution.
The identity of any virus isolates should be confirmed by VN, immunofluorescence (Crawford & Henson,
1973) or ABC technique, using a monospecific antiserum to EAV or MAbs to the structural proteins, N or
GP5 of the virus (Balasuriya et al., 1998; Del Piero, 2000; Little et al., 1995), or by standard RT-PCR
(Balasuriya et al., 1998; Gilbert et al., 1997) or real-time RT-PCR assay (Balasuriya et al., 2002; Lu et al.,
2007; Westcott et al., 2003).
In the one-way neutralisation test, serial decimal dilutions of the virus isolate are tested against a
neutralising MAb or monospecific antiserum prepared against the prototype Bucyrus strain of EAV (ATCC
VR 796) and also a serum negative for neutralising antibodies to the virus. Corresponding titrations of the
prototype Bucyrus virus with the same reference antibody reagents are included as test controls. The test is
performed in either 25 cm2 tissue culture flasks or multiwell plates. Appropriate quantities of the known EAV
positive and negative antibody reagents are inactivated for 30 minutes in a water bath at 56C and diluted
1/4 in phosphate buffered saline, pH 7.2; then 0.3 ml of diluted antibody reagent is dispensed into five tubes
for each virus isolate to be tested. Serial decimal dilutions (101 to 105) of each virus are made in Eagles
Minimal Essential Medium containing 10% fetal bovine serum, antibiotics and 10% freshly diluted guinea-pig
complement. Then, 0.3 ml of each virus dilution is added to the tubes containing positive and negative
antibody reagents. The tubes are shaken and the virus/antibody mixtures are incubated for 1 hour at 37C.
The mixtures are then inoculated onto 3- to 5-day-old confluent monolayer cultures of RK-13 cells, either in
25 cm2 flasks or multiwell plates, using two flasks or wells per virus dilution. Each flask is inoculated with
0.25 ml of virus/antibody mixture; the inoculum size is pro-rated where multiwell plates are used. Inoculated
flasks or plates are incubated for 2 hours at 37C, gently rocking after 1 hour to disperse the inoculum over
the cell monolayers. Without removing any of the inoculum or washing the cell monolayers, the latter are
overlaid with 0.75% carboxymethyl cellulose containing medium and incubated for 45 days at 37C, either
902
in an aerobic incubator or an incubator containing a humidified atmosphere of 5% CO2 in air. After removal
of the medium, monolayers are stained with 0.1% formalin-buffered crystal violet solution. Plaques are
counted and the virus infectivity titre is determined both in the presence and absence of EAV antibodies
using the SpearmanKrber method. Confirmation of the identity of an isolate is based on a reduction in
plaque count of at least 102 logs of virus in the presence of antibody positive serum against the Bucyrus
strain of EAV.
The vast majority of EAV isolates from carrier stallions are made in the first passage in cell culture using the
described test procedure (Timoney & McCollum, 1993; 2000). The occurrence of nonviral cytotoxicity or
bacterial contamination of specimens is not considered significant problems when attempting isolation of this
virus from stallion semen. Nonviral cytotoxicity, if observed, usually affects monolayers inoculated with the
101 and, much less frequently, the 102 dilution of seminal plasma. Treatment of seminal plasma with
polyethylene glycol (Mol. wt 6000) prior to inoculation has been used with some success in overcoming this
problem (Fukunaga et al., 2000). The method described involves the addition of polyethylene glycol to the
101 to 103 dilutions of seminal plasma to give a final concentration of 10% in each dilution. The mixtures
are held overnight at 4C with gentle stirring, after which they are centrifuged at 2000 g for 30 minutes and
the supernatants are discarded. The precipitates are suspended in cell culture maintenance medium to onetenth the volume of the original dilutions and the mixtures are homogenised. They are then centrifuged at
2000 g for 30 minutes and the supernatants are taken off and used for inoculation. There is no evidence to
indicate that pretreatment of seminal plasma in this manner reduces sensitivity of the virus isolation
procedure (Fukunaga et al., 2000). Where bacterial contamination of a sample is a problem, it is preferable
to request a repeat semen collection from the individual stallion. If this is not possible, an attempt can be
made to control the contamination by pre-treatment of the sample with antibiotic containing viral transport
medium, holding overnight at 4C followed by ultracentrifugation and resuspension of the pellet before
diluting and inoculating the specimen into cell culture.
The presence of anti-EAV antibody activity in the seminal plasma of certain virus-shedding stallions has not
been found to prevent detection of the carrier state in these animals.
b)
Nucleic acid recognition methods

The standard two-step RT-PCR, single-step RT-PCR and real-time RT-PCR (rRT-PCR) assays are gaining
greater acceptance and being more widely used as an alternative to virus isolation in cell culture for the
detection of EAV in diagnostic materials. The RT-PCR-based assays provide a means of identifying virusspecific RNA in clinical specimens, namely nasopharyngeal swab filtrates, buffy coats, raw and extended
semen and urine, and in post-mortem tissue samples (Balasuriya et al., 2002; Gilbert et al., 1997; Westcott
et al., 2003). Standard two-step RT-PCR, single-step RT-PCR, RT-nested PCR (RT-nPCR), and one tube
TaqMan rRT-PCR assays have been developed and evaluated for the detection of various strains of the
virus in tissue culture fluid, semen and nasal secretions (Balasuriya et al., 2002; Gilbert et al., 1997; Lu et
al., 2007; Westcott et al., 2003). These assays targeted six different open reading frames (ORFs) in the EAV
genome (ORFs 1b, 37). However, there is considerable variation in the sensitivity and specificity among
RT-PCR assays incorporating different primer pairs targeting various ORFs. Results comparable to virus
isolation have been obtained with some but not all standard single-step RT-PCR, two-step RT-PCR, RTnPCR or one tube TaqMan rRT-PCR assays (Balasuriya et al., 2002; Gilbert et al., 1997; Lu et al., 2007).
Compared with traditional virus isolation, these RT-PCR-based assays are frequently more sensitive, less
expensive and considerably more rapid to perform, the majority taking less than 24 hours to complete. In
addition, RT-PCR assays have the advantage of not requiring viable virus for performance of the test. The
one-tube rRT-PCR assay for EAV provides a simple, rapid and reliable method for the detection and
identification of viral nucleic acid in equine semen and tissue culture fluid (Balasuriya et al., 2002; Lu et al.,
2007). The one tube rRT-PCR has the following important advantages over the standard two-step RT-PCR:
1) eliminating the possibility of cross contamination between samples with previously amplified products as
the sample tube is never opened; and 2) reducing the chance of false-positive reactions because the rRTPCR product is detected with a sequence-specific probe. Because of the high sensitivity of the RT-PCR
assay, however, and in the absence of appropriate safeguards in the laboratory, there is the potential for
cross-contamination between samples, giving rise to false-positive results. For example, the RT-nPCR
assay, while it provides enhanced sensitivity for the detection of EAV, it also increases the likelihood of
false-positive results. The risk of cross-contamination is greater using the RT-nPCR assay because of the
second PCR amplification step involving the product from the first RT-PCR reaction. To minimise the risk of
cross-contamination, considerable care needs to be taken, especially during the steps of RNA extraction and
reaction setup. Relevant EAV positive and negative template controls and, where appropriate, nucleic acid
extracted from the tissue culture fluid of uninfected cells, need to be included in each RT-PCR assay. Thus,
in many circumstances, use of the single-step RT-PCR or the one tube rRT-PCR assay would largely
circumvent the problems associated with cross contamination.
Primer selection is critical to the sensitivity of the RT-PCR assay with primers (and probe in the case of the
rRT-PCR assay) preferably designed from the most conserved region(s) of the EAV genome. Comparative
903
nucleotide sequence analysis has shown that ORF 1b (encodes the viral polymerase), ORF 6 (M protein)
and 7 (N protein) are more conserved than the other ORFs among EAV strains so far analysed from North
America and Europe (Balasuriya et al., 2004; Lu et al., 2007; Westcott et al., 2003). The most conserved
gene among different strains of EAV is ORF7 and primers specific for ORF7 (and probe for rRT-PCR) have
detected a diversity of strains of the virus of European and North American origin (Balasuriya et al., 2002; Lu
et al., 2007). Furthermore, the use of multiple primer pairs specific for different ORFs 1b ([forward: 5-GATGTC-TAT-GCT-CCA-TCA-TT-3 and reverse: 5-GGC-GTA-GGC-TCC-AAT-TGA-A-3]) and/or [forward: 5CCT-GAG-ACA-CTG-AGT-CGC-GT-3 and reverse 5-CCT-GAT-GCC-ACA-TGG-AAT-GA-3]) (Gilbert et
al., 1997), ORF 6 ([forward: 5-CTG-AGG-TAT-GGG-AGC-CAT-AG-3 and reverse: 5-GCA-GCC-AAA-AGCACA-AAA-GC-3]) and ORF 7 ([forward 5-ATG-GCG-TCA-AGA-CGA-TCA-CG-3 and reverse 5-AGA-ATATCC-ACG-TCT-TAC-GGC-3]) markedly increases the likelihood of detecting North American and European
strains of EAV in the RT-PCR assay. The two primer pairs specific for ORF 1b are suitable for use in the RTrPCR assay (Gilbert et al., 1997). While the RT-PCR has been found to be highly sensitive for viral nucleic
acid detection in raw semen, there is evidence to that it is not of equivalent reliability when testing extended
or cryopreserved semen of very low virus infectivity.
In addition to the foregoing RT-PCR assays, 2 TaqMan fluorogenic probe-based one-tube rRT-PCR
assays have been described for the detection of EAV nucleic acid (Balasuriya et al., 2002); primers
([forward: 5-GGC-GAC-AGC-CTA-CAA-GCT-ACA-3, reverse: 5-CGG-CAT-CTG-CAG-TGA-GTG-A-3] and
probe [5FAM-TTG-CGG-ACC-CGC-ATC-TGA-CCA-A-TAMRA-3] and (Westcott et al., 2003); primers
[forward: 5-GTA-CAC-CGC-AGT-TGG-TAA-CA-3, reverse: 5-ACT-TCA-ACA-TGA-CGC-CAC-AC-3] and
probe [5FAM-TGG-TTC-ACT-CAC-TGC-AGA-TGC-CGG-TAMRA-3]). It should be noted, however, that
genomic variation among field isolates of EAV could reduce the sensitivity of both RT-PCR and rRT-PCR
assays, even when the primers and probe are based on the most conserved region of the EAV genome
(ORF 7 [Lu et al., 2007]).
In the absence of general agreement on a complete consensus or universal primer set for EAV, and since
no RT-PCR assay can determine the actual infectivity of a sample, there is a value to performing virus
isolation in conjunction with RT-PCR or rRT-PCR for the identification of virus in clinical or post-mortem
specimens.
Strains of EAV isolated from different regions of the world have been classified into different phylogenetic
groups by sequence analysis of the GP3, GP5 and M envelope protein genes (ORFs 3, 5 and 6,
respectively) and the nucleocapsid (N) protein gene (ORF 7 [Balasuriya et al., 1998; Stadejek et al., 1999]).
The GP5 gene has been found to be most useful and reliable for this purpose (Stadejek et al., 1999). The
relationships between strains demonstrated by nucleotide sequencing are a useful molecular
epidemiological tool for tracing the origin of outbreaks of EVA (Balasuriya et al., 1998; Balasuriya et al.,
2004).
c)
Histopathological and immunohistochemical methods

Where mortality is associated with a suspected outbreak of EVA, a wide range of tissues should be
examined for histological evidence of panvasculitis that is especially pronounced in the small arteries
throughout the body, particularly in the caecum, colon, spleen, associated lymphatic glands and adrenal
cortex (Crawford & Henson, 1973; Del Piero, 2000; Jones et al., 1957). The presence of a disseminated
necrotising arteritis involving endothelial and medial cells of affected vessels is considered to be
pathognomonic of EVA. The characteristic vascular lesions present in the mature animal are not, however, a
prominent a feature in many cases of EAV-related abortion.
EAV antigen can be identified in various tissues of EVA-affected animals either in the presence or absence
of lesions (Del Piero, 2000). Antigen has been demonstrated in lung, heart, liver and spleen and the
placenta of aborted fetuses (Del Piero, 2000). Immunohistochemical examination of biopsied skin specimens
has also been investigated as a means of confirming acute EAV infection. Though of some value, it is not
entirely reliable for the diagnosis of the disease. Viral antigen can be detected within the cytoplasm of
infected cells by immunofluorescence using conjugated equine polyclonal anti-EAV serum (Crawford &
Henson, 1973), or by the ABC technique using mouse MAbs to the GP5 (Glaser et al., 1995) or N proteins of
the virus (Del Piero, 2000).
2.
Serological tests
A variety of serological tests including neutralisation (microneutralisation [Senne et al., 1985] and plaque
reduction [McCollum, 1970] (VN)), the complement fixation (CF) test (Fukunaga & McCollum, 1977), the indirect
fluorescent antibody test (Crawford & Henson, 1973), the agar gel immunodiffusion (Crawford & Henson, 1973),
the ELISA (Cho et al., 2000; Hedges et al., 1998; Kondo et al., 1998; Nugent et al., 2000) and the fluorescent
microsphere immunoassay (MIA) (Go, pers. comm.) have been used to detect antibody to EAV.
904
The test currently in widest international use to diagnose infection, carry out seroprevalence studies, and test
horses for export, is a microneutralisation test in the presence of complement. It has also been used to screen
fetal heart blood for the retrospective diagnosis of cases of EVA-related abortion. Apart from the VN test, the CF
test has been used for diagnosing recent EAV infection as complement-fixing antibodies are relatively short-lived
in duration (Fukunaga & McCollum, 1977). In contrast, neutralising antibody titres to EAV frequently persist for
several years after natural infection (Timoney & McCollum, 1993). Although a number of ELISAs have been
developed (Cho et al., 2000; Hedges et al., 1998; Kondo et al., 1998; Nugent et al., 2000), none has as yet been
as extensively validated as the VN, though some appear to offer nearly comparable sensitivity and specificity
(Hedges et al., 1998; Nugent et al., 2000). Unlike the VN test, a positive reaction in the ELISA is not necessarily
reflective of the protective immune status of an individual horse to EAV as both non-neutralising and neutralising
antibodies are involved.
Antiserum to unpurified EAV has been prepared in horses and in rabbits using conventional immunisation
protocols. Also, mouse monoclonal and monospecific rabbit antibodies have been developed to the nucleocapsid
protein (N) major envelope glycoprotein (GP5), and unglycosylated envelope protein (M) of EAV (Balasuriya et
al., 1997; Glaser et al., 1995).
OIE Standard Sera for EAV are available2 and these can facilitate international standardisation of the
microneutralisation test and ELISA.
Only one major serotype of EAV has been recognised so far (McCollum, 1970; Timoney & McCollum, 1993). This
is represented by the prototype Bucyrus strain (ATCC VR 796). The reference virus used in the EAV VN test is
the CVL-Bucyrus (Weybridge) strain. Virus stock is grown in the RK-13 cell line, clarified of cellular debris by lowspeed centrifugation and stored in aliquots at 70C. Several frozen aliquots are thawed and the infectivity of the
stock virus is determined by titration in RK-13 cells.
a)
Virus neutralisation (a prescribed test for international trade)

The VN test is used to screen stallions for evidence of EAV infection and to determine whether there is a
need to attempt virus detection in semen using cell culture or RT-PCR assay. It is also used for diagnostic
purposes to confirm infection in suspect cases of EVA. The VN test procedure in current widest use is that
developed by the National Veterinary Service Laboratories of the United States Department of Agriculture
(Senne et al., 1985). It is important to obtain a sterile blood sample as bacterial contamination of serum can
interfere with the test result. It is recommended that the test be carried out in RK-13 cells using the approved
CVL-Bucyrus (Weybridge) strain of EAV as reference virus3 (Edwards et al., 1999). Although originally
derived from the prototype Bucyrus virus, the passage history of the CVL (Weybridge) strain is not fully
documented. The sensitivity of the VN test for detection of antibodies to EAV can be significantly influenced
by several factors, especially the source and passage history of the strain of virus used (Edwards et al.,
1999). The CVL-Bucyrus (Weybridge) strain and the highly attenuated MLV vaccine strain of EAV are of
comparable sensitivity for detecting low-titred positive sera, especially from EVA-vaccinated horses. Efforts
are continuing to bring about greater uniformity in the testing protocol and serological results among
laboratories providing the VN or other comparable serological assays for this infection.
2
3
Test procedure
i)
Sera are inactivated for 30 minutes in a water bath at 56C (control sera, only once).
ii)
Serial twofold dilutions of the inactivated test sera in serum-free cell culture medium (25 l volumes)
are made in a 96-well, flat-bottomed, cell-culture grade microtitre plate starting at a 1/2 serum dilution
and using duplicate rows of wells for each serum to be tested. Most sera are screened initially at a 1/4
and 1/8 serum dilution (i.e. final serum dilution after addition of an equal volume of the appropriate
dilution of stock virus to each well). Positive samples at the 1/8 dilution can, if desired, be retested and
titrated out for end-point determination. Individual serum controls, together with negative and known
low- and high-titred positive control sera must also be included in each test.
iii)
A dilution of stock virus to contain from 100 to 300 TCID50 (50% tissue culture infective dose) per 25 l
is prepared using as diluent, serum-free cell culture medium containing antibiotics and fresh guinea-pig
or rabbit complement at a final concentration of 10%.
iv)
25 l of the appropriate dilution of stock virus is added to every well containing 25 l of each serum
dilution, except the test serum control wells.
OIE Reference Laboratory for Equine viral arteritis at the Maxwell H. Gluck Equine Research Center, University of
Kentucky, United States of America.
Available from the OIE Reference Laboratory for Equine viral arteritis at the Animal Health Veterinary Laboratories
Agency, Weybridge, United Kingdom.
905
v)
A virus back titration of the working dilution of stock virus is included, using four wells per tenfold
dilution, to confirm the validity of the test results.
vi)
The plates are covered and shaken gently to facilitate mixing of the serum/virus mixtures.
vii)
The plates are incubated for 1 hour at 37C in a humid atmosphere of 5% CO2 in air.
viii) A suspension of cells from 3- to 5-day-old cultures of RK-13 cells are prepared using a concentration
that will ensure confluent monolayers in the microtitre plate wells within 1824 hours after seeding.
ix)
100 l of cell suspension is added to every well, the plates covered with plate lids or sealed with tape
and shaken gently.
x)
The plates are incubated at 37C in a humid atmosphere of 5% CO2 in air.
xi)
The plates are read microscopically for nonviral CPE after 1218 hours and again for viral CPE after
4872 hours incubation. The validity of the test is confirmed by establishing that the working dilution of
stock virus contained 30300 TCID50 virus and that the titres of the positive serum controls are within
0.3 log10 units of their predetermined titres.
A serum dilution is considered to be positive if there is an estimated 75% or preferably a 100% reduction in
the amount of viral CPE in the serum test wells compared with that present in the wells of the lowest virus
control dilution. End-points are then calculated using the SpearmanKrber method. A titre of 1/4 or greater
is considered to be positive. A negative serum should only have a trace (less than 25%) or no virus
neutralisation at the lowest dilution tested. Antibody titres may, on occasion, be difficult to define as partial
neutralisation may be observed over a range of several serum dilutions. Infrequently, sera will be
encountered that cause toxic changes in the lower dilutions tested. In such cases it may not be possible to
establish whether the sample is negative or a low-titred positive. The problem may be overcome by testing
another serum sample from the animal in question or by retesting the toxic sample using microtitre plates
with confluent monolayers of RK-13 cells that had been seeded the previous day. It has been reported that
the toxicity of the serum can, in some cases, be reduced or eliminated if the sample is adsorbed with packed
RK-13 cells prior to testing or by substituting rabbit in place of guinea-pig complement in the virus diluent.
Vaccination status for equine herpesviruses should be considered when evaluating sera causing non-viral
cytotoxicity. One of the equine herpesvirus vaccines currently available in Europe has been shown to
stimulate antibodies to rabbit kidney cells used in the vaccine production. These, in turn, can give rise to
cytotoxicity, usually in the 1/4 and/or 1/8 serum dilutions and cause difficulties in interpretation of the test
results (Newton et al., 2004).
b)

A number of direct or indirect ELISAs have been developed for the detection of antibodies to EAV (Cho et
al., 2000; Hedges et al., 1998; Kondo et al., 1998; Nugent et al., 2000). These have been based on the use
of purified virus or recombinant-derived viral antigens. The usefulness of earlier assays was compromised
by the frequency of false-positive reactions. The latter were associated with the presence of antibodies to
various tissue culture antigens in the sera of horses that had been vaccinated with tissue-culture-derived
antigens. Identification of the importance of the viral GP5 protein in stimulation of the humoral antibody
response to EAV led to the development of several ELISAs that employ a portion of, or the entire
recombinant protein produced in a bacterial or baculovirus expression system (Cho et al., 2000; Hedges et
al., 1998). Most recently, an ovalbumin-conjugated synthetic peptide representing amino acids 81106 of the
GP5 protein has been used (Nugent et al., 2000). Some of these assays appear to offer nearly comparable
sensitivity and specificity to the VN test and may detect EAV-specific antibodies prior to a positive reaction
being obtainable in the VN test. False-negative reactions can occur, however, with some of these assays.
Screening a random peptide-phage library with polyclonal sera from EAV-infected horses led to the
identification of ligands, which were purified and used as antigen in an ELISA for EAV. No correlation was
found, however, between absorbency values obtained with this assay and neutralising antibody titres,
indicating that the antibodies being detected were largely against nonsurface epitopes of the virus. An ELISA
based on the use of a combination of the GP5, M or N structural proteins of EAV expressed from
recombinant baculoviruses successfully detected viral antibody in naturally or experimentally infected horses
but not in EVA-vaccinated animals (Hedges et al., 1998). Of major importance with respect to any GP5
protein-based ELISA for EAV is the fact that test sensitivity will vary depending on the ectodomain
sequence(s) of this viral protein used in the assay. Considerable amino acid sequence variation within this
domain has been found between isolates of EAV. To maximise sensitivity of a GP5-based ELISA, it may be
necessary to include multiple ectodomain sequences representative of known phenotypically different
isolates of EAV rather than depend on a single ectodomain sequence. Two more recently described ELISAs
appear to offer most promise as reliable serodiagnostic tests for EAV infection (Cho et al., 2000; Nugent et
al., 2000). A blocking ELISA involving MAbs produced against the GP5 protein was reported to have a
sensitivity of 99.4% and a specificity of 97.7% compared with the VN test (Cho et al., 2000). Another assay,
a GP5 ovalbumin-conjugated synthetic peptide ELISA was shown to have a sensitivity and specificity of
96.75% and 95.6%, respectively, using a panel of 400 VN positive sera and 400 VN negative samples
906
(Nugent et al., 2000). It is expected that an ELISA will soon be available, having very similar if not equivalent
sensitivity and specificity to the VN test.

A number of experimental and commercial vaccines have been developed against EVA. Currently, there are two
commercially available vaccines, both tissue-culture derived. The first is a modified live virus (MLV) vaccine
prepared from virus that has been attenuated for horses by multiple serial transfers in equine and rabbit cell
cultures (Doll et al., 1968; McCollum, 1970). This vaccine is licensed for use in stallions, nonpregnant mares and
in nonbreeding horses. Whereas nonbreeding horses can be vaccinated at any time, stallions and mares should
be vaccinated not less than 3 weeks prior to breeding. The vaccine is not recommended for use in pregnant
mares, especially in the last 2 months of gestation, nor in foals under 6 weeks of age unless in the face of
significant risk of exposure to natural infection. The vaccine is commercially available in the USA and Canada. It
has also been used in New Zealand, subject to ministerial controls, to aid in that countrys EVA eradication
programme.
The second commercially available vaccine against EVA is an inactivated product prepared from virus grown in
equine cell culture, which is filtered, chemically inactivated and then combined with a metabolisable adjuvant. This
vaccine is licensed for use in nonbreeding and breeding horses. In the absence of appropriate safety data, the
vaccine is currently not recommended for use in pregnant mares. The initial vaccination regimen involves two
doses of vaccine administered intramuscularly 36 weeks apart. Booster vaccination at 6-month intervals is
recommended by the manufacturer. The inactivated vaccine is licensed for commercial use in certain European
countries, including Denmark, France, Germany, Hungary, Ireland, Sweden and the United Kingdom.
An additional inactivated vaccine against EVA has been developed in Japan for use should an outbreak of EVA
occur in that country. It is an aqueous formalin-inactivated vaccine that has been shown to be safe and effective
for use in nonbreeding and breeding horses. For optimal immunisation with this vaccine, horses require a primary
course of two injections given at an interval of 4 weeks, with a booster dose administered every 612 months. As
the vaccine is currently not commercially available, no details can be provided on its production.
1.
Seed management
a)

Both MLV and inactivated commercial vaccines are derived from the prototype Bucyrus strain of EAV (ATCC
VR 796). Available evidence points to the existence of only one major serotype of the virus, and strain
variation is not considered to be of significance in relation to vaccine efficacy (McCollum, 1970; Timoney &
McCollum, 1993).
In the case of the MLV vaccine, the prototype virus was attenuated by serial passage in primary cultures of
horse kidney (HK-131), rabbit kidney (RK-111), and a diploid equine dermal cell line, ATCC CCL57 (ECID24) (Doll et al., 1968; McCollum, 1970). The indications from the use of this vaccine are that the virus is safe
and immunogenic between its 80th and 111th passage in primary rabbit kidney (Doll et al., 1968; McCollum,
1970; Timoney et al., 1988).
The inactivated adjuvanted vaccine is prepared from the unattenuated prototype Bucyrus strain of EAV
(ATCC VR 796) that has been plaque purified and in its fourth serial passage in the diploid equine dermal
cell line (ECID-4). After growth in cell culture, the virus is then purified by filtration before being chemically
inactivated and adjuvanted.
Suitable lots of master seed virus for each vaccine should be maintained in liquid nitrogen or its equivalent.
b)
Method of culture
The virus for both MLV and inactivated vaccines should be grown in a stable cell culture system, such as
equine dermal cells, using an appropriate medium supplemented with sterile bovine serum or bovine serum
albumin as replacement for bovine serum in the growth medium. Cell monolayers should be washed prior to
virus inoculation to remove traces of bovine serum. Extensive virus growth as evidenced by the appearance
of cytopathic changes in 80100% of the cells should be obtained within 23 days.
907
c)
In the case of both MLV and inactivated vaccines, the respective virus strains should be grown in an
appropriate cell culture system that has been officially approved for vaccine production and confirmed to be
free from extraneous bacteria, fungi, mycoplasmas and viruses (Moore, 1986). The identity of the vaccine
virus in the master seed should be confirmed by neutralisation with homologous anti-EAV serum. Incomplete
neutralisation of EAV by homologous horse or rabbit antisera has been scientifically documented (Moore,
1986; Senne et al., 1985) and is a problem when screening master seed virus for extraneous viruses and
when attempting to confirm the identity of the vaccine virus. The problem has been circumvented by
reducing the infectivity titre of the master seed virus below that required for seed virus production before
conducting a neutralisation test on the diluted virus. Virus/serum mixtures are tested for residual live virus by
serial passage in cell culture. No evidence of cytopathic viruses, haemadsorbing viruses, or noncytopathic
strains of bovine virus diarrhoea virus should be found, based on attempted virus isolation in cell culture. If
cells of equine origin are used, they should be confirmed to be free from equine infectious anaemia virus.
The newer technologies of PCR and antigen-capture ELISA may be used as adjuncts to virus isolation in
screening for adventitious agents.
The MLV vaccine has been shown to be both safe and effective for use in stallions and nonpregnant mares
(Timoney et al., 1988). Although not recommended by the manufacturer for pregnant mares, especially in
the last 2 months of gestation, the vaccine has been used to immunise pregnant mares in the face of high
risk of natural exposure to EAV, with minimal, if any, reported adverse effects. Vaccination confers a high
level of protective immunity that persists for at least several years (McCollum, 1970; Timoney & McCollum,
1993). Based on experimental studies and extensive field use of the vaccine since 1985, there is no
evidence of back reversion to virulence of the vaccine virus, nor of recombination of the vaccine virus with
naturally occurring strains of EAV. Furthermore, there is no confirmed evidence that the attenuated strain of
EAV in the current vaccine localises and sets up the carrier state in the reproductive tract of the vaccinated
stallion (Timoney & McCollum, 1993; 2000; Timoney et al., 1988).
The commercial inactivated vaccine has been shown to be nonreactive and safe for use in healthy
nonbreeding and breeding horses. Transient local reactions may be observed in less than 10% of horses
vaccinated with the inactivated vaccine. Limited field studies of this vaccine indicate that it is immunogenic,
stimulating a satisfactory degree of immunity, the duration of which has yet to be reported.
Although there are no published reports on the efficacy of either commercial vaccine in preventing
establishment of the carrier state in the stallion, an experimental aqueous formalin inactivated vaccine
against EVA has been shown to prevent virus persistence in the reproductive tract of vaccinated stallions
following subsequent challenge with EAV (Fukunaga et al., 1992).
2.
Both the MLV and inactivated vaccines are produced by cultivation of the respective seed viruses in an equine
dermal cell system. Cell monolayers should be washed prior to inoculation with seed virus to remove traces of
bovine serum in the growth medium. Inoculated cultures should be maintained on an appropriate maintenance
medium. Harvesting of infected cultures should take place when almost the entire cell sheet shows the
characteristic CPE. Supernatant fluid and cells are harvested and clarified of cellular debris and unwanted
material by filtration. In the case of the inactivated vaccine, the purified virus is then chemically inactivated and
adjuvanted with a metabolisable adjuvant.
3.
In-process control
The MLV and inactivated vaccines should be produced in a stable cell line that has been tested for identity and
confirmed to be free from contamination by bacteria, fungi, mycoplasmas or other adventitious agents. In addition
to the preproduction testing of the master seed virus for each vaccine and the cell line for adventitious
contaminants, the cell cultures infected with the respective vaccine viruses should be examined macroscopically
for evidence of microbial growth or other extraneous contamination during the incubation period. If growth in a
culture vessel cannot be reliably determined by visual examination, subculture, microscopic examination, or both
should be carried out.
4.
Batch control
a)
Sterility
908
b)
Safety
In the case of both MLV and inactivated vaccines, each production lot of vaccine should be checked for
extraneous bacterial, fungal and mycoplasmal contaminants. The vaccine should be safety tested by the
intramuscular inoculation of at least two horses seronegative for neutralising antibodies to EAV with one
vaccine dose of lyophilised virus each (Moore, 1986). None of the inoculated horses should develop any
clinical signs of disease other than mild pyrexia during the ensuing 2-week observation period. In addition,
nasopharyngeal swabs should be collected daily from each horse for attempted virus isolation; white blood
cell counts and body temperatures should also be determined on a daily basis. No significant febrile or
haematological changes should supervene following vaccination (Timoney & McCollum, 1993; Timoney et
al., 1988). Limited shedding of vaccine virus by the respiratory route for at most 7 days may be
demonstrated in the occasional vaccinated horse (Timoney et al., 1988). There is no evidence of persistence
of the vaccine virus in the reproductive tract after vaccination of stallions (Timoney & McCollum, 1993; 2000;
Timoney et al., 1988).
To ensure complete inactivation of the vaccine virus, each serial lot of the inactivated vaccine should be
checked for viable virus by three serial passages in equine dermal cells and by direct fluorescent antibody
staining with specific EAV conjugate before being combined with adjuvant. This should be followed by a
safety test in guinea-pigs and mice.
c)
Potency
Potency of the vaccine in the final containers is determined by plaque infectivity assay in monolayer cultures
of equine dermal cells and by a vaccination challenge test in horses (Moore, 1986). The vaccine must be
tested in triplicate in cell culture, the mean infectivity titre calculated and the dose rate determined on the
basis that each dose of vaccine shall contain not less than 3 104 plaque-forming units of attenuated EAV.
The in-vivo potency of the MLV and inactivated vaccines is evaluated in a single vaccination challenge test
using 1720 vaccinated and 57 control horses or in two tests each comprising ten vaccinates and five
controls.
The viral antigen concentration in the inactivated vaccine is over one-thousand times the concentration of
viral antigen present in the MLV vaccine.
d)
Detectable neutralising antibody titres to EAV should develop in the majority of horses within 12 weeks of
vaccination with the MLV vaccine (Timoney & McCollum, 1993; Timoney et al., 1988). Reported responses
to primary vaccination have been variable in a couple of studies. In one stallion vaccination study, there was
a rapid fall in antibody titres with a significant number of animals reverting to seronegativity 13 months after
vaccination (Timoney et al., 1988). On the other hand, other studies have been characterised by an
excellent durable response, with persistence of high VN levels for at least 12 years. Revaccination with this
vaccine results in an excellent anamnestic response, with the development of high antibody titres that
remain relatively undiminished for several years (Timoney & McCollum, 1993).
Experimental studies have shown that most horses vaccinated with the inactivated vaccine develop low to
moderate neutralising antibody titres to EAV by day 14 after the second vaccination. There is no published
information on the duration of immunity conferred by this vaccine.
e)
Stability
The lyophilised MLV vaccine can be stored for at least 34 years at 27C without loss in infectivity,
provided it is kept in the dark. Infectivity is preserved for much longer periods if vaccine is frozen at 20C or
below. Once rehydrated, however, the vaccine should be used within 1 hour or else destroyed. The
inactivated vaccine is stored as a liquid suspension at 28C, with no loss of potency for at least 1 year,
provided it is protected from light.
f)
Preservatives
The preservatives added to the MLV and inactivated vaccines are neomycin, polymyxin B and amphotericin B.
g)
Pregnant mares should not be vaccinated with the MLV vaccine during the last 2 months of gestation, as
there is a risk, albeit minimal, of fetal invasion by the vaccine virus. The possibility of a vaccinally induced
anaphylactic reaction, though very rare, could result from the administration of either the MLV or inactivated
vaccine. In the absence of appropriate safety data, the inactivated vaccine is currently not recommended for
use in pregnant mares.
909
5.
a)
Safety
With the exception of the inactivated vaccine, which needs to be sterility tested a second time to ensure
freedom from contamination, no further safety tests are required on the inactivated or MLV vaccines.
b)
Potency
No potency tests additional to those conducted on each production lot of the MLV or inactivated vaccines
are required on either final product.
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BALASURIYA U.B.R., PATTON J.F., ROSSITO P.V., TIMONEY P.J., MCCOLLUM W.H. & MACLACHLAN N.J. (1997).
Neutralization determinants of laboratory strains and field isolates of equine arteritis virus: Identification of four
neutralization sites in the amino-terminal ectodomain. Virology, 232, 114128.
CAVANAGH D. (1997). Nidovirales: A new order comprising Coronaviridae and Arteriviridae. Arch. Virol., 142, 629
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CHO H.J., ENTZ S.C., DEREGT D., JORDAN L.T., TIMONEY P.J. & MCCOLLUM W.H. (2000). Detection of antibodies to
equine arteritis virus by a monoclonal antibody-based blocking ELISA. Can. J. Vet. Res., 64, 3843.
CRAWFORD T.B. & HENSON J.B. (1973). Immunofluorescent, light microscopic and immunologic studies of equine
viral arteritis. Proceedings of the Third International Conference on Equine Infectious Diseases, Paris, 1972.
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DEL PIERO F. (2000). Equine viral arteritis. Vet. Pathol., 37, 287296.
DOLL E.R., BRYANS J.T., WILSON J.C. & MCCOLLUM W.H. (1968). Immunisation against equine viral arteritis using
modified live virus propagated in cell cultures of rabbit kidney. Cornell Vet., 48, 497524.
EDWARDS S., CASTILLO-OLIVARES J., CULLINANE A., LABLE J., LENIHAN P., MUMFORD J.A., PATON D.J., PEARSON J.E.,
SINCLAIR R., WESTCOTT D.G.F., WOOD J.L.N., ZIENTARA S. & NELLY M. (1999). International harmonisation of
laboratory diagnostic tests for equine viral arteritis. Proceedings of the Eighth International Conference on Equine
Infectious Diseases, Dubai, UAE, 1998, 359362.
FUKUNAGA Y. & MCCOLLUM W.H. (1977). Complement fixation reactions in equine viral arteritis. Am. J. Vet. Res.,
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FUKUNAGA Y., WADA R., MATSUMURA T., ANZAI T., IMAGAWA H., SUGIURA T., KUMANOMIDO T., KANEMARU T. & KAMADA
M. (1992). An attempt to protect against persistent infection of equine viral arteritis in the reproductive tract of
stallions using formalin inactivated-virus vaccine. Proceedings of the Sixth International Conference on Equine
Infectious Diseases, Cambridge, UK, 1991, 239244.
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FUKUNAGA Y., WADA R., SUGITA S., FUJITA Y., NAMBO Y., IMAGAWA H., KANEMARU T., KAMADA M., KOMATSU N. &
AKASHI H. (2000). In vitro detection of equine arteritis virus from seminal plasma for identification of carrier
stallions. J. Vet. Med. Sci., 62, 643646.
GILBERT S.A., TIMONEY P.J., MCCOLLUM W.H. & DEREGT D. (1997). Detection of equine arteritis virus in the semen
of carrier stallions using a sensitive nested PCR assay. J. Clin. Microbiol., 35, 21812183.
GLASER A.L., DE VRIES A.A.F. & DUBOVI E.J. (1995). Comparison of equine arteritis virus isolates using neutralizing
monoclonal antibodies and identification of sequence changes in GL associated with neutralization resistance. J.
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HEDGES J.F., BALASURIYA U.B.R., SHABBIR A., TIMONEY P.J., MCCOLLUM W.H., YILMA T. & MACLACHLAN N.J. (1998).
Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing GL, M and N
proteins expressed from recombinant baculoviruses. J. Virol. Methods, 76, 127137.
JONES T.C., DOLL E.R. & BRYANS J.T. (1957). The lesions of equine viral arteritis. Cornell Vet., 47, 5268.
KONDO T., FUKUNAGA Y., SEKIGUCHI K., SUGIURA T. & IMAGAWA H. (1998). Enzyme-linked immunosorbent assay for
serological survey of equine arteritis virus in racehorses. J. Vet. Med. Sci., 60, 10431045.
LITTLE T.V., DEREGT D., MCCOLLUM W.H., & TIMONEY P.J. (1995). Evaluation of an immunocytochemical method for
rapid detection and identification of equine arteritis virus in natural cases of infection. Proceedings of the Seventh
International Conference on Equine Infectious Diseases, Tokyo, Japan, 1994, 2731.
LU Z., BRANSCUM A., SHUCK K.M., ZANG J., DUBOVI E., TIMONEY P.J. & BALASURIYA U.B.R. (2007). Detection of equine
arteritis virus nucleic acid in equine semen and tissue culture fluid. J. Vet. Diagn. Invest.
MCCOLLUM W.H. (1970). Vaccination for equine viral arteritis. Proceedings of the Second International Conference
on Equine Infectious Diseases, Paris, 1969, Karger, Basle, Switzerland, 143151.
MCCOLLUM W.H., PRICKETT M.E. & BRYANS J.T. (1971). Temporal distribution of equine arteritis virus in respiratory
mucosa, tissues and body fluids of horses infected by inhalation. Res. Vet. Sci., 2, 459464.
MOORE B.O. (1986). Development and evaluation of three equine vaccines. Irish Vet. J., 40, 105107.
NEWTON J.R., GERAGHTY R.J., CASTILLO-OLIVARES J., CARDWELL M. & MUMFORD J.A. (2004). Evidence that use of an
inactivated equine herpesvirus vaccine induces serum cytotoxicity affecting the equine arteritis virus neutralisation
test. Vaccine, 22, 41174123.
NUGENT J., SINCLAIR R., DEVRIES A.A.F., EBERHARDT R.Y., CASTILLO-OLIVARES J., DAVIS POYNTER N., ROTTIER P.J.M.
& MUMFORD J.A. (2000). Development and evaluation of ELISA procedures to detect antibodies against the major
envelope protein (GL) of equine arteritis virus. J. Virol. Methods, 90, 167183.
SENNE D.A., PEARSON J.E. & CABREY E.A. (1985). Equine viral arteritis: A standard procedure for the virus
neutralisation test and comparison of results of a proficiency test performed at five laboratories. Proc. U.S. Anim.
Health Assoc., 89, 2934.
STADEJEK T., BJORKLUND H., BASCUNANA C.R., CIABATTI I.M., SCICLUNA M.T., AMADDEO D., MCCOLLUM W.H.,
AUTORINO G.L., TIMONEY P.J., PATON D.J., KLINGEBORN B. & BELAK S. (1999). Genetic diversity of equine arteritis
virus. J. Gen. Virol., 80, 691699.
TIMONEY P.J., BRUSER C.A., MCCOLLUM W.H., HOLYOAK G.R. & LITTLE T.V. (2004). Comparative sensitivity of LLCMK2, RK-13, vero and equine dermis cell lines for primary isolation and propagation of equine arteritis virus. In:
Proceedings of the International Workshop on the Diagnosis of Equine Arteritis Virus Infection, Timoney P.J., ed.
M.H. Cluck Equine Research Center, 2021 October 2004, Lexington, Kentucky, USA, pp 2627.
TIMONEY P.J. & MCCOLLUM W.H. (1993). Equine viral arteritis. Vet. Clin. North Am. Equine Pract., 9, 295309.
TIMONEY P.J. & MCCOLLUM W.H. (2000). Equine viral arteritis: Further characterization of the carrier state in the
stallion. J. Reprod. Fertil. (Suppl.), 56, 311.
911
TIMONEY P.J., UMPHENOUR N.W. & MCCOLLUM W.H. (1988). Safety evaluation of a commercial modified live equine
arteritis virus vaccine for use in stallions. Proceedings of the Fifth International Conference on Equine Infectious
Diseases, Lexington, 1987, University Press of Kentucky, Lexington, Kentucky, USA, 1927.
VAALA W.E., HAMIR A.N., DUBOVI E.J., TIMONEY P.J. & RUIZ B. (1992). Fatal congenitally acquired equine arteritis
virus infection in a neonatal foal. Equine Vet. J., 24, 155158.
WEBER H., BECKMANN K. & HAAS L. (2006). Fallbericht. Equines arteritisvirus (EAV) als aborterreger bei alpacas?
Dtsch. Tierarztl. Wschr., 113, 162163.
WESTCOTT D.G., KING D.P., DREW T.W., NOWOTNY N., KINDERMANN J., HANNANT D., BELAK S. & PATON D.J. (2003).
Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with
fluorescent probes. Vet. Res., 34, 165176.
*
* *
NB: There are OIE Reference Laboratories for Equine viral arteritis
diagnostic tests, reagents and vaccines for equine viral arteritis
912
NB: Version amended following delisting of the mallein test by the World Assembly in May 2012
CHAPTER 2.5.11.
GLANDERS
SUMMARY
Glanders is a contagious and fatal disease of horses, donkeys, and mules, and is caused by
infection with the bacterium Burkholderia mallei (the name recently changed from Pseudomonas
mallei and was previously classified as Pfeifferella, Loefflerella, Malleomyces or Actinobacillus).
The disease causes nodules and ulcerations in the upper respiratory tract and lungs. A skin form
also occurs, known as farcy. Control of glanders requires testing of suspect clinical cases,
screening of apparently normal equids, and elimination of positive reactors. It is transmitted to
humans and all infected/contaminated or potentially infected/contaminated material must be
handled in a laboratory that meets the requirements for Containment Group 3 pathogens.
Identification of the agent: Smears from fresh material may reveal Gram-negative nonsporulating,
nonencapsulated rods. The presence of a capsule-like cover has been demonstrated by electron
microscopy. The bacteria grow aerobically and prefer media that contain glycerol. Unlike the
Pseudomonas species and the closely related bacterium Burkholderia pseudomallei, Burkholderia
mallei is nonmotile. Guinea-pigs are highly susceptible, and males can be used, if strictly
necessary, to recover the organism from a heavily contaminated sample. Commercially available
biochemical identification kits lack diagnostic sensitivity. Specific monoclonal antibodies and
polymerase chain reaction (PCR) as well as real-time PCR assays are available. The latter have
also been evaluated in recent outbreaks.
Mallein and serological tests: Complement fixation test and enzyme-linked immunosorbent
assays are the most accurate and reliable serological tests for diagnostic use. The mallein test is a
sensitive and specific clinical test for hypersensitivity against Burkholderia mallei. Mallein, a water
soluble protein fraction of the organism, is injected subcutaneously, intradermo-palpebrally or given
by eyedrop. In infected animals, the skin or the eyelid swells markedly within 12 days. A rose
bengal plate agglutination test has recently been developed in Russia; it has been validated in
Russia only.
Requirements for vaccines and diagnostic biologicals: There are no vaccines. Mallein purified
protein derivative is currently available commercially from the Central Veterinary Control and
Research Institute, 06020 Etlik, Ankara, Turkey.
A. INTRODUCTION
Glanders is a bacterial disease of perissodactyls or odd-toed ungulates with zoonotic potential known since
ancient times. It is caused by the bacterium Burkholderia mallei (the name recently changed from Pseudomonas
mallei (Yabuuchi et al., 1992) and has been classified in the past as Pfeifferella, Loefflerella, Malleomyces or
Actinobacillus). Outbreaks of the disease may occur in felines living in the wild or in zoological gardens.
Susceptibility to glanders has been proved in camels, bears, wolves and dogs. Carnivores may become infected
by eating infected meat, but cattle and pigs are resistant (Minett, 1959). Small ruminants may also be infected if
kept in close contact to glanderous horses (Wittig et al., 2006). Glanders in the acute form occurs most frequently
in donkeys and mules with high fever and respiratory signs (swollen nostrils, dyspnoea, and pneumonia); death
occurs within a few days. In horses, glanders generally takes a more chronic course and they may survive for
several years. Chronic and subclinical occult cases are dangerous sources of infection due to permanent or
intermittent shedding of bacteria (Wittig et al., 2006).
In horses, inflammatory nodules and ulcers develop in the nasal passages and give rise to a sticky yellow
discharge, accompanied by enlarged firm submaxillary lymph nodes. Stellate scarring follows upon healing of the
ulcers. The formation of nodular abscesses in the lungs is accompanied by progressive debility, febrile episodes,
913
Chapter 2.5.11. Glanders
coughing and dyspnoea. Diarrhoea and polyuria can also occur. In the skin form (farcy), the lymphatics are
enlarged and nodular abscesses (buds) of 0.52.5 cm develop, which ulcerate and discharge yellow oily pus.
Nodules are regularly found in the liver and spleen. Discharges from the respiratory tract and skin are infective,
and transmission between animals, which is facilitated by close contact, by inhalation, ingestion of contaminated
material (e.g. from infected feed and water troughs), or by inoculation (e.g. via a harness) is common. The
incubation period can range from a few days to many months (Monlux, 1984; Wittig et al., 2006).
Glanders is transmissible to humans by direct contact with diseased animals or infected/contaminated material. In
the untreated acute disease. 95% mortality can occur within 3 weeks (Neubauer et al., 1997). However, survival is
possible if the infected person is treated early and aggressively with multiple systemic antibiotic therapies (Kahn &
Ashford, 2001; Srinivasan et al., 2001). A chronic form with abscessation also occurs (Neubauer et al., 1997).
When handling suspect or known infected animals or fomites, stringent precautions should be taken to prevent
self-infection or transmission of the bacterium to other equids. Laboratory samples should be securely packaged,
kept cool and shipped as outlined in Chapter 1.1.1 Collection and shipment of diagnostic specimens. All
manipulations with potentially infected/contaminated material must be performed in a laboratory that meets the
requirements for Containment Group 3 pathogens as outlined in Chapter 1.1.3 Biosafety and biosecurity in the
veterinary microbiology laboratory and animal facilities.
Glanders has been eradicated from many countries by statutory testing, elimination of infected animals, and
import restrictions. It persists in some Asian, African and South American countries. It can be considered a reemerging disease and may be imported by pet or racing equids into glanders free areas (Neubauer et al., 2005).
1.
Cases for specific glanders investigation should be differentiated on clinical grounds from other chronic infections
of the nasal mucosae or sinuses, and from strangles (Streptococcus equi infection), ulcerative lymphangitis
(Corynebacterium pseudotuberculosis), pseudotuberculosis (Yersinia pseudotuberculosis) and sporotrichosis
(Sporotrichium spp.). Glanders should be excluded positively from suspected cases of epizootic lymphangitis
(caused by Histoplasma farciminosum), with which it has many clinical similarities. In humans in particular,
glanders should be distinguished from melioidosis (B. pseudomallei infection), which is caused by an organism
with close similarities to B. mallei (Minett, 1959).
a)
Morphology of Burkholderia mallei

The organisms are fairly numerous in smears from fresh lesions, but in older lesions they are scanty (Wilson
& Miles, 1964). They should be stained by methylene blue or Gram stain. The organisms are mainly
extracellular, fairly straight Gram-negative rods with rounded ends, 25 m long and 0.30.8 m wide with
granular inclusions of various size. They often stain irregularly and do not have a readily visible capsule,
under the light microscope, or form spores. The presence of a capsule-like cover has been established by
electron microscopy. This capsule is composed of neutral carbohydrates and serves to protect the cell from
unfavourable environmental factors. Unlike other organisms in the Pseudomonas group and its close relative
Burkholderia pseudomallei, Burkholderia mallei has no flagellae and are therefore nonmotile (Krieg & Holt,
1984; Sprague & Neubauer, 2004). The organisms are difficult to demonstrate in tissue sections, where they
may have a beaded appearance (Miller et al., 1948). In culture media, they vary in appearance depending
on the age of the culture and type of medium. In older cultures, there is much pleomorphism. Branching
filaments form on the surface of broth cultures (Neubauer et al., 2005).
b)
Cultural characteristics
It is preferable to attempt isolation from unopened uncontaminated lesions (Miller et al., 1948). The organism
is aerobic and facultatively anaerobic only in the presence of nitrate (Gilardi, 1985; Krieg & Holt, 1984),
growing optimally at 37C (Mahaderan et al., 1987). It grows well, but slowly, on ordinary culture media, 72hour incubation of cultures is recommended; glycerol enrichment is particularly useful. After a few days on
glycerol agar, there is a confluent, slightly cream-coloured growth that is smooth, moist, and viscuous. With
continued incubation, the growth thickens and becomes dark brown and tough. It also grows well on glycerol
potato agar and in glycerol broth, on which a slimy pellicle forms. On plain nutrient agar, the growth is much
less luxuriant, and growth is poor on gelatin (Steele, 1980). In samples not obtained under sterile conditions
B. mallei is regularly overgrown by other bacteria.
Alterations to characteristics may occur in vitro, so fresh isolates should be used for identification reactions.
Litmus milk is slightly acidified by B. mallei, and coagulation may occur after long incubation. The organism
reduces nitrates. Although some workers have claimed that glucose is the only carbohydrate that is
fermented (slowly and inconstantly), other workers have shown that if an appropriate medium and indicator
are used, glucose and other carbohydrates, such as arabinose, fructose, galactose and mannose, are
914
consistently fermented by B. mallei (Evans, 1966). Indole is not produced, horse blood is not haemolysed
and no diffusible pigments are produced in cultures (Krieg & Holt, 1984). A commercial laboratory test kit
(e.g. API [Analytical Profile Index] system: Analytab Products, BioMerieux or Biolog [Hayward, California])
can be used for easy confirmation that an organism belongs to the Pseudomonas group. In general,
commercially available systems are not suited to unambiguously identifying members of the steadily growing
number of species of the genus Burkholderia (Glass & Popovic, 2005). Lack of motility is then of special
relevance. A bacteriophage specific for B. mallei is available (Woods et al., 2002).
All culture media prepared should be subjected to quality control and must support growth of the suspect
organism from a small inoculum. The reference strain should be cultured in parallel with the suspicious
samples to ensure that the tests are working correctly.
In contaminated samples, supplementation of media with substances that inhibit the growth of Gram-positive
organisms (e.g. crystal violet, proflavine) has proven to be of use, as has pretreatment with penicillin
(1000 units/ml for 3 hours at 37C) (Minett, 1959). A selective medium has been developed (Xie et al., 1980)
composed of polymyxin E (1000 units), bacitracin (250 units), and actidione (0.25 mg) incorporated into
nutrient agar (100 ml) containing glycerine (4%), donkey or horse serum (10%), and ovine haemoglobin or
tryptone agar (0.1%).
Outside the body, the organism has little resistance to drying, heat, light or chemicals, so that survival
beyond 2 weeks is unlikely (Neubauer et al., 1997). Under favourable conditions, however, it can probably
survive a few months. Burkholderia mallei can remain viable in tap water for at least 1 month (Steele, 1980).
For disinfection, benzalkonium chloride or roccal (1/2000), sodium hypochlorite (500 ppm available
chlorine), iodine, mercuric chloride in alcohol, and potassium permanganate have been shown to be highly
effective against B. mallei (Mahaderan et al., 1987). Phenolic disinfectants are less effective.
c)
Laboratory animal inoculation

Guinea-pigs, hamsters and cats have been used for diagnosis when necessary. If isolation in a laboratory
animal is considered unavoidable, suspected material is inoculated intraperitoneally into a male guinea-pig.
As this technique has a sensitivity of only 20%, the inoculation of at least five animals is recommended
(Neubauer et al., 1997). Positive material will cause a severe localised peritonitis and orchitis (the Strauss
reaction). The number of organisms and their virulence determines the severity of the lesions. Additional
steps are used when the test material is heavily contaminated (Gould, 1950). The Strauss reaction is not
specific for glanders, and other organisms can elicit it. Bacteriological examination of infected testes should
confirm the specificity of the response obtained.
d)
Confirmation by polymerase chain reaction and real-time PCR

In the past few years, several PCR and real-time PCR assays for the identification of Burkholderia mallei
have been developed (Bauernfeind et al., 1998; Lee et al., 2005; Sprague et al., 2002; Thibault et al., 2004;
Ulrich et al., 2006; URen et al., 2005), but only a PCR and a real-time PCR assay were evaluated using
samples from a recent outbreak of glanders in horses (Scholz et al., 2006; Tomaso et al., 2006). These two
assays will be described in more detail here. However, the robustness of these assays will have to be
demonstrated in the future by interlaboratory studies. The guidelines and precautions outlined in Chapter
1.1.5 Principles and methods of validation of diagnostic assays for infectious diseases have to be taken into
account.
DNA preparation
Single colonies are transferred from an agar plate to 200 l lysis buffer (5 buffer D [PCR Optimation Kit,
Invitrogen, DeShelp, The Netherlands, 1/5 diluted in ultra-pure water]; 0.5% Tween 20 [ICI, American
Limited, Merck, Hohenbrunn, Germany]; 2 mg/ml proteinase K [Roche Diagnostics, Mannheim, Germany]).
After incubation at 56C for 1 hour and inactivation for 10 minutes at 95C, 2 and 4 l of the cleared lysate
are used as template in the PCR or the real-time PCR assay, respectively.
Tissue samples of horses (skin, liver, spleen, lung, and conchae) inactivated and preserved in formalin
(48 hours, 10% v/v) are cut with a scalpel into pieces of 0.5 0.5 cm (approximately 500 mg). The
specimens are washed twice in deionised water (10 ml), incubated over night in sterile saline at 4C, and
minced using liquid nitrogen, a mortar and a pestle. Total DNA is prepared from 50 mg tissue using the
TM
QIAamp Tissue Kit according to the manufacturers instructions (Qiagen, Hilden, Germany). DNA is eluted
with 80 l dH2O of which 4 l are used as template.
PCR assay (Scholz et al., 2006)
The assay may have to be adapted to the PCR instrument used with minor modifications to the cycle
conditions and the concentration of the chemicals used.
915
The oligonucleotides used in by Scholz et al. (2006) are designed based on differences of the fliP
sequences from B. mallei ATCC 23344T (accession numbers NC_006350, NC_006351) and B. pseudomallei K96243 (accession numbers NC_006348, NC_006349). Primers Bma-IS407-flip-f (5-TCA-GGT-TTGTAT-GTC-GCT-CGG-3) and Bma- IS407-flip-r (5-CTA-GGT-GAA-GCT-CTG-CGC-GAG-3) are used to
amplify a 989 bp fragment. PCR is done using 50 l ready-to-go mastermix (Eppendorf, Hamburg,
Germany) and 15 pmol of each primer. Thermal cycling conditions are 94C for 30 seconds; 65C for
30 seconds and 72C for 60 seconds. This cycle is repeated for 35 times. A final elongation step is added
(72C for 7 minutes). Visualisation of the products is done by UV light after agarose gel (1% w/v in TAE
buffer) electrophoresis and staining with ethidium bromide. No template controls containing PCR-grade
water instead of template and positive controls containing DNA of B. mallei have to be included in each run
to detect contamination by amplicons of former runs or amplification failure.
The lower detection limit is 10 fg or 2 genome equivalents.
Real-time PCR assay (Tomaso et al., 2006)
The assay has to be adapted to the real-time PCR instrument used with minor modifications, e.g. the cycling
vials have to be chosen according to the manufacturers recommendations and the concentration of the
oligonucleotides may have to be doubled or the labelling of the probes has to be changed. The authors used
TM
real-time PCR system (Stratagene, Amsterdam, The Netherlands) and 96-well plates
a MX3000P
(ThermoFast 96 ABGeneTM, Rapidozym, Berlin, Germany).
The oligonucleotides used by Tomaso et al. (2006) were designed based on differences of the fliP
sequences from B. mallei ATCC 23344T (accession numbers NC_006350, NC_006351) and B. pseudomallei K96243 (accession numbers NC_006348, NC_006349). The fluorogenic probe is synthesised with 6carboxy-fluorescein (FAM) at the 5-end and black hole quencher 1 (BHQ1) at the 3-end. Oligonucleotides
used were Bma-flip-f (5-CCC-ATT-GGC-CCT-ATC-GAA-G-3), Bma-flip-r (5-GCC-CGA-CGA-GCA-CCTGAT-T-3) and probe Bma-flip (5-6FAM-CAG-GTC-AAC-GAG-CTT-CAC-GCG-GAT-C-BHQ1-3).
The 25 l reaction mixture consists of 12.5 l 2 TaqManTM Universal MasterMix (Applied Biosystems,
Foster City, USA), 0.1 l of each primer (10 pmol/l), 0.1 l of the probe (10 pmol/l) and 4 l sample.
Thermal cycling conditions are 50C for 2 minutes; 95C for 10 minutes; 45 (50) cycles at 95C for
25 seconds and 63C for 1 minute. Possible contaminations with amplification products from former
reactions are inactivated by an initial incubation step using uracil N-glycosilase.
The authors suggest to include an internal inhibition control based on a bacteriophage lambda gene target
(Lambda-F [5-ATG-CCA-CGT-AAG-CGA-AAC-A-3] Lambda-R [5-GCA-TAA-ACG-AAG-CAG-TCG-AGT-3],
Lam-YAK [5-YAK-ACC-TTA-CCG-AAA-TCG-GTA-CGG-ATA-CCG-C-DB-3]), which can be titrated to give
reproducible cycle threshold values. However, depending on the sample material a house keeping gene
targeting PCR may be used additionally or as an alternative. No template controls containing 4 l of PCRgrade water instead and positive controls containing DNA of B. mallei have to be included in each run to
detect amplicon contamination or amplification failure.
The linear range of the assay was determined to cover concentrations from 240 pg to 70 fg bacterial
DNA/reaction. The lower limit of detection defined as the lowest amount of DNA that was consistently
detectable in three runs with eight measurements each is 60 fg DNA or four genome equivalents (95%
probability). The intra-assay variability of the fliP PCR assay for 35 pg DNA/reaction is 0.68 % (based on Ct
values) and for 875 fg 1.34%, respectively. The inter-assay variability for 35 pg DNA/reaction is 0.89%
(based on Ct values) and for 875 fg DNA 2.76 %, respectively.
e)
Other methods
The genome of the Burkholderia mallei type strain ATCC 23344T was sequenced in 2004 (Nierman et al.,
2004). Several genomes of other isolates followed and revealed a wide genomic plasticity. Passages in
different host species or culture media may provoke considerable sequence alterations (Romero et al.,
2006). The loss of the ability to produce LPS and/or capsule polysaccharide upon ongoing culture due to
mutation is a well known fact and results in reduced or absent virulence and influences serologic tests
(Neubauer et al., 2005). Several molecular typing techniques have successfully been introduced. Simple
molecular techniques like PCR-restriction fragment length polymorphism (Tanpiboonsak et al., 2004) and
pulsed field gel electrophoresis (Chantratita et al., 2006) can be used for further discrimination of isolates.
Ribotyping using restriction enzymes PstI and EcoRI in combination with an E. coli 18-mer rDNA probe
produced 17 distinct ribotypes within 25 B. mallei isolates (Harvey & Minter, 2005). These techniques are
still the in-house tests of specialised laboratories as an extensive strain collection is necessary. Multilocus
sequence typing (MLST) can be done with purified DNA so there is no need for excessive cultivation of the
agent or the keeping of strain collections. Web-based analysis might even enhance diagnostics (Godoy et
916
al., 2003). No specific histopathology features can be described for lesions caused by B. mallei. For
immunuhistochemical analysis, B. mallei specific hyperimmune sera of rabbits can be used.
2.
Serological tests and the mallein test
a)
Complement fixation test (a prescribed test for international trade)

Although not as sensitive as the mallein test, the CF test is an accurate serological test that has been used
for glanders diagnosis for many years (Blood & Radostits, 1989). It is reported to be 9095% accurate,
serum being positive within 1 week of infection and remaining positive in the case of exacerbation of the
chronic process (Steele, 1980). Recently, however, the specificity of CF testing has been questioned
(Neubauer et al., 2005).
Antigen preparation (Kelser & Reynolds, 1935)
i)
Flasks of beef infusion broth with 3% glycerol are inoculated with log-phase growth B. mallei and
incubated at 37C for 812 weeks.
ii)
The cultures are inactivated by exposing the flasks to flowing steam (100C) for 60 minutes.
iii)
The clear supernatant is decanted and filtered. The filtrate is heated again by exposure to live steam
for 75 minutes on 3 consecutive days, and clarified by centrifugation.
iv)
The clarified product is concentrated to one-tenth the original volume by evaporation on a steam or hot
water bath.
v)
Concentrated antigen is bottled in brown-glass bottles to protect from light and stored at 4C. Antigen
has been shown to be stable for at least 10 years in this concentrated state.
vi)
Lots of antigen are prepared by diluting the concentrated antigen 1/20 with sterile physiological saline
with 0.5% phenol. The diluted antigen is dispensed into brown-glass vials and store at 4C. The final
working dilution is determined by a block titration. The final working dilution for CF test use is made at
the time the CF test is performed.
The resulting antigen is primarily lipopolysaccharide. An alternative procedure is to use young cultures by
growing the organism on glycerolagar slopes for 12 hours and washing off with normal saline. A
suspension of the culture is heated for 1 hour at 70C and the heat-treated bacterial suspension is used as
antigen. The disadvantage of this antigen preparation method is that the antigen contains all the bacterial
cell components. The antigen should be safety tested by inoculating blood agar plates.
Test procedure (NVSL, 2006)
i)
Serum is diluted 1/5 in veronal (barbiturate) buffered saline containing 0.1% gelatin (VBSG) or CFD
(complement fixation diluent available as tablets) without gelatine.
ii)
Diluted serum is inactivated for 30 minutes at 56C. The USDA complement fixation protocol calls for
inactivation for 35 minutes (NVSL, 2006). (Serum of equidae other than horses should be inactivated at
63C for 30 minutes.)
iii)
Twofold dilutions of the sera are prepared in 96-well round-bottom microtitre plates.
iv)
Guinea-pig complement is diluted in the chosen buffer and 5 (or optionally 4) complement haemolytic
units-50% (CH50) are used.
v)
Sera, complement and antigen are reacted in the plates and incubated for 1 hour at 37C. (An alternate
acceptable procedure is overnight incubation at 4C.)
vi)
A 2% suspension of sensitised washed sheep red blood cells is added. The USDA protocol calls for
confirmation of positive reactions in a tube test using 3% sheep red blood cells (NVSL, 2006).
vii)
Plates are incubated for 45 minutes at 37C, and then centrifuged for 5 minutes at 600 g.
A sample that produces 100% haemolysis at the 1/5 dilution is negative, 2575% haemolysis is suspicious,
and no haemolysis (100% fixation) is positive. Unfortunately, false-positive results can occur, and
B. pseudomallei and B. mallei cross react and cannot be differentiated by serology (Blood & Radostits, 1989;
Neubauer et al., 1997). Also healthy horses can have a false positive CF reaction for a variable period
following a mallein intradermal test.
b)
Enzyme-linked immunosorbent assays

Both plate and membrane (blot) enzyme-linked immunosorbent assays (ELISAs) have been reported for the
serodiagnosis of glanders, but none of these procedures has been shown to differentiate serologically
between B. mallei and B. pseudomallei. Blotting approaches have involved both dipstick dot-blot and
917
electrophoretically separated and transferred western blot methods (Katz et al., 1999; Verma et al., 1990). A
competitive ELISA that uses an uncharacterised anti-lipopolysaccharide monoclonal antibody has also been
developed and found to be similar to the CF test in performance (Katz et al., 2000). Continuing development
of monoclonal antibody reagents specific for B. mallei antigenic components offers the potential for more
specific ELISAs in the foreseeable future that will help resolve questionable test results of quarantined
imported horses (Burtnick et al., 2002; Feng et al., 2006; Khrapova et al., 1995; Neubauer et al., 1997). At
this time, none of these tests has been validated.
c)
The mallein test

The mallein purified protein derivative (PPD), which is available commercially, is a solution of water-soluble
protein fractions of heat-treated B. mallei. The test depends on infected horses being hypersensitive to
mallein. Advanced clinical cases in horses and acute cases in donkeys and mules may give inconclusive
results requiring additional methods of diagnosis to be employed (Allen, 1929).
The intradermo-palpebral test
This is the most sensitive, reliable and specific test for detecting infected perissodactyls or odd-toed
ungulates, and has largely displaced the ophthalmic and subcutaneous tests (Blood & Radostits, 1989):
0.1 ml of concentrated mallein PPD is injected intradermally into the lower eyelid and the test is read at
24 and 48 hours. A positive reaction is characterised by marked oedematous swelling of the eyelid, and
there may be a purulent discharge from the inner canthus or conjunctiva. This is usually accompanied by a
rise in temperature. With a negative response, there is usually no reaction or only a little swelling of the
lower lid.
d)
Other serological tests

The avidinbiotin dot ELISA has been described (Verma et al., 1990), but has not yet been widely used or
validated. The antigen is heat-inactivated bacterial culture that has been concentrated and purified. A dot of
this antigen is placed on a nitrocellulose dipstick that is then used to test for antibody against B. mallei in
equine serum. Using antigen-dotted, preblocked dipsticks, the test can be completed in approximately
1 hour. Serum or whole blood can be used for the test, and partial haemolysis does not impart any
background colour to the antigen-coated area on the nitrocellulose. Recently, polysaccharide microarray
technology has offered a new promising approach to improve sensitivity in serology (Parthasarathy et al.,
2006).
The rose bengal plate agglutination test (RBT) has been described for the diagnosis of glanders in horses
and other susceptible animals; this test has been validated in Russia only. The antigen is a heat-inactivated
bacterial suspension coloured with rose bengal, which is used in a plate agglutination test.
The accuracy of other agglutination tests and precipitin is unsatisfactory for use in control programmes.
Horses with chronic glanders and those in a debilitated condition give negative or inconclusive results.

No vaccines are available.
Mallein PPD for use in performing the intradermo-palpebral and ophthalmic tests is produced commercially by the
Central Veterinary Control and Research Institute, 06020 Etlik, Ankara, Turkey.
The ID-Lelystad has provided the following information on requirements for mallein PPD.
1.
Seed management
Three strains of Burkholderia mallei are employed in the production of mallein PPD, namely Bogor strain
(originating from Indonesia), Mukteswar strain (India) and Zagreb strain (Yugoslavia). The seed material is kept
as a stock of freeze-dried cultures. The strains are subcultured on to glycerol agar at 37C for 12 days. For
maintaining virulence and antigenicity, the strains may be passaged through guinea-pigs.
2.
DorsetHenley medium, enriched by the addition of trace elements, is used for production of mallein PPD. The
liquid medium is inoculated with a thick saline suspension of B. mallei, grown on glycerol agar. The production
medium is incubated at 37C for about 10 weeks. The bacteria are then killed by steaming for 3 hours in a Kochs
918
steriliser. The fluid is then passed through a layer of cotton wool to remove coarse bacterial clumps. The resulting
turbid fluid is cleared by membrane filtration, and one part trichloroacetic acid 40% is immediately added to nine
parts culture filtrate. The mixture is allowed to stand overnight and a light brownish to greyish precipitate settles.
The supernatant fluid is pipetted off and discarded. The precipitate is centrifuged for 15 minutes at 2500 g and the
layer of precipitate is washed three or more times in a solution of 5% NaCl, pH 3, until the pH is 2.7. The washed
precipitate is dissolved by stirring with a minimum of an alkaline solvent. The fluid is dark brown and a pH of 6.7
will be obtained. This mallein concentrate has to be centrifuged thoroughly and the supernatant is diluted with an
equal amount of a glucose buffer solution. The protein content of this product is estimated by the Kjeldahl method
and freeze-dried after it has been dispensed into ampoules.
3.
In-process control
During the period of incubation, the flasks are inspected frequently for any signs of contamination, and suspect
flasks are discarded. A typical growth of the B. mallei cultures comprises turbidity, sedimentation, some surface
growth with a tendency towards sinking, and the formation of a conspicuous slightly orange-coloured ring along
the margin of the surface of the medium.
4.
Batch control
Each batch of mallein PPD is tested for sterility, safety, preservatives, protein content and potency.
Sterility testing is performed according to the European Pharmacopoeia guidelines.
The examination for safety is conducted on from five to ten normal healthy horses by carrying out the intradermopalpebral test. The resulting swelling should be, at most, barely detectable and transient, without any signs of
conjunctival discharge.
Preparations containing phenol as a preservative should not contain more than 0.5% (w/v) phenol. The protein
content should be not less than 0.95 mg/ml and not more than 1.05 mg/ml.
Potency testing is performed in guinea-pigs and horses. The animals are sensitised by subcutaneous inoculation
with a concentrated suspension of heat-killed B. mallei in paraffin oil or incomplete Freunds adjuvant. Cattle can
also be used instead of horses. The production batch is bioassayed against a standard mallein PPD by
intradermal injection in 0.1 ml doses in such a way that complete randomisation is obtained.
In guinea-pigs, the different areas of erythema are measured after 24 hours, and in horses the increase in skin
thickness is measured by calipers. The results are statistically evaluated, using standard statistical methods for
parallel-line assays.
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*
* *
NB: There are OIE Reference Laboratories for Glanders
diagnostic tests, reagents and diagnostic biologicals for glanders
922
CHAPTER 2.5.12.
HORSE MANGE
See Chapter 2.9.8. Mange
*
* *
923
CHAPTER 2.5.13.
VENEZUELAN EQUINE ENCEPHALOMYELITIS
SUMMARY
Venezuelan equine encephalomyelitis (VEE) viruses, of the genus Alphavirus of the family
Togaviridae, cause disease ranging from mild febrile reactions to fatal encephalitic zoonoses in
Equidae and humans. They are transmitted by haematophagous insects, primarily mammalophilic
mosquitoes.
The VEE complex of viruses includes six antigenic subtypes (IVI). Within subtype I there are five
antigenic variants (variants ABF). Originally, subtypes I-A and I-B were considered to be distinct
variants, but they are now considered to be identical (I-AB). Antigenic variants I-AB and I-C are
associated with epizootic activity in equids and human epidemics. Historically, severe outbreaks
have involved many thousands of human and equine cases. The other three variants of subtype I
(I-D, I-E, I-F) and the other five subtypes of VEE (IIVI) circulate in natural enzootic cycles. Equidae
serve as amplifying hosts for epizootic VEE strains while enzootic VEE viruses cycle primarily
between sylvatic rodents and mosquitoes. Enzootic variants and subtypes have been considered to
be nonpathogenic for equids, but can cause clinical disease in humans. During 1993 and 1996
however, limited outbreaks of encephalitis in horses in Mexico were shown to be caused by
enzootic VEE viruses of subtype I-E.
Identification of the agent: Diagnosis of VEE virus infection can be confirmed by the isolation,
identification, and antigenic classification of the isolated virus.
A presumptive diagnosis of equine encephalomyelitis can be made when susceptible animals in
tropical or subtropical areas display clinical signs of encephalomyelitis where haematophagous
insects are active. VEE virus can be isolated in cell cultures or in laboratory animals using the blood
or serum of febrile animals in an early stage of infection. It is recovered less frequently from the
blood or brain tissue of encephalitic animals.
VEE virus can be identified by complement fixation, haemagglutination inhibition, plaque reduction
neutralisation (PRN), or immunofluorescence tests using VEE-specific antibodies. Specific
identification of epizootic VEE variants can be made by the indirect fluorescent antibody test, or a
differential PRN test using subtype- or variant-specific monoclonal antibody, or by nucleic acid
sequencing.
Serological tests: Specific antibodies may be demonstrated by PRN tests against epizootic VEE
virus variants or by IgM capture enzyme-linked immunosorbent assay. Antibody can also be
demonstrated by the haemagglutination inhibition or the complement fixation tests.
Any diagnosis of VEE in an individual that is based on seroconversion in the absence of an
epizootic should be made with care. Infections of equids with enzootic VEE viruses produce a low
level viraemia accompanied by antibody development, but without clinical disease in most cases.
Antibody induced by such subclinical infections may be reactive to epizootic VEE virus variants.
Requirements for vaccines and diagnostic biologicals: The only acceptable vaccines against
VEE are an attenuated virus vaccine, made with strain TC-83, or inactivated virus preparations also
made from this strain. Attenuated virus is immunogenic when given by intramuscular injection, but
sometimes causes adverse reactions in the recipient.
Formalin-inactivated virulent VEE virus preparations should never be used in equids, as residual
virulent virus can remain after formalin treatment, and thereby cause severe illness in both animals
and humans. Epizootics of VEE have occurred from the use of such formalin-treated viruses.
924
Chapter 2.5.13. Venezuelan equine encephalomyelitis
A. INTRODUCTION
Venezuelan equine encephalomyelitis (VEE) is an arthropod-borne inflammatory viral infection of equines and
humans, resulting in mild to severe febrile and, occasionally fatal, encephalitic disease.
VEE viruses form a complex within the genus Alphavirus, family Togaviridae. The VEE virus complex is
composed of six subtypes (IVI). Subtype I includes five antigenic variants (ABF), of which variants 1-AB and 1C are associated with epizootic VEE in equids and concurrent epidemics in humans (Calisher et al., 1980; Monath
& Trent, 1981; Pan-American Health Organization, 1972; Walton, 1981; Walton et al., 1973; Walton & Grayson,
1989). The epizootic variants 1-AB and 1-C are thought to originate from mutations of the enzootic 1-D serotype
(Weaver et al., 2004); 1-AB and 1-C isolates have only been obtained during equine epizootics. The enzootic
strains include variants 1-D, 1-E and 1-F of subtype I, subtype II, four antigenic variants (AD) of subtype III, and
subtypes IVVI. Normally, enzootic VEE viruses do not produce clinical encephalomyelitis in the equine species
(Walton et al., 1973), but in 1993 and 1996 in Mexico, the 1-E enzootic subtype caused limited epizootics in
horses. The enzootic variants and subtypes can produce clinical disease in humans (Monath & Trent, 1981; PanAmerican Health Organization, 1972; Powers et al., 1997; Walton, 1981; Walton & Grayson, 1989).
Historically, epizootic VEE was limited to northern and western South America (Venezuela, Colombia, Ecuador,
Peru and Trinidad) (Pan-American Health Organization, 1972). From 1969 to 1972, however, epizootic activity
(variant 1-AB) occurred in Guatemala, El Salvador, Nicaragua, Honduras, Costa Rica, Belize, Mexico, and the
United States of America (USA) (Texas). Epizootics of VEE caused by I-AB or I-C virus have not occurred in
North America and Mexico since 1972. Recent equine and human isolations of epizootic VEE virus were subtype
1-C strains from Venezuela in 1993, 1995 and 1996 and Colombia in 1995.
The foci of enzootic variants and subtypes are found in areas classified as tropical wet forest, i.e. those areas with
a high water table or open swampy areas with meandering sunlit streams. These are the areas of the Americas
where rainfall is distributed throughout the year or areas permanently supplied with water. Enzootic viruses cycle
among rodents, and perhaps birds, by the feeding of mosquitoes (Monath & Trent, 1981; Pan-American Health
Organization, 1972; Walton, 1981; Walton & Grayson, 1989). Enzootic VEE strains have been identified in the
Florida Everglades (subtype II), Mexico (variant I-E), Central American countries (variant I-E), Panama (variants ID and I-E), Venezuela (variant I-D), Colombia (variant I-D), Peru (variants 1-D, III-C, and III-D), French Guiana
(variant III-B and subtype V), Ecuador (variant I-D), Suriname (variant III-A), Trinidad (variant III-A), Brazil
(variants I-F and III-A and subtype IV), and Argentina (subtype VI). In an atypical ecological niche, variant III-B
has been isolated in the USA (Colorado and South Dakota) in an unusual association with birds (Monath & Trent,
1981; Pan-American Health Organization, 1972; Walton, 1981; Walton & Grayson, 1989).
A tentative diagnosis of viral encephalomyelitis in equids can be based on the occurrence of acute neurological
disease during the summer in temperate climates or in the wet season in tropical or subtropical climates. These
are the seasons of haematophagous insect activity. Virus infection will result in clinical disease in many equids
concurrently rather than in isolated cases. Epizootic activity can move vast distances through susceptible
populations in a short time (Monath & Trent, 1981; Pan-American Health Organization, 1972; Walton, 1981;
Walton & Grayson, 1989). Differential diagnoses include eastern or western equine encephalomyelitis (chapter
2.5.5), Japanese encephalitis (chapter 2.1.7), West Nile fever (chapter 2.1.20), rabies (chapter 2.1.13) and other
infectious, parasitic, or non-infectious agents producing similar signs.
Human VEE virus infections have originated by aerosol transmission from the cage debris of infected laboratory
rodents and from laboratory accidents. Infections with both epizootic and enzootic variants and subtypes have
been acquired by laboratory workers (American Committee on Arthropod-Borne Viruses [ACAV], 1980). Severe
clinical disease or death can occur in humans. Those who handle infectious VEE viruses or their antigens
prepared from infected tissues or cell cultures should be vaccinated and shown to have demonstrable immunity in
the form of VEE virus-specific neutralising antibody (Berge et al., 1961; Pan-American Health Organization,
1972). All procedures producing aerosols from VEE virus materials should be conducted in biosafety cabinets at
containment level 3 (see Chapter 1.1.3 Biosafety and biosecurity in the veterinary microbiological laboratory and
animal facilities) (ACAV, 1980; United States Department of Health and Human Services, 1999).
1.
A confirmatory diagnosis of VEE is based on the isolation and identification of the virus or on the demonstration of
seroconversion. The period of viraemia coincides with the onset of pyrexia within 1224 hours of infection.
Viraemia terminates 56 days after infection, and coincides with the production of neutralising antibodies and the
appearance of clinical neurological signs. Frequently, VEE viruses cannot be isolated from the brains of infected
equids. Blood samples for virus isolation should be collected from febrile animals that are closely associated with
clinical encephalitic cases.
925
Virus may be isolated from the blood or sera of infected animals by inoculating 14-day-old mice or hamsters
intracerebrally or by the inoculation of other laboratory animals, such as guinea-pigs and weaned mice. It may
also be isolated by the inoculation of various cell cultures including African green monkey kidney (Vero), rabbit
kidney (RK-13), baby hamster kidney (BHK-21), or duck or chicken embryo fibroblasts, or by inoculation of
embryonated chicken eggs. Details of virus identification techniques are described in chapter 2.5.5.
Isolates can be identified as VEE virus by complement fixation (CF), haemagglutination inhibition (HI), or plaque
reduction neutralisation (PRN) tests, or by immunofluorescence as described in chapter 2.5.5. The VEE virus
isolates can be characterised by the indirect fluorescent antibody or PRN tests using monoclonal antibody or by
nucleic acid sequencing. The VEE virus characterisation should be carried out in a reference laboratory (see
Table given in Part 4 of this Terrestrial Manual).
2.
Serological tests
Diagnosis of VEE virus infection in equids requires the demonstration of specific antibodies in paired serum
samples collected in the acute and convalescent phases. After infection, PRN antibodies appear within 57 days,
CF antibodies within 69 days, and HI antibodies within 67 days. The second convalescent phase serum sample
should be collected 47 days after the collection of the first acute phase sample or at the time of death. The
serological procedures are described in detail in chapter 2.5.5. Vaccination history must be taken into account
when interpreting any of the VEE serological test results. In horses not recently vaccinated with an attenuated live
virus strain, demonstration of VEE-specific serum IgM antibodies in a single serum sample supports recent virus
exposure.
Any diagnosis of VEE in an individual that is based on seroconversion in the absence of an epizootic should be
made with care. Although enzootic subtypes and variants are nonpathogenic for equids, infection will stimulate
antibody production to epizootic VEE virus variants.

The acceptable vaccines against VEE infection are an attenuated virus vaccine, strain TC-83, and an inactivated
virus preparation made from that strain (Monath & Trent, 1981; Pan-American Health Organization, 1972; Walton,
1981; Walton & Grayson, 1989). The inactivated vaccine is now the most widely used, and is marketed in
EEE/VEE, EEE/WEE/VEE, EEE/WEE//VEE/tetanus toxoid, and EEE/WEE/VEE/West Nile virus/tetanus toxoid
combinations.
Inactivated vaccine should be administered in two doses with an interval of 24 weeks between doses. Annual
revaccination is recommended.
Attenuated vaccine should be reconstituted with physiological saline and used immediately. Multidose vials are
kept on ice while the vaccine is being used. Any vaccine not used within 4 hours of reconstitution should be safely
discarded. Foals under 2 weeks of age and pregnant mares should not be vaccinated. Animals are vaccinated
subcutaneously in the cervical region with a single dose. Revaccination is not recommended.
NOTE: Formalin-treated preparations of virulent epizootic VEE virus should never be used in equids. Residual
virulent virus can remain after formalin treatment, and result in severe illness. Epizootics of VEE have occurred in
Central and Southern America from the use of such preparations (Walton, 1981; Weaver et al., 1999).
1.
Seed management1
a)

Attenuated VEE virus vaccine strain TC-83 originated from the Trinidad donkey strain (a variant of I-AB) of
epizootic VEE virus isolated in 1944. This strain was derived by serial passage of the Trinidad donkey strain
in fetal guinea pig heart cells. It is safe and immunogenic at the established passage levels, and induces
protective immunity in vaccinated equids, although adverse reactions can sometimes occur. The vaccine
was originally developed for use in personnel involved in high-risk VEE virus research. Suitable seed lots
should be maintained at 70C in a lyophilised state.
926
Sections on Seed management, Manufacture, In-process control, and Batch control are taken from the Biotechnology,
Biologics, and Environmental Protection Division of the United States Department of Agricultures Animal and Plant Health
Inspection Service (APHIS).
b)
Method of culture
The virus is grown in fetal guinea pig heart cell cultures in a suitable medium.
c)
The cells used for vaccine production must be free from bacterial, fungal, mycoplasmal, and viral
contamination. VEE virus is identified in batches of vaccine by PRN tests against hyperimmune serum. For
inactivated vaccines of cell culture origin, strain TC-83 virus is treated with formaldehyde.
2.
Method of manufacture (see footnote 1)
Vaccine is produced by harvesting supernatant fluid from fetal guinea pig heart monolayers in which the
replication of attenuated VEE virus has occurred. The monolayers are maintained at approximately 37C. The
time of harvesting is determined by the occurrence of characteristic cytopathic changes when approximately 70
100% of the cell sheet is affected, typically 13 days after infection. The supernatant fluid is clarified by low speed
centrifugation and suitable stabilisers are added to protect the virus during freezing and lyophilisation.
3.
In-process control (see footnote 1)
Cultures should be examined daily for cytopathic changes. After harvesting, the virus suspension should be
tested for the presence of microbial contaminants.
Inactivated vaccines derived from attenuated strain TC-83 virus should be checked to exclude the presence of
viable virus after formalin treatment.
4.
Batch control (see footnote 1)
a)
Sterility
b)
Safety
Safety tests of the inactivated vaccine are described in chapter 2.5.5.
Safety tests of the attenuated vaccine are conducted in mice. A 0.5 ml dose is injected intraperitoneally or
subcutaneously into each of eight mice, and the animals are kept under observation for 7 days. If adverse
reactions attributable to the product occur during this period, the product is considered to be unsatisfactory.
c)
Potency
Potency tests of the inactivated vaccine are described in chapter 2.5.5 except that antibody titre in
inoculated guinea-pigs will be 1/4.
Potency of the attenuated vaccine can be determined by testing in horses. Each of 20 susceptible horses is
inoculated subcutaneously with 1 ml of lyophilised vaccine that has a reconstituted virus titre of at least
2.5 log10 TCID50 (50% tissue culture infective dose) per ml. For a valid test, at least 19 of 20 vaccinated
horses must have HI antibody titres of at least 1/20 or serum neutralising antibody titres of at least 1/40
within 2128 days of vaccination.
When tested at any time within the expiration period following lyophilisation, the product must have a virus
titre of 0.7 log10 greater than that used to test horses as described above, but no less than
2.5 log10 TCID50/dose.
The final product must be free from bacterial, fungal, mycoplasmal, or extraneous viral contaminants.
d)
Comprehensive studies on duration of immunity are not available. An annual revaccination is recommended
for the inactivated vaccine. Foals that are vaccinated at under 1 year of age should be revaccinated before
the next vector season. Revaccination with the attenuated vaccine is not recommended.
927
e)
Stability
The lyophilised vaccine is stable and immunogenic for 3 years if kept refrigerated at 27C. After 3 years,
vaccine should be discarded. The vaccines should be used immediately after reconstitution. Multidose vials
of the attenuated vaccine should be kept on ice while being used. All unused vaccine should be safely
discarded 4 hours after reconstitution.
f)
Preservatives
The preservatives used are thimerosal at a 1/1000 dilution and antibiotics (neomycin, polymyxin,
amphotercin B, and gentamicin).
g)
Pregnant mares and foals under 2 weeks of age should not be vaccinated.
All personnel handling infectious VEE viruses or their antigens prepared from infected tissues or cell cultures
should be vaccinated and shown to have demonstrable immunity in the form of VEE virus-specific
neutralising antibody. All procedures producing aerosols from VEE virus materials should be conducted in
biosafety cabinets with biocontainment and efficient filtration of the exhaust air from the laboratory (ACAV,
1980; United States Department of Health and Human Services, 1999).
5.
a)
Safety and potency

Safety and potency tests are as outlined above under Batch control (Sections C.4.b and C.4.c). The
attenuated vaccine must have a virus titre of no less than 2.5 log10 TCID50/dose.
REFERENCES
AMERICAN COMMITTEE ON ARTHROPOD-BORNE VIRUSES (ACAV), SUBCOMMITTEE ON ARBOVIRUS LABORATORY SAFETY
(1980). Laboratory safety for arboviruses and certain viruses of vertebrates. Am. J. Trop. Med. Hyg., 29, 1359
1381.
BERGE T.O., BANKS I.S. & TIGERTT W.D. (1961). Attenuation of Venezuelan equine encephalomyelitis virus by in
vitro cultivation in guinea pig heart cells. Am. J. Hyg., 73, 209218.
CALISHER C.H., SHOPE R.E., BRANDT W., CASALS J., KARABATSOS N., MURPHY F.A., TESH R.B. & WIEBE M.E. (1980).
Proposed antigenic classification of registered arboviruses. I. Togavirus Alphavirus. Intervirology, 14, 229232.
MONATH T.P. & TRENT D.W. (1981). Togaviral diseases of domestic animals. Comp. Diagn. Vir. Dis., 4, 331440.
PAN-AMERICAN HEALTH ORGANIZATION (1972). Venezuelan encephalitis. In: Proceedings of a Workshop/
Symposium on Venezuelan Encephalitis Virus. Sci. Publ. No. 243, Washington DC, USA, 416 pp.
POWERS A.M., OBERSTE M.S., BRAULT A.C., RICO-HESSE R., SCHMURA S.M., SMITH J.F., KANG W, SWEENEY W.P. &
WEAVER S.C. (1997). Repeated emergence of epidemic/epizootic Venezuelan equine encephalitis from a single
genotype of enzootic subtype ID virus. J. Virol., 71, 66976705.
UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES (1999). Biosafety in Microbiological and Biomedical
Laboratories. US Government Printing Office, Washington DC, USA, 190196.
WALTON T.E. (1981). Chapter 24. Equine encephalomyelitis. In: Virus Diseases of Food Animals. A World
Geography of Epidemiology and Control. Disease Monographs, Vol. 2, Gibbs E.P.J., ed. Academic Press, New
York, USA, 587625.
WALTON T.E., ALVAREZ O. Jr, BUCKWALTER R.M. & JOHNSON K.M. (1973). Experimental infection of horses with
enzootic and epizootic strains of Venezuelan equine encephalomyelitis virus. J. Infect. Dis., 128, 271282.
WALTON T.E. & GRAYSON M.A. (1989). Chapter 46. Venezuelan equine encephalomyelitis. In: The Arboviruses:
Epidemiology and Ecology, Vol. 4, Monath T.P., ed. CRC Press, Boca Raton, Florida, USA, 203231.
928
WEAVER S.C., FERRO C., BARREREA R. BOSCHELL J. & NAVARRO J.C. (2004) Venezuelan equine encephalitis. Annu.
Rev. Entomol., 49, 141174.
WEAVER S.C., PFEFFER M., MARRIOTT K., KANG W. & KINNEY R.M. (1999) Genetic evidence for the origins of
Venezuelan equine encephalitis virus subtype IAB outbreaks. Am. J. Trop. Med. Hyg., 60, 441448.
*
* *
NB: There is an OIE Reference Laboratory for Venezuelan equine encephalomyelitis
diagnostic tests, reagents and vaccines for Venezuelan equine encephalomyelitis
929
SECTION 2.6.
LEPORIDAE
CHAPTER 2.6.1.
MYXOMATOSIS
SUMMARY
Myxomatosis is a major disease of the European rabbit caused by the Myxoma virus, a member of
the Poxviridae family. The diagnosis of myxomatosis, regardless of its clinical form, depends on the
isolation and identification of the virus or the demonstration of its antigens. The presence of a
humoral immune response facilitates a retrospective diagnosis of a mild form of the disease, and
can provide an indication of the prevalence of infection in a rabbit population. The disease is
characterised by gross myxomatous skin lesions.
Identification of the agent: When skin lesions are present on a dead rabbit, the viral antigen may
be demonstrated by immunodiffusion tests on lesion fragments. Monolayer cell cultures of rabbit
kidney inoculated with lesion material will show the characteristic cytopathic effects of poxviruses.
The presence of virus can be confirmed by immunofluorescence and negative-staining electron
microscopy.
The inoculation of rabbits with suspect material takes longer to identify infection, but this will serve
to confirm the presence of infective virus and indicate its pathogenicity.
Serological tests: Identification and titration of specific antibodies arising from natural infection or
from immunisation are done by traditional complement fixation or by a recently developed and more
sensitive enzyme-linked immunosorbent assay (ELISA), which is not affected by pro- or anticomplementary factors. The difficulty in obtaining blood samples from representative members of a
population can be obviated by collecting blood dried on filter paper; this can later be extracted and
examined by the indirect fluorescent antibody test or ELISA. Microcapillary blood sampling can also
be used for the ELISA.
Qualitative agar gel immunodiffusion tests have the advantage of detecting both antigen and
humoral antibodies.
Requirements for vaccines and diagnostic biologicals: Modified live virus vaccines prepared
from fibroma virus or modified Myxoma virus strains are available for immunisation of rabbits.
A. INTRODUCTION
Myxomatosis is a major viral disease of wild and domestic European rabbits (Oryctolagus cuniculus) caused by
the Myxoma virus (MV), a member of the Poxviridae family. The aetiological agent was first isolated from a colony
of laboratory rabbits in Uruguay in 1898 and identified as a poxvirus in 1927. The natural hosts are two species of
leporid: Sylvilagus brasiliensis in South America (South American strains) and Sylvilagus bachmani (Californian
strains) in California (Fenner, 1994). In its natural hosts, the viral strains produce only a benign fibroma,
generalised disease occurring only in juvenile animals. In the European rabbits, two forms of the disease have
been identified to date: the nodular (classical) form and the amyxomatous (respiratory) form.
Florid skin lesions and severe immunodysfunction, accompanied by supervening Gram-negative bacterial
infections of the respiratory tract, characterise the nodular myxomatosis syndrome caused by a virulent MV strain.
931
Chapter 2.6.1. Myxomatosis
Prototype strains of virus deriving from the Australian and European outbreaks have been designed that
characterise the various virulence grades (from grade I to grade V) as determined in laboratory rabbits (Fenner &
Ratcliffe, 1965). After infection with a grade I (the most virulent) strain, the first sign of infection is a lump at the
site of infection, which increases in size and usually becomes protuberant and ulcerates. An acute blepharoconjunctivitis and an oedematous swelling of the perineum and scrotum gradually develop. The secondary skin
lesions appear on about the sixth or the seventh day (Fenner, 1994). Death usually occurs between the eighth
and fifteenth day post-infection. After infection with grade II to V strains, the clinical signs are usually the same
with the exception that they evolve more slowly and are less severe. When animals survive, the lesions
progressively heal. The mortality rate fluctuates between 20 and 100%, according to the viral strain. The natural
mode of transmission of the nodular form is by biting insects, but limited transmission from rabbit to rabbit is
possible if they are closely confined. This form is mainly observed in small-scale rabbitries (Arthur & Louzis,
1988). The clinical signs of amyxomatous myxomatosis are mainly respiratory, skin nodules being few and small.
It might be thought that amyxomatous myxomatosis would not spread via vectors but through direct contact, and
would arise predominantly in intensive enclosed rabbitries. However, this last notion must be viewed with caution
as the disease has also been observed in wild rabbits. So far, these forms of myxomatosis have been reported
only in France (Brun et al., 1981; Joubert et al., 1982), Spain (Rosell et al., 1984) and more recently in Belgium
(Marlier & Vindevogel, 1996).
As MV has a very narrow host range (it only infects leporides), there is no health risk to humans.
As the signs of the disease become less distinct with the attenuation of virus strains, the submission of samples
for laboratory diagnosis becomes more important. Moreover, the expression of the ectodermotropism is clearly
reduced for amyxomatous MV strains, and the clinical diagnosis of the amyxomatous form of myxomatosis is
clearly more difficult than for the classical one. The different techniques available vary in their ability to detect MV
in typical myxomatous lesions, oedema of the eyelids or genital oedema. Nevertheless, the diagnosis of
attenuated typical myxomatosis or of atypical (amyxomatous) forms most often requires the isolation of the virus
by inoculation of sensitive cell lines such as the RK-13 cell line (ATCC CCL37) and identification of the virus by
immunological methods. In both cases, the agent can also be identified by demonstration of MV nucleic acid by
polymerase chain reaction, molecular techniques were not specifically evaluated for diagnosis.
1.
A portion of lesion (myxoma or pieces of organs or tissues, especially eyelids) is excised with scissors. Myxoma
are separated from the epidermis and superficial dermis. This is washed with phosphate buffered saline (PBS)
with antibiotics as defined below and homogenised with ground glass at a dilution rate of 1 g tissue/4.59.0 ml of
PBS. Cells are disrupted by two freezethaw cycles, or by ultrasonication to liberate virions and viral antigens.
This suspension is centrifuged for 510 minutes at 1500 g. The supernatant fluid is used for the tests.
a)
Culture
Isolation of the virus in cell culture is accomplished using primary cultures of rabbit kidney (RK) cells, or with
established cell lines, such as RK-13, in Opti-MEM containing 2% calf serum, 300 international units (IU)/ml
penicillin; 300 g/ml streptomycin; 100 g/ml gentamycin; 50 IU/ml nystatin (mycostatin); and 5 g/ml
amphotericin (fungizone). The inoculum consists of the supernatant fluid from a homogenised lesion or
oculo-respiratory discharge in Opti-MEM with 2% calf serum and antibiotics. This is removed from the cell
layer after 2 hours. The cell layer is washed in a small volume of medium and then replenished with
maintenance medium (Opti-MEM).
A cytopathic effect (CPE) typical of poxviruses (Joubert, 1973) usually develops after 2448 hours (37C
and 5% CO2), but with some strains, it may take up to 7 days for CPE to be observed. According to the viral
strain, groups of cells with a confluent cytoplasm form syncytia that vary in size from 2 to 50 or even
100 nuclei together. The nuclei of some cells change, the chromatin forming basophilic aggregations that
vary in size and number and give the culture a leopard-skin appearance. Eosinophilic intracytoplasmic
inclusions remain discrete, if present at all. Affected cells round up, contract and become pyknotic. They
then lyse and become detached from the glass or plastic support. Later, all cells are affected and the cell
monolayer detaches completely.
Shopes fibroma virus at first produces well-defined voluminous masses of rounded cells, which proliferate
and pile up (Joubert, 1973). At the edge, cells just becoming infected show discrete nuclear changes and
acidophilic cytoplasmic inclusions that are numerous at an early stage. The cell layer is destroyed after
several days.
932
b)
Immunological methods
Agar gel immunodiffusion (AGID) tests are simple and rapid to perform results can be obtained within
24 hours. Agar plates are prepared with Noble agar (0.6 g), ethylene diamine tetra-acetic acid (EDTA)
(2.5 g), sodium chloride (4.5 g), and distilled water (500 ml) containing thiomersal (merthiolate) at 1/100,000
dilution. Standard antiserum (see below), and the test sample are placed in opposing wells that are 6 mm in
diameter and 5 mm apart. Another technique is to deposit a small portion of the lesion directly into the agar,
5 mm away from a filter paper disk impregnated with the antiserum. A number of lines of precipitation,
usually up to three, appear within 48 hours, indicating the presence of myxoma viral antigens. Only one line
forms in the presence of heterologous reactions with Shopes fibroma virus.
Indirect fluorescent antibody (IFA) tests can be applied to cultures from 24 hours onwards. IFA tests reveal
intracytoplasmic multiplication of virus, without being able to distinguish MV from Shopes fibroma virus. The
inoculation of chicken embryo cells (trypsinised at day 11 of egg incubation) does not result in CPE, but it is
useful for detecting the viral antigens by IFA tests.
c)
Electron microscopy
Negative-staining electron microscopy (EM) can be applied to a portion of skin lesion. The technique is
simple and rapid to perform, giving results in 1 hour. About 1 mm3 of the tissue is laid in a watch glass and
three drops of distilled water are added. After 12 minutes at room temperature an EM grid coated with
formvar and carbon is laid over the liquid. After 1 minute any excess liquid is removed with filter paper and
immediately a 2% aqueous solution of ammonium molybdate, pH 7.0, is dropped on to the grid. After
10 seconds the excess liquid is removed with filter paper and the grid is prepared for the electron
microscope. In a positive case, typical poxvirus particles can be seen. MV cannot be distinguished from
Shopes fibroma virus using this method.
d)
Inoculation tests
Rabbit intradermal inoculation also offers a means of identifying the virus through its special characteristics
and pathogenicity (virulence grade, classical or amyxomatous forms). It should be avoided if possible but
has the advantage of being a gauge of virulence, from the type of inflammation in lesions (local or systemic
infection) to the extent of lesions and survival time, and can distinguish Shopes fibroma virus (with its simple
fibromatous local lesion) from MV (capable of causing generalised infection in adults). Rabbits should be of
a domestic breed, weighing approximately 2 kg, unvaccinated and previously tested for the absence of
antibodies (Joubert, 1973).
The inoculum may be the supernatant fluid from a homogenised lesion (with antibiotics) or the product of a
cell culture. Between 0.1 and 0.2 ml is administered intradermally behind the ear or into the dorso-lumbar
region, which has previously been depilated. The inoculum may be assayed by injecting serial dilutions in
saline buffer at one site for each dilution. A primary lesion will appear at the sites within 25 days, followed
by conjunctivitis. Using five sites for each dilution allows a 50% infective dose (ID50) to be obtained. If the
animal survives, the disease can be confirmed serologically after 15 days.
2.
Serological tests
Antibodies develop within 813 days. In the nonlethal forms and in vaccinated rabbits, the titre is highest after 20
60 days; it declines thereafter, disappearing after 68 months in the absence of reinfection (serological response
evaluated by use of the complement fixation [CF] test) (Saurat et al., 1980).
Various serological tests may be used, but agar gel immunodiffusion (AGID), CF, IFA and enzyme-linked
immunosorbent assay (ELISA) (in order of increasing sensitivity) are the most appropriate tests for international
trade and other applications. These tests require standard antigens and antisera. The antigen can be prepared
from the Lausanne strain, or some antigenically related strain, propagated in rabbits or cell cultures.
Preparation of standard reagents (AGID, CF and IFA tests)
Preparation of antigen
Myxomatous lesions are removed from rabbits at 67 days after inoculation and homogenised in veronal
buffer to a dilution of 1/5. The antigen is the supernatant fluid that is obtained following centrifugation (5
10 minutes, 1500 g). Any anticomplementary activity is abolished by adding 0.6% chloroform. The antigen
fluid can be frozen at30C or 70C for stock purposes or used directly in CF tests after titration against a
standard antiserum.
933
Antigen is made from cell cultures using the RK-13 cell line. RK-13 (rabbit kidney cells CCL-37) are cultured
in Dulbeccos modified Eagles medium (DMEM, Gibco) containing penicillin and streptomycin and 10% fetal
calf serum (FCS) for 48 hours before infection. Cells are cultured at 37C in 5% CO2. Cells are infected by
the MV strain at a multiplicity of infection (m.o.i.) of 15. After incubation for 2 hours at 37C, the inoculum is
removed and the cells are maintained in DMEM with 5% FCS for 48 hours at 37C with 5% CO2. The
monolayer is harvested about 48 hours after infection, when the cells clearly show CPE (80%), and is
centrifuged (1000 g). The supernatant fluid is retained. Infected cells are frozen and thawed three times to
release additional virus and the viral suspension is clarified at 1000 g. The newly harvested supernatant is
added to the original supernatant. The final supernatant fluid is the antigen, and is stored at 20C or 70C
(for longer conservation). It is titrated in cell cultures before use.
Titration of standard antigen by complement fixation test

i)
Inactivate standard antiserum for 30 minutes in a water bath at 56C. No international standard serum
for myxomatosis is available, but internal positive standards should be prepared and the titre estimated
in the appropriate range, using the CF test or ELISA. After this, the following procedure is used to
standardise batches of antigen.
ii)
Make doubling dilutions of standard serum in calcium/magnesium/veronal buffer (CMV)

(BIOMERIEUX, ref 72171), pH 7.2, from 1/2 to 1/4096, using a 96-well round-bottomed microtitre plate,
one row (column) per dilution and 25 l per well.
iii)
Using tubes, make doubling dilutions of antigen in CMV, from 1/10 to 1/1280.
iv)
Add 25 l of the first antigen dilution to each well in the first line of the plate. Similarly add succeeding
dilutions of antigen to subsequent lines of the plate so that a checkerboard titration of antigen and
antibody is created.
v)
Add 25 l (6 H50 units [50% haemolysis]) of complement per well.
vi)
Incubate the plate, covered with a plastic film, for 1 hour at 37C or 14 hours at 4C.
vii)
Add 50 l per well of the haemolytic system (2.5% sheep red blood cells [RBCs] and an equal volume
of anti-sheep RBC serum diluted both in CMV) (for optimal working dilution of anti-sheep RBC, follow
the recommendations of the producer; alternatively it should be determined individually for each lot of
serum used).
viii) Cover the plate again and incubate for 30 minutes at 37C.
ix)
Read the highest dilution of antigen giving complete haemolysis (H100) with the highest dilution of
standard serum. There is 1 antigenic unit (AgU) in 25 l of antigen of this dilution.
Myxoma virus titration of infectious particles in cell cultures

i)
MV is diluted from 101 to 105 in DMEM + antibiotics + 2% FCS.
ii)
Confluent RK-13 cells in P6 (Falcon plates) are infected in triplicate with 200 l of serial dilutions of
interest and incubated at 37C for 2 hours in a 5% CO2 incubator.
iii)
Inoculum is then removed and 2 ml of DMEM + 5% FCS is added to each well.
iv)
Incubation is performed at 37C in a 5% CO2 incubator for 2 days.
v)
Medium is then removed and replaced by solid medium with 1% LMP agarose and 2% FCS.
vi)
Incubation is performed for 1 or 2 days more, then plaques are counted without coloration for each
dilution (coloration is possible with an overlay containing neutral red).
vii)
After average calculation, the titre of the viral suspension is expressed in plaque-forming units (PFU)
per ml.
Preparation and titration of standard positive serum

For the standard antiserum, an adult serologically negative rabbit is vaccinated with an attenuated strain of
MV, or with the Shopes fibroma virus. After 34 weeks, the rabbit is inoculated intradermally with virulent
myxoma virus (Lausanne strain or a related one) (5 103 PFU). Serum is obtained 3 weeks later and
titrated by the CF test or ELISA. If the titre is >1/640 or > 1/1000, respectively for CF test and ELISA, the
animal is bled and the serum is stored at 20C.
a)
Complement fixation test

CF tests (Saurat et al., 1980) are done in tubes or in microtitre plates (Chantal et al., 1993) by conventional
methods, recording 100% or 50% haemolysis. This is the standard method at the present time.
934
i)
Titrate the complement in haemolysis tubes, in the presence of 1 AgU, in order to determine the
H50 unit.
ii)
Inactivate the test serum and the positive and negative control sera in a water bath for 30 minutes at
56C.
iii)
Make doubling dilutions of test and control sera in CMV, from 1/4 to 1/1024, using a 96-well roundbottomed microtitre plate and 25 l per well. Use the first well for the initial 1/4 dilution and the second
as a serum control (anticomplementary control at 1/4 dilution). Provide antigen (without serum),
complement, and RBC control wells (see below) (two wells of each).
iv)
Add 1 AgU of MV antigen in 25 l per well (except to serum, complement and RBC control wells), then
add 6 H50 of complement in 25 l per well (except to RBC control wells).
v)
Incubate the plates, covered with plastic film, for 14 hours (overnight) at 4C.
vi)
Add 50 l per well of the haemolytic system
vii)
Cover the plates again and incubate for 30 minutes at 37C.
viii) Prepare H100, H75, H50, H25 haemolysis controls using complement controls (H100) and CMV.
b)
ix)
Read after centrifugation (1000 g, 10 minutes) or passive sedimentation at 4C. The test sera results
are determined as the highest dilution of serum that gives at least 50% haemolysis inhibition.
x)
A negative serum should give haemolysis inhibition <50% at 1/4 dilution.

IFA test (Gilbert et al., 1989) is carried out using chicken embryo or RK-13 cell cultures in flat-bottomed wells
of microtitre plates: cell s suspension, 4 104 cells diluted in medium, is distributed into all wells and a
confluent cell sheet is formed within 24 hours. The medium is discarded and 100 l of viral suspension (with
a multiplicity of infection of 0.05) is added to each well. After 2 hours, 100 l of Minimal essential medium
(MEM) containing 2% calf serum is added. After 48 hours of incubation, the plates are washed with PBS and
fixed with acetone containing 50% ethanol for 30 minutes at 20C, or paraformaldehyde (4% in PBS) at
room temperature. The plates are then dried at 37C for 15 minutes. The plates can be stored at 30C or
70C for 3 months. Sera are tested by IFA using anti-rabbit IgG conjugated to fluorescein isothiocyanate.
The test results may be qualitative with sera diluted 1/20, or quantitative with serial dilutions of serum.
c)

An ELISA has been developed (Gelfi et al., 1999) that uses a semi-purified myxomatosis virus (French
hypervirulent T1 strain antigenically related to Lausanne strain) produced in RK-13. The virus is harvested
as a suspension of cells 48 hours after infection, and is centrifuged. The cell pellet is homogenised in TL20
(20 mM Tris, pH 8.6, 150 mM NaCl, and 1 mM EDTA), disrupted in ground glass and centrifuged at 1200 g
at 4C for 10 minutes.
The supernatant fluid is laid down on an equal volume of a 36% sucrose cushion in TL20 and centrifuged at
200,000 g for 2 hours in an SW 41 rotor at 4C. The pellet is homogenised in 412 ml TL20 and again laid
down on a 36% sucrose cushion.
The new pellet is homogenised in 0.51 ml TL20 and quantified by the Bradford method (colorimetric
reaction with Coomassie brilliant blue) (Bertagnoli et al., 1996) or spectrophotometry (viral proteins account
for around three-fifths of total protein). It can be stored at 30C before use.
i)
Coat probind (Falcon) assay plates for 16 hours (overnight at 37C) with 1 g per well viral proteins in
100 l PBS, pH 7.6, leaving one column blank (i.e. coat with PBS only). Note that different batches of
antigen vary in activity, and should be titrated against known standards to select antigen with high
optical densities (OD).
ii)
After three washes in PBS, block free binding sites by incubation in 25 mg/ml gelatin in PBS for 1 hour
at 37C.
iii)
Wash the plate three times in PBS0.01% Tween 20, and add 100 l serial twofold dilutions of serum
in PBSTween. Include positive and negative serum standards in each plate.
iv)
After 60 minutes incubation at 37C and three washes in PBSTween, add 100 l of a dilution in PBS
Tween of goat anti-rabbit IgG serum (previously tested) conjugated to alkaline phosphatase for 1 hour
at 37C.
v)
After four washes in PBSTween and one more in PBS alone add, as substrate, 100 l of disodium pnitrophenyl phosphate at a concentration of 1 mg/ml in 10% diethanolamine.
935
vi)
After 12 minutes in the dark at room temperature, the enzymatic reaction is stopped by the addition of
50 l of 2 N NaOH.
vii)
Read OD in a spectrophotometer at a wavelength of 405 nm.
viii) Express the serum sample titre as the inverse of the highest dilution for which the OD value is bigger
than three times the OD value of the negative serum standard at the same dilution.
The detection of specific MV antibodies by ELISA has been shown to be a highly sensitive and specific
method for kinetic studies in experimental infection (Bertagnoli et al., 1996). Evaluation of the test has shown
its great value for diagnostic application in wild rabbit populations (Gelfi et al., 1999).
For epidemiological surveys, the IFA test and the indirect ELISA can also be carried out using blood dried on
blotting or filter paper: discs are cut (paper punch size) and two discs are placed in each well, to which is
added 100 l PBS to extract the serum. The extract product is about 1/20 diluted and can be used as a fresh
sample for testing (Gilbert et al., 1989). Blood samples collected in anticoagulant-coated capillary tubes can
be used for the ELISA. The sample is washed in the diluting solution to obtain the required dilution (Rodak et
al., 1985).
d)
Agar gel immunodiffusion test

Agar gel immunodiffusion (AGID) (Sobey et al., 1966) is qualitative and can detect antigen or antibody. Agar
is prepared as described previously (Section B.1) using 6 ml per 10 cm Petri dish. Strips of filter paper
containing the standard antigen and antiserum, and discs containing test sera are arranged on the surface of
the agar (discs between the strips). The plates are incubated in a humid atmosphere at 37C and read after
2448 hours. Three precipitation lines should appear. If the test sera contain MV-specific antibody, at least
one of the three lines is bent towards the antigen band; otherwise it remains straight. If sera contain MV
antigen, at least one of the lines is bent towards the standard serum strip. The test can also be carried out in
a more conventional manner using liquid reagents in wells cut in the agar.

Two types of live vaccine are used for vaccination against myxomatosis: a heterologous vaccine prepared from
Shopes fibroma virus (Fenner & Woodroofe, 1954; Jobert, 1983; Shope, 1932), and a homologous vaccine
prepared from an attenuated strain of MV (Arguello Villares, 1986; Gorski & Mizak, 1985; Saurat et al., 1978;
Tozzini & Mani, 1975; Von Der Ahe et al., 1981). They are administered subcutaneously or intradermally.
A new recombinant MV expressing rabbit haemorrhagic disease virus (RHDV) capsid protein and conferring
double protection against myxomatosis and RHDV (Bertagnoli et al., 1996) has been developed, but is not yet
available commercially.
A master seed virus (MSV) must be established and used according to a seed-lot system. A record must be kept
of its origin, passage history and characteristics.
1.
Seed management
a)

The viruses employed are Shopes fibroma virus or MV. The strains of Shopes fibroma virus are usually the
original Shopes OA strain (1932), Boerlages strain or various closely related strains. The strains of MV are
modified by passaging in embryonated chicken eggs, rabbit kidney cells at decreasing temperatures, or
chicken embryo cells. The strains usually result from having been cloned several times.
b)
Method of culture
Shopes fibroma virus strains are maintained by passage in specific pathogen free (SPF) rabbits or in
unvaccinated rabbits from a stock known to be free from myxomatosis. Skin on the backs of healthy adult
rabbits is shaved, and multiple sites are inoculated with a 1% suspension of virulent material. Fibromas are
fully developed within 810 days, at which time the rabbits are killed and the tumours are removed
aseptically and homogenised with distilled water. The suspension is stored at 30C or 70C in 50%
buffered glycerol, or as a 5% dilution in a protein solution (bovine albumin). The production of the Shopes
fibroma virus is also possible in rabbit dermal cell line.
936
MV is grown on chicken embryo cell culture obtained from flocks free from specified pathogens, or on
suitable cell lines (rabbit dermal cell line). Virus can also be cultivated on RK-13 cells.
c)
i)
Identity
Specific antigenic characteristics of the Shopes fibroma virus strains are verified by AGID using sera
against fibroma and myxomatosis.
The identity of MV is confirmed by neutralisation tests in RK-13 cells, or in a suitable cell line using a
monospecific antiserum (produced by vaccination of rabbits with the specific vaccine viral strain).
ii)
Purity
The Master seed must be free from bacterial, fungal, mycoplasmal and viral contamination.
iii)
Safety
Samples for safety testing are taken from a batch produced according to the manufacturing process.
The dose to be used shall contain the maximum titre or potency established by the manufacturer
(release titre).
Several tests are performed, at the Master Seed level, to demonstrate different aspects of safety. The
safety of 10 times the normal dose must be demonstrated. Also, it is necessary to examine the
dissemination of vaccine virus within the vaccinated animal, the ability of vaccine virus to spread from
the vaccinated animal to in-contact animals and to test whether there is reversion-to-virulence of the
vaccine virus, following serial passage in rabbits.
The pathogenicity of the Shopes fibroma virus strains is tested by inoculating rabbits with serial
dilutions of supernatant fluids obtained by centrifugation of tumour preparations. Macroscopic and
histopathological features and the course of development of fibromas are tested in SPF rabbits
periodically. (Numerous serial passages in rabbits may induce mutation to the inflammatory IA strain,
which produces severe lesions that are more inflammatory than neoplastic.)
The residual pathogenicity of the MV strains is tested by intradermal inoculation into SPF rabbits or
unvaccinated rabbits free from myxomatosis. These rabbits should not develop more than a local
reaction with perhaps small secondary lesions on the head that disappear within a few days.
For both strains, the rectal temperature and the body weight should be recorded as additional
parameters.
iv)
Efficacy
Different trials must be undertaken from representative batches of final product containing the minimum
titre or potency. The protective effect is demonstrated as follows:
A minimum of ten adult rabbits are inoculated with a dose of fibroma vaccine, and three rabbits serve
as unvaccinated controls. After 14 days, all rabbits are inoculated, intradermally into the eyelids, with a
pathogenic strain of MV (example: 0.1 ml inoculum containing 103 ID50 [median infectious dose]).
During the following 21 days, the controls will die from myxomatosis, and at least seven of the ten
vaccinated rabbits must present no signs of generalised infection.
Similarly, myxoma vaccine is tested in ten rabbits with three controls. After 14 days, all the rabbits are
challenged with a sufficient quantity of virulent strain (example: 0.1 ml of the Lausanne virus strain
containing 103 ID50). After 21 days, seven of ten vaccinated rabbits must have survived, while controls
must have died from myxomatosis.
The manufacturer shall have established a minimum titre or potency taking into account loss in potency
during the shelf life.
2.
Shopes fibroma virus is produced by multiple intradermal inoculations of seed virus into the skin on the back of a
number of rabbits. The product of fibroma homogenate can be stored by freezing or used immediately. Production
is also possible in rabbit dermal cell line. Only the second (and perhaps the third) viral passage can be used if
modification of the virus is to be avoided. After clarification by centrifugation, the supernatant fluid is mixed with a
stabiliser containing antibiotics and is distributed into ampoules or bottles for Iyophilisation. Kaolin may be added
as an adjuvant (40 mg/ml), in which case the vaccine is administered subcutaneously.
MV is produced in chicken embryo cells (derived from SPF eggs) or a suitable cell line, limiting the passage
number to a maximum of five. Virus is harvested after 26 days. The viral suspension may be stored at 70C.
937
The vaccine is prepared by diluting in specified proportions the viral preparation with a stabiliser for Iyophilisation.
After homogenisation, the product is distributed into bottles for Iyophilisation, the bottles being sealed under
vacuum or in sterile nitrogen.
Each virus can also be produced in RK-13 cells.
3.
In-process control
The Shopes fibroma virus titre is measured by calculating the ID50 after intradermal inoculation of serial dilutions
of the clarified supernatant fluid into several sites (e.g. five) on up to six rabbits. A dilution of a standard
preparation of Shopes fibroma virus is also inoculated into each rabbit to confirm the animals correct response to
inoculation. The titration can also be performed in a rabbit cell line. In each case the titre should correlate with the
required potency as defined by the test for efficacy, see Section C.1.c.
The identity of MV is checked in RK cells. Titration of each virus can also be done in RK-13 cells (TCID50).
Testing for contaminating viruses is done by inoculating a confluent monolayer of Vero cells. Vaccine, adjusted to
the equivalent of 20 doses/ml, is neutralised with an equal volume of monospecific hyperimmune serum for
30 minutes at 37C. The mixture is filtered through a 0.22 m membrane filter, and 1 ml volumes are inoculated
into five 25 ml bottles of cell cultures. These are kept under observation for 7 days. After harvesting, the cells are
suspended in medium and subjected to several freezethaw cycles, followed by centrifugation and filtration, and
the material is inoculated into fresh cultures and observed for 7 days. There should be no evidence of CPE or
haemadsorption to chicken RBCs.
4.
Batch control
a)
Sterility
b)
Safety
After rehydration, ten doses of the lyophilised fibroma vaccine are injected subcutaneously into each of three
susceptible rabbits, which are then observed for 21 days. Local reactions should be slight, with no
generalisation and no effect on general health.
Myxoma vaccine is tested using ten doses injected intradermally into the ears of three susceptible rabbits,
which are then observed for 21 days. The primary myxoma lesion should remain mild.
c)
Potency
The batch potency is determined by measurement of virus content. Serial dilutions of the vaccine are
inoculated into suitable cell cultures. One dose of vaccine shall contain not less than the minimum titre
established in Section C.1.c.
If the vaccine strain is not adapted to cultures, an efficacy test in rabbits shall be carried out (see Section C.1.c).
d)
Several groups of ten susceptible rabbits are vaccinated. One batch is tested by challenge infection (as in
the batch potency test), at 1, 2, 3, etc., months post-vaccination for Shopes fibroma virus, and at 1, 3, 6, and
9 months for MV. The duration of immunity is deduced from the time during which at least seven of the ten
rabbits prove to be resistant to infection.
e)
Stability
Titrations of vaccine virus are carried out at intervals until 3 months beyond the requested shelf life on at
least three batches of vaccine.
5.
a)
Safety
See Section C.4.b.
b)
Potency
See Section C.4.c.
938
REFERENCES
ARGUELLO VILLARES J.L. (1986). Contribucin a la profilaxis de la mixomatosis del conejo mediante el uso de una
ceba homologa. Medicina Veterinaria, 3, 91103.
ARTHUR C.P. & LOUZIS C. (1988). A review of myxomatosis among rabbits in France. Rev. sci. tech. Off. int. Epiz.,
7, 937976.
BERTAGNOLI S., GELFI J., LEGALL G., BOILLETOT E., VAUTHEROT J.F., RASSCHAERT D., LAURENT S., PETIT F., BOUCRAUTBARALON C. & MILON A. (1996). Protection against myxomatosis and rabbit viral hemorrhagic disease with
recombinant myxoma virus expressing rabbit hemorrhagic disease virus capsid protein. J. Virol., 70, 50615066.
BRUN A., SAURAT P., GILBERT Y., GODART A. & BOUQUET J.F. (1981). Donnes actuelles sur lpidmiologie, la
pathognie et la symptomatologie de la myxomatose. Rev. Med. Vet., 132, 585590.
CHANTAL J., BOUCRAUT-BARALON C., GANIERE J.P., PETIT F., PY R. & PICAVET D.P. (1993). Raction de fixation du
complment en plaques de microtitration: application la srologie de la myxomatose. Etude comparative des
rsultats avec la raction dimmunofluorescence indirecte. Rev. sci. tech. Off. int. Epiz., 12, 895907.
FENNER F. (1994). Myxoma virus. In: Virus Infections of Vertebrates, Vol. 5. Virus Infections of Rodents and
Lagomorphs, Osterhaus A.D.M.E., ed. Elsevier Science B.V., Amsterdam, Netherlands, 5971.
FENNER F. & RATCLIFFE F.N. (1965). Myxomatosis. Cambridge University Press, London, UK.
FENNER F. & WOODROOFE G.M. (1954). Protection of laboratory rabbits against myxomatosis by vaccination with
fibroma virus. Aust. J. Exp. Biol., 32, 653668.
GELFI J., CHANTAL J., PHONG T.T., PY R. & BOUCRAUT-BARALON C. (1999). Development of an ELISA for detection of
myxoma virus-specific rabbit antibodies; test evaluation for diagnostic applications on vaccinated and wild rabbit
sera. J. Vet. Diagn. Invest., 11, 240245.
GILBERT Y., PICAVET D.P. & CHANTAL J. (1989). Diagnostic de la myxomatose: mise au point dune technique
dimmunofluorescence indirecte. Utilisation de prlvements sanguins sur papier buvard pour la recherche
danticorps. Rev. sci. tech. Off. int. Epiz. 8, 209220.
GORSKI J. & MIZAK B. (1985). Polish vaccine against myxomatosis in rabbits. Med. Weter., 41, 113116.
JOBERT R. (1983). Contribution ltude de la vaccination contre la myxomatose, vaccination laide du virus
fibromateux. Thse Doctorat Vtrinaire Toulouse n 82.
JOUBERT L. (1973). La Myxomatose T.II. Srie : Les Maladies Animales Virus. LExpansion diteur, Paris,
France.
JOUBERT L., DUCLOS P.H. & TUAILLON P. (1982). La myxomatose des garennes dans le Sud-Est: la myxomatose
amyxomateuse. Rev. Med. Vet., 133, 739753.
MARLIER D. & VINDEVOGEL H. (1996). La myxomatose amyxomateuse: isolement de trois souches en Belgique.
Ann. Med. Vet., 140, 343346
RODAK L., SMID B., VALICEK L. & JURAK E. (1985). Collection of microvolume blood samples into glass capillaries for
the detection of antibody against Aujeszkys disease virus in pigs by enzyme-linked immunosorbent assay
(ELISA) and solid-phase radioimmunoassay (RIA). Acta Vet. (Brno), 54, 207216.
ROSELL J.M., GONZALES J.L., RUEDA A., GALLEGO E. & FLORES J.M. (1984). Mixomatosis atipica en Espana.
Medicina Veterinaria, 1, 401412.
SAURAT P., CHANTAL J., GANIERE J.P., GILBERT Y., PICAVET D.P & LEFORT C. (1980). La rponse immunitaire dans la
myxomatose. Etude de la rponse humorale. Bull. Mens. Off. Nation. Chasse, 12, 297309.
SAURAT P., GILBERT Y. & GANIERE J.P. (1978). Etude dune souche de virus myxomateux modifi. Rev. Med. Vet.,
129, 415451.
SHOPE R.E. (1932). A filtrable virus causing a tumor like condition in rabbits and its relationship to virus
myxomatosis. J. Exp. Med., 56, 803.
939
SOBEY W.R., CONOLLY D. & ADAMS K.M. (1966). Myxomatosis: a simple method of sampling blood and testing for
circulating soluble antigens or antibodies to them. Aust. J. Sci., 28, No. 9, 354.
TOZZINI F. & MANI P. (1975). Studio su alcune carrateristiche di crescita del virus della mixomatosi ceppo Pisa 73.
Arch. Vet. Ital., 26, 1926.
VON DER AHE C., DEDEK J. & LOEPELMANN H. (1981). Ergebnisse und Erfahrungen in der DDR bei der staatlichen
Prfung und Praxiserpropung der aus der CSSR emportierten Myxomatose-Vakzine. Monatshe. Veterinarmed.,
36, 492495.
*
* *
NB: There is an OIE Reference Laboratory for Myxomatosis
diagnostic tests, reagents and vaccines for myxomatosis
940
CHAPTER 2.6.2.
RABBIT HAEMORRHAGIC DISEASE
SUMMARY
Rabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal disease of the European
rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). A similar disease, termed
European brown hare syndrome (EBHS), has been described in the hare (Lepus europaeus). Its
aetiological agent is a different species of lagovirus, antigenically related to the RHD virus (RHDV).
RHD is characterised by high morbidity and high mortality (7090%), and spreads very rapidly by
direct and indirect transmission. Infection can occur by nasal, conjunctival or oral routes.
Transmission of RHD is facilitated by the high stability of the virus in the environment. The
incubation period varies from 1 to 3 days, and death usually occurs 1236 hours after the onset of
fever. The clinical manifestations have been described mainly in the acute infection and are
nervous and respiratory signs, apathy and anorexia. Clear and specific lesions, both gross and
microscopic, are present. There is primary liver necrosis and a massive disseminated intravascular
coagulopathy in all organs and tissues. The most severe lesions are in the liver, trachea and lungs.
Petechiae are evident in almost all organs and are accompanied by poor blood coagulation. In
rabbits younger than 45 weeks, the RHDV infection course is subclinical.
Identification of the agent: The liver, spleen and blood of rabbits that died of acute RHD contain a
very high concentration of virus. Direct diagnosis is therefore easy and several tests can be used
However, as no sensitive cell substrates are established in-vitro isolation cannot be used. As a
consequence, the only available biological assay is reproduction of the disease in sensitive rabbits.
The main laboratory tests used are RNA amplification (reverse-transcription polymerase chain
reaction [RT-PCR]) and sandwich enzyme-linked immunosorbent assay (ELISA) based on the use
of monoclonal antibodies (MAbs). As RHDV haemagglutinates human Group O red blood cells very
efficiently, the haemagglutination (HA) test can also be used. False-negative results could arise in
the few rabbits that die from the chronic form of RHD (i.e. 48 days post-infection). In these rabbits
both an anti-RHDV IgM and virus-like particles (VLPs), made by the inner shell of RHDV, heavily
interfere with HA and some ELISAs. Moreover, using highly sensitive PCR in blood, intestinal
contents or faeces from healthy rabbits suspected to be RHD convalescent or RHDV re-infected,
some non-pathogenic RHDV-related caliciviruses were isolated and partially characterised;
importantly these RHDV-related viruses seem quite widespread in both commercial and wild rabbit
populations. The detection of calicivirus particles in liver homogenates or VLPs by electron
microscopy is also possible.
Serological tests: The level of RHDV-specific antibodies in the serum is a direct indication of the
state of protection of rabbits from RHD. In addition, determinating the levels for each class of
immunoglobulins (IgM, IgA and IgG) helps in distinguishing the first RHDV infection from reinfection or vaccination. Several types of ELISAs were developed and are currently used as a
replacement for the haemagglutination inhibition (HI) test initially used. The total level of RHDV-Ig is
semi-quantified using three main ELISAs: a) with the virus adsorbed on the solid phase; b) with the
virus and the serum both in liquid phase (competition ELISA), c) with the antigen (virus or subunits)
linked to the solid phase throughout MAbs. The different degree of exposition of the internal viral
epitopes obtained in these ELISAs helps to distinguish the Ig induced by RHDV from those induced
by the non-pathogenic related viruses.
Requirements for vaccines: Indirect control of the disease is achieved by vaccination using a
killed vaccine prepared from clarified liver suspensions of experimentally infected rabbits and
subsequently inactivated and adjuvanted. Vaccinated animals quickly produce solid protective
941
Chapter 2.6.2. Rabbit haemorrhagic disease
immunity against RHDV infection (within 710 days) and experimental data indicate that protection
lasts for a long period (over 1 year).
A. INTRODUCTION
Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease of wild and domestic European
rabbits (Oryctolagus cuniculus) older than 2 months. RHD is caused by a calicivirus (genus Lagovirus, family
Caliciviriade), a non-enveloped small round RNA virus with only one major capsid protein (VP60) (Capucci et al.,
1990; Meyers et al., 1991; Ohlinger et al., 1990). The genus Lagovirus also includes the European brown hare
syndrome virus (EBHSV), the causative agent of a disease of brown hare (Lepus europaeus) termed EBHS, very
similar to RHD. Despite their high genetic relation (VP60 nucleotide similarity of 70%), RHDV and EBHSV are two
distinct viral species (Lavazza et al., 1996; Wirblich et al., 1994). There is only one serotype of RHDV and it
consists of two subtypes: RHDV (wild type) and RHDVa (Capucci et al., 1998; Schirrmeier et al., 1999).
RHD was first reported in 1984 in China (Peoples Rep. of) (Lui et al., 1984) and 2 years later in Europe. To date,
RHD has been reported in over 40 countries in Asia, Africa, Americas, Europe and Oceania and is endemic in
most parts of the world (McIntosh et al., 2007). The RHDVa subtype has been identified in Europe since 199697
(Capucci et al., 1998; Schirrmeier et al., 1999) and is replacing the original RHDV in several parts of the world.
However, considering the RHDV genetic sequences deposited at the NCBI databank, the presence of RHDVa in
China (Peoples Rep. of) dates back to 1985.
RHD is characterised by high morbidity and a mortality rate that usually ranges between 70 and 90%. The
incubation period varies from 1 to 3 days, and death usually occurs 1236 hours after the onset of fever. In the
few rabbits that survive (510%), a specific and relevant IgM response appears within 3 days immediately
followed by an IgA and by an IgG response 23 days later (Barbieri et al., 1997). This antibody response triggers
the virus-clearance mechanism. In spite of this prompt humoral response, 510% of infected rabbits die between
4 and 8 days post-infection from a chronic form of RHD. In the liver and spleen of these rabbits, an RHD virus-like
particle (VLP) has been detected instead of RHDV (Barbieri et al., 1997; Capucci et al., 1991; Granzow et al.,
1996). This VLP is characterised by the lack of the outer shell on the viral capsid made up by the half C-terminal
portion of the VP60. As a consequence, this VLP is negative in the haemagglutination (HA) test as well as with
anti-RHDV monoclonal antibodies (MAbs) directed to outer conformational epitopes (Capucci et al., 1995). In the
blood and faeces of convalescent rabbits, as well as in rabbits infected with RHDV but already protected by
specific antibodies previously acquired (i.e. vaccinated or survivors of infection), the viral RNA is detected using
polymerase chain reaction (PCR) up to 15 weeks after the infection (Gall et al., 2007; Gall & Schirrmeier, 2006).
Whether this is the consequence of a slow viral clearance or of a real and prolonged virus replication
(persistence) is still to be established.
As a result of RHD serology testing (Capucci et al., 1991; 1997; Collins et al., 1995; Cooke et al., 2000), several
rabbit caliciviruses related to RHDV but non-pathogenic have been isolated and partially characterised in Europe
and Oceania (Capucci et al., 1996b; 1997; Forrester et al., 2002; Moss et al., 2002; Strive et al., 2009; White et
al., 2004). These caliciviruses induce a serological response that may interfere with and complicate RHD
serological diagnosis (Capucci et al., 1991; Cooke et al., 2000; Marchandeau et al., 2005; Nagesha et al., 2000;
Robinson et al., 2002).
1.
The liver contains the highest viral titre (from 103 LD50 [50% lethal dose] to 106.5 LD50/ml of 10% homogenate)
and is the organ of choice for viral identification for both RHDV and EBHSV. The amount of virus present in other
parts of the body is directly proportional to vascularisation; thus spleen and serum may serve as alternative
diagnostic materials, albeit suboptimal. Higher levels of VLPs have been reported in the spleen than in the liver of
animals that died from a subacute/chronic form of RHD (Barbieri et al., 1997; Capucci et al., 1991; Granzow et al.,
1996). The initial treatment of the diagnostic samples is almost identical irrespective of the diagnostic method to
be applied, with the exception of immunostaining techniques. An organ fragment is mechanically homogenised in
520% (w/v) phosphate buffered saline solution (PBS), pH 7.27.4, filtered through cheesecloth and clarified by
centrifugation at 5000 g for 15 minutes. At this stage, the supernatant can be directly examined by the HA test or
enzyme-linked immunosorbent assay (ELISA). If the sample is to be observed by electron microscopy (EM), it is
advisable to perform a second centrifugation at 12,000 g for 15 minutes, before the final ultracentrifugation. For
detection by PCR, viral RNA from the samples may be also directly extracted from tissues.
942
a)

Virus detection by ELISA relies on a sandwich technique and several variations of this have been
described. One procedure uses the reagents, solutions, times and temperature that are used in the
competitive ELISA (C-ELISA) for serology (see Section B.2.b), except that the Tween 20 concentration is
twofold (0.1% [v/v]). The microplate used should be of high adsorption capability (e.g. Nunc Maxisorp
immunoplate). The liver homogenate is a 10% (w/v) suspension in standard PBS; 50 l/well is the standard
volume to use in each step. The ELISA buffer used for all steps is PBS with 1% yeast extract (or bovine
serum albumin [BSA]), and 0.1% Tween 20, pH 7.4. All incubation steps are for 5060 minutes at 37C with
gentle agitation. After all steps three washes of 35 minutes must be performed using PBS with 0.05%
Tween 20. A positive and negative RHD rabbit liver homogenate must be used as controls. The horseradish
peroxidase (HRPO) conjugate could be purified lgG from a specific polyclonal serum or MAbs (see Section
B.2.b). Anti-RHDV MAbs have been produced in several laboratories and can be used instead of rabbit
polyclonal sera. MAbs recognising specific epitopes expressed only by the RHDVa variant were also
produced (Capucci, pers. data).
To better characterise the antigenicity of the RHD isolates by sandwich ELISA, it is advisable to test each
sample in four replicates, and then to use four different HRPO conjugates, i.e. two MAbs recognising the
same antigenic determinant present on the virus surface and expressed alternatively by the classical strain
or by the RHDVa variant, a polyclonal hyperimmune anti-RHDV serum (which could identify potential new
variant or correlated calicivirus, such as EBHSV) and a pool of MAbs recognising internal epitopes that can
detect smooth, degraded VLPs as well as EBHSV. An alternative antigen-capture ELISA has been
described using a sheep anti-RHDV as the capture antibody and a MAb for detection of RHDV (Collins et al.,
1996).
Test procedure (example)
For steps that are not specifically indicated see the procedure of the C-ELISA for serology (Section B.2.b).
i)
Coat the plate with anti-RHDV hyperimmune serum and the negative RHDV serum. The latter serves
as control for nonspecific reactions (false-positive samples). For each sample, four wells must be
sensitised with the positive serum and four wells with the negative one.
ii)
Dilute the liver extract to 1/5 and 1/30 (two replicates for each dilution) in ELISA buffer (see above),
directly in the wells of the plate (e.g. add 45 l of the buffer into all the wells of the plate, add 10 l of
the sample to the first two wells and then, after rocking, transfer 9 l to the second wells). Treat the
controls, both positive and negative, in the same way as the samples.
iii)
After incubation and washing (see above), incubate with the HRPO conjugate.
iv)
After a last series of washing, add the chromogenic substrate. Orthophenylene-diamine (OPD) can be
used as peroxidase substrate for the final development of the reaction. Use 0.15 M citrate phosphate
buffer, pH 5.0, with 0.5 mg/ml OPD and 0.02% H2O2. The reaction is stopped after 5 minutes by the
addition of 50 l of 1 M H2SO4.
v)
Absorbance is read at 492 nm. Positive samples are those showing a difference in absorbance >0.3,
between the wells coated with RHDV-positive serum and wells coated with the negative serum.
Usually, at the dilution 1/30, positive samples taken from rabbits with the classical acute form of RHD
give an absorbance value >0.8, while the absorbance value of the negative sample, at the dilution 1/5,
ranges from 0.1 to 0.25.
For diagnosis of EBHSV, it is possible to use this RHDV-specific sandwich ELISA, but, due to the high
antigenic difference existing between the two viruses, there is a risk of obtaining false-negative results.
Therefore, the adoption of an EBHSV-specific sandwich ELISA technique using either a high-titre positive
anti-EBHSV hare serum, or cross-reacting RHDV MAbs (Capucci et al., 1991; 1995), or specific EBHSV
MAbs, instead of rabbit serum, is highly recommended (Capucci et al., 1991).
b)

The application of the RT-PCR to the detection of RHDV-specific nucleic acid has been described by several
authors (Gould et al., 1997; Guittre et al., 1995). Owing to the low level of sequence variation among RHDV
isolates and the high sensitivity of PCR, reverse transcription (RT)-PCR represents an ideal rapid diagnostic
test for RHD. This method is carried out on organ specimens (optimally liver), urine, faeces and sera using
different oligonucleotide primers derived from the capsid region of the RHDV genome (N-terminal portion).
The RHD OIE Reference Laboratory uses a single-step RT-PCR with the following primers specific for the
PV60 gene: [forward: 5-CCT-GTT-ACC-ATC-ACC-ATG-CC-3], [reverse: 5-AAC-CCT-CCA-GGT-ACTGGT-TG-3], whereas Guittre et al. used [forward: 5-GAG-CTC-GAG-CGA-CAA-CAG-GC-3], [reverse: 5CAA-ACA-CCT-GAC-CCG-CGA-AC-3]. cDNA obtained from the RT reaction is usually PCR amplified as
943
described (Guittre et al., 1995) or using one step standard RT-PCR methodology. To reveal the PCR
product, the amplified DNA reaction mixture is subjected to electrophoresis on agarose gel. If needed,
specificity of the PCR product can be determined by sequencing or by Southern blot and hybridisation with a
radioactively labelled internal probe. A similar RT-PCR method has been used to identify the nonpathogenic
RCV (Capucci et al., 1998). Several primers, specific for the RHDV RNA polymerase gene and
complementary to the VP60 and ORF2 genes, are used and the amplified fragments are subjected to
Southern blot analysis. RT-PCR represents an extremely sensitive method for the detection of RHDV, and is
104-fold more sensitive than ELISA (Guittre et al., 1995). It is not strictly necessary for routine diagnosis, but
it is more sensitive, convenient and rapid than other tests.
Similarly an RT-PCR for the detection of EBHSV has been applied to the detection and characterisation of
EBHSV stains (Le Gall-Recul et al., 2001]).
An internally controlled multiplex real-time RT-PCR using TaqMan fluorogenic probes and external
standards for absolute RNA quantification has been developed recently as a further diagnostic tool for the
detection of RHDV. The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a
linearity over a range from 101 to 1010 copies. The method has been used to quantify RHDV RNA in
experimental infection of vaccinated rabbits and in RHD convalescent rabbits (Gall et al., 2007; Gall &
Schirrmeier, 2006). The oligonucletides used in this method are: [VP60-7_forward: 5-ACY-TCA-CTG-AAC
TYA-TTG-ACG-3, vp60-8_reverse: 5 TCA-GAC-ATA-AGA-AAA-GCC-ATT-GG-3] and probe [VP60-9_fam
5-FAM-CCA-ARA-GCA-CRC-TCG-TGT-TCA-ACC-T-TAMRA-3].
An in-situ hybridisation technique using either sense or antisense DNA probes has been developed for
investigating the presence of RHDV in tissue samples (Gelmetti et al., 1998). This method is highly sensitive
and can be used for early diagnosis of RHD as it gives positive results as soon as 68 hours post-infection.
However, it is expensive and difficult to carry out, and thus it is mainly indicated for research studies.
c)
Electron microscopy
Negative-staining EM can be performed using the so-called drop method. A formvar/carbon-coated grid is
placed on a drop of organ suspension (prepared as described in Section B.1), and left for 5 minutes. After
removing excess fluid with the edge of a torn piece of filter paper, the grid is put to float on a drop of 2%
sodium phosphotungstate (NaPT), pH 6.8, for 1.5 minutes. Excess stain is removed and the grid is finally
observed at 25,000 magnification.
Due to the lower sensitivity of the drop method, it is advisable to ultracentrifuge the sample in order to
concentrate the viral particles. The pellet obtained after ultracentrifugation (at least 100,000 g for 30 minutes
or, alternatively, using Beckman Airfuge at 21 psi for 5 minutes) is resuspended in PBS or distilled water, put
on to a grid for a few minutes, and then stained as described. RHD virions are visible as uncoated particles,
3235 nm in diameter, presenting an inner shell (2527 nm in diameter), delineated by a rim from which
radiate ten short regularly distributed peripheral projections. Smooth (s-RHDV) particles are identified by the
complete loss of external portions, becoming perfectly hexagonal and smaller, with only the capsid rim
visible (Barbieri et al., 1997; Capucci et al., 1991; Granzow et al., 1996).
For diagnostic purposes and especially when other methods give doubtful results, the best EM method is an
immuno-EM technique (IEM). This method uses either a hyperimmune anti-RHDV serum, obtained from
rabbit or other species, or specific MAbs, which are incubated with an equal volume of the sample for 1 hour
at 37C before ultracentrifugation. The immunological reaction induces the clumping of the viral particles into
aggregates that are quickly and easily identified by EM. Immunogold methods can also be applied to better
identify virions and viral proteins.
EBHSV can also be identified in diagnostic samples by EM examination. In addition, the IEM method using
convalescent anti-EBHSV serum or specific anti-EBHS MAbs can be used to identify EBHSV. By using
antisera that is specific for EBHSV and RHDV, it is possible to differentiate between the two viruses.
d)
Haemagglutination test
HA was the first test to be used for routine laboratory diagnosis of RHD (Lui et al., 1984|]). It should be
performed with human Group O red blood cells (RBCs), freshly collected, stored overnight in Alsevers
solution, and washed in 0.85% PBS at pH 6.5 (range 67.2). HA is less evident or non-existent when RBCs
of other species are used. Washed RBCs are suspended at 0.75% in PBS. A twofold dilution of the clarified
supernatant of a 10% tissue homogenate of liver or spleen is incubated with an equal volume of washed
RBCs in a sealed round-bottom microtitre plate at, preferably, 4C. After 1 hour (range from 20 minutes to
2 hours) of incubation, agglutination at an end-point dilution of >1/160 is considered to be positive. Lower
titres should be regarded as suspicious, and should be checked using other methods. Around 10% of
samples found to be positive by ELISA or EM give negative results in HA (HA false-negative). Some RHD
944
isolates may exhibit temperature-dependent differences in haemagglutinating characteristics (Capucci et al.,

1996a) and could show HA activity only when the test is performed at 4C. Nevertheless, the HA false
negativity is mainly detected in organs of rabbits showing a subacute/chronic form of the disease and it
depends on the characteristics of the VLPs.
Hare organs rarely give a significant titre when the RHDV HA protocol is used. To demonstrate HA activity in
organs from EBHSV-infected rabbits, a modified procedure should be adopted: all steps are carried out at
4C, the organ suspension is treated with an equal volume of chloroform, and RBCs are used at a pH not
higher than 6.5 (Capucci et al., 1991). Even using this method, only about 50% of the samples give positive
results. This is because the disease of hares is often subacute or chronic and therefore the virus has the
antigenic and structural characteristics typical of the VLPs particles (Capucci et al., 1991).
Because of the practical difficulty of obtaining and keeping and the risk from working with human Group O
blood cells, and because of the difficulty of obtaining consistent results, this test has been replaced by the
virus-detection ELISA.
e)
Immunostaining
Tissue fixed in 10% buffered formalin and embedded in paraffin can be immunostained using an avidin
biotin complex (ABC) peroxidase method. The sections are first deparaffinised in xylene and alcohol,
counter-stained with haematoxylin for 1 minute and rinsed in tap water. They are then put in a methanol bath
containing 3% H2O2 and washed in PBS three times for 5 minutes each. To limit background interference
caused by nonspecific antibody binding, the samples are incubated with normal rabbit serum for 1 hour at
room temperature prior to the addition of biotin. The slides are incubated overnight in a humid chamber at
room temperature with biotinylated rabbit anti-RHDV serum or MAbs, are washed as before and incubated
again for 30 minutes at 37C with an ABC peroxidase. The slides are then washed three times. Amino-ethylcarbazole is used as substrate. Finally, the slides are rinsed in tap water and mounted (Stoerckle-Berger et
al., 1992).
Intense nuclear staining and diffuse cytoplasmic staining of necrotic cells in the liver, mainly in the periportal
areas, are characteristic and specific. Positive staining of macrophages and Kupffers cells is also observed,
as well as hepatocellular reactions. Positive reactions can also be detected in the macrophages of the lungs,
spleen and lymph nodes, and in renal mesangial cells (Stoerckle-Berger et al., 1992).
Tissue cryosections fixed in methanol or acetone can be directly immunostained by incubation for 1 hour
with fluorescein-conjugated rabbit anti-RHDV serum or MAbs. Specific fluorescence can be detected in the
liver, spleen, and renal glomeruli.
f)
Western blotting
When other tests such as HA or ELISA give doubtful results (low positivity) or the samples are suspected of
containing s-RHDV particles, western blotting analysis is useful for determining the final diagnosis.
Homogenates are prepared as described previously, and virus particles are further concentrated (tenfold) by
ultracentrifugation (100,000 g for 90 minutes) through a 20% (w/w) sucrose cushion.
Both the supernatant and the pellet can be examined to detect, respectively, the RHDV 6S subunits
(Capucci et al., 1995) and the denatured VP60 structural protein of RHDV or its proteolytic fragments, which
can range in size from 50 to 28 kDa. A positive and negative control samples should be used on each
occasion.
RHDV proteins could be detected with polyclonal antibodies or MAbs. If MAbs are used, they should
recognise continuous epitopes. RHDV-specific MAbs recognising internal or buried epitopes could be used
also to detect EBHSV. Rabbit anti-RHDV hyperimmune sera are less efficient than MAbs at recognising the
same band patterns (Capucci et al., 1996b).
Sample proteins are denatured for 2 minutes at 100C in the presence of 60 mM Tris, pH 6.8, 2% sodium
dodecyl sulphate (SDS), 2% beta-mercaptoethanol, and 5% glycerol, separated on 10% SDS/PAGE
(polyacrylamide gel electrophoresis), and then transferred by electroblotting to nitrocellulose or PVDF
(polyvinylidene flouride) membranes, in 25 mM Tris, 192 mM glycine pH 8.3 and 20% (v/v) methanol at 1.5
for 60 minutes with cooling or at 0.15 A overnight. After transfer the membranes are saturated for 30
60 minutes in blocking buffer or PBS, pH 7.4 containing 2% bovine serum albumin (BSA), and subsequently
incubated for 2 hours at room temperature with the appropriate serum dilution in PBS, pH 7.4, and 1% BSA.
The filters are washed thoroughly with PBS and incubated for 1 hour at room temperature with anti-species
alkaline phosphatase-labelled immunoglobulins at a dilution predetermined by titration. Finally, the filters are
again washed and the chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate nitro blue tetrazolium)
is added.
945
Positive test samples and the positive control will produce a pattern consistent with reaction to proteins of
molecular weights of, respectively, 60 kDa (the single structural protein of RHDV) or 4128 kDa (the
fragments of the VP60 associated with the transition from RHDV to s-RHDV), when examining the pellet,
and 6 kDa (the subunits) when examining the supernatant.
Western blot analysis can also be used to identify EBHSV. The test procedure is identical. The pattern of
protein bands, detected using either an anti-EBHSV polyclonal serum or cross-reacting anti-RHDV MAbs, is
similar. However, the percentage of samples showing viral degradation is higher and therefore several
fragments of lower molecular weight, originating from the VP60 structural protein, are often observed.
g)
Rabbit inoculation
As no efficient in-vitro replication system has been established for RHDV and EBHSV, cell culture isolation
cannot be included among the diagnostic methods. Rabbit inoculation therefore remains the only way of
isolating, propagating and titrating the infectivity of the RHDV. However, experimental infection of rabbits is
not a practical method for the routine diagnosis of RHD and should be considered only when all other
methods give inconclusive results. When this occurs, the rabbits involved must be fully susceptible to the
virus, i.e. they should be over 2 months old and have no RHDV antibodies (see serological methods). RHD
can be reproduced by using filtered and antibiotic-treated liver suspensions, inoculated either by the
intramuscular, intravenous or oro-nasal route. When the disease is clinically evident, the signs and postmortem lesions are similar to those described after natural infection. A rise in body temperature is registered
between 18 and 24 hours post-infection, followed, in 7090% of cases, by death between Robinson et al.,
2002 and 72 hours post-infection. A few individuals may survive until 68 days after infection. Animals that
overcome the disease show only a transient hyperthermia, depression and anorexia, but present a striking
seroconversion that can be detected easily 34 days post-infection.
2.
Serological tests
Infection by RHDV can be diagnosed through detection of a specific antibody response. As the humoral response
has great importance in protecting animals from RHD, determination of the specific antibody titre after vaccination
or in convalescent animals is predictive of the ability of rabbits to resist RHDV infection.
Three basic techniques are applied for the serological diagnosis of RHDV: haemagglutination inhibition (HI) (Lui
et al., 1984), indirect ELISA (I-ELISA) and C-ELISA (Capucci et al., 1991). Each of these methods has
advantages and disadvantages. With respect to the availability of reagents and the technical complexity of
carrying out the test, HI is the most convenient method, followed by the I-ELISA and C-ELISA, respectively. On
the other hand, both ELISAs are quicker and easier than HI, particularly when a large number of samples are
tested. The specificity of the C-ELISA is markedly higher than those achieved with the other two methods
(Capucci et al., 1991). An alternative C-ELISA method has been described (Collins et al., 1995). For improved
serological interpretation and for correctly classifying the immunological status of rabbits, a combination of ELISA
techniques that distinguish IgA, IgM and IgG antibody responses is also available.
Some other additional tests (Capucci, unpublished data; Cooke et al., 2000) could be used for particular
investigations and when a higher level of sensitivity is needed to detect antibodies in non-target species or
antibodies induced by cross-reacting RHDV-like agents (see Section A. Introduction).
They are:
I-ELISA: the antigen is linked to the solid phase by a RHDV-specific MAb (1H8). It has a slightly higher
sensitivity than C-ELISA, making possible measurement of highly cross-reactive antibodies and it can detect
antibodies with low avidity.
Solid-phase ELISA (SP-ELISA): the purified antigen is directly adsorbed to the solid phase and because of
virus deformation, internal epitopes are exposed. Therefore it detects a wider spectrum of antibodies and
has high sensitivity and low specificity. For these reasons it can also be used for EBHSV serology.
Sandwich ELISA to detect IgM and IgG in liver or spleen samples already examined with the virological test:
such a test is particularly useful in those animals that die from the chronic form of the disease, when
detection of the virus may be difficult. In this case, a high level of RHDV-specific IgM and a low level, if any,
of IgG are the unambiguous markers of positivity for RHD.
a)
Antigen: The antigen is prepared using infected rabbit liver collected freshly at death. The liver is
homogenised in 10% (w/v) PBS, pH 6.4, and clarified by two consecutive low speed centrifugations (500 g
for 20 minutes and 6000 g for 30 minutes). The supernatant, drawn from the tube so as to avoid the
946
superficial lipid layer, is filtered through a 0.22 m pore size mesh, titrated by HA, and divided into aliquots,
which are stored at 70C.
Serum samples: Before testing, sera are inactivated by incubation at 56C for 30 minutes. The sera are then
treated with 25% kaolin (serum final dilution: 1/10) at 25C for 20 minutes and centrifuged. This is followed
by a second kaolin treatment, also at 25C for 20 minutes, this time with 1/10 volume of approximately 50%
packed human Group O RBCs. These are freshly collected, stored overnight in Alsevers solution and
washed in 0.85% PBS, pH 6.5. The sera are clarified by centrifugation.
Test procedure
i)
Dispense 50 l of serum into the first well of a round-bottom microtitre plate and make double dilutions
into wells 28 using PBS with 0.05% BSA.
ii)
Add 25 l of RHDV antigen containing 8 HA units to each well and incubate the plate at 25C for 30
60 minutes.
iii)
Add 25 l of human Group O RBCs at 23% concentration to each well and allow to settle at 25C for
3060 minutes.
iv)
Titrate the antigen with each test to ensure that 8 HA/ 25 l were used, and include positive and
negative serum controls.
The serum titre is the end-point dilution showing inhibition of HA. The positive threshold of serum titres is
correlated to the titre of the negative control sera; it usually is in the range 1/201/80.
Because of the practical difficulty of obtaining, keeping and the risk from working with human Group O blood
cells, and because of the difficulty in obtaining consistent results, this test is being superseded by the
serological or antibody-detection ELISA.
b)
Competitive enzyme-linked immunosorbent assay

Antigen: An international standard strain is not yet available; however, as only one serotype has been
identified so far world-wide, reliable results can be obtained by different laboratories each using their own
standard virus. Even the antibodies induced by the identified RHDV variants are recognised by the standard
method described here. In addition, the test can also easily detect antibodies originating from infection of
rabbits with the nonpathogenic RCV, because of its high genetic correlation with RHDV (Capucci et al.,
1996b; 1997).
The antigen can be prepared as described previously for HI (Section B.2.a), taking care to store it at 20C
in the presence of glycerol at 50% (v/v) to prevent freezing. If necessary, the virus can be inactivated before
the addition of glycerol, using 1.0% binary ethylenimine (BEI) at 33C for 24 hours. Antigen must be
pretitrated in ELISA and then used as the limiting reagent: i.e. the dilution that corresponds to 6070% of the
plateau height (absorbance value at 492 nm in the range 1.11.3).
Anti-RHDV serum: specific polyclonal sera with high anti-RHDV titre can be obtained in different ways. Two
possible and currently used methods are as follows:
i)
Rabbits are infected with a RHDV-positive 10% liver extract diluted 1/100 in PBS to obtain
convalescent sera (2125 days) containing a high level of anti-RHDV IgG. Due to the high mortality
rate associated with RHDV, it is necessary to infect at least 1015 seronegative rabbits or to infect
rabbits that are only partially protected (e.g. 48 rabbits infected from 3 to 7 days post-vaccination).
Rabbits that survive the infection must be bled 2125 days post-infection to obtain the convalescent
sera. Alternatively, convalescent rabbits can be re-infected 34 months post-infection and bled 10
15 days later to obtain RHDV hyperimmune sera.
ii)
RHDV is purified from the livers of experimentally infected rabbits that died from an acute form of the
disease (between 28 and 40 hours post-Infection), using one of the methods that has been published
(Capucci et al., 1990; 1991; 1995; Meyers et al., 1991; Ohlinger et al., 1990). Then the purified RHDV
can be used to immunise sheep or goats according to classical protocols using oil adjuvants. The same
procedure can also be used to inoculate rabbits if the purified virus is inactivated before inoculation.
Anti-RHDV MAbs may be used instead of rabbit polyclonal sera. Purification of rabbit IgG and conjugation to
HRPO can be done following the standard protocols. The conjugated antibody is titred in a sandwich ELISA
in the presence and absence of RHDV antigen (negative rabbit liver). It is then used at the highest dilution
showing maximum (plateau high) absorbance (if the serum had a good anti-RHDV titre, the value of the
HRPO conjugate should range from 1/1000 to 1/3000).
Control sera: negative serum is taken from rabbits fully susceptible to RHDV infection. Positive serum is
either a convalescent serum diluted 1/100 in a negative serum or a serum taken from a vaccinated animal.
947
i)
The rabbit anti-RHDV serum diluted to a predetermined titre, e.g. 1/5000 in 0.05 M carbonate/
bicarbonate buffer, pH 9.6, should be adsorbed to an ELISA microplate of high adsorption capability
(e.g. Nunc Maxisorb Immunoplate) at 4C overnight.
ii)
Wash the plate three times for 35 minutes each time, in PBS, pH 7.4, with 0.05% Tween 20 (PBST).
When the plates are not immediately used, they can be stored, closed in a plastic bag, for 1 month at
20C.
iii)
Distribute 25 l/well PBST with 1% yeast extract (PBSTY) or 1% BSA (PBST-BSA) to all the wells
needed on the plate (see below). Add 7 l of the first serum sample to the first two wells (A1 and B1),
7 l of the second serum to the second two wells (C1 and D1), and continue with the third (E1 and F1)
and the fourth (G1 and H1) sera, thus completing the first column. If qualitative data (positive/negative)
are needed, repeat the operation in the second column with sera samples from 5 to 8, and in the third
column with sera samples from 9 to 12, and so on. If the titre of the serum needs to be determined, the
serum must be diluted further. Agitate the plate and then use an eight-channel micropipette to transfer
7 l from the wells in column 1 to the wells in column 2. This corresponds to a four-fold dilution of the
sera. This last operation can be repeated once (titre 1/160), twice (titre 1/640), or four times (titre
1/10,240). Either in the case of testing sera for qualitative data (single dilution), or for getting the final
titre (several dilutions), complete each plate leaving 12 wells free for the control sera. Add 7 l of
positive sera to wells G7 and H7, and 7 l of negative sera to wells G10 and H10, then dilute them
once and twice (1/401/160).
iv)
Add 25 l/well antigen suspended in PBSTY to all the wells on the plate, at a dilution that is double the
decided dilution, as described above in the antigen section (see the first part of this ELISA method
description).
v)
Incubate the plate at 37C on a rocking platform for 5060 minutes.
vi)
Wash the plate as described in step ii.
vii)
Add 50 l/well rabbit IgG anti-RHDV conjugated with HRPO at the decided dilution, as described above
in the anti-RHDV serum section (see the first part of this ELISA test description).
viii) Incubate the plate at 37C on a rocking platform for 5060 minutes, and wash as described in step ii
adding a fourth wash of 3 minutes duration.
ix)
Use 50 l/well OPD as hydrogen donor under the following conditions: 0.5 mg/ml OPD in 0.15 M
phosphate/citrate buffer, pH 5, and 0.02% H2O2. Stop the reaction after 5 minutes by addition of
50 l/well 1 M H2SO4.
x)
Read the plate on a spectrophotometer using a 492 nm filter.
The serum is considered to be negative when the absorbance value of the first dilution (1/10) decreases by
less than 15% of the reference value (dilution 1/10 of the negative control serum), while it is positive when
the absorbance value decreases by 25% or more. When the absorbance value of the 1/10 dilution
decreases by between 15% and 25% of the reference value, the sera is considered to be doubtful.
The serum titre corresponds to the dilution giving an absorbance value equal to 50% (10) of the average
value of the three negative serum dilutions.
A wide range of titres will be found, depending on the origin of the sample. Positive sera range from 1/640 to
1/10,240 in convalescent rabbits, from 1/80 to 1/640 in vaccinated rabbits and from 1/10 to 1/160 in nonpathogenic infection. Knowing the origin of the sample allows a choice to be made between testing one or
more dilutions. Testing only the first dilution gives a positive or negative result. The titre is established by
testing all dilutions, up to the sixth one.
Due to the significant antigenic differences existing between RHDV and EBHSV (Capucci et al., 1990;
Stoerckle-Berger et al., 1992), the serological techniques described above, which use RHDV as antigen, are
not recommended for the serological diagnosis of EBHS. However, a direct ELISA method could be
employed for the detection of positive and negative EBHSV hare sera; in fact, the adsorption of RHDV on to
the solid phase of an ELISA microplate exposes cross-reactive antigenic determinants. Alternatively, a
specific C-ELISA for EBHSV can be arranged in a similar way, using specific reagent (antigen and antisera)
prepared as described above for RHDV.
948
c)
Isotype enzyme-linked immunosorbent assays (isoELISAs)

These isoELISAs enable the detection and titration of isotypes IgA, IgM and IgG (Capucci et al., 1997).
Isotype titres are critical for the interpretation of field serology in four main areas: cross-reactive antibodies,
natural resilience of young rabbits, maternal antibodies, antibodies in previously infected rabbits (Cooke et
al., 2000). In fact in the case of passive antibodies, only IgG are detected; in vaccinated animals, no IgA are
detected and in recently infected rabbits, first IgM and then IgA and IgG are detected (Cooke et al., 2000).
To detect RHDV-specific IgG, one RHDV-specific MAb is adsorbed to the Maxisorp plate at a concentration
of 2 g/mI by the method described above for the polyclonal serum in the C-ELISA (see above Section
B.2.b, test procedure step i). Virus is added to the plates at a concentration double that used in the C-ELISA
and after incubation and washing; sera are added and serially diluted four-fold starting from 1/40. A MAb
anti-rabbit IgG HRPO conjugate is used to detect IgG bound to the virus. The final step for the isoELISAs for
IgG, lgM and IgA is the addition of OPD and H2SO4 as for the C-ELISA. To detect IgM and IgA isotypes the
phases of the ELISA reaction are inverted in order to avoid competition with IgG, which is usually the
predominant isotype. MAb anti-rabbit IgM or anti-rabbit IgA is adsorbed to the wells and then the sera are
diluted as described above. Incubation with the antigen follows and then HRPO-conjugated MAb is used to
detect the RHDV bound to the plate. Sera are considered to be positive if the OD492 (optical density) value
at the 1/40 dilution is more than 0.2 OD units (two standard deviations) above the value of the negative
serum used as a control. The titre of each serum is taken as the last dilution giving a positive value. Because
isoELISA tests do not follow identical methodology, equivalent titres do not imply that isotypes are present in
the same amounts.

1.
Background
a)

In countries where RHD is endemic, indirect control of the disease in farmed animals and pet rabbits is
achieved by vaccination using the appropriate type of vaccine one that is prepared from clarified liver
suspension of experimentally infected rabbits, and that is subsequently inactivated and adjuvanted. The
methods of inactivation (formaldehyde, beta-propiolactone or other substances) and the adjuvants used
(incomplete mineral oil or aluminium hydroxide), can vary according to the protocol used by the different
manufacturers.
Most vaccine manufacturers recommend a single basic vaccination, with yearly booster. Normally, a 1-ml
dose is inoculated subcutaneously in the neck region, or intramuscularly. The first injection should be given
at 23 months. In those units with no history of disease, where the anamnesis for RHD is negative, it is
advisable to vaccinate only the breeding stock. Considering the high restocking rate in industrial rabbit
farms, the usual vaccination programme is to administer the vaccine to all breeders, independently of their
age, every 6 months. This should ensure that all animals get at least one vaccination per year. Booster
vaccination is strongly recommended to ensure a good level of protection, although experimental data
indicate that protection usually lasts for a long time (over 1 year).
Given the short life-cycle (approximately 80 days) of fattening rabbits and their natural resistance to the
disease until 3540 days of age, vaccinating these rabbits is not necessary if the situation on the farm is
normal, i.e. good biosecurity measures are applied and there are no outbreaks of the disease in the area.
Following an outbreak of RHD, even if strict hygiene and sanitary measures are adopted, including cleaning
and disinfection, safe disposal of carcasses and an interval before restocking, it is strongly recommended to
vaccinate meat animals at the age of 40 days, because the incidence of re-infection is very high. Only after
several production cycles is it advisable to stop vaccination of meat animals. To verify the persistence of
infective RHD inside the unit, a variable number of rabbits, starting with a small sentinel group, should not be
vaccinated.
Given that immunity starts after about 710 days, vaccination could also be considered a quite effective
post-exposure treatment. In some situations in particular it may be included in the emergency strategies
applied when RHD occurs on those farms having separate sheds and where good biosecurity measures are
regularly applied. Indeed, better results in limiting the diffusion of the disease and reducing economic losses
could be obtained by using seroterapy through the parenteral administration of anti-RHDV hyperimmune
sera, which produces a rapid, but short-lived, protection against RHDV infection.
Vaccine should be stored at 28C and it should not be frozen, or exposed to bright light or high
temperatures.
949
2.
a)

i)
At present, RHDV replication can be obtained exclusively in susceptible animals. Therefore, the source
of seed virus for the production of inactivated tissue vaccines is infected liver homogenates obtained by
serial passages in rabbits that have been inoculated with a partially purified RHD viral suspension. The
rabbits used for inoculation are selected from colonies shown to be healthy and susceptible to the
disease by periodic serological testing.
ii)

The partially purified RHD viral suspension is obtained by centrifuging the 1/5 liver suspension (w/v) in
PBS at 10,000 g for 20 minutes at 4C. The resulting supernatant is treated with 8% (v/v) polyethylene
glycol (PEG 6000) overnight at 4C. The pellet is resuspended at a dilution of 1/10 in PBS, and
subsequently centrifuged at 10,000 g for 20 minutes at 4C. The supernatant is ultracentrifuged at
80,000 g for 2 hours at 4C through a 20% cushion of sucrose. The pellet is resuspended in PBS
(1/100 of the starting volume).
This viral suspension is then characterised by negative-stain EM examination, determination of
reactivity in ELISA, and capability of HA at room temperature (HA titre against RBCs of human Group
O higher than 1/1280).
The absence of viable bacteria, fungi or mycoplasma should be determined by using common
laboratory bacteriological methods. PCR methods may be used for the detection of extraneous viruses
(e.g. Myxoma virus [MV]).
Seed virus is controlled by direct inoculation into susceptible rabbits followed by evaluation of the
clinical signs in the course of the experimental infection. Suitable seed virus should cause the death of
7080% of the rabbits within 2472 hours post-inoculation, with the internal organ lesions characteristic
of RHD. To validate the test, gross and histopathological examination of all rabbits should be
performed to exclude undercurrent diseases.
Seed virus is titrated before use and should contain at least 105 LD50. It should be stored frozen
(70C), better with the addition of 1:1 volume of glycerol or freeze-dried.
b)
i)
Procedure
Following inoculation of susceptible rabbits, the liver and spleen of those rabbits that die between
24 and 72 hours post-inoculation are collected. Rabbits that died later must be discarded. The organs
are minced in 1/10 (w/v) sterile PBS, pH 7.27.4, and the mixture is homogenised for 10 minutes in a
blender in a refrigerated environment. The mixture is then treated with 2% chloroform (18 hours at
4C), followed by centrifugation at 6000 g for 1 hour at 4C. The supernatant is collected by high
pressure continuous pumping and is subsequently inactivated. The viral suspension is assayed by HA
test and ELISA and, once the number of HA units from the initial titration is known, more sterile PBS is
added in sufficient volume to provide, after inactivation and adsorption/addition of the adjuvant , a
concentration of 6401280 HA units/ ml in the commercial product. Various agents have proved
effective at abolishing viral infectivity. The most frequently used are formaldehyde and betapropiolactone, which can be used at different concentrations and temperatures, for variable periods of
time and also in combination. During inactivation, it is advisable to continuously agitate the fluid.
Aluminium hydroxide, Freunds incomplete adjuvant or another oil emulsion is then incorporated into
the vaccine as adjuvant. A preservative, thiomersal (merthiolate), is finally added at a dilution of
1/10,000 (v/v) before distribution into bottles.
ii)

As the virus cannot be grown in vitro, the only requirements are those concerning infected animals.
Rabbits must be free from RHDV and myxomatosis virus and should not have anti-RHDV antibodies,
including cross-reactive antibodies induced by the non-pathogenic RHDV-related rabbit calicivirus
(RCV).
The animals (at least 4 months old) must be kept in strict quarantine on arrival, in a separate area and
reared under satisfactory health conditions (see Laboratory animal facilities in Chapter 1.1.3 Biosafety
and biosecurity in the veterinary microbiology laboratory and animal facilities).
Seed virus propagation and production of vaccine batches rely on the same protocol of experimental
infection, involving intramuscular injection of a dose of at least 100 LD50.
950
iii)
In-process controls
Antigen content
The RHD titre is determined before inactivation by calculating the HA titre, which should be higher than
1/1280, and the ELISA reactivity. Both values are again determined after inactivation and
adsorption/addition of the adjuvant. Negative-staining EM confirms the identity of RHD.
Sterility
The organs are tested for the presence of viable bacteria, viruses, fungi or mycoplasma according to
the protocol used for testing master seed virus. PBS solution and aluminium hydroxide gel are
sterilised by autoclaving; oil emulsion is sterilised by heating at 160C for 1 hour.
Inactivation
Before incorporation of the adjuvant, the inactivating agent and the inactivation process must be shown
to inactivate the vaccine virus under the conditions of manufacture. Thus, a test is carried out on each
batch of the bulk harvest as well as on the final product.
Thirty adult rabbits (>4 months of age) are used in three groups of 10. The first and second group are
injected with concentrated antigen and kept under observation for 15 and 7 days, respectively. The
second group is humanely killed after 7 days. The third group is injected with the liver of rabbits from
the second group and kept under observation for 21 days. The dose of the inoculum, administered
parenterally (intramuscular or subcutaneous), is 1 ml of concentrated antigen (PEG precipitation)
corresponding to at least 10 doses (HA 20480). The observation period is: 10 rabbits for 7 days,
10 rabbits for 15 days and 10 rabbits for 21 days. All the rabbits kept under observation must survive
without any clinical signs. The liver should give negative results using the HA test and sandwich ELISA.
The rabbits inoculated with antigen should have a positive serological titre (e.g. >1/80 using
competitive ELISA) and those injected with livers obtained after the first passage should be
serologically negative.
iv)

Sterility, safety and potency tests should be carried out on each batch of final vaccine; tests for
duration of immunity should be carried out once using a typical batch of vaccine, and stability tests
should be carried out on three batches.
Sterility/purity
Each batch of vaccine must be tested for the presence of viable bacteria, viruses, fungi or mycoplasma
according to the same protocol recommended for testing master seed virus.
Safety
Before proceeding with field trials, the safety of this new vaccine is tested in laboratory studies. The
following safety tests in particular should be carried out:
a)
The safety of the administration of one dose;
b)
The safety of the administration of an overdose (at least two doses of inactivated vaccine);
c)
The safety of the repeated administration of one dose.
The test is carried out for each approved route of administration. Use at least 10 adults (>4 months of
age) that are RHDV antibody free. Observe these animals for 21 days by evaluating the following life
parameters: general conditions and reactions, sensory condition, water and food consumption,
characteristics of faeces, and local abnormal reactions at the inoculum point. Record the body
temperature the day before vaccination, at vaccination, 4 hours after vaccination and then daily for
4 days; note the maximum temperature increase for each animal. No abnormal local or systemic
reaction should occur; the average body temperature increase should not exceed 1C and no animal
should have a temperature rise greater than 2C. A local reaction lasting less than 21 days may occur.
If the vaccine is intended for use in pregnant rabbits, administer the vaccine to not less than
10 pregnant does according to the schedule to be recommended. Prolong the observation period until
1 day after parturition. The does should remain in good health and there should not be abnormal local
or systemic reactions. No adverse effects on the pregnancy or on the offspring should be noted.
Batch potency
Use susceptible adult rabbits (>4 months old), free from antibodies against RHDV and reared in
suitable isolation conditions to ensure absence of contact with RHDV. Ten rabbits are vaccinated with
one full dose of vaccine given by the recommended route. Two other groups of five animals each are
vaccinated with 1/4 and 1/16 of the full dose, respectively. A fourth group of 10 unvaccinated rabbits is
951
maintained as controls. All animals are challenged not less than 21 days post-vaccination by
intramuscular inoculation of a dose of RHDV containing at least 100 LD50 or presenting a HA titre
higher than 1/2560. Observe the rabbits for a further 21 days. The test is not valid if: a) during the
period between vaccination and challenge more than 10% of the vaccinated or more than 20% of
control rabbits show abnormal clinical signs or die from causes not attributable to the vaccine; b) less
than 70% of control rabbits died with typical signs of RHD. The vaccine complies with the test if: a) not
less than 90% of vaccinated rabbits show no signs of RHD; b) the mean antibody level of vaccinated
animals, is not significantly less than the level recorded in the protection test performed using as
vaccine the inactivated seed virus.
c)

The tests for safety, potency and sterility of the final product must be performed after bottling and packaging.
Thus, it is important that these two last manufacturing steps be performed following standardised good
manufacturing procedures. The tests are conducted by removing samples from a statistically determined
number of randomly taken multi-dose containers (20, 50 or 100 doses) of vaccine.
i)
Safety requirements
Target and non-target animal safety
Rabbit is the sole species susceptible to RHDV and in the interest of animal welfare, tests and trials
must be held only on target animals. The safety requirements of the final product for rabbits should be
verified in field studies on both fattening and breeder rabbits. At least 30 breeder rabbits, >4 months of
age, and 70 rabbits 3045 days of age should be used. Breeder rabbits are vaccinated subcutaneously
at the back of the neck twice (at an interval of 3 weeks) with one dose. Fattening rabbit are vaccinated
either at 30 or 45 days of age. Animals are observed for 4 months from the first vaccination.
Unvaccinated animals are kept as controls.
The control of the safety of the vaccine in breeder rabbits is done by evaluating their reproductive
performance. The following parameters are considered: local or general reactions; total number of born
rabbits and the number of live rabbits born; percentage of mortality at the weaned period; average
weight of bunnies at the weaned period; daily consumption of food. The control of the safety of the
vaccine in fattened rabbits is done by evaluating their daily health. The following parameters are
considered: local or general reactions; individual weight increase from weaning (30 days) and every
15 days; daily consumption of feed; conversion index; mortality during the fattening period. Vaccinated
rabbits should not show any changes in their general conditions or abnormal local or systemic
reactions for the whole test duration.
The vaccine should not contain any ingredients that are likely to pose a risk for consumers of
vaccinated rabbits. However, as the inactivated vaccine contains a mineral oil adjuvant, there is an
associated risk that might arise from accidental self injection. Accidental injection can cause intense
swelling and severe consequences if expert medical advice is not sought promptly.
Reversion-to-virulence for attenuated/live vaccines
Reversion-to-virulence does not occur because it is an inactivated vaccine.
Environmental consideration
During the safety and efficacy field trials, interactions with other vaccines (e.g. vaccine against
myxomatosis) or pharmaceutical products (medicated feeding-stuffs containing antibiotics against
respiratory diseases and bacterial enteritis) should be checked and recorded. No interactions have
been reported to date.
The inactivated vaccine does not spread in the environment and, in previous trials, there were no signs
of ecotoxicity problems for the viral antigens. The risk of ecotoxicity caused by the use of vaccine is
zero because of the nature of the vaccine (inactivated vaccine for parenteral use). The vaccine
contains no ingredients likely to pose a risk to the environment. In addition, the vaccine is administered
by injection so environmental contamination is unlikely. To achieve the highest standard of safety in
accordance with good hygiene rules, the bottles must be dipped in an antiseptic solution after use.
ii)
The efficacy should be tested in the laboratory with both challenge and serology tests. Forty rabbits
(20 vaccinated and 20 unvaccinated), at least 4 months of age, are challenged with virulent virus: all
the vaccinated animals must be protected, giving positive serological titres and all the control
unvaccinated animals must have died within the observation period.
The in-field efficacy of the vaccine may be determined by evaluating the seroconversion in blood
samples taken from both fattening and breeder rabbits at different check-points from vaccination. Titres
are measured by C-ELISA and anti-isotype IgM, IgA and IgG ELISAs.
952
Before the first vaccination, the C-ELISA should confirm, in all rabbits, the total absence of anti-RHDV
antibodies or the presence of titres at the lower acceptance limit of the protection titre 1/10.
Vaccinated animals develop an RHDV protective immunity in a short period of time: in the serum of
infected animals, circulating antibodies are present just 34 days post-infection (IgM and IgA), whereas
in rabbits vaccinated with the inactivated and adjuvated vaccine, the first antibodies usually appear
after 710 days (only IgM). IgG appear after approximately 1520 days. After vaccination there is very
low or no IgA production. As it is produced only during infection with the live virus following oro-nasal
dissemination, IgA could be considered to be a marker of contact with the field virus. The mucosal
immuno-system may also be involved in protection to the disease even if the vaccine is parentally and
not orally administered. This is suggested by oral challenge experiments in vaccinated rabbits when
IgA but no IgM appear very quickly in the serum. This suggests that B memory cells able to produce
IgA are already present at the mucosal level, which is usually the first site of replication of RHDV.
There is a definite correlation between the titre in the C-ELISA and the state of protection from the
disease, i.e. rabbits with titres higher than 1/10 for antibodies specifically induced by RHDV did not
show any sign of disease when challenged with virulent RHDV. In convalescent rabbits, serological
titres could be as high as 1/20480, whereas in vaccinated rabbits they are usually between 1/40 and
1/640 according to the time elapsed since vaccination. Maternal antibodies (IgG only) usually
disappear within 30 days of age in young rabbits born to vaccinated healthy does, but they last longer
(until 4555 days of age) when rabbits are born to convalescent does, as the passive titres of young
are directly related to that of their mothers. This is true of young rabbits from industrial farms that are
weaned quite early (2535 days of age), whereas in young wild rabbits, maternal antibodies can last
for 80 days (Forrester et al., 2002). In young rabbits (<3540 days old), a low level of antibody (1/80
1/320) could also be induced by an active infection not leading to disease, as commonly occurs in
animals of this age.
The data reported in the literature indicate the long-term duration of immunity induced by a single
vaccination (up to 15 months). At 912 months post-vaccination, titres are 24 times lower than
observed 23 weeks after vaccination. The booster effect, in the case of natural infection or revaccination, depends on the time elapsed since vaccination, i.e. it is lower 57 months post-vaccination
and higher in animals vaccinated before that time.
To exactly determine the duration and efficacy of immunity, it is advisable to carry out the following
test: 20 rabbits vaccinated once are divided into four groups and are serologically tested at monthly
intervals over a period of 1 year. Each group is inoculated with virulent RHDV at 3, 6, 9 months or
1 year post-vaccination. Challenge infection should produce increasing seroconversion, which is
directly related to the time that has elapsed since vaccination. The absence of clinical signs of disease
and mortality supports the efficacy of the vaccine.
iii)
Stability
Evidence should be provided to show that the vaccine passes the batch potency test at 3 months
beyond the suggested shelf life.
A suitable preservative is normally required for vaccine in multi-dose containers. Its persistence
throughout shelf life should be checked.
3.
Several studies have been carried out on the expression of RHDV capsid protein in Escherichia coli, in vaccinia
virus, and in attenuated Myxoma virus (MV). Moreover, it has been shown by various authors that a recombinant
capsid protein, VP60, expressed in the baculovirus/Sf9 cell expression system, self assembled into VLPs that are
structurally and antigenically identical to RHD virions. While the fusion protein expressed in E. coli is highly
insoluble and of low immunogenicity, active immunisation can be achieved with VLPs obtained in the baculovirus
system or by using recombinant vaccinia, MV and canarypox, administered either intramuscularly or orally. In
particular, rabbits vaccinated with recombinant MV expressing the RHDV capsid protein were protected against
lethal RHDV and MV challenges. The resulting recombinant virus was also capable of spreading horizontally and
promoting protection of contact animals, thus providing the opportunity to immunise a wild rabbit population.
Similarly, the immunogenicity of VLPs administered by the oral route as an alternative to parenteral immunisation
offers an economical and practical way to administer a vaccine for mass immunisation of wild animals.
The VP60 structural protein has been also expressed in transgenic plants, either with a new plum pox virus
(PPV)-based vector (PPV-NK), or in transgenic potato plants under the control of a cauliflower mosaic virus 35S
promoter or a modified 35S promoter. In both cases the immunisation of rabbits with extracts of Nicotiana
clevelandii plants infected with the PPV-NK VP60 chimera and with leaf extracts from potatoes carrying this
modified 35S promoter, respectively, induced an efficient immune response that protected animals against a
lethal challenge with RHDV. However, at the present time, recombinant vaccines are not yet registered and
953
In France, a vaccine has been commercialised that is a combination of a traditional inactivated liver-derived RHD
vaccine and a live attenuated Myxoma virus vaccine, and which can be administered by the intradermal route.
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syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses. J. Virol., 68, 51645173.
*
* *
NB: There is an OIE Reference Laboratory for Rabbit haemorrhagic disease
diagnostic tests, reagents and vaccines for rabbit haemorrhagic disease
955
SECTION 2.7.
OVIDAE AND CAPRINAE

CHAPTER 2.7.1.
BORDER DISEASE
SUMMARY
Border disease (BD) is a viral disease of sheep and goats first reported in sheep in 1959 from the
border region of England and Wales, and since recorded world-wide. Distribution of the virus is
world-wide. Prevalence rates vary in sheep from 5% to 50% between countries and from region to
region within countries. Clinical signs include barren ewes, abortions, stillbirths and the birth of
small weak lambs. Affected lambs can show tremor, abnormal body conformation and hairy fleeces
(so-called hairy-shaker or fuzzy lambs) and the disease has been referred to as hairy shaker
disease. Vertical transmission plays an important role in the epidemiology of the disease. Infection
of fetuses can result in the birth of persistently infected (PI) lambs. These PI lambs are viraemic,
antibody negative and constantly excrete virus. The virus spreads from sheep to sheep, with PI
animals being the most potent source of infection. Infection in goats is less common with abortion
being the main presenting sign. In many regions the commonest cause of BD is the pestivirus
border disease virus (BDV), but in some parts of the world, bovine viral diarrhoea virus (BVDV) may
be a more common cause of BD. The source of BVDV for sheep often is close contact with cattle.
It is important to identify the viraemic PI animals so that they will not be used for breeding or trading
purposes. Serological testing is insufficient. It is generally considered that serologically positive,
nonviraemic sheep are safe, as latent infections are not known to occur in recovered animals.
Identification of the agent: BD virus (BDV) is a Pestivirus in the family Flaviviridae and is closely
related to classical swine fever virus and BVDV. Nearly all isolates of BDV are noncytopathogenic
in cell culture. There are no defined serotypes but virus isolates exhibit considerable diversity.
Three distinct antigenic groups, plus two further separate genotypes, have been identified.
Apparently healthy PI sheep resulting from congenital infection can be identified by isolation and
immunostaining of noncytopathogenic virus from blood or sera in laboratory cell cultures. Rapid
direct methods to identify PI sheep include detection of viral antigen or viral RNA in leukocytes and
immunohistochemical demonstration of viral antigen in skin biopsies. The demonstration of virus is
less reliable in lambs younger than 2 months that have received colostral antibody. Acute infection
is usually subclinical and viraemia is transient and difficult to detect. From dead animals, the
isolation of virus from tissues of aborted or stillborn lambs is difficult, but tissues from PI sheep
contain high levels of virus, which can be easily detected by isolation and direct methods.
Serological tests: Acute infection with BDV is best confirmed by demonstrating seroconversion
using paired or sequential samples from several animals in the group. The enzyme-linked
immunosorbent assay and virus neutralisation test are the most commonly used antibody detection
methods.
Requirements for vaccines and diagnostic biologicals: There is no standard vaccine for BDV,
but a commercial killed whole-virus vaccine has been produced. Ideally, such a vaccine should be
suitable for administration to females before breeding for prevention of transplacental infection. The
957
Chapter 2.7.1. Border disease
use of BVDV vaccines has been advocated, but the antigenic diversity of BD viruses must be
considered.
BD viruses have contaminated several modified live veterinary vaccines produced in sheep cells or
containing sheep serum. This potential hazard should be recognised by manufacturers of biological
products.
A. INTRODUCTION
Border disease virus (BDV) is a Pestivirus of the family Flaviviridae and is closely related to classical swine fever
virus (CSFV) and bovine viral diarrhoea virus (BVDV). Pestivirus taxonomy is quite problematic at present. There
are four recognised species, namely CSFV, BVDV types 1 and 2 and BDV (Theil et al., 2005). While CSF
viruses are predominantly restricted to pigs, examples of the other three species have all been recovered from
sheep, with the majority of isolates being BD viruses (Vilcek et al., 1997). Nearly all virus isolates of BDV are
noncytopathogenic, although occasional cytopathic viruses have been isolated (Vantsis et al., 1976). BDV
spreads naturally among sheep by the oro-nasal route and by vertical transmission. It is principally a cause of
congenital disease in sheep and goats, but can also cause acute and persistent infections. Infection is less
common in goats, in which persistent infection is rare as abortion is the main presenting sign. Sheep can become
infected with BVDV from cattle (Carlsson, 1991), and in some countries, BVDV can be a more common cause of
BD than BDV. Pigs may also be infected by pestiviruses other than CSFV and antibodies to BDV in pigs may
interfere with tests for the diagnosis of CSF (Oguzoglu et al., 2001). Several new BD viruses from sheep, goats
and Pyrenean chamois (Rupicapra pyrenaica pyrenaica) have been described recently. Phylogenetic analysis
using computer-assisted nucleotide sequence analysis suggests that genetic variability among BD viruses is
greater than within each of the other pestivirus species. Four distinguishable genogroups of BDV have been
described as well as putative novel pestivirus genotypes from Tunisian sheep and a goat (Becher et al., 2003;
Vilcek & Nettleton, 2006). The chamois BD virus is similar to isolates from sheep in the Iberian Peninsula
(Valdazo-Gonzalez et al., 2007). This chapter describes BDV infection in sheep.
a)
Acute infections
Healthy newborn and adult sheep exposed to BDV experience only mild or inapparent disease. Slight fever
and a mild leukopenia are associated with a short-lived viraemia detectable between days 4 and 11 postinfection, after which virus neutralising antibody appears in the serum (Thabti et al., 2002).
Acute infections are best diagnosed serologically using paired sera from a representative number of sheep.
Occasional BDV isolates have been shown to produce high fever, profound and prolonged leukopenia,
anorexia, conjunctivitis, nasal discharge, dyspnoea and diarrhoea, and 50% mortality in young lambs. One
such isolate was recovered from a severe epidemic of BD among dairy sheep in 1984 (Chappuis et al.,
1986). A second such isolate was a BDV contaminant of a live CSFV vaccine (Wensvoort & Terpstra, 1988).
b)
Fetal infection
The main clinical signs of BD are seen following the infection of pregnant ewes. While the initial maternal
infection is subclinical or mild, the consequences for the fetus are serious. Fetal death may occur at any
stage of pregnancy, but is more common in fetuses infected early in gestation. Small dead fetuses may be
resorbed or their abortion may pass unnoticed as the ewes continue to feed well and show no sign of
discomfort. As lambing time approaches, the abortion of larger fetuses, stillbirths and the premature births of
small, weak lambs will be seen. Confirmation that an abortion or stillbirth is due to BDV is often difficult to
establish, but virus may be isolated from fetal tissues in some cases. In aborted fetuses, it is also possible to
detect virus by immunohistochemistry of brain, thyroid and other tissues (Thur et al., 1997). Samples of fetal
fluids or serum should be tested for BDV antibody.
During lambing, an excessive number of barren ewes will become apparent, but it is the diseased live lambs
that present the main clinical features characteristic of BD. The clinical signs exhibited by BD lambs are very
variable and depend on the breed of sheep, the virulence of the virus and the time at which infection was
introduced into the flock. Affected lambs are usually small and weak, many being unable to stand. Nervous
signs and fleece changes are often apparent. The nervous signs of BD are its most characteristic feature.
The tremor can vary from violent rhythmic contractions of the muscles of the hindlegs and back, to barely
detectable fine trembling of the head, ears, and tail. Fleece abnormalities are most obvious in smoothcoated breeds, which develop hairy fleeces, especially on the neck and back. Abnormal brown or black
pigmentation of the fleece may also be seen in BD-affected lambs. Blood samples to be tested for the
presence of BDV and/or antibody should be collected into anticoagulant from suspect lambs before they
have received colostrum. Once lambs have ingested colostrum, it is difficult to detect virus until they are
2 months old and maternal antibody levels have waned. However, during this period, it may be possible to
detect viral antigen in skin biopsies, by immunohistochemistry, in washed leukocytes by enzyme-linked
immunosorbent assay (ELISA) or by reverse-transcription polymerase chain reaction (RT-PCR).
958
With careful nursing, a proportion of BD lambs can be reared, although deaths may occur at any age. The
nervous signs gradually decline and can disappear by 36 months of age. Weakness, and swaying of the
hind-quarters, together with fine trembling of the head, may reappear at times of stress. Affected lambs often
grow slowly and under normal field conditions many will die before or around weaning time. In cases where
losses at lambing time have been low and no lambs with obvious signs of BD have been born, this can be
the first presenting sign of disease.
Some fetal infections occurring around mid-gestation can result in lambs with severe nervous signs,
locomotor disturbances and abnormal skeletons. Such lambs have lesions of cerebellar hypoplasia and
dysplasia, hydranencephaly and porencephaly resulting from necrotising inflammation. The severe
destructive lesions appear to be immune mediated, and lambs with such lesions frequently have high titres
of serum antibody to BDV. Most lambs infected in late gestation are normal and healthy and are born free
from virus but with BDV antibody. Some such lambs can be weak and may die in early life (Barlow &
Patterson, 1982).
c)
Persistent viraemia
When fetuses survive an infection that occurs before the onset of immune competence, they are born with a
persistent viraemia. The ovine fetus can first respond to an antigenic stimulus between approximately
60 and 85 days of its 150-day gestation period. In fetuses infected before the onset of immune competence,
viral replication is uncontrolled and 50% fetal death is common. In lambs surviving infection in early
gestation, virus is widespread in all organs. Such lambs appear to be tolerant of the virus and have a
persistent infection, usually for life. A precolostral blood sample will be virus positive and antibody negative.
Typically, there is no inflammatory reaction and the most characteristic pathological changes are in the
central nervous system (CNS) and skin. Throughout the CNS, there is a deficiency of myelin, and this
causes the nervous signs. In the skin, primary wool follicles increase in size and the number of secondary
wool follicles decreases, causing the hairy or coarse fleece.
Persistently viraemic sheep can be diagnosed by virus isolation/detection in a blood sample. Viraemia is
readily detectable at any time except within the first 2 months of life, when virus is masked by colostral
antibody; however, the virus may be detected in washed leukocytes during this period, and in animals older
than 4 years, some of which develop low levels of anti-BDV antibody (Nettleton et al., 1992). Although virus
detection in blood during an acute infection is difficult, persistent viraemia should be confirmed by retesting
animals after an interval of at least 3 weeks.
Some viraemic sheep survive to sexual maturity and are used for breeding. Lambs born to these infected
dams are always persistently viraemic. Persistently viraemic sheep are a continual source of infectious virus
to other animals and their identification is a major factor in any control programme. Sheep being traded
should be screened for the absence of BDV viraemia.
Usually persistently infected (PI) rams have poor quality, highly infective semen and reduced fertility. All
rams used for breeding should be screened for persistent BDV infection on a blood sample. Semen samples
can also be screened for virus, but virus isolation is much less satisfactory than from blood due to the toxicity
of semen for cell cultures. RT-PCR for detecting pestivirus nucleic acid may be justifiable on semen from
some rams.
d)
Late-onset disease in persistently viraemic sheep

Some PI sheep housed apart from other animals spontaneously develop intractable diarrhoea, wasting,
excessive ocular and nasal discharges, sometimes with respiratory distress. At necropsy such sheep have
gross thickening of the distal ileum, caecum and colon resulting from focal hyperplastic enteropathy.
Cytopathic BDV can be recovered from the gut of these lambs. With no obvious outside source of cytopathic
virus, it is most likely that such virus originates from the lamb's own virus pool. Other PI sheep in the group
do not develop the disease. This syndrome, which has been produced experimentally and recognised in
occasional field outbreaks of BD, has several similarities with bovine mucosal disease (Nettleton et al.,
1992).
1.
Identification of the agent (the prescribed test for international trade)
There is no designated OIE reference laboratory for BDV, but the reference laboratories for BVDV or CSFV will
be able to provide advice (see Table given in Part 4 of this Terrestrial Manual). One of the most sensitive proven
methods for identifying BDV remains virus isolation. Direct immunofluorescence or other immunohistochemical
959
techniques on frozen tissue sections as well as antigen-detecting ELISA and conventional and real-time RT-PCR
are also valuable methods for identifying BDV-infected animals.
a)
Virus isolation
It is essential that laboratories undertaking virus isolation have a guaranteed supply of pestivirus-free
susceptible cells and fetal bovine serum (FBS), or equivalent, that contain no anti-pestivirus activity and no
contaminating virus. It is important that a laboratory quality assurance programme be in place.
The virus can be isolated in a number of primary or secondary ovine cell cultures (e.g. kidney, testes, lung).
Ovine cell lines for BDV growth are rare. Semicontinuous cell lines derived from fetal lamb muscle (FLM),
whole embryo (Thabti et al., 2002) or sheep choroid plexus can be useful, but different lines vary
considerably in their susceptibility to the virus. Ovine cells have been used successfully for the isolation and
growth of BD viruses and BVDV types 1 and 2 from sheep. In regions where sheep may become infected
with BVD viruses from cattle, a virus isolation system using both ovine and bovine cells could be optimal.
Several bovine cell cultures may be suggested, including testicular, embryonic tracheal or turbinate cells, or
a susceptible continuous kidney cell line. However, bovine cells are insensitive for the primary isolation and
growth of some BD viruses, so reliance on bovine cells alone is inadvisable.
From live animals, serum can be tested for the presence of infectious virus, but the most sensitive way to
confirm pestivirus viraemia is to wash leukocytes repeatedly (at least three times) in culture medium before
co-cultivating them with susceptible cells for 57 days. Cells are frozen and thawed once and an aliquot
passaged onto further susceptible cells grown on cover-slips, chamber slides or plastic plates. The cells are
stained, 34days later, for the presence of pestivirus using an immunofluorescence or immunoperoxidase
test. Tissues should be collected from dead animals in virus transport medium (10% [w/v]). In the laboratory,
the tissues are ground, centrifuged to remove debris, and the supernatant passed through 0.45 m filters.
Spleen, thyroid, thymus, kidney, brain, lymph nodes and gut lesions are the best organs for virus isolation.
Semen can be examined for the presence of BDV, but raw semen is strongly cytotoxic and must be diluted,
usually at least 1/10 in culture medium. As the major threat of BDV-infected semen is from PI rams, blood is
a more reliable clinical sample than semen for identifying such animals. There are many variations in virus
isolation procedures. All should be optimised for maximum sensitivity using a standard reference virus
preparation and, whenever possible, recent BDV field isolates. A practical sensitive isolation procedure is
outlined below:
i)
Cultures with subconfluent or newly confluent monolayers of susceptible ovine cells are washed at
least twice with Hanks balanced salt solution to remove growth medium before being inoculated with
approximately 0.2 ml of sample, which is allowed to adsorb for 2 hours at 37C.
ii)
Cultures are washed with at least 2 ml medium. This is discarded and an appropriate volume of culture
maintenance medium is added.
iii)
Cultures are incubated for 57 days at 37C. They are examined microscopically on a daily basis and
evidence of cytopathic effect (CPE) is recorded.
iv)
Cultures are frozen at 70C and then thawed for passage, as before, on to fresh cultures.
v)
34 days later, cells growing on glass are fixed in cold acetone for 15 minutes while cells growing on
plastic are fixed as described in the virus neutralisation test section below. Fixed cells are stained using
an indirect or direct immunofluorescence method. Essential controls must include known negative cells
and cells growing standard cytopathic and noncytopathic BDV strains.
vi)
The cells are examined under a UV microscope for the diffuse cytoplasmic fluorescence that is
characteristic of pestiviruses.
Immunoperoxidase staining can also be used on cover-slips, chamber slides as well as microtitre plates (see
method under virus neutralisation [VN] test below). Frozen and thawed cultures can also be tested in an
antigen detection ELISA system that employs monoclonal antibodies (MAbs) against epitopes on the
conserved nonstructural NS 2-3 protein. Staining for noncytopathic pestiviruses will usually detect virus at
the end of the first passage, but in order to detect slow-growing viruses in poorly permissive cells two
passages are desirable.
b)
Immunohistochemistry
Viral antigen demonstration is possible in most of the tissues of PI animals (Braun et al., 2002; Thur et al.,
1997). This should be done on acetone-fixed frozen tissue sections (cryostat sections) or paraffin wax
embedded samples using appropriate antibodies. Panpestivirus-specific antibodies with NS2-3 specificity
are suitable. Tissues with a high amount of viral antigen are brain, thyroid gland and oral mucosa. Skin
biopsies have been shown to be useful for in-vivo diagnosis of persistent BDV infection.
960
c)
Enzyme-linked immunosorbent assay for antigen detection

The first ELISA for pestivirus antigen detection was described for detecting viraemic sheep. This has now
been modified into a double MAb capture ELISA for use in sheep and cattle. Two capture MAbs are bound
to wells in microtitre plates, and two other MAbs, conjugated to peroxidase, serve as detector MAbs
(Entrican et al., 1994). The test is most commonly employed to identify PI viraemic sheep using washed,
detergent-lysed blood leukocytes. The sensitivity is close to that of virus isolation and it is a practical method
for screening high numbers of blood samples. As with virus isolation, high levels of colostral antibody can
mask persistent viraemia. The ELISA is more effective than virus isolation in the presence of antibody, but
may give false-negative results in viraemic lambs younger than 2 months old. The ELISA is usually not
sensitive enough to detect acute BDV infections on blood samples. As well as for testing leukocytes, the
antigen ELISA can also be used on tissue suspensions, especially spleen, from suspected PI sheep and, as
an alternative to immunofluorescence and immunoperoxidase methods, on cell cultures. Several pestivirus
ELISA methods have been published and commercial kits are now available for detecting BDV. ELISAs
employing MAbs recognising epitopes on the conserved non-structural NS2-3 should recognise all strains of
BDV. ELISAs relying on MAbs recognising epitopes on structural proteins such as Erns, that are used for
BVDV detection in cattle, are unsuitable for the diagnosis of BDV viraemia in sheep.
d)
Nucleic acid detection methods

The complete genomic sequences of three BD viruses have been determined and compared with those of
other pestiviruses (Becher et al., 1998; Ridpath & Bolin, 1997). Phylogenetic analysis shows BD viruses to
be more closely related to CSFV than to BVDV (Becher et al., 2003; Van Rijn et al., 1997; Vilcek & Nettleton,
2006; Vilcek et al., 1997). RT-PCR for diagnosing pestivirus infection is now used widely. Various formats
are described. Basic RT-PCR protocols involve the following stages:
i)
Total RNA is isolated by phenol-chloroform, TRIZOL, guanidine isothiocyanate (GITC), or a

commercially available spin column or magnetic bead separation methods. NOTE: many of these
chemicals are highly toxic; adhere to the manufacturers safety procedures.
ii)
RT-PCR is performed. This is a two-stage reaction that consists of:

a)
Reverse transcription to produce single-stranded cDNA from viral RNA;
b)
Subsequent PCR amplification of the cDNA to produce readily detectable amounts of doublestranded DNA.
This process may be done as two separate reactions, each done in a separate PCR tube (two-step
RT-PCR), or as two stages in a single PCR tube (one-step RT-PCR). In a two-step format, either
random hexamers or specific primers may be used to prime the RT stage; in the one-step format only
specific primers may be used.
iii)
Specific product is detected by one of the following methods:

a) Use of BD-specific primers in the RT-PCR, with visualisation by agarose gel electrophoresis,
ethidium bromide staining and UV transillumination to demonstrate the correct sized amplicon.
NOTE: ethidium bromide is highly toxic; adhere to manufacturers recommendations for handling.
NOTE: UV transillumination must be carried out taking appropriate precautions to minimise skin
exposure.
b) Nested PCR, using pan-pestivirus primers (usually directed to the 5UTR region) in a primary PCR,
followed by specific BD virus primers in a secondary (nested) PCR. Such assays typically employ
approximately 25 cycles in the primary PCR and 3035 cycles in the nested PCR. Amplicons are
detected by visualisation by agarose gel electrophoresis as above. These assays increase
specificity and sensitivity but are more susceptible to contamination.
c) Real-time RT-PCR, using specific BD primers and/or fluorophore-labelled oligonucleotide probe to
detect BD. This method has advantages in specificity and prevention of contamination. It is also
possible to carry out a nested form of real-time RT-PCR.
Oligonucleotide primer/probe design is critical to the success of these assays, due to the genetic variability
of BDV isolates. Panpestivirus primers are valuable for detecting and typing all species of Pestivirus
(Sandvik et al., 1997; Vilcek et al., 1994), and can be combined with sequencing if required for specificity or
epidemiological investigation. Specific primers for the specific recognition of BD viruses have also been
described (Fulton et al., 1999; Vilcek & Paton, 2000; Willoughby et al., 2006). Using a closed one-tube RTPCR with fluorescent probes reduces the potential for cross-contamination of diagnostic samples
(McGoldrick et al., 1999; Willoughby et al., 2006). The development of a real-time RT-PCR allows the rapid
simultaneous detection and typing of ovine pestiviruses (Willoughby et al., 2006). Important applications of
RT-PCR methods include the detection of viral RNA in fetal tissues and in cell culture constituents or
vaccines (Vilcek, 2001); it may also prove valuable for detecting virus when BDV-specific antibodies are
961
present. Validation of the RT-PCR is in process. The precautions to be taken with RT-PCR have been
covered in Chapter 1.1.5 Principles and methods of validation of diagnostic assays for infectious diseases.
2.
Serological tests
Antibody to BDV is usually detected in sheep sera using VN or an ELISA. The less sensitive agar gel
immunodiffusion (AGID) test may also be used. Control positive and negative reference sera must be included in
every test. These should give results within predetermined limits for the test to be considered valid. Single sera
can be tested to determine the prevalence of BDV in a flock, region or country. For diagnosis, however, acute and
convalescent sera are the best samples for confirming acute BDV infection. Repeat sera from one animal should
always be tested alongside each other on the same plate.
a)

A standard cytopathic strain of BDV (e.g. Moredun strain) can be used for the VN test with semicontinuous
cells such as FLM. An outline protocol is given below.
i)
Test and control sera are heat-inactivated for 30 minutes at 56C.
ii)
From a starting serum dilution of 1/4, serial twofold dilutions of the test sera are made in cell culture
growth media in a cell culture-grade flat-bottomed 96-well microtitre plate. For each sample two or four
wells are used at each dilution depending on the accuracy required. The range of dilution can also
vary. It is common to screen sera initially at a dilution of 1/4 and titrate out positive sera. To screen
sera a minimum of four wells is required. The standard working volume is 25 l: 25 l of the diluted
serum is added to each well; 25 l of media is added to each of the lower two control wells and 25 l of
media containing 100 TCID50 (50% tissue culture infective dose) of virus is added to each of the two
upper test wells. Control positive and negative sera and a virus titration are included in every test.
iii)
Plates are sealed with nontoxic plate sealers or lids, and incubated at 37C for 1 hour.
iv)
100 l of a cell suspension with a count of 2 105 cells/ml is added to every well. The FBS or
equivalent serum used for cell growth must be free from antibody to BDV.
v)
The plate is sealed or incubated in a humid chamber in 5% CO2 for 4 days at 37C.
vi)
The wells are examined microscopically for CPE. In the control wells of the test sera, cell degeneration
will be due to toxicity. Further dilution of toxic sera can be attempted, but it may not be possible to
obtain reliable results with occasional sera. The VN titre for each serum is the dilution at which the
virus is neutralised in 50% of the wells. This can be calculated by the SpearmanKrber method. A
seronegative animal will show no neutralisation at the lowest dilution (i.e. 1/4).
The choice of test virus is difficult due to antigenic diversity among pestiviruses (Dekker et al., 1995;
Nettleton et al., 1998) Standard strains of cytopathic BVD viruses and bovine cells can be used. Oregon
C24V results correlate better with Moredun BDV than results with the NADL strain. No single strain is ideal.
A local strain that gives the highest antibody titre with a range of positive sheep sera should be used. The
VN test can also be used with noncytopathic viruses when the following immunoperoxidase staining system
is used after step v above:
i)
The culture medium is removed and the cells are washed gently with warm phosphate buffered saline
(PBS), air-dried and cooled to 4C.
ii)
The cells are fixed by quickly adding to all wells 95% acetone (in water) previously cooled to 20C.
The plates are held at 20C for 30 minutes and should not be stacked or allowed to warm as etching
of the plastic may occur.
iii)
The acetone is removed and the plates are dried quickly in a cool environment.
iv)
50 l of BDV antiserum is added to all wells at a predetermined dilution in PBS with 1% Tween 80
(PBST). The plates are incubated at 37C for 30 minutes in a humid atmosphere.
v)
The plates are emptied and washed three times with PBST.
vi)
The wells are drained and an appropriate anti-species serum conjugated to peroxidase at a
predetermined dilution is added, and the plates are left for 30 minutes at 37C in a humid atmosphere.
vii)
The plates are emptied and washed three times with PBST.
viii) The plates are drained well and 50 l of activated substrate, e.g. 3-amino-9-ethyl carbazole (AEC) is
added. AEC stock solution is: AEC (0.1 g) dissolved in dimethyl formamide (15 ml). For use add stock
962
(0.3 ml) to membrane-filtered 0.05 M acetate buffer, pH 5.0, (4.7 ml), and then add 30% H2O2 (5 l).
NOTE. This solution is toxic and should be handled with adequate precautions.
ix)
The plates are incubated at room temperature and known virus-positive control wells are monitored for
development of specific red-brown cytoplasmic staining. When staining is complete the substrate is
removed carefully and the wells are washed thoroughly with tap water. Leaving the tap water in the
wells, the plates are examined microscopically for virus-containing wells.
x)
The VN titre is calculated as above using the SpearmanKrber method.
xi)
Alternatively, the test can be performed using direct fluorescein-isothiocyanate conjugate staining.
Occasionally there may be a need to determine whether antibody in a flock is against a virus belonging to a
particular Pestivirus serogroup. A differential VN test can be used in which sera are titrated out against
representative viruses from each of the four Pestivirus groups, i.e. BDV, BVDV types 1 and 2, and CSFV.
Maximum titre will identify the infecting serotype and the spectrum of cross-reactivity with the other
serotypes will also be revealed.
b)

An MAb-capture ELISA for measuring BDV antibodies has been described. Two panpestivirus MAbs that
detect different epitopes on the immunodominant nonstructural protein NS 2/3 are used to capture
detergent-lysed cell-culture grown antigen. The results correlate qualitatively with the VN test (Fenton et al.,
1991).
Antigen is prepared as follows: Use eight 225 cm2 flasks of newly confluent FLM cells; four flasks will be
controls and four will be infected. Wash the flasks and infect four with a 0.010.1 m.o.i. (multiplicity of
infection) of Moredun cytopathic BDV. Allow the virus to adsorb for 2 hours at 37C. Add maintenance media
containing 2% FBS (free from BDV antibody), and incubate cultures for 45 days until CPE is obvious. Pool
four control flask supernatants and separately pool four infected flask supernatants. Centrifuge at 3000 g for
15 minutes to pellet cells. Discard the supernatants. Retain the cell pellets. Wash the flasks with 50 ml of
PBS and repeat the centrifugation step as above. Pool all the control cell pellets in 8 ml PBS containing 1%
Nonidet P40 and return 2 ml to each control flask to lyse the remaining attached cells. Repeat for infected
cells. Keep the flasks at 4C for at least 2 hours agitating the small volume of fluid on the cells vigorously
every 30 minutes to ensure total cell detachment. Centrifuge the control and infected antigen at 12,000 g for
5 minutes to remove the cell debris. Supernatant antigens are stored at 70C in small aliquots.
Test procedure
i)
The two MAbs are diluted to a predetermined dilution in 0.05 M bicarbonate buffer, pH 9.6. All wells of a
suitable ELISA-grade microtitre plate (e.g. Nunc maxisorb, Greiner 129b) are coated overnight at 4C.
ii)
After washing three times in PBST, a blocking solution of PBST containing 10% horse serum (PBSTH)
is added to all wells, which are incubated at 37C for 1 hour.
iii)
The antigen is diluted to a predetermined dilution in PBSTH and alternate rows of wells are coated with
virus and control antigens for 1 hour at 37C. The plates are then washed three times in PBST before
addition of test sera.
iv)
Test sera are diluted 1/50 in PBSTH and added to duplicate virus and duplicate control wells for 1 hour
at 37C. The plates are then washed three times in PBST.
v)
Anti-ovine IgG peroxidase conjugate is diluted to a predetermined dilution in PBSTH and added to all
wells for 1 hour at 37C. The plates are washed three times in PBST.
vi)
A suitable activated enzyme substrate, such as ortho-phenylene diamine (OPD) or tetramethyl blue
(TMB), is added noting the manufacturers toxicity warning. After colour development, the reaction is
stopped with sulphuric acid and the absorbance read on an ELISA plate reader. The mean value of the
two control wells is subtracted from the mean value of the two virus wells to give the corrected
absorbance for each serum. Results are expressed as corrected absorbance with reference to the
corrected absorbance of known positive and negative sera. Alternatively, ELISA titres can be
extrapolated from a standard curve of a dilution series of a known positive reference serum.
If antigens of sufficient potency can be produced the MAb capture stage can be omitted. In this case
alternate rows of wells are coated with virus and control antigen diluted to a predetermined dilution in
0.05 M bicarbonate buffer, pH 9.6, overnight at +4C. The plates are washed and blocked as in step ii
above. After washing, diluted test sera are added and the test proceeds from step iv as above.
c)

The AGID test was first used to demonstrate an immunological relationship between BD, BVD and CSF
viruses.
963
The Oregon C24V strain of BVDV grown on calf testis cells has been used to detect antibody in sheep.
Suitable antigen can be prepared using medium harvested from cells showing early CPE. Concentration of
the medium approximately 100-fold by dialysis against polyethylene glycol (PEG) is required. Alternatively,
PEG 6000 can be added to sonicated virus/cell suspensions at the rate of 8% (w/v). After constant stirring
overnight at 4C, the precipitate is removed by centrifugation at 1800 g for 1 hour. The supernatant is
decanted thoroughly and the precipitate resuspended to 1% of the original virus/cell culture volume in
distilled water. The resuspended precipitate is centrifuged at 286,000 g for 2 hours and the supernatant
withdrawn for use as antigen. The precipitate is discarded.

To be useful, a BDV vaccine should be effective when administered to female sheep before breeding to prevent
transplacental infection. Experimental and commercial inactivated whole virus BDV vaccines have been produced
in Europe (Brun et al., 1993; Vantsis et al., 1980).
Pestivirus contaminants of modified live virus vaccines have been found to be a cause of serious disease
following their use in pigs, cattle, sheep and goats. Contaminated vaccines have included those used for the
control of Aujeskys disease, CSF, rotavirus, coronavirus, rinderpest, sheep pox and contagious pustular
dermatitis. The insidious ability of pestiviruses to cross the placenta, and thus establish PI animals, gives them
the potential to contaminate vaccines through cells, serum used as medium supplement, or seed stock virus. As
nearly all isolates of pestiviruses are noncytopathic, they will remain undetected unless specific tests are carried
out.
1.
Seed management
a)
Characterisation of the seed

An ideal vaccine should contain a strain or strains of virus that give protection against all sheep pestiviruses.
Recent evidence is that three antigenically distinguishable groups of pestiviruses infect sheep. One group is
represented by the Moredun reference strain of BDV; the second group contains viruses similar to the
majority of cattle BVDV strains (BVDV type 1); and the third group contains the less common BVDV (type 2)
strains (Wensvoort et al., 1989). More recently, ovine pestivirus isolates have been divided on the basis of
phylogenetic and antigenic analysis into BDV-1, BDV-2 and BDV-3 genotypes (Becher et al., 2003).
Phylogenetic analysis alone suggests that a BDV Italian caprine isolate and the chamois/Iberian sheep
isolates represent two further genotypes (Vilcek & Nettleton, 2006). Further cross-neutralisation studies are
required to determine the significance of these findings. Nevertheless it would appear that any BDV vaccine
should contain at least a representative of the BDV and BVDV (type 1) groups. Characterisation of the
biologically cloned vaccine viruses should include typing with MAbs and genotyping (Paton et al., 1995).
b)
Culture
A variety of ruminant cell cultures can be used. Optimal yields depend on the cell type and isolate used. A
commercial BDV vaccine containing two strains of virus is prepared on ovine cell lines (Brun et al., 1993).
Cells must be produced according to a seed-lot system from a master cell seed (MCS) that has been shown
to be free from all contaminating microorganisms. Vaccine should only be produced in cells fewer than
20 passages from the MCS. Control cells from every passage should be checked for pestivirus
contamination.
c)
All vaccines should pass standard tests for safety and efficacy. Safety testing of inactivated BDV vaccines
should include monitoring of all vaccine components for contaminating pestiviruses.
Efficacy tests of BDV vaccines should demonstrate their ability to prevent transplacental spread of virus.
Effective challenge of vaccinated pregnant ewes at 5060 days gestation has been achieved by intranasal
installation of virus or by mixing with PI sheep (Brun et al., 1993).
2.
Inactivated vaccines have been prepared using conventional laboratory techniques with stationary or rolled cell
cultures. Inactivants have included formalin and beta-propriolactone. Adjuvants have included aluminium
hydroxide and oil (Brun et al., 1993; Vantsis et al., 1980).
964
3.
In-process control
Cultures should be inspected daily to ensure they are free from gross bacterial contamination and that any CPE
observed is appropriate to the cytopathic virus being grown. No CPE should be observed in cultures being used
to grow noncytopathic strains of virus.
4.
Batch control
a)
Sterility
b)
Safety
Samples from inactivated vaccines should be tested rigorously for viable virus. Samples of the product
should be passaged several times in sensitive cell cultures to ensure absence of live BDV. This in-vitro
monitoring can be augmented by injecting two BDV-seronegative sheep with 20 doses of unformulated
antigen as part of a standard safety test. Presence of live virus will result in the development of a more
convincing serological response than will occur with inactivated virus alone. The sheep sera can also be
examined for antibody to other prescribed agents.
c)
Potency
Vaccine potency is also best tested in seronegative sheep in which the development and level of antibody is
measured. An indirect measure of potency is given by the level of virus infectivity prior to inactivation. The
antigen content following inactivation can be assayed by MAb-capture ELISA and related to the results of
established in-vivo potency results. As recommended for potency testing of BVDV vaccine in cattle it should
be demonstrated that the vaccine can prevent transplacental transmission of BDV in pregnant sheep.
d)
No information is available on duration of immunity following vaccination. Inactivated vaccines are unlikely to
provide sustained levels of immunity and it is likely that after an initial course of two or three injections
annual booster doses may be required. Insufficient information is available to determine any correlation
between vaccinal antibody titres in the dam and fetal protection.
e)
Stability
There is little information on the stability of BDV vaccines. Inactivated vaccines could be expected to have at
least a 1 year shelf life when protected from light and stored at 4C.
f)
Preservatives
Preservatives may be added to multidose vaccine containers subject to the approval of the Control Authority.
g)
BDV is not considered to be a hazard to human health. Standard good microbiological practice should be
used when handling the virus.
5.
a)
Safety
In-vitro test only.
b)
Potency
In-vitro antigen content test.
REFERENCES
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Vet. Med. (Suppl. J. Vet. Med.), 36, 187.
965
BECHER P., AVALOS-RAMIREZ R., ORLICH M., CEDILLO ROSALES S., KONIG, M., SCHWEIZER M., STALDER H., SCHIRRMEIR
H & THIEL H.-J. (2003). Genetic and antigenic characterisation of novel pestivirus genotypes; Implications for
classification. Virology, 311, 96104.
BECHER P., ORLICH M. & THIEL H.-J. (1998). Complete genomic sequence of border disease virus a pestivirus from
sheep. J. Virol., 72, 51655173.
BRAUN U., HILBE M., EHRENSPERGER F., SALIS F., ALTHER P., STRASSER M., STALDER H.P. & PETERHANS E. (2002).
Border Disease in einem Schafbetrieb. Schweiz. Arch. Tierheilk., 144, 419426.
BRUN A., LACOSTE F., REYNAUD G., KATO F. & SAINT-MARC B. (1993). Evaluation of the potency of an inactivated
vaccine against border disease pestivirus infection in sheep. In: Proceedings of the Second Symposium on
Pestiviruses, Edwards S., ed. Fondation Marcel Merieux, Annecy, France, 13 October 1992, 257259
CARLSSON U. (1991). Border disease in sheep caused by transmission of virus from cattle persistently infected
with bovine virus diarrhoea virus. Vet. Rec., 128, 145147.
CHAPPUIS G., BRUN A., KATO F., DAUVERGNE M., REYNAUD G. & DURET C. (1986). Etudes serologiques et
immunologiques realisees a la suite de Iisolement dun pestivirus dans un foyer ovina chez des moutons de
LAveyron. In: Pestiviroses des Ovins et des Bovins, Espinasse J. & Savey M. eds. Ste Franoise de Buiatrie,
Paris, France, 55, 66.
DEKKER A., WENSVOORT G. & TERPSTRA C. (1995). Six antigenic groups within the genus pestivirus as identified by
cross-neutralisation assays. Vet. Microbiol., 47, 317329.
ENTRICAN G., DAND A. & NETTLETON P.F. (1994). A double monoclonal-antibody ELISA for detecting pestivirus
antigen in the blood of viraemic cattle and sheep. Vet. Microbiol., 43, 6574.
FENTON A., SINCLAIR J.A., ENTRICAN G., HERRING J.A. & NETTLETON P.F. (1991). A monoclonal antibody capture
ELISA to detect antibody to border disease virus in sheep sera. Vet. Microbiol., 28, 327333.
FULTON R.W., DOFFAY J.M., SALIKI J.T., BURGE L.J., HELMAN R.G., CONFER A.W., BOLIN S.R. & RIDPATH J.F. (1999).
Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typing ruminant pestiviruses: bovine viral
diarrhea viruses and border disease virus. Can. J. Vet. Res., 63, 276281.
MCGOLDRICK A., BENSAUDE E., IBATA G., SHARP G. & PATON D.J. (1999). Closed one-tube reverse transcription
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NETTLETON P.F., GILMOUR J.S., HERRING J.A. & SINCLAIR J.A. (1992). The production and survival of lambs
persistently infected with border disease virus. Comp. Immunol. Microbiol. infect. Dis., 15, 179188.
NETTLETON P.F., GILRAY J.A., RUSSO P. & DLISSI E. (1998). Border disease of sheep and goats Vet. Res., 29, 327
340.
OGUZOGLU T.C., FLOEGEL-NIESMANN G., FREY H.R. & MOENNIG V. (2001). Differential diagnosis of classical swine
fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine
farm. Dtsch Tierarztl. Wochenschr., 108, 210213.
PATON D.J., SANDS J.J., LOWINGS J.P., SMITH J.E., IBATA G. & EDWARDS S. (1995). A proposed division of the
pestivirus genus into subgroups using monoclonal antibodies, supported by cross-neutralization assays and
genetic sequencing. Vet. Res., 26, 92109.
RIDPATH J.F. & BOLIN S.R. (1997). Comparison of the complete genomic sequence of the border disease virus,
BD31, to other pestiviruses. Virus Res., 50, 237243.
SANDVIK T., PATON D.J & LOWINGS P.J. (1997). Detection and identification of ruminant and porcine pestiviruses by
nested amplification of the 5 untranslated cDNA region. J. Virol. Methods, 64, 4356.
THABTI F., FRONZAROLI L., DLISSI E., GUIBERT J.M., HAMMAMI S., PEPIN M. & RUSSO P. (2002). Experimental model of
border disease virus infection in lambs: comparative pathogenicity of pestiviruses isolated in France and Tunisia.
Vet. Res., 33, 3545.
966
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Flaviviridae. In: Virus Taxonomy. Eighth report of the International Committee on Taxonomy of Viruses, Fauquet
C.M., Mayo M.A., Maniloff J., Desselberger U. & Ball L.A., eds. Elsevier Academic Press, 981998.
THUR B., HILBE M., STRASSER M. & EHRENSPERGER F. (1997). Immunohistochemical diagnosis of pestivirus infection
associated with bovine and ovine abortion and perinatal death. Am. J. Vet. Res., 58, 13711375.
VALDAZO-GONZALEZ B., ALVAREZ-MARTINEZ M. & SANDVIK T. (2007). Genetic and antigenic typing of border Disease
virus isolates in sheep from the Iberian peninsula. Vet. J., 174, 316324.
VAN RIJN P.A., VAN GENNIP H.G.P., LEENCLERSE C.H., BRUSCHKE C.J.M., PATON D.J., MOORMANN R.J.M. & VAN
OIRSCHOT J.T. (1997). Subdivision of the pestivirus genus based on envelope glycoprotein E2 Virology, 237, 337
348.
VANTSIS J.T., BARLOW R.M., FRASER J. & MOULD D.L. (1976). Experiments in border disease VIII. Propagation and
properties of a cytopathic virus. J. Comp. Pathol., 86, 111120.
VANTSIS J.T., RENNIE J.C., GARDINER A.C., WELLS P.W., BARLOW R.M. & MARTIN W.B. (1980). Immunisation against
Border disease. J. Comp. Path., 90, 349354.
VILCEK S. (2001). Identification of pestiviruses contaminating cell lines and fetal calf sera. Acta Virol., 45, 8186.
VILCEK S., HERRING A.J., HERRING J.A., NETTLETON P.F., LOWINGS J.P.L. & PATON D.J (1994) Pestiviruses isolated
from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and
restriction endonucelase analysis. Arch. Virol., 136, 309323.
VILCEK S. & NETTLETON P.F. (2006). Pestiviruses in wild animals Vet. Microbiol., 116, 112.
VILCEK S., NETTLETON P.F., PATON D.J. & BELAK S. (1997). Molecular characterization of ovine pestiviruses. J.
Gen. Virol., 78, 725735.
VILCEK S. & PATON D.J. (2000). A RT-PCR assay for the rapid recognition of border disease virus. Vet. Res., 31,
437445.
WENSVOORT G. & TERPSTRA C. (1988). Bovine viral diarrhoea virus infection in piglets born to sows vaccinated
against swine fever with contaminated virus. Res. Vet. Sci., 45, 143148.
WENSVOORT G., TERPSTRA C. & DE KLUYVER E.P. (1989). Characterisation of porcine and some ruminant
pestiviruses by cross-neutralisation. Vet. Microbiol., 20, 291306.
WILLOUGHBY K., VALDAZO-GONZALEZ, B., MALEY M., GILRAY J. & NETTLETON P.F. (2006). Development of a real time
RT-PCR to detect and type ovine pestiviruses. J. Virol. Methods, 132, 187194.
*
* *
967
CHAPTER 2.7.2.
CAPRINE AND OVINE BRUCELLOSIS

(excluding Brucella ovis)
SUMMARY
Brucella melitensis (biovars 1, 2 or 3) is the main causative agent of caprine and ovine brucellosis.
Sporadic cases caused by B. abortus have been observed, but cases of natural infection are rare in
sheep and goats. Brucella melitensis is endemic in the Mediterranean region, but infection is
widespread world-wide. North America (except Mexico) is believed to be free from the agent, as are
Northern and Central Europe, South-East Asia, Australia and New Zealand.
Clinically, the disease is characterised by one or more of the following signs: abortion, retained
placenta, orchitis, epididymitis and, rarely, arthritis, with excretion of the organisms in uterine
discharges and in milk. Diagnosis depends on the isolation of Brucella from abortion material, udder
secretions or from tissues removed at post-mortem. Presumptive diagnosis of Brucella infection can
be made by assessing specific cell-mediated or serological responses to Brucella antigens.
Brucella melitensis is highly pathogenic for humans, causing Malta fever, one of the most serious
zoonoses in the world. All infected tissues, cultures and potentially contaminated materials should
therefore be handled at containment level 3.
Identification of the agent: Presumptive evidence of Brucella is provided by the demonstration, by
modified acid-fast staining of organisms typical of Brucella in abortion material or vaginal discharge,
especially if supported by serological tests. The polymerase chain reaction (PCR) methods provide
additional means of detection. Whenever possible, Brucella spp. should be isolated using selective
or non-selective media by culture from uterine discharges, aborted fetuses, udder secretions or
selected tissues, such as lymph nodes, spleen, uterus, testes and epididymes. Species and biovars
should be identified by phage lysis, and by cultural, biochemical and serological criteria. Molecular
methods have been developed that could also be used for complementary identification based on
specific genomic sequences.
Serological and allergic skin tests: The buffered Brucella antigen tests (BBAT) and the
complement fixation test (CFT) are usually recommended for screening flocks and individual
animals. The serum agglutination test is not considered to be reliable for use in small ruminants.
The indirect enzyme-linked immunosorbent assay (I-ELISA) and fluorescence polarisation assay
(FPA) can also be used for screening purposes. For pooled samples, there are no useful tests such
as the milk ring test for cattle. The brucellin allergic skin test can be used as a screening or
complementary test in unvaccinated flocks, provided that a purified, lipopolysaccharide (LPS)-free,
standardised antigen preparation is used. Results must then be interpreted in relation to the clinical
signs, history, and results of serological or cultural examination.
Requirements for vaccines and diagnostic biologicals: Brucella melitensis strain Rev.1 remains
the reference vaccine to immunise sheep and goats at risk of infection from B. melitensis and is the
vaccine with which any other vaccines should be compared. Production of Brucella antigens or
Rev.1 vaccine is based on a seed-lot system. Seed cultures to be used for antigens for serological
and allergic skin tests and for vaccines should originate from reference centres. They must conform
to minimal standards for viability, smoothness, residual infectivity and immunogenicity, purity,
identity and safety, if applicable. Brucellin preparations for the intradermal test must be free of
smooth lipopolysaccharide and must not produce nonspecific inflammatory reactions or interfere
with serological tests. Antigens for BBAT and CFT must be prepared from smooth strains of
B. abortus, strain 1119-3 or strain 99. Antigens for I-ELISA are prepared from B. abortus strain
1119-3 or strain 99 or B. melitensis biovar 1 reference strain 16M or antigens prepared from
968
Chapter 2.7.2. Caprine and ovine brucellosis (excluding Brucella ovis)
different smooth Brucella strains. All antigens must comply with minimum standards for purity,
sensitivity and specificity.
A. INTRODUCTION
Brucellosis in sheep and goats (excluding Brucella ovis infection) is primarily caused by one of the three biovars
of B. melitensis. Sporadic infections caused by B. abortus or B. suis have been observed in sheep and goats, but
such cases are rare. Pathologically and epidemiologically, B. melitensis infection in sheep and goats is very
similar to B. abortus infection in cattle (see Chapter 2.4.3 Bovine brucellosis). In most circumstances, the primary
route of transmission of Brucella is the placenta, fetal fluids and vaginal discharges expelled by infected ewes and
goats when they abort or have a full-term parturition. Shedding of Brucella is also common in udder secretions
and semen, and Brucella may be isolated from various tissues, such as lymph nodes from the head, spleen and
organs associated with reproduction (uterus, epididymides and testes), and from arthritic lesions (Alton et al.,
1988).
Brucella melitensis infection in domestic and wild susceptible species (see chapter 2.4.3) is not rare when these
species are reared in close contact with sheep and goats in enzootic areas. The manifestations of brucellosis in
these animals are similar to those in cattle or sheep and goats.
The World Health Organization (WHO) laboratory biosafety manual classifies Brucella (and particularly
B. melitensis) in Risk group III. Brucellosis is readily transmissible to humans, causing acute febrile illness
undulant fever which may progress to a more chronic form and can also produce serious complications affecting
the musculoskeletal, cardiovascular, and central nervous systems. Infection is often due to occupational
exposure and is essentially acquired by the oral, respiratory, or conjunctival routes, but ingestion of dairy products
constitutes the main risk to the general public. There is an occupational risk to veterinarians, abattoir workers and
farmers who handle infected animals and aborted fetuses or placentas. Brucellosis is one of the most easily
acquired laboratory infections, and strict safety precautions should be observed when handling cultures and
heavily infected samples, such as products of abortion. Specific recommendations have been made for the safety
precautions to be observed with Brucella-infected materials (for further details see Chapter 1.1.3 Biosafety and
biosecurity in the veterinary microbiology laboratory and animal facilities, and refs 1, 39, 94 and 95 of chapter
2.4.3). Laboratory manipulation of live cultures or contaminated material from infected animals is hazardous, as is
handling large volumes of Brucella, and must be done under containment level 3 or higher conditions, as outlined
in chapter 1.1.3, to minimise occupational exposure.
The classification, microbiological and serological properties of the genus Brucella and related species and
biovars are given in the chapter 2.4.3.
1.
Refer to chapter 2.4.3 for the detailed agent identification procedure for Brucella.
2.
Serological tests
In situations where bacteriological examination is not practicable, diagnosis of Brucella infection must often be
based on serological methods (Alton et al., 1988; MacMillan, 1990). In routine tests, anti-Brucella antibodies are
detected in serum. The most widely used serum-testing procedures for the diagnosis of smooth Brucella
infections in sheep and goats are the buffered Brucella antigen tests (BBAT), and the complement fixation test
(CFT). The bulk milk ring test, which has been very useful in cattle, is ineffective in small ruminants.
In small ruminants, the BBAT and the CFT are the most widely used methods (Joint FAO/WHO Expert Committee
on Brucellosis, 1986). The indirect enzyme-linked immunosorbent assay (I-ELISA) and the fluorescence
polarisation assay (FPA) have shown similar diagnostic performance. All these tests are prescribed for
international trade. The BBAT is not completely specific, but is adequate as a screening test for detecting infected
flocks or for guaranteeing the absence of infection in brucellosis-free flocks. However, due to the relative lack of
sensitivity of both tests, discrepancies between results obtained using the Rose Bengal test (RBT) and the CFT
are not rare in infected sheep and goats (Blasco et al., 1994). The results of the two tests should therefore be
considered simultaneously to increase the likelihood of detecting infected individuals and to improve control of the
disease in areas where it has not been completely eradicated (Alton, 1990; Blasco, 1992; Blasco et al., 1994).
When, for practical or economic reasons, the CFT cannot be used simultaneously with the RBT in eradication
programmes, it is recommended to improve the sensitivity of the RBT by using three volumes of serum and one
volume of antigen (e.g. 75 l and 25 l, respectively) in place of an equal volume of each. This simple
969
modification increases RBT sensitivity and minimises the discrepancies between RBT and CFT results (Blasco et
al., 1994). Because antibodies induced after Rev.1 vaccination cannot be differentiated in both tests from those
induced by B. melitensis infection, RBT and CFT results should be carefully interpreted according to the
vaccination status in the flock. In addition, both tests are not specific enough to discriminate serological reactions
due to B. melitensis from the false-positive reactions (FPSR) due to cross-reacting bacteria such as Yersinia
enterocolitica O:9.
Good diagnostic results have been obtained in sheep and goats with indirect (I-) or competitive (C-) enzymelinked immunosorbent assays (ELISAs) using various antigens, but generally the ELISAs that use antigens with a
high content of smooth lipopolysaccharide (sLPS) are the most useful. The C-ELISA provides similar sensitivity to
the classical tests, RBT and CFT, and the I-ELISA has greater sensitivity. Like these classical tests, both ELISAs
are unable to differentiate B.-melitensis-infected animals from those recently vaccinated with the Rev.1 vaccine
(Marin et al., 1999) or infected with cross-reacting bacteria. Some of these ELISAs have potential advantages in
sensitivity and/or specificity with respect to both BBAT and CFT (Garin-Bastuji et al., 1998). Preliminary C-ELISAs
studies with a periplasmic protein from B. abortus (Rossetti et al., 1996) or B. melitensis (Cloeckaert et al., 1996)
as antigen have been applied in sheep and reported to be promising in differentiating Rev.1 vaccinated from
B. melitensis infected animals (Cloeckaert et al., 2001; Debbarh et al., 1996).
Reference sera
The OIE reference standards are those against which all other standards are compared and calibrated. For the
BBAT and CFT, please refer to chapter 2.4.3 for antigen standardisation and test protocols. A caprine reference
standard for ELISAs and FPA for sheep and goat antibodies has been developed and will be available to national
reference laboratories soon1.
Production of cells
Please refer to chapter 2.4.3. Brucella abortus biovar 1 strains 99 or 1119 are the only strains recommended for
the preparation of BBAT and CFT in sheep and goats.
a)
Brucella-buffered antigen test (a prescribed test for international trade)

Please refer to Chapter 2.4.3 Bovine brucellosis.
Antigen production
Please refer to chapter 2.4.3. Note that RB antigen made with B. abortus is usually used to test for
B. melitensis. The standardisation of RB antigen, as it is prescribed in chapter 2.4.3, provides a sufficient
sensitivity to the BBAT for international trade purposes. Moreover, it helps assure an adequate specificity in
free areas where FPSR occur because of cross-reacting bacteria such as Yersinia enterocolitica O:9.
However this standardisation is probably the main cause of the reduced sensitivity of some RB antigen
batches and of the discrepancies with the CFT (Blasco et al., 1994). Therefore, when RBT is used in
eradication programmes in endemic areas, it could be advisable to adjust the RB antigen titre so that it is
positive at a 1/45 OIEISS dilution and negative at a 1/55 dilution, without affecting the specificity of the test.
The discrepancies with the CFT can also be minimised by using three volumes of serum and one volume of
antigen (e.g. 75 l and 25 l, respectively) in place of an equal volume of each as mentioned in the standard
test procedure.
Test procedure
Please refer to chapter 2.4.3.
b)
Antigen production
Please refer to chapter 2.4.3. Note that CF antigen made with B. abortus is used to test for B. melitensis.
Test procedure
970
Obtainable from the OIE Reference Laboratory for Brucellosis at Animal Health and Veterinary Laboratories Agency
(AHVLA) Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
c)
Enzyme-linked immunosorbent assays (a prescribed test for international trade)

Several variations of the I-ELISA have been described using different antigen preparations, antiglobulinenzyme conjugates, and substrate/chromogens. Several commercial I-ELISAs are available but before being
used for international trade, their respective cut-off should have been properly established using the
appropriate validation techniques (see Chapter 1.1.5 Principles and methods of validation of diagnostic assays
for infectious diseases) and these tests should be standardised against the above-mentioned Standard.
The test method is described in chapter 2.4.3.
d)
Fluorescence polarisation assay (a prescribed test for international trade)

The FPA for detection of caprine and ovine antibody to Brucella sp. is essentially the same as that described
for cattle (for more details see chapter 2.4.3); an example serum dilution used is 1/25 for the tube test and
1/10 for the plate test (Nielson et al., 1999; 2004; 2005; Ramirez-Pfeiffer et al., 2006). It is a simple
technique for measuring antigen/antibody interaction. The FPA may be used as a screening and/or
confirmatory test. Before being used for international trade, the FPA cut-off should be properly established
using the appropriate validation techniques (see chapter 1.1.5) and the test should be standardised against
the above-mentioned Standard.
3.
Other tests
a)
Brucellin skin test (an alternative test for international trade)

An alternative diagnostic test is the brucellin skin test, which can be used for screening unvaccinated flocks,
provided that a purified (free of sLPS) and standardised antigen preparation (e.g. brucellin INRA) is used.
The brucellin skin test has a high sensitivity for the diagnosis of B. melitensis infection in small ruminants
and, in absence of vaccination, is considered one of the most specific diagnostic tests (Alton et al., 1988;
Blasco, 1992; Garin-Bastuji et al., 1998; Joint FAO/WHO Expert Committee on Brucellosis, 1986). This test
is of particular value for the interpretation of FPSR due to infection with cross-reacting bacteria (FPSR
affected animals are always negative in the skin test), especially in brucellosis-free areas.
Rev.1 vaccinated animals can react in this test for years (Garin-Bastuji et al., 1998). Therefore this test
cannot be recommended either as the sole diagnostic test or for the purposes of international trade in areas
where Rev.1 vaccine is used.
To obtain suitable results it is essential to use standardised brucellin preparations that do not contain sLPS,
as this antigen may provoke antibody-mediated inflammatory reactions or induce antibodies that interfere
with subsequent serological screening. One such preparation is brucellin INRA, which is prepared from a
rough strain of B. melitensis that is commercially available2.
Test procedure
i)
A volume of 0.1 ml of brucellin is injected intradermally into the lower eyelid.
ii)
The test is read after 48 hours.
iii)
Any visible or palpable reaction of hypersensitivity, such as an oedematous reaction leading to an

elevation of the skin or thickening of the eyelid ( 2 mm), should be interpreted as a positive reaction.
Although in the absence of vaccination the brucellin intradermal test is one of the most specific tests in
brucellosis, diagnosis should not be made exclusively on the basis of positive intradermal reactions and
should be supported by adequate serological tests. The intradermal inoculation of brucellin might induce a
temporary anergy in the cellular immune response. Therefore an interval of 6 weeks is generally
recommended between two tests repeated on the same animal.
b)
Native hapten tests

The native hapten-based gel precipitation tests3 (as described in chapter 2.4.3.) are also of interest in sheep
and goats as they are very specific for discriminating the serological responses of infected animals (positive)
from those induced in Rev.1 vaccinated animals (usually negative after a given time after vaccination).
2
3
Brucellergne OCB, Synbiotics Europe, 2 rue Alexander Fleming, 69007 Lyon, France.
The detailed procedure could be obtained from the Departamento de Sanidad Animal, Centro de Investigacion y
Tecnologia Agroalimentaria/Gobierno de Aragon, Avenida Montaana 930, 50059. Zaragoza. Spain.
971
The optimal diagnostic sensitivity (around 90%) is obtained in the double gel diffusion (DGD) or reverse
radial immunodiffusion tests for sheep and goats, respectively (Debbarh et al., 1996; Joint FAO/WHO Expert
Committee on Brucellosis, 1986).

C1. Brucellin
C2. Vaccines
Brucella melitensis strain Rev.1 vaccine
The most widely used vaccine for the prevention of brucellosis in sheep and goats is the Brucella melitensis
Rev.1 vaccine, which remains the reference vaccine with which any other vaccines should be compared. The
RB51 vaccine is not effective in sheep against B. melitensis infection (El-Idrissi et al., 2001). In addition, other
rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in the IELISA with sLPS and are less effective than Rev.1 vaccine against B. melitensis infection in sheep (Barrio et al.,
2009). The Rev.1 vaccine is used as a freeze-dried suspension of live B. melitensis biovar 1 Rev.1 strain for the
immunisation of sheep and goats. It should be normally given to lambs and kids aged between 3 and 6 months as
a single subcutaneous or conjunctival inoculation. The standard dose is between 0.5 109 and 2.0 109 viable
organisms. The subcutaneous vaccination induces strong interferences in serological tests and should not be
recommended in combined eradication programmes (Daz-Aparicio et al., 1994; Marin et al., 1999). However,
when this vaccine is administered conjunctivally, it produces a similar protection without inducing a persistent
antibody response, thus facilitating the application of eradication programmes combined with vaccination (DazAparicio et al., 1994; Marin et al., 1999). Care must be taken when using Rev.1 vaccine to avoid the risk of
contaminating the environment or causing human infection. In many developing countries and endemic areas,
vaccination of the whole population has to be considered as the best option for the control of the disease (Blasco,
1997). However, Rev.1 vaccine is known to often cause abortion and excretion in milk when animals are
vaccinated during pregnancy, either with a full or reduced dose (Blasco, 1997). These side-effects are
considerably reduced when adult animals are vaccinated conjunctivally (full dose) before mating or during the last
month of pregnancy. Therefore, when mass vaccination is the only means of controlling the disease, a
vaccination campaign should be recommended using the standard dose of Rev.1 administered by the conjunctival
route when the animals are not pregnant or during the late lambing and prebreeding season (Blasco, 1997).
The subcutaneous vaccination of young animals and the vaccination of adult animals, even at reduced doses,
may lead to long-term persistence of vaccinal antibodies in a significant proportion of vaccinated animals that
creates serious interferences in the serological diagnosis of brucellosis. As indicated above, conjunctival
vaccination minimises these problems and thus it is the recommended method for combined eradication
programmes. Therefore, the serological diagnosis of brucellosis should take into account the vaccinal state of the
herd and the overall frequency distribution of antibody titres detected in the group of animals tested.
1.
Seed management
a)

Brucella melitensis biovar 1 strain Rev.1 original seed for vaccine production can be obtained commercially4.
A European reference Rev.1 strain that possesses the characteristics of the Rev.1 original seed is also
obtainable from the European Pharmacopoeia5.
Production of Brucella live vaccines is based on the seed-lot system described above (Section B.2) for
BBAT and CFT antigens. Strains should be cultured in a suitable medium. Strain Rev.1 must conform to the
characteristics of B. melitensis biovar 1, except that it should grow more slowly. Additionally, when incubated
in air (atmospheres containing CO2 alter the results) at 37C, it should grow on agar containing streptomycin
(2.5 g/ml), and it should be inhibited by the addition to a suitable culture medium of sodium benzylpenicillin
(3 g [5 International Units (IU)]/ml), thionin (20 g/ml) or basic fuchsin (20 g/ml). Recently, polymerase
chain reaction and molecular techniques have been used to further characterise the vaccine (Bardenstein et
al., 2002; Cloeckaert et al., 2002). It must also conform to the characteristics of residual virulence and
immunogenicity in mice of the original seed.
4
5
972
Obtainable from the OIE Reference Laboratory for Brucellosis at Anses Maisons-Alfort, 94706, France.
Obtainable from the European Pharmacopoeia, BP 907, 67029 Strasbourg Cedex 1, France.
b)
Method of culture
Serumdextrose agar, and trypticasesoy agar, to which 5% serum or 0.1% yeast extract may be added,
are among the solid media that have been found to be satisfactory for propagating the Rev.1 strain (Alton et
al., 1988; WHO, 1977). Rev.1 strain does not grow well on potato agar.
For vaccine production, Rev.1 may be grown under conditions similar to those described for S99 and S1119-3
(see chapter 2.4.3), except that Rev.1 generally needs 35 days to grow, the phenol saline is replaced by a
freeze-drying stabiliser, and the organisms are not killed but are stored at 4C while quality control examinations
are carried out as described below. Moreover, the specific requirements for Rev.1 vaccine production recommend
that: each seed lot (i.e. the culture used to inoculate medium for vaccine production) should be no more than three
passages removed from an original seed culture and that the harvest of a vaccine lot should be no more than
three passages from a seed lot or an original seed. The original seed culture should always be checked for the
absence of dissociation before use. The recommended method for preparing seed material is given in ref. 2. The
following freeze-drying stabiliser (sterilised by filtration) is of proven value: enzymatic digest of casein (2.5 g);
sucrose (5 g); sodium glutamate (1 g); distilled water (100 ml).
c)
Numerous independent studies have confirmed the value of B. melitensis strain Rev.1 as a vaccine for
protecting sheep and goats from brucellosis. Its virulence is unchanged after passage through pregnant
sheep and goats. Abortions may result when the Rev.1 vaccine is inoculated into pregnant ewes or goats.
The vaccine-induced abortions are not avoided using reduced doses, and doses as low as 106, used either
subcutaneously or conjunctivally, have been demonstrated to induce abortions and milk excretion of the
vaccine strain (Blasco, 1997).
A Rev.1 vaccine is efficient if it possesses the characteristics of the Rev.1 original strain, i.e. those of
B. melitensis biovar 1 reference strain 16M (ATCC No. 23456), except those specific for the strain Rev.1
(Alton et al., 1988; Joint FAO/WHO Expert Committee on Brucellosis, 1986), and if it proves to be
satisfactory with respect to immunogenicity and residual virulence in the mouse model (Bosseray, 1992)
(see below).
2.
Method of manufacture (Alton et al., 1988; WHO, 1977)
For production of B. melitensis strain Rev.1 vaccine, the procedures described above for antigens (Alton et al.,
1988) can be used except that the cells are collected in a freeze-drying stabiliser and deposited by centrifugation.
The yield from one fermenter run or the pooled cells from a batch of Roux flask cultures inoculated on the same
occasion from the same seed lot constitutes a single harvest. More than one single harvest may be pooled to
form the final bulk that is used to fill the final containers of a batch of vaccine. Before pooling, each single harvest
must be checked for purity, cell concentration, dissociation and identity. The volume of the final bulk is adjusted
by adding sufficient stabiliser so that a dose contains an appropriate number of viable organisms. After adjusting
the cell concentration of the final bulk, tests for identity, dissociation and absence of contaminating organisms are
conducted (see below).
3.
In-process control
In-process checks should be made on the growth of Rev.1 vaccine from either solid or liquid medium to verify
identity and to ensure purity and freedom from dissociation to rough forms during preparation of seed lots, single
harvests, final bulks and the final (filling) lots. At least 99% of cells in seed lots and 95% of cells in final lots should
be in the smooth phase.
Cell concentration should be estimated on the bulks and precisely determined on final lots. Immunogenicity and
the residual virulence (50% persistence time or 50% recovery time) should also be determined on seed lots and
final lots. If these tests have been done with good results on a representative batch of the test vaccine, it does not
have to be repeated routinely on other vaccine lots prepared from the same seed lot and with the same
manufacturing process.
4.
Batch control
With freeze-dried vaccine, the control tests should be conducted on the vaccine reconstituted in the form in which
it will be used.
a)
Sterility (or absence of extraneous microorganisms)

The Rev.1 vaccine should be checked for bacterial and fungal contamination as prescribed in Chapter 1.1.7
Tests for sterility and freedom from contamination of biological materials.
973
b)
Safety
The Rev.1 vaccine is a virulent product per se, and it should keep a minimal virulence to be efficacious (see
Section C2.4.c in chapter 2.4.3).
c)
Potency
A Rev.1 vaccine is efficient if it possesses the characteristics of the Rev.1 original strain, i.e. if it is
satisfactory with respect to immunogenicity, and residual virulence (Bosseray, 1993). Batches should also
be checked for the number of viable organisms.
Identity
The reconstituted Rev.1 vaccine should not contain extraneous microorganisms. Brucella melitensis
present in the vaccine is identified by suitable morphological, serological and biochemical tests and by
culture: when incubated in air at 37C, Rev.1 strain is inhibited by addition to the suitable culture
medium of 3 g (5 IU) per ml of sodium benzyl-penicillin, thionin (20 g/ml) or basic fuchsin (20 g/ml);
the strain grows on agar containing 2.5 g per ml of streptomycin.
Smoothness (determination of dissociation phase)

Sometimes slight and difficult to observe differences, can be seen in the size of Rev.1 colonies. The
small colonies (11.2 mm in diameter) are typical for Rev.1, but larger Rev.1 colonies can appear
depending on the medium used, the amount of residual moisture in the incubator atmosphere, and the
presence or absence of CO2. The frequency of variation in colony size occurs normally at a ratio of
1 large to 103 small colonies. Both Rev.1 variants are of the S (smooth) type. To avoid an increase in
this colony size variation along successive passages, it is important to always select small colonies for
preparation of seed lots.
Enumeration of live bacteria

Residual virulence (50% persistence time or 50% recovery time) (Bosseray, 1991; Grillo et al., 2000)
The same technical procedures indicated for 50% recovery time (RT50) calculation of S19 vaccine (see
chapter 2.4.3) have to be applied for Rev.1, except that B. abortus S19 seed lot or batch to be tested
(test vaccine) and the S19 original seed culture (used as a reference strain), respectively, are replaced
by the corresponding B. melitensis Rev.1 seed lot or batch to be tested (test vaccine) and the
B. melitensis Rev.1 original seed culture as the reference strain. For the reference original Rev.1 strain,
RT50 and confidence limits are around 7.9 1.2 weeks. A given Rev.1 vaccine seed lot or batch should
keep similar residual virulence to be acceptable.
If this test has been done with good results on a representative batch of the test vaccine, it does not have to
be repeated routinely on other vaccine lots prepared from the same seed lot and with the same
Immunogenicity in mice
The same technical procedures indicated for immunogenicity calculation of S19 vaccine (see chapter
2.4.3) have to be applied for Rev.1, except that B. abortus S19 seed lot or batch to be tested (test
vaccine) and the B. abortus S19 original seed culture (used as a reference strain), respectively, are
replaced by the corresponding B. melitensis Rev.1 seed lot or batch to be tested (test vaccine) and the
B. melitensis Rev.1 original seed culture as the reference strain.
Conditions of the control experiment are satisfactory when: i) the response in unvaccinated mice (mean
of Y) is at least of 4.5; ii) the response in mice vaccinated with the reference Rev.1 vaccine is lower
than 2.5; and iii) the standard deviation calculated on each lot of six mice is lower than 0.8.
If this test has been done with good results on a representative batch of the test vaccine, it does not have to
be repeated routinely on other vaccine lots prepared from the same seed lot and with the same
d)
It is accepted that subcutaneous or conjunctival vaccination with standard doses of Rev.1 confers a solid
and durable immunity in sheep and goats. However, growing field evidence shows that the immunity
conferred declines with time, and revaccination could be advisable in endemic areas.
974
The use of reduced doses of Rev.1 produces a less efficient immunity, while side-effects, such as antibody
responses or induction of abortion, are not fully avoided.
e)
Stability
Strain Rev.1 vaccine prepared from seed stock from appropriate sources is stable in characteristics provided
that the in-process and batch control requirements described above are fulfilled, and shows no tendency to
reversion to virulence. The lyophilised vaccine shows a gradual loss of viable count, but should retain its
potency for the recommended shelf life. Allowance for this phenomenon is normally made by ensuring that
the viable count immediately following lyophilisation is well in excess of the minimum requirement.
Maintenance of a cold chain during distribution of the vaccine will ensure its viability.
f)
Preservatives
Antimicrobial preservatives must not be used in live Rev.1 vaccine. For preparation of the freeze-dried
vaccine, a stabiliser as described in Section C2.4.f of chapter 2.4.3 is recommended.
g)
Please refer to chapter 2.4.3. Brucella melitensis Rev.1, although an attenuated strain, is still capable of
causing disease in humans. Accordingly, cell cultures and suspensions must be handled under appropriate
conditions of biohazard containment (see chapter 1.1.3). Reconstitution and subsequent handling of the
reconstituted vaccine should be done with care to avoid accidental injection or eye or skin contamination.
Vaccine residues and injection equipment should be decontaminated with a suitable disinfectant. Medical
advice should be sought in the event of accidental exposure. The efficacy of the antibiotic treatment of
infections caused by Rev.1 (a streptomycin-resistant strain) in humans has not been adequately established
but data in mice suggest that if Rev.1 contamination occurs, a combined treatment with doxycycline plus
rifampicin or gentamycin could be recommended (Grillo et al., 2006).
5.
a)
Safety
See Section C2.4.b of chapter 2.4.3.
b)
Potency
For the freeze-dried vaccine, the potency must be determined on the final product. The tests are as
described in Section C2.4.c of chapter 2.4.3.
In order to assess the vaccine efficiency, a representative sample of previously seronegative animals
vaccinated with each new vaccine batch should be bled 1520 days after vaccination and the serum
samples submitted to BBAT. If adequate and independently of the vaccination route used, more than 80% of
vaccinated animals should be BBAT positive.
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Boca Raton, Florida, USA, 383409.
ALTON G.G., JONES L.M., ANGUS, R.D. & VERGER J.M. (1988). Techniques for the Brucellosis Laboratory. INRA,
Paris, France.
BARDENSTEIN S., MANDELBOIM M., FICHT T.A., BAUM M. & BANAI M. (2002). Identification of the Brucella melitensis
vaccine strain Rev.1 in animals and humans in Israel by PCR analysis of the PstI site polymorphism of its omp2
gene. J. Clin. Microbiol., 40, 14751480.
BARRIO M.B., GRILL M.J., MUOZ P.M., JACQUES I., GONZALEZ D., DE MIGUEL M.J., MARN C.M., BARBERN M.,
LETESSON J.J., GORVEL J.P., MORIYN I., BLASCO J.M. & ZYGMUNT M.S. (2009). Rough mutants defective in core and
O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth
lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep.
Vaccine, 27, 17411749.
BLASCO J.M. (1992). Diagnosis of Brucella melitensis infection in small ruminants. In: Prevention of Brucellosis in
the Mediterranean Countries, Plommet M., ed. Pudoc Scientific, Wageningen, The Netherlands, 272277.
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BLASCO J.M. (1997). A review on the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Prev. Vet.
Med., 31, 275283.
BLASCO J.M., GARIN-BASTUJI B., MARIN C.M., GERBIER G., FANLO J., JIMENEZ DE BAGUES M.P. & CAU C. (1994).
Efficacy of different rose bengal and complement fixation antigens for the diagnosis of Brucella melitensis
infection in sheep and goats. Vet. Rec., 134, 415420.
BOSSERAY N. (1991). Brucella melitensis Rev.1 attenuated vaccine: Stability of markers, residual virulence and
immunogenicity in mice. Biologicals, 19, 355363.
BOSSERAY N. (1992). Le vaccin Rev.1: drive des caractres dimmunognicit et de virulence indpendante des
marqueurs classiques. In: Prevention of Brucellosis in the Mediterranean Countries, Plommet M., ed. Pudoc
Scientific, Wageningen, The Netherlands, 182186.
BOSSERAY N. (1993). Control methods and thresholds of acceptability for anti-Brucella vaccines. Dev. Biol. Stand.,
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CLOECKAERT A., BAUCHERON S., VIZCAINO N. & ZYGMUNT M.S. (2001). Use of recombinant BP26 protein in
serological diagnosis of Brucella melitensis infection in sheep. Clin. Diag Lab. Immunol., 8, 772775.
CLOECKAERT A., DEBBARH H.S., VIZCAINO N., SAMAN E., DUBRAY G. & ZYGMUNT M.S. (1996). Cloning, nucleotide
sequence and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected
sheep. FEMS Microbiol. Lett., 140, 139144.
CLOECKAERT A., GRAYON M. & GRPIENT O. (2002). Identification of Brucella melitensis vaccine strain Rev.1 by
PCR-RFLP based on a mutation in the rpsL gene. Vaccine, 20, 25462550.
DEBBARH H.S., ZYGMUNT M.S., DUBRAY G. & CLOECKAERT A. (1996). Competitive ELISA using monoclonal
antibodies to the Brucella melitensis bp26 protein to evaluate antibody response in infected and B. melitensis
Rev1 vaccinated sheep. Vet. Microbiol., 53, 325337.
DAZ-APARICIO E., MARN C., ALONSO B., ARAGN V., PREZ S., PARDO M., BLASCO J.M., DAZ R. & MORIYN I.
(1994). Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. J. Clin. Microbiol., 32,
11591165.
EL-IDRISSI A.H., BENKIRANE A., EL-MAADOUDI M., BOUSLIKHANE M., BERRADA J. & ZEROUALI A. (2001). Comparison
of the efficacy of Brucella abortus strain RB51 and Brucella melitensis Rev. 1 live vaccines against experimental
infection with Brucella melitensis in pregnant ewes. Rev. sci. tech. Off. int. Epiz., 20, 741747.
GARIN-BASTUJI B., BLASCO J.M., GRAYON M. & VERGER J.M. (1998). Brucella melitensis infection in sheep: present
and future. Vet. Res., 29, 255274.
GRILLO M.J., BOSSERAY N. & BLASCO J.M. (2000). In vitro markers and biological activity in mice of seed lot strains
and commercial Brucella melitensis Rev.1 and Brucella abortus B19 vaccines. Biologicals, 28, 119127.
GRILLO M.J., DE MIGUEL M.J., MUOZ P.M., MARIN C.M., ARIZA J. & BLASCO J.M. (2006). Efficacy of several antibiotic
combinations against Brucella melitensis Rev 1 experimental infection in BALB/c mice J. Antimicrob. Chemother.,
58, 622626.
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COMMITTEE ON BRUCELLOSIS (1986). Technical Report Series 740, Sixth Report. WHO, Geneva, Switzerland.
MACMILLAN A. (1990). Conventional serological tests. In: Animal Brucellosis, Nielsen K.H. & Duncan J.R., eds.
CRC Press, Boca Raton, Florida, USA, 153197.
MARIN C.M., MORENO E., MORIYON I., DIAZ R. & BLASCO J.M. (1999) Performance of competitive and indirect
enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide, and standard
serological tests in diagnosis sheep brucellosis. Clin. Diagn. Lab. Immunol., 6, 269272.
NIELSEN K., GALL D., SMITH P., BALSEVICIUS S., GARRIDO F., DURAN FERRER M., BIANCIFIORI F., DAJER A., LUNA E.,
SAMARTINO L., BERMUDEZ R., MORENO F., RENTERIA T. & CORRAL A. (2004). Comparison of serological tests for
detection of ovine and caprine antibody to Brucella melitensis. Rev. sci. tech. Off. Int. Epiz., 23, 979987.
NIELSEN K., GALL D., SMITH P., BERMUDEZ R., MORENO F., RENTERIA T., RUIZ A., APARICIO L., VAZQUEZ A., DAJER A.,
LUNA E., SAMARTINO L. & HALBERT G. (2005). Evaluation of serological tests for the detection of caprine antibody to
Brucella melitensis. Small Rumin. Res., 56, 253258.
976
NIELSEN K., GALL D., SMITH P., VIGLIOCCO A., PERREZ B., SAMARTINO L., DAJER A., ELZER P. & ENRIGHT F. (1999).
Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine
brucellosis. Vet. Microbiol., 68, 245253.
RAMIREZ-PFEIFFER C., NIELSEN K., MARIN-RICALDE F., RODRIGUES-PADILLA C., GOMEZ-FLORES R. (2006). Comparison
of fluorescence polarization assay with card and complement fixation tests for the diagnosis of goat brucellosis in
a high prevalence area. Vet. Immunol. Immunopathol., 110, 121127.
ROSSETTI O., ARESE A., BOSCHIROLI L. & CRAVERO S. (1996). Cloning of Brucella abortus gene and characterisation
of expressed 26 kilodalton periplasmic protein: potential use for diagnosis. J. Clin. Microbiol., 34, 165169.
WORLD HEALTH ORGANIZATION (1977). Requirements for Brucella melitensis strain Rev.1 vaccine (Live for
veterinary use). World Health Organization (WHO) Technical Report Series No. 610, 28th Report, Annex 4. WHO,
Geneva, Switzerland, 8597.
*
* *
NB: There are OIE Reference Laboratories for Caprine and ovine brucellosis (excluding Brucella ovis)
diagnostic tests, reagents and vaccines for caprine and ovine brucellosis (excluding Brucella ovis)
977
CHAPTER 2.7.3/4.
CAPRINE ARTHRITIS-ENCEPHALITIS
& MAEDI-VISNA
SUMMARY
Maedi-visna (MV) and caprine arthritis-encephalitis (CAE) are persistent lentivirus infections of
sheep and goats and are often grouped together as the small ruminant lentiviruses (SRLVs).
Maedi-visna is also known to as ovine progressive pneumonia (OPP). Phylogenetic analyses
comparing nucleotide sequences of MV virus (MVV) and CAE virus (CAEV) has demonstrated that
these are closely related lentiviruses. One source of CAEV and MVV transmission is colostrum and
milk. The source of horizontal transmission in the absence of lactation remains unknown; however,
faeces and lung fluids are known to harbour infectious virus. Ovine lentiviruses have been identified
in most of the sheep-rearing countries of the world, with the notable exceptions of Australia and
New Zealand. The distribution of CAEV is highest in industrialised countries, and seems to have
coincided with the international movement of European breeds of dairy goats. Clinical and
subclinical MV and CAE are associated with progressive, mononuclear cell inflammatory lesions in
the lungs, joints, udder and central nervous system. Indurative mastitis is common in both species,
and its economic significance may be underestimated. Laboured breathing associated with
emaciation caused by progressive pneumonitis is the predominant features in clinically affected
sheep, whereas polyarthritis is the main clinical sign in goats. However, most lentivirus-infected
sheep and goats are largely asymptomatic, but remain persistent carriers of virus and are capable
of transmitting infection via colostrum or milk and respiratory secretions. The most practical and
reliable approach to confirming a diagnosis of MV or CAE is a combination of serology and clinical
evaluation. Although serology represents the most cost-effective method of diagnosing persistently
infected, clinically normal animals, it should be understood that testing errors occur. The frequency
of error depends on several factors including but not limited to: 1) the assay format, 2) the
homology between the strain of virus used in the assay and the strains of virus present in the
testing populations, and 3) the viral antigen used in the assay.
Identification of the agent: Virus isolation can be attempted from live clinical or subclinical cases
by co-cultivating peripheral blood or milk leukocytes with appropriate ovine or caprine cell cultures,
such as chorod plexus (MVV) or synovial membrane (CAEV) cells. Virus isolation is very specific
but has variable sensitivities. Following necropsy, virus isolation is most readily accomplished by
establishing explant cultures of affected tissues, e.g. lung, chorod plexus, synovial membrane or
udder. Also, alveolar macrophages may be obtained from the lung at post-mortem and co-cultivated
with susceptible cells. The cytopathic effects are characteristic, consisting of the appearance of
refractile stellate cells and syncytia. The presence of MVV or CAEV can be confirmed by
immunolabelling methods and electron microscopy.
Nucleic acid recognition methods: Many standard and a few quantitative polymerase chain
reaction (PCR) assays for detecting MV and CAE provirus have been described and are now being
used routinely in many laboratories for the rapid detection, quantitation, and identification of the
small ruminant lentivirus strains. Cloning and/or sequencing of PCR products is the most direct
method to confirm specificity of PCR products.
Serological tests: Most infected sheep and goats possess detectable specific antibodies that can
be assayed by a number of different serological tests. The two most commonly used are the agar
gel immunodiffusion test and the enzyme-linked immunosorbent assay (ELISA). Western blot
analysis and radio-immunoprecipitation are also performed, but only in specialised laboratories. A
milk antibody assay may be appropriate in dairy goat herds. The time required for seroconversion
following infection can be relatively prolonged and unpredictable, being measured in months rather
978
Chapter 2.7.3/4. Caprine arthritis/encephalitis & Maedi-visna
than in weeks. However, after seroconversion, the antibody response usually persists and antibodypositive sheep and goats are regarded as virus carriers.
available.
A. INTRODUCTION
Maedi-visna (MV), of sheep and caprine arthritis/encephalitis (CAE) of goats are persistent virus infections
caused by closely related lentiviruses (Peterhans et al., 2004). Maedi-visna is also known as ovine progressive
pneumonia (OPP). Sheep can be experimentally infected with CAE and goats can be experimentally infected with
MV (Banks et al., 1983). In addition, phylogenetic analyses comparing nucleotide sequences of MV virus (MVV)
and CAE virus (CAEV) show clear indications of the existence and epidemiological importance of cross-species
transmission between sheep and goats without demonstrating clearly that one virus has emerged from the other
(Herrmann et al., 2004; Karr et al., 1996; Leroux et al., 1997; Roland et al., 2002; Shah et al., 2004a; 2004b;
Valas et al., 1997; Zanoni, 1998). MV and CAE are characterised by lifelong persistence of the causal agent in
host monocytes and macrophages, and a variable length of time between infection and induction of a
serologically detectable antiviral antibody response. Most infected sheep and goats do not exhibit clinical disease,
but remain persistently infected and are capable of transmitting virus (Adams et al., 1983; Cork, 1990; Crawford et
al., 1980).
Maedi-visna is an Icelandic name that describes two of the clinical syndromes recognised in MV virus (MVV)infected sheep. Maedi means laboured breathing and describes the disease associated with a progressive
interstitial pneumonitis, and visna means shrinkage or wasting, the signs associated with a paralysing
meningoencephalitis. Whereas progressive lung disease is the primary finding with MVV infection, chronic
polyarthritis, with synovitis and bursitis is the primary clinical outcome of CAE virus (CAEV) infection. Encephalitis
occurs primarily in kids aged between 2 and 6 months following CAEV infection, but careful differential diagnoses
need to be conducted to rule out other syndromes or infections in kids. Indurative mastitis occurs in both
syndromes. The lungs of sheep affected by MV do not collapse when removed from the thorax and often retain
the impression of the ribs. The lungs and lymph nodes increase in weight (up to 23 times the normal weight).
The lesions are uniformly distributed throughout the lungs, which are uniformly discoloured or mottled grey-brown
in colour and of a firm texture. Udders affected by MV are diffusely indurated and associated lymph nodes may be
enlarged.
When MV or CAE is the suspected cause of clinical disease, confirmation of the diagnosis can be achieved by a
combination of clinical evaluation, serology and, when necessary, histological examination of appropriate tissues
collected at necropsy. Important tissues to examine include lung for progressive interstitial pneumonitis, brain and
spinal cord for meningoencephalitis, udder for indurative mastitis, affected joints and synovium for arthritis, and
kidney for vasculitis (Crawford & Adams, 1981; Cutlip et al., 1985; 1986; Knowles et al., 1990; Oliver et al., 1981a;
1981b). The nature of the inflammatory reaction in each site is similar, consisting of an interstitial, mononuclear
cell reaction, sometimes with large aggregates of lymphoid cells and follicle formation.
1.
Isolation and characterisation of MVV or CAEV would not normally be attempted for routine diagnostic purposes.
Due to the persistent nature of these infections, the establishment of a positive antibody status is sufficient for the
identification of virus carriers. However, due to a late seroconversion after infection, negative serology may occur
in recently infected animals.
There are two approaches to the isolation of MVV and CAEV: one for use with the live animal, and the second for
use with necropsy tissues.
a)
Isolation from the live animal
Maedi-visna virus
The MV provirus DNA is carried in circulating monocytes and tissue macrophages. Virus isolation from the
live animal therefore requires the establishment of leukocyte preparations, with aseptic precautions, from
peripheral blood or milk during lactation, culturing them together with indicator cells. Sheep chorod plexus
(SCP) cells are commonly used for this purpose. These indicator cells can be prepared as primary explant
cultures from fetal or newborn virus-free lambs, and their number can be multiplied over three to four
passages for storage in liquid nitrogen. The recovered SCP cells are suitable for co-cultivation for up to 10 or
979
15 passages. Although the cells continue to grow well thereafter, their susceptibility to MVV may become
reduced.
Leukocyte preparations can be made from peripheral blood as buffy coats by the centrifugation at 1000 g of
heparinised, ethylene diamine tetra-acetic acid (EDTA) or citrated samples for 15 minutes. The cells are
aspirated, suspended in Hanks balanced salt solution (HBSS), and further purified by centrifugation at 400 g
on to a suitable cushion (e.g. Ficoll Paque [Pharmacia]) for 40 minutes. The interface cells are spin-washed
once or twice in HBSS at 100 g for 10 minutes, and the final cell pellet is resuspended in medium to a
concentration of approximately 106 cells/ml; cells are generally cultured for 1012 days in Teflon bags and
are then added to a washed monolayer of slightly subconfluent SCP cells in a flask with an area of 25 cm2.
Leukocytes can be similarly deposited from milk by centrifugation, when they are spin-washed, resuspended
and finally added to SCP monolayer cultures.
These cultures are maintained at 37C in a 5% CO2 atmosphere, changing the medium and passaging as
necessary. They are examined for evidence of a cytopathic effect (CPE), which is characterised by the
appearance of refractile stellate cells with dendritic processes accompanied by the formation of syncytia.
The cultures should be maintained for several weeks before being discarded as uninfected. Once a CPE is
suspected, cover-slip cultures should be prepared. These are fixed, and evidence of viral antigen is sought
by immunolabelling, usually by means of indirect fluorescent antibody or by the use of indirect
immunoperoxidase methods. In addition, the cells of any suspect monolayers are deposited by
centrifugation, and preparations are made for the identification of any characteristic lentivirus particles by
transmission electron microscopy. Reverse transcriptase in the supernatant of the cell culture is indicative of
the presence of retroviruses.
Caprine arthritis/encephalitis virus
The same principles that apply to the isolation of MVV also apply to the isolation of CAEV. CAEV was
originally isolated by explantation of synovial membrane from an arthritic goat (Crawford & Adams, 1981).
With live CAEV-infected goats, peripheral blood, milk, and possibly joint fluid aspirate represent the most
suitable specimens from which leukocyte preparations can be established. Goat synovial membrane (GSM)
cells are suitable indicator cells. If a CPE is suspected, tests for detection of viral antigen should be carried
out, as described above.
b)
Isolation from necropsy tissues
Caprine arthritis/encephalitis virus and Maedi-visna virus
Samples of suspect tissues, collected as fresh as possible, such as lung, synovial membranes, udder, etc.,
are collected aseptically into sterile HBSS or cell culture medium and minced finely in a Petri dish using
scalpel blades. Individual fragments are collected by Pasteur pipette and transferred to flasks of 25 cm2,
approximately 2030 fragments per flask, and a drop of growth medium is placed carefully on each. The
flasks are then incubated at 37C in a humid 5% CO2 atmosphere, and left undisturbed for a few days to
allow the individual explants to adhere to the plastic. Fresh medium can be added with care, after which rafts
of cells will gradually grow out from the fragments. When there is sufficient cell out-growth, the cultures are
trypsin dispersed to allow the development of cell monolayers. These can be examined for CPE, and any
suspected virus growth is confirmed in the same way as for the co-cultivations.
Adherent macrophage cultures are easy to establish from lung-rinse material (post-mortem broncho-alveolar
lavage) and can be tested for virus production by serology, electron microscopy, or reverse transcriptase
assay within 12 weeks. Virus isolations can be done by co-cultivation of macrophages and SCP or GSM
cells as described for leukocytes above.
c)

Most virus disease diagnostic laboratories will be equipped for the basic cell culture procedures described
above. Many laboratories can now also perform nucleic acid recognition methods for the detection,
quantitation, and identification of MV and CAE proviral DNA using the standard polymerase chain reaction
(PCR) followed by Southern blotting, in situ hybridisation, or cloning and/or sequencing of the PCR product
(Alvarez et al., 2006; Herrmann-Hoesing et al., 2007; Johnson et al., 1992). Standard PCR techniques for
the detection of MV and CAE proviral DNA in cells and tissues are routinely used in many laboratories and
are generally used as supplemental tests for determining infection status of those animals that cannot be
definitively diagnosed by serology (Deandres et al., 2005; Knowles, 1997). Real-time or quantitative PCR
techniques are beginning to be used in a few laboratories and these tests, in addition to determining
infection status, also quantify the amount of MV or CAE provirus in an animal (Alvarez et al., 2006;
Herrmann-Hoesing et al., 2007). Furthermore, molecular techniques such as PCR, cloning and sequencing
also provide knowledge on a countrys or regions specific MV and CAE strains, which may influence which
serological assay and corresponding MV or CAE antigen to use. Phylogenetic analyses of MV and CAE
980
proviral DNAs from SRLV strains throughout the world have suggested that in some areas, MV may have
naturally infected goats, and CAE may have naturally infected sheep (Shah et al., 2004a; 2004b). In the
future, molecular diagnostic tests along with phylogenetic analyses of MV and CAE provirus may be used to
track transmission.
An important issue in the use of PCR is specificity. Because of the possibility of amplifying unrelated
sequences from the hosts genomic DNA (false positives), the amplified product should be checked by either
hybridisation, restriction endonuclease digestion patterns, or sequencing. Sequencing provides the best
proof of specificity in the validation of PCR-based tests and is recommended by the OIE. Sensitivity of PCRbased tests can be improved by the use of nested PCR, but specificity of the nested PCR test should be
checked using hybridisation, restriction endonuclease digestion patterns, or sequencing methods.
2.
Serological tests
Ovine and caprine lentivirus infections are persistent, so antibody detection is a valuable serological tool for
identifying virus carriers. The close antigenic relationship between MVV and CAEV does not extend to
detection of heterologous antibody in some serological assays (Knowles et al., 1994).
The assays most commonly used to serologically diagnose the presence of a small ruminant lentivirus
infection are agar gel immunodiffusion (AGID) and the enzyme-linked immunosorbent assay (ELISA). AGID
was first developed and reported in 1973 (Terpstra & De Boer, 1973), and the ELISA was first developed
and reported in 1982 (Houwers et al., 1982). The AGID is specific, reproducible and simple to perform, but
experience is required for reading the results. The ELISA is economical, quantitative and can be automated,
thus making it useful for screening large numbers of sera. The sensitivity and specificity of both the AGID
assay and ELISA depend upon the virus strain used in the assay, viral antigen preparation, and the
standard of comparison assay. Western blot analysis and/or radio-immunoprecipitation are the standards of
comparison used to access sensitivity and specificity of new AGID tests and ELISAs.
a)
Agar gel immunodiffusion (a prescribed test for international trade)

There are two MV and CAE viral antigens of major importance in routine serology, a viral surface envelope
glycoprotein commonly referred to as SU or gp135, and a nucleocapsid protein referred to as CA or p28.
These are both conserved in an antigen preparation consisting of medium harvested from infected cell
cultures and concentrated approximately 50-fold by dialysis against polyethylene glycol. As an example the
WLC-1 strain of MV virus is commonly used in the AGID assay in the United States (Cutlip et al., 1977)1 and
a Canadian MV field strain is used for AGID tests in Canada (Simard & Briscoe, 1990b).
It is important to recognise that the sensitivity of the AGID test for detecting anti-CAEV antibody is
dependent on both the virus strain and the viral antigen used (Adams & Gorham, 1986; Knowles et al.,
1994). It was demonstrated that an AGID test with CAEV gp135 afforded greater sensitivity than an AGID
test with CAEV p28 (Adams & Gorham, 1986). Also, it was shown that when compared with radioimmunoprecipitation, the sensitivity of the AGID test for anti-CAEV antibody was 35% greater using CAE
virus antigen over using MV virus antigen (Knowles et al., 1994). The most likely explanation for this
difference in sensitivity between the CAE and MV virus antigen for the detection of anti-CAEV antibody is
that although the radio-immunoprecipitation assay requires only the binding of a single epitope by antibody
to obtain a positive result, precipitation in an agar gel requires multiple epitopeantibody interactions.
Although the MV and CAE viruses have 7374.4% nucleotide sequence identity in the envelope gene
(Herrmann et al., 2004), this amount of identity may not be sufficient to produce sufficient antibody to CAE
and MV mutually common epitopes resulting in undetectable antibody/antigen precipitin lines using MV virus
antigen. When the appropriate antigen is used, the AGID test performance is high. When compared with
immunoprecipitation, the AGID for the detection of anti-CAEV antibody, if CAEV antigen was used, had 92%
sensitivity and 100% specificity (Knowles et al., 1994). In addition, the AGID for detection of anti-MVV
antibody, if MVV antigen was used, had 99.3 and 99.4% sensitivity and specificity, respectively (Herrmann et
al., 2003b).
In adult persistently MVV-infected sheep and CAEV-infected goats, the predominant immunoprecipitating
antibody response is directed against gp135 antigen (Herrmann et al., 2005; Knowles et al., 1990). An antip28 response is usually present at a lower titre than the anti-gp135 response in persistently infected adult
small ruminants using immunoprecipitation. In some CAEV-infected goats there is evidence to suggest that
an anti-gp135 antibody response is produced, in the absence of an anti-p28 response and vice versa, in a
proportion of individuals (Dawson, 1985; Rimstad et al., 1994). Hence, for validation of a test, standard sera
producing both anti-gp135 and anti-p28 precipitin lines are required.
This virus has been distributed by Dr Howard Lehmkuhl, National Animal Disease Center, United States Department of
Agriculture, P.O. Box 70, Ames, Iowa, USA.
981
The gel medium is 0.71% agarose in 0.05 M Tris buffer, pH 7.2, with 8.0% NaCl. The test is conveniently
performed in plastic Petri dishes, or in 10 cm2 plastic trays. The pattern and size of the wells will determine
the number of sera tested per plate. Various well patterns can be adopted, but a hexagonal arrangement
with a central well is usual: for example, a pattern with alternating large (5 mm in diameter) and small (3 mm
in diameter) peripheral wells, 2 mm apart and 2 mm from a central antigen well that is 3 mm in diameter. The
large peripheral wells are used for test sera and the small ones for standard sera. A weak positive control
must be included in each test. The plates are incubated overnight at 2025C in a humid chamber, and then
examined for precipitin lines. Plates may be incubated at 28C for another 24 hours to enhance the
precipitin lines.
An important consideration is the need for experienced personnel to interpret the AGID. Interpretation of
AGID results is dependent on the antigen used. Examples of AGIDs with different antigen preparations and
a guide for interpretation of the results can be found in Adams et al., 1983.
b)

Currently, there are over 30 different ELISAs reported for the detection of anti-MVV or anti-CAEV antibodies
in the sera of sheep or goats, respectively (Deandres et al., 2005). Most of these ELISAs are indirect ELISAs
(I-ELISA) although there are three reported competitive ELISAs (C-ELISA) using monoclonal antibodies
(Frevereiro et al., 1999; Herrmann et al., 2003a; 2003b; Houwers & Schaake, 1987). Half of I-ELISAs use
purified whole virus preparations for antigen whereas the other half use recombinant protein and/or synthetic
peptide antigens. A few of the I-ELISAs have shown high sensitivity and specificity against a standard of
comparison, western blot analysis or radio-immunoprecipitation (Kwang et al., 1993; Rosati et al., 1994;
Saman et al., 1999). When compared with radio-immunoprecipitation, one C-ELISA has shown high
sensitivity and specificity both in sheep and goats in the USA suggesting that this one test can be used for
both MVV and CAEV US surveillance (Herrmann et al., 2003a; 2003b). Although ELISAs have been used for
several years in some European countries (Ppin et al., 1998) in control and eradication schemes of MVV in
sheep (Motha & Ralston, 1994) and CAEV in goats, AGID remains the most frequently used test.
Whole-virus antigen preparations are produced by differential centrifugation of supernatants from infected
cell cultures and detergent treatment of purified virus, and are coated on microplates (Dawson et al., 1982;
Simard & Briscoe, 1990a; Zanoni et al., 1994). Whole-virus preparations should contain both gp135 and
p28. Recombinant antigens or synthetic peptides are usually produced from whole or partial segments of the
gag or envelope genes and may be used in combination (Kwang et al., 1993; Power et al., 1995; Rosati et
al., 1994; Saman et al., 1999). Thus, recombinant gag or envelope gene products fused with glutathione Stransferase fusion protein antigen that have been produced in Escherichia coli provide a consistent source of
antigen for international distribution and standardisation.
The ELISA technique is also applicable to colostrum or milk, and some studies have evaluated paired serum
and milk samples. Because colostrum and milk are sources of CAEV transmission, the testing of milk
samples for anti-CAEV or anti-MVV antibody would not provide timely information for the prevention of
transmission, especially to offspring from the immediate gestation (Knowles, 1997).
The ELISA is performed at room temperature (~25C) and is easy to perform in laboratories that have the
necessary equipment (microplate reader) and reagents. It is convenient for large-scale screening, as it is a
reliable and quantitative technique for demonstrating small ruminant lentiviruses (SRLVs) antibodies in
sheep and goats. It requires relatively pure antigen. One disadvantage of several ELISAs is that many have
not been validated against a standard of comparison such as western blot analysis or radioimmunoprecipitation. The OIE recommends the validation of 1000 negatives and 300 positives using a
standard of comparison such as western blot analysis or radio-immunoprecipitation, and to date, only one
ELISA has met these testing standards (Zanoni et al., 1994).
For I-ELISA, wells of the microplate are coated with antigen. Diluted serum samples are added to the wells
and react to antigens bound to the solid support. Unbound material is removed by washing after a suitable
incubation period. Conjugate (e.g. horseradish-peroxidase-labelled anti-ruminant Ig) reacts with specific
antibodies bound to the antigen. Unreacted conjugate is removed by washing after a suitable incubation
period. Enzyme substrate is added. The rate of conversion of substrate is proportional to the amount of
bound antibodies. The reaction is terminated after a suitable time and the amount of colour development is
measured spectrophotometrically. A disadvantage of the I-ELISA is that test sera typically need to be diluted
1/50 or greater in order to lower the number of false positives.
Specific MAbs have been used in a C-ELISA for SRLVs to capture gp135 or p28 as antigen (Frevereiro et
al., 1999; Herrmann et al., 2003a; 2003b; Houwers & Schaake, 1987; Ozyoruk et al., 2001). C-ELISA
overcomes the problem of antigen purity, as the specificity of this test depends on the MAb epitope. For CELISA, sample sera containing anti-SRLV antibodies inhibit binding of enzymelabelled MAb to SRLV
antigen coated on the plastic wells. Binding of the enzyme-labelled MAb conjugate is detected by the
addition of enzyme substrate and quantified by subsequent colour product development. Strong colour
development indicates little or no blockage of enzyme-labelled MAb binding and therefore the absence of
982
SRLV antibodies in sample sera. In contrast, weak colour development due to the inhibition of the enzymelabelled MAb binding to the antigen on the solid phase indicates the presence of SRLV antibodies in sample
sera. The format of the C-ELISA requires that serum antibodies must bind to or bind in close proximity to the
specific MAb epitope.
Materials and reagents
Microtitre plates with 96 flat-bottomed wells, freshly coated or previously coated with SRLV antigen;
microplate reader (equipped with 405, 450, 490 and 620 nm filters); 37C humidified incubator; 1-, 8- and
12-channel pipettes with disposable plastic tips; microplate shaker (optional); fridge; freezer.
Positive and negative control sera; conjugate (e.g. ruminant anti-immunoglobulin labelled with peroxidase);
tenfold concentration of diluent (e.g. phosphate buffered saline/Tween); distilled water; 10 wash solution;
substrate or chromogen (e.g. ABTS [2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] or TMB
[3,3,5,5-tetramethylbenzidine]); stop solution (e.g. detergent, sulfuric acid).
Indirect ELISA: test procedure
i)
Dilute the serum samples, including control sera, to the appropriate dilution (e.g. 1/20) and distribute
0.10.2 ml per well (in duplicate if biphasic ELISA). Control sera are positive and negative sera
provided by the manufacturer and an internal positive reference serum from the laboratory in order to
compare the titres between different tests.
ii)
Cover the plate with a lid and incubate at room temperature or 37C for 3090 minutes. Empty the
contents and wash three times in washing solution at room temperature.
iii)
Add the appropriate dilution of freshly prepared conjugate to the wells (0.1 ml per well). Cover each
plate and incubate as in step ii. Wash again three times.
iv)
Add 0.1 ml of freshly prepared or ready-to-use chromogen substrate solution to each well (e.g. ABTS in
citrate phosphate buffer, pH 5.0, and 30% H2O2 solution [0.1 l/ml]).
v)
Shake the plate; after incubation, stop the reaction by adding stopping solution to each well (e.g. 0.1 ml
sulphuric acid).
vi)
Read the absorbance of each well with the microplate reader at 405 nm (ABTS) or 450620 nm (TMB).
The absorbance values will be used to calculate the results.
viii) Interpretation of the results

For commercial kits, interpretations and validation criteria are provided with the kit.
For example: calculate the mean absorbance (Ab) of the sample serum and of the positive (Abpos) and
negative (Abneg) control sera, and for each serum, calculate the percentage:
Ab - Abneg
x 100
Abpos - Abneg
Interpret the results as follows:
Ab <30%
negative serum
Ab 3040%
doubtful serum
Ab >40%
positive serum
Competitive ELISA: test procedure
i)
Add 0.05 ml of undiluted serum and positive/negative controls to antigen-coated plate.
ii)
Incubate for 1 hour at room temperature.
iii)
Empty the plate and wash the plate three with diluted wash solution.
iv)
Add 0.05 ml of diluted antibody-peroxidase conjugate to each well. Mix well and incubate for
30 minutes at room temperature.
v)
After the 30-minute incubation, empty the plate and repeat the washing procedure described in step iii.
vi)
Add 0.05 ml of substrate solution (ex: TMB) to each well. Mix and cover plate with aluminium foil.
Incubate for 20 minutes at room temperature. Do not empty wells.
983
vii)
Add 0.05 ml of stop solution to each well. Mix. Do not empty wells.
viii) Immediately after adding the stop solution, the plate should be read on a plate reader (620, 630 or
650 nm).
ix)
Interpretation of results
Example: Calculation: 100 [(Sample Ab 100)/(Mean negative control Ab)] = % inhibition.
For goats, if a test sample causes >33.2% inhibition, it is positive; if a test sample causes <33.2%
inhibition, it is negative. For sheep, if a test sample causes >20.9% inhibition, it is positive; if a test
sample causes <20.9% inhibition, it is negative.

There are no biological products available. MAbs recognising conformational epitopes of the CAEV gp135 have
been described (Ozyoruk et al., 2001).
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BADIOLA J.J. (1999). A new sensitive serological assay for detection of lentivirus infections in small ruminants, Clin.
Diagn. Lab. Immunol., 6, 734740.
SHAH C., BNI J., HUDER J.B., VOGT H.R., MLHERR J., ZANONI R., MISEREZ R., LUTZ H. & SCHPBACH J. (2004a).
Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence
for regular sheep-to-goat transmission and worldwide propagation through lifestock trade. Virology, 319, 1226.
SHAH C., HUDER J.B., BNI J., SCHNMANN M., MHLHERR J., LUTZ H. & SCHPBACH J. (2004b). Direct evidence for
natural transmission of small ruminant lentiviruses of subtype A4 from goats to sheep and vice versa. J. Virol., 78,
75187522.
SIMARD C.L. & BRISCOE M.R. (1990a). An enzyme-linked immunosorbent assay for detection of antibodies to
maedi-visna virus in sheep. A simple technique for production of antigen using sodium dodecyl sulfate treatment.
Can. J. Vet. Res., 54, 446450.
SIMARD C.L. & BRISCOE M.R. (1990b). An enzyme-linked immunosorbent assay for detection of antibodies to
Maedi-visna virus in sheep. Comparison to conventional agar gel immunodiffusion test. Can. J. Vet. Res., 54,
451456.
TERPSTRA C. & DE BOER G.F. (1973). Precipitating antibodies against maedi-visna virus in experimentally infected
sheep. Archiv fr die gesamte Virusforschung, 43, 5362.
VALAS S., BENOIT C., GUIONAUD C., PERRIN G. & MAMOUN R.Z. (1997). North American and French caprine arthritisencephalitis viruses emerge from ovine maedi-visna viruses. Virology, 237, 307318.
ZANONI R.G. (1998). Phylogenetic analysis of small ruminant lentiviruses. J. Gen. Virol., 79, 19511961.
ZANONI R.G., VOGT H.R., POHL B., BOTTCHER J., BOMMELI W. & PETERHANS E. (1994). An ELISA based on whole
virus for the detection of antibodies to small-ruminant lentiviruses. J. Vet. Med. B, 41, 662669.
*
* *
NB: There are OIE Reference Laboratories for Caprine arthritis/encephalitis & Maedi-visna
diagnostic tests, reagents and vaccines for caprine arthritis/encephalitis & Maedi-visna
986
CHAPTER 2.7.5.
CONTAGIOUS AGALACTIA
SUMMARY
Contagious agalactia is a serious disease syndrome of sheep and goats that is characterised by
mastitis, arthritis, keratoconjunctivitis and, occasionally, abortion. Mycoplasma agalactiae is the
main cause of the disease in sheep and goats, but M. capricolum subsp. capricolum (Mcc),
M. mycoides subsp. capri (Mmc) (formerly named M. mycoides subsp, mycoides LC [LC = large
colonies]) and M. putrefaciens produce a clinically similar disease, more often in goats, which may
be accompanied by pneumonia. Antibodies to Mmc and Mcc have been detected in South
American camelids (alpacas, llamas and vicunas), but no mycoplasmas have yet been isolated.
Identification of the agent: Definitive diagnosis requires the isolation of the causative
mycoplasmas from the affected animals, which are then identified by biochemical, serological and,
increasingly, molecular tests such as the polymerase chain reaction. Samples of choice include
milk, conjunctival and ear swabs, and joint fluid. All four mycoplasmas grow relatively well in most
mycoplasma media although M. agalactiae shows a preference for organic acids such as pyruvate
as substrates.
Serological tests: Detection of antibodies in serum by complement fixation test or enzyme-linked
immunosorbent assay (ELISA) provides rapid diagnosis of disease, but may not be very sensitive in
chronically affected herds and flocks. Indirect ELISAs have been used routinely in control
programmes for screening herds for M. agalactiae. Confirmation of infection by isolation and
identification is usually necessary in areas believed to be free of contagious agalactia. Serological
tests are not widely available for M. putrefaciens.
Requirements for vaccines and diagnostic biologicals: Commercial vaccines for M. agalactiae,
inactivated with formalin, are widely used in southern Europe, but are not considered to be very
efficacious. Under experimental conditions, M. agalactiae vaccines inactivated with saponin or
phenol have been shown to be more protective than formalised preparations. Live vaccines for
M. agalactiae are used in Turkey, where they are reported to be more protective than inactivated
vaccines. A commercial vaccine containing M. agalactiae, Mmc and Mcc is available. Autogenous
vaccines for Mmc and, occasionally, for Mcc are believed to be used in some countries. No
vaccines exist for M. putrefaciens, as the disease it causes is not considered to be sufficiently
serious or widespread.
A. INTRODUCTION
Contagious agalactia is a disease of sheep and goats that is characterised by mastitis, arthritis and
keratoconjunctivitis, and has been known for nearly 200 years. It occurs in Europe, western Asia, the United
States of America (USA) and North Africa, and is mainly caused by Mycoplasma agalactiae (Bergonier et al.,
1997). In recent years, M. capricolum subsp. capricolum (Mcc) and M. mycoides subsp. capri1 (formerly
M. mycoides subsp. mycoides LC [LC = large colonies]) have also been isolated in many countries from sheep
and goats with mastitis and arthritis. The clinical signs of these infections are sufficiently similar to be considered
indistinguishable from contagious agalactia. In addition, M. putrefaciens also causes mastitis and arthritis in
goats, which is very similar to that caused by M. agalactiae, Mmc and Mcc (Rodriguez et al., 1994). Furthermore,
the consensus of the working group on contagious agalactia of the EC COST2 Action 826 on ruminant
1
2
International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Mollicutes has proposed the
merging of these two subspecies into the single subsp: M. mycoides subsp. capri; a decision is pending.
European Cooperation in the field of Scientific and Technical Research.
987
Chapter 2.7.5. Contagious agalactia
mycoplasmoses, which met in Toulouse, France, in 1999, was that all four mycoplasmas should be considered as
causal agents of contagious agalactia.
Clinically, the disease caused by M. agalactiae is recognised by elevated temperature, inappetence and alteration
in the consistency of the milk in lactating ewes with decline and subsequent failure of milk production, often within
23 days, as a result of interstitial mastitis (Bergonier et al., 1997); lameness and keratoconjunctivitis affects
about 510% of infected animals. Fever is common in acute cases and may be accompanied by nervous signs,
but both signs are rare in the more frequently observed subacute and chronic infections. Pregnant animals may
abort. Mycoplasma agalactiae may occasionally be found in lung lesions (Loria et al., 1999), but pneumonia is not
a consistent finding. Bacteraemia is common, particularly for Mmc and Mcc and could account for the isolation of
the organism from sites where it is only transiently present.
Mastitis, arthritis, pleurisy, pneumonia, and keratoconjunctivitis may all result from infection with Mmc, which has
one of the widest geographical distribution of ruminant mycoplasmas, being found on all continents where small
ruminants are kept and wherever contagious agalactia and caprine pleuropneumonia are reported (Da Massa et
al., 1983; Nicholas, 2002); however the lack of diagnostic facilities for mycoplasma diseases in many countries
means that it is probably under reported. Mmc is mostly confined to goats but has occasionally been isolated from
sheep with reproductive disease and cattle with arthritis or respiratory disease. Cases usually occur sporadically,
but the disease may persist and spread slowly within a herd. After parturition, the opportunity for spread in milking
animals increases, and kids ingesting infected colostrum and milk become infected. The resulting septicaemia,
with arthritis and pneumonia, causes high mortality in kids (Bergonier et al., 1997; Da Massa et al., 1983).
Mcc is widely distributed and highly pathogenic, particularly in North Africa but the frequency of occurrence is low
(Bergonier et al., 1997). Goats are more commonly affected than sheep, and clinical signs of fever, septicaemia,
mastitis, and severe arthritis may be followed rapidly by death (Bergonier et al., 1997; Bolske et al., 1988).
Pneumonia may be seen at necropsy. The severe joint lesions seen in experimental infections with this organism
are accompanied by intense periarticular subcutaneous oedema affecting tissues some distance from the joint
(Bolske et al., 1988).
Mycoplasma putrefaciens is common in milking goat herds in western France where it can be isolated from
animals with and without clinical signs (Mercier et al., 2001). It has also been associated with a large outbreak of
mastitis and agalactia leading to severe arthritis in goats accompanied by abortion and death without pyrexia in
California, USA (Bergonier et al., 1997). Mycoplasma putrefaciens was the major finding in an outbreak of
polyarthritis in kids in Spain (Rodriguez et al., 1994).
Antibodies to Mmc and Mcc, but not M. agalactiae, have been detected in South American camelids, including
llamas, alpacas and vicunas, but as yet no mycoplasmas have been isolated (Nicholas, 1998). These camelids
are affected by a range of mycoplasma-like diseases, including polyarthritis and pneumonia, so it is likely that
mycoplasmas including Mmc and Mcc may be found in the future.
1.
Identification of the agents
a)
Selection of samples
Preferred samples from living animals include: nasal swabs and secretions; milk from mastitic females or
from apparently healthy females where there is a high rate of mortality/morbidity in kids; joint fluid from
arthritic cases; conjunctival swabs from cases of ocular disease; and blood for antibody detection from
affected and non-affected animals (Nicholas & Baker, 1998). The ear canal has also been shown to be a
source of pathogenic mycoplasmas, although in practice the presence of nonpathogenic mycoplasmas at
this site may make confirmation difficult (Nicholas & Baker, 1998). Mycoplasmas may be isolated from the
blood during the acute stage of the disease when there is mycoplasmaemia. From dead animals, samples
should include: udder and associated lymph nodes, joint fluid, lung tissue (at the interface between diseased
and healthy tissue) and pleural/pericardial fluid. Samples should be dispatched quickly to a diagnostic
laboratory in a moist and cool condition. All four causative mycoplasmas are relatively easy to isolate from
internal organs, joints and milk and grow well in most mycoplasma media, producing medium to large
colonies in 34 days.
b)
Mycoplasma medium
The usual techniques used in the isolation of mycoplasmas apply to all four causative organisms (Nicholas &
Baker, 1998). Many media have been reported to grow the causative mycoplasmas. Improved growth rates
of M. agalactiae have been seen in media containing organic acids such as pyruvate and isopropanol (Khan
et al., 2004). The formulation of PRM medium (Khan et al., 2004) is as follows:
988
Heat inactivated porcine serum 100 ml/litre, special peptone 20 g/litre, yeast extract 5 g/litre, glycerol
5 g/litre, sodium chloride 5 g/litre, HEPES 9 g/litre, fresh yeast extract 100 ml/litre, sodium pyruvate 5 g/litre,
12.5 ml of 0.2% phenol red and ampicillin (200,000 International Units/ml. Make up to 1 litre in distilled water
and sterilise by filtration. Adjust the pH of the broth medium to 7.6. Prepare solid medium by adding 10 g of
LabM agar No. 1 (Bury UK, or agar of equivalent quality) and dispense into sterile Petri dishes.
Thallium acetate (250 mg/litre), which is toxic and inhibitory to some mycoplasmas but not those causing
contagious agalactia, may be a necessary component of the transport medium to reduce bacterial
contamination from clinical samples, but should be omitted once the mycoplasmas begin to grow in vitro. A
satisfactory alternative to thallium acetate may be colistine sulphate (37.5 mg/litre).
Test procedure
i)
Make tenfold dilutions (101106) of the liquid sample (milk, synovial fluid, conjunctival and ear swabs)
or tissue homogenate in appropriate broth medium.
ii)
Spread a few drops of each sample on the agar medium and dispense a 10% (v/v) inoculum into broth
medium.
iii)
Streak swabs directly on to agar medium.
iv)
Incubate inoculated broths (optimally with gentle shaking) and agar media at 37C in humidified
atmosphere with 5% carbon dioxide.
v)
Examine broths daily for signs of growth (indicated by a fine cloudiness or opalescence) or changes in
pH indicated by a colour change and examine agar media under 35 magnification for typical fried
egg colonies.
vi)
If no mycoplasma growth is seen after 7 days, subculture a 10% (v/v) inoculum of broth into fresh broth
and spread about 50 l of this on to agar media.
vii)
Repeat as for step v. If no mycoplasmas are seen after 21 days incubation, consider the results to be
negative.
viii) If bacterial contamination results (seen as excessive turbidity), filter sterilise by passing 1 ml of
contaminated broth through a 0.45 m filter into fresh broth medium.
Clinical samples frequently contain more than one mycoplasma species so clone purification of colonies is
often considered necessary before performing biochemical and serological identification, in particular the
growth and film inhibition tests (GIT and FIT, respectively). However, cloning is a lengthy procedure taking at
least 2 weeks. The immunofluorescence test (Bradbury, 1998), dot immunobinding tests (Poumarat, 1998)
and, more recently, polymerase chain reaction (PCR) tests (see Section B.1.e) do not require cloning as
these tests can detect the pathogenic mycoplasmas in mixed cultures, saving a great deal of time.
c)
Biochemical tests
The first test that should be performed on the cloned isolates is sensitivity to digitonin, which separates
mycoplasmas from acholeplasmas; the latter are ubiquitous contaminants that can overgrow the
mycoplasmas of interest. Growth in liquid medium containing glucose (1%), arginine (0.2%), and
phenolphthalein diphosphate (0.01%), on solid medium containing horse serum or egg yolk for the
demonstration of film and spots, and on casein agar or coagulated serum agar to test for proteolysis, are
among the most useful tests for differentiating the four mycoplasmas (Poveda, 1998). These biochemical
characteristics, however have been increasingly found to be variable for the individual mycoplasmas and
have little diagnostic value. The most impressive biochemical characteristic that differentiates
M. putrefaciens from all other mycoplasmas is the odour of putrefaction it produces in broth culture. Other
features that may be helpful include: film and spot production seen on the surface of the broth and solid
media caused by M. agalactiae and to a lesser extent by M. putrefaciens; and the proteolytic activity of Mcc
and MmmLC on casein and coagulated serum.
A rapid and highly convenient biochemical test that exploits the C8-esterase activity of M. agalactiae has
been reported (Khan et al., 2001). The mycoplasma forms red colonies on agar media within 1 hour of
adding the chromogenic substrate, SLPA-octanoate (a newly synthesised ester formed from a C8 fatty acid
and a phenolic chromophore). This activity is shared with M. bovis, although this mycoplasma is rarely found
in small ruminants. Isolates need not be cloned as M. agalactiae can be detected easily in mixed cultures. If
necessary PCRs can be used to distinguish rapidly M. agalactiae from M. bovis (see Section B.1.e).
d)
Serological identification
Identification of isolates using specific antisera is usually carried out with the GIT, FIT (Poveda & Nicholas,
1998) or the indirect fluorescent antibody (IFA) test (Bradbury, 1998). A recently developed dot
immunobinding test, which is carried out in microtitre plates, offers many improvements over the other
serological tests such as rapidity and higher throughputs (Poumarat, 1998) but requires subjective
989
judgements of staining intensity. For M. agalactiae, film inhibition may often be more reliable as growth
inhibition is not seen with all isolates; it can also be used for serodiagnosis. Film production by the
mycoplasma may be enhanced by the incorporation of 10% egg yolk suspension into the solid medium.
Test procedure
i)
Inoculate at least two dilutions of 48-hour cloned broth cultures (101 and 102) on to predried agar
media by allowing 50 l of the cultures to run down the tilted plates using the running drop technique
(Poveda & Nicholas, 1998). Remove any excess liquid with a pipette.
ii)
Allow the plates to dry. It is possible to apply two or three well separated running drops to each 90 mm
diameter plate.
iii)
Apply predried filter paper discs containing 30 l of specific antiserum to the culture; ensure good
separation of discs (at least 30 mm).
iv)
Incubate the plates as for mycoplasma culture and examine daily by eye against a light background.
Interpretation of the results
A zone of inhibition over 2 mm, measured from the paper disc to the edge of mycoplasma growth is
considered to be significant. Partial inhibition can occur with weak antiserum or where there are mixed
cultures. Stronger reactions can be obtained if about 60 l of antisera is added to 6 mm diameter wells made
in the agar with a cork borer or similar device (Poveda & Nicholas, 1998).
In the IFA test, specific antisera are applied to colonies on solid medium. Homologous antiserum remains
attached after washing and is demonstrated by adding fluorescein-conjugated antiglobulin, washing, and
viewing the colonies with an epifluorescence microscope (Bradbury, 1998). Controls should include known
positive and known negative control organisms, and a negative control serum. However like the
immunobinding tests subjective judgements are required to assess staining intensity.
Antisera for these serological tests have traditionally been prepared against the type strains of the various
Mycoplasma species, and most field isolates have been readily identified using these antisera. As more
strains have been examined, however, some have been found to react poorly with these antisera, while
reacting well with antisera to other representative strains of the species. Intraspecies variation in antigenic
composition has not been reported for M. putrefaciens, but occurs to some degree with M. agalactiae and
with Mcc strains. Thus, diagnostic laboratories may need to have several antisera to enable all strains of the
species to be identified.
e)

PCR assays are routinely used in many laboratories and are extremely sensitive. They can provide a rapid
early warning system when carried out on clinical samples, enabling a full investigation to take place when
results are positive. However negative results should not be considered definitive. Several PCRs specific for
M. agalactiae have been developed and show similar levels of sensitivity, although they are based on
different gene sequences (Bashiruddin et al., 2005; Dedieu et al., 1995; Subrahamaniam et al., 1998; Tola et
al., 1997a). They can be used directly on nasal, conjunctival, synovial and tissue samples; they have been
used on milk samples where they have been reported to be more sensitive than culture (Tola et al., 1997a),
although occasionally undefined inhibitors may interfere with the test. PCRs can also be used, more reliably,
on mycoplasmas growing in culture; a 24 hour enrichment of the mycoplasma in the appropriate medium
greatly facilitates PCR detection even in the presence of bacterial contamination (Nicholas, 2002). A newly
described PCR based method called denaturing gradient gel electrophoresis (DGGE) that uses
mycoplasma-specific primers is capable of identifying the majority of small ruminant mycoplasmas including
all the causative agents of contagious agalactia by their migration pattern (McAuliffe et al., 2005). A positive
PCR result, particularly in an area previously free of contagious agalactia, should be confirmed by isolation
and identification of the mycoplasma using standard procedures.
Individual PCRs have been reported for Mmc and Mcc (Bashiruddin et al., 1994) and M. putrefaciens
(Peyraud et al., 2003) respectively. In addition a multiplex test has been described which can detect
simultaneously M. agalactiae, Mcc and Mmc (Greco et al., 2001).
Test procedure
The following primers based on the uvrC gene have been shown to be specific for M. agalactiae
(Subrahamanian et al., 1998). PCRs may need to be optimised in each laboratory. Positive and negative
control DNA should be run in each assay.
MAGAUVRC1-L
MAGAUVRC1-R
990
CTC-AAA-AAT-ACA-TCA-ACA-AGC
CTT-CAA-CTG-ATG-CAT-CAT-AA
i)
Extract DNA from Mycoplasma isolates or clinical material using the appropriate method (Bolske et al.,
1988).
ii)
Carry out PCR methods in 50 l reaction mixtures containing: 1 l of sample DNA, 20 pmol of each
primer (see above), 1 mM each dNTP, 10 mM Tris/HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl and
1.25 mM U Taq DNA polymerase.
iii)
Subject the mixture to 35 amplification cycles in a thermal cycler with the following parameters:
30 seconds at 94C, 30 seconds at 50C annealing temperature and 1 minute at 72C.
iv)
Analyse the PCR products by electrophoresis on a 0.7% agarose at 110 V for 2 hours and visualise by
staining with ethidium bromide. A 1.7 kb fragment indicates the presence of M. agalactiae.
2.
Serological tests
a)
Complement fixation
A standard complement fixation test (CFT) for M. agalactiae has also been applied to other mycoplasmas
involved in the contagious agalactia syndrome (Bergonier et al., 1997). Antigens are prepared from washed
organisms, standardised by opacity, and lysed, either ultrasonically or by using sodium lauryl sulphate followed
by dialysis. Sera are inactivated at 60C for 1 hour, and the test is carried out in microtitre plates with overnight
fixation in the cold or at 37C for 3 hours. The haemolytic system is added, and the test is read after complete
lysis is shown by the antigen control. A positive result is complete fixation at a serum dilution of 1/40 or greater
for the following mycoplasmas: M. agalactiae, Mcc, and Mmc. The CFT is regarded as a herd test and at least
ten sera are tested from each herd, preferably from acute and convalescent cases.
Some sera from healthy flocks react in the CFT using M. agalactiae up to a serum dilution of 1/20, but rarely
react with the other two antigens. However, in flocks infected with M. agalactiae, sera giving a homologous
reaction at 1/80 may cross-react at up to 1/40, the positive threshold, with the other two antigens. It is often
difficult to perform the CFT if the quality of the test sera is poor; where possible, the enzyme-linked
immunosorbent assay (ELISA) is preferred.
b)

ELISAs using sonicated or Tween-20-treated antigens have been reported to be more sensitive than the
CFT for the detection of antibody to M. agalactiae (Bergonier et al., 1997). Problems of nonspecificity have
been overcome by the use of monoclonal or protein G conjugates in the ELISA (Lambert et al., 1998). The
use of these conjugates enables the testing of sera from a wide range of mammalian species, including
camelids. A number of commercial ELISA kits are now available and these are being used for large-scale
surveys in France and the United Kingdom (Bergonier et al., 1997; Nicholas, 1998). In a ring trial of
serological tests for M. agalactiae organised in 1998 under the auspices of the EC COST Action 826 on
ruminant mycoplasmoses, commercial ELISAs performed better than home-made kits.
ELISAs are not widely available for the other three causative mycoplasmas although home-made assays
are carried out by some laboratories.
c)
Immunoblotting test
Immunoblotting tests have been reported as the most sensitive and specific test for M. mycoides subsp.
mycoides SC, the cause of contagious bovine pleuropneumonia (see chapter 2.4.9). Immunoblotting tests
have also been described for M. agalactiae (Nicholas, 1998; Tola et al., 1997b). Strong bands at
approximately 80 and 55 kDa were seen with sera with antibodies to M. agalactiae, while sera from healthy
flocks show no bands or very faint bands of different sizes. Diluting the sera to 1/50 improves the
discrimination between positive and negative sera (Nicholas, 1998).

Vaccines for the prevention of contagious agalactia due to M. agalactiae are used widely in the Mediterranean
countries of Europe and in western Asia. No single vaccine has been universally adopted, and no standard
methods of preparation and evaluation have been applied.
1.
Vaccines for Mycoplasma agalactiae infection
a)
Inactivated vaccines for Mycoplasma agalactiae infection

In Europe, where live vaccines for M. agalactiae are not acceptable, attention has focused on the use of
killed organisms, mostly using formalin and an adjuvant such as aluminium hydroxide in an oil emulsion. The
991
titres of the preparations, before inactivation, are very high (1081010 colony-forming units per ml) and are
derived from laboratory strains. Some products are available commercially including a trivalent preparation
containing M. agalactiae, Mcc and Mmc but there are few data on their efficacy. A formalin-inactivated oil
emulsion vaccine was shown to be immunogenic and protective in a small trial in lactating sheep and also
prevented transmission of M. agalactiae (Greco et al., 2002).
It is possible that in some instances the apparent lack of protection given by vaccines could be the result of
animals being infected with one of the other four mycoplasmas involved in the contagious agalactia
syndrome (Gil et al., 1999). A multivalent formalin inactivated vaccine incorporating all four causative
mycoplasmas and adjuvanted with saponin and aluminium hydroxide appears beneficial in preliminary trials
(Ramirez et al., 2001).
More recently vaccines inactivated with phenol or with saponin have given superior protection against
experimental infections compared with formalin, sodium hypochlorite or heat-inactivated vaccines (Tola et
al., 1999).
b)
Live attenuated vaccines for Mycoplasma agalactiae infection

Live attenuated vaccines against M. agalactiae have been used in Turkey for many years and have been
reported to provide better protection in ewes and their lambs than inactivated vaccines (Nicholas, 2002).
However they can produce a transient infection with shedding of mycoplasma. Live vaccines should not be
used in lactating animals and should be part of a regional plan in which all flocks from which animals are
likely to come into contact be vaccinated at the same time.
2.
Vaccines for Mycoplasma mycoides subsp. capri infection
There is little recent published information on the availability of vaccines for Mmc although it is believed that
inactivated vaccines are widely used in many Mediterranean countries and in Asia suggesting that their
production and use is localised (Bergonier et al., 1997). Saponised vaccines have been reported in India which
provoke a strong antibody response and show some protection (Sunder et al., 2002).
3.
Mycoplasma capricolum subsp. capricolum and M. putrefaciens
Although infections with Mcc and M. putrefaciens can be severe, their prevalence is relatively low and, as might
be expected, little or no work appears to have been carried out on preventive vaccination for these infections.
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Vet. Sci., 75, 37.
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Belgium, 130133.
MCAULIFFE L., ELLIS R., LAWES J., AYLING R.D. & NICHOLAS R.A.J (2005). 16S rDNA and DGGE: a single generic
test for detecting and differentiating Mycoplasma species. J. Med. Microbiol., 54, 19.
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mycoplasmas. Small Rumin. Res., 45, 145149.
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R.J. & Nicholas R.A.J. eds. Humana Press, Totowa, USA, 3744.
PEYRAUD A., WOUBIT S., POVEDA J.B., DE LA FE C., MERCIER P. & THIAUCOURT F. (2003). A specific PCR for the
detection of Mycoplasma putrefaciens, one of the agents of the contagious agalactia syndrome of goats. Mol.
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POUMARAT F. (1998). Identification of mycoplasmas by dot immunobinding on membrane filtration (MF Dot). In:
Mycoplasma Protocols, Miles R.J. & Nicholas R.A.J., eds. Humana Press, Totowa, USA, 113118.
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R.J. & Nicholas R.A.J., eds. Humana Press, Totowa, USA, 6978.
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inhibition tests. In: Mycoplasma Protocols, Miles R.J. & Nicholas R.A.J., eds. Humana Press, Totowa, USA, 105
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inactivated polyvalent vaccine against Mycoplasma spp on infected goats. In: Mycoplasmas of Ruminants:
Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, Vol. 5, Poveda J.B., Fernandez A., Frey J. &
Johansson K.-E., eds. European Commission, Brussels, Belgium, 154157.
RODRIGUEZ J.L., POVEDA J.B., GUTIERREZ C., ACOSTA B. & FERNANDEZ A. (1994). Polyarthritis in kids associated with
Mycoplasma putrefaciens. Vet. Rec., 135, 406407.
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SUBRAHAMANIAM S., BERGONIER D., POUMARAT F., CAPUAL S., SCHLATTER Y., NICOLET J. & FREY J. (1998). Species
identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC gene by PCR. Mol. Cell.
Probes, 12, 161169.
SUNDER J., SRIVASTAVA N.C. & SINGH V.P. (2002) Preliminary trials on development of vaccine against Mycoplasma
mycoides subsp. mycoides type LC infection in goats. J. Appl. Anim. Res., 21, 7580.
TOLA S., ANGIOI A., ROCCHIGIANI A.M., IDINI G., MANUNTA D., GALLERI G. & LEORI G. (1997a). Detection of
Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction. Vet. Microbiol., 54, 1722.
TOLA S., MANUNTA D., COCCO M., TURRININ F., ROCCHIGIANI A.M., IDINI G., ANGIOI A. & LEORI G. (1997b).
Characterisation of membrane surface proteins of Mycoplasma agalactiae during natural infection. FEMS
Microbiol. Lett., 154, 355362.
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*
* *
NB: There is an OIE Reference Laboratory for contagious agalactia
diagnostic tests, reagents and vaccines for contagious agalactia
994
CHAPTER 2.7.6.
CONTAGIOUS CAPRINE PLEUROPNEUMONIA
SUMMARY
Contagious caprine pleuropneumonia (CCPP) is a severe disease of goats caused by Mycoplasma
capricolum subspecies capripneumoniae (Mccp). This organism is closely related to three other
mycoplasmas: M. mycoides subsp. mycoides large colonies (LC), M. mycoides subsp. capri, and
M. capricolum subsp. capricolum. Unlike true CCPP, which is confined to the thoracic cavity, the
disease caused by the latter three mycoplasmas is accompanied by prominent lesions in other
organs and/or parts of the body besides the thoracic cavity.
Typical cases of CCPP are characterised by extreme fever (4143C), high morbidity and mortality
rates in susceptible herds affecting all ages and both sexes, and abortions in pregnant goats. It
appears to be transmitted by an infective aerosol. After approximately 23 days of high fever,
respiratory signs become apparent: respiration is accelerated and painful, and in some cases is
accompanied by a grunt. Coughing is frequent, violent and productive. In the terminal stages,
animals are unable to move they stand with their front legs wide apart, the neck is stiff and
extended, and sometimes saliva continuously drips from the mouth. Post-mortem examination
reveals fibrinous pleuropneumonia with massive lung hepatisation and pleurisy, accompanied by
accumulation of straw-coloured pleural fluid.
The disease has been shown recently to affect wild ruminants such as the wild goats (Capra
aegagru), Nubian Ibex (Capra ibex Nubian) and Laristan Mouflon (Ovis orientalis laristanica) and
Gerenuk (Litocranius walleri). Clinical disease and seropositivity have been reported in sheep in
contact with affected goats, but the role of sheep as reservoirs of infection is unclear.
Identification of the agent: Definitive diagnosis requires culture of the causative organism from
lung tissue samples and/or pleural fluid taken at post-mortem. After cloning and purification,
isolates can be identified by several biochemical, immunological and molecular tests. Isolating the
causative agent is a difficult task. Recently polymerase chain reaction based tests have been
described and shown to be specific, sensitive and can be applied directly to clinical material, such
as lung and pleural fluid.
Serological tests: Serological tests have been applied for the diagnosis of CCPP in outbreaks in
Eritrea and Turkey. Such tests are best used on a herd basis rather than for diagnosis in individual
animals. The complement fixation test remains the most widely used serological test for CCPP,
although the latex agglutination test is being increasingly used in the diagnostic laboratories as well
as a pen side test; it can used to test whole blood as well as serum. Indirect hemagglutination is
also used. A specific competitive enzyme-linked immunosorbent assay has been developed, but is
not widely available. As with the other serological tests, it does not detect all reactors, but its
specificity and suitability for large-scale testing make it an appropriate test for epidemiological
investigations.
Requirements for vaccines and diagnostic biologicals: Vaccine against CCPP caused by Mccp
is available commercially.
A. INTRODUCTION
Contagious caprine pleuropneumonia (CCPP) is a severe disease of goats occurring in many countries in Africa
and Asia where the total goat population is more than 500 million (Acharya, 1992). The first description of the
disease was in 1873 in Algeria. Shortly after, in 1881, the disease was introduced in the Cape Colony of South
Africa by a shipment of Angora goats (Hutcheon, 1881; 1889). The disease was eradicated using a policy of
995
Chapter 2.7.6. Contagious caprine pleuropneumonia
slaughter of the infected goats coupled with a traditional vaccination procedure for the in-contact goats. Classical,
acute CCPP is caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp) (Leach et al., 1993), originally
known as the F38 biotype. This organism was first isolated and shown to cause CCPP in Kenya (MacMartin et al.,
1980; MacOwan & Minette, 1976; 1977a; 1977b); it has subsequently been isolated in the Sudan, Tunisia, Oman,
Turkey, Chad, Uganda, Ethiopia, Niger, Tanzania, Eritrea and the United Arab Emirates. CCPP was first reported
in mainland Europe in 2004, when outbreaks were confirmed in Thrace, Turkey, with losses of up to 25% of kids
and adults in some herds (Ozdemir et al., 2005). However, the exact distribution of the disease is not known and
it may be much more widespread than the zone represented by the countries where Mccp has been isolated as
CCPP is often confused with other respiratory infections and also because the isolation of the causative organism
is difficult.
In CCPP outbreaks in mixed goat and sheep herds, sheep may also be affected, as verified by isolation of Mccp
(Blske et al., 1995) or detection of antibodies from clinically affected sheep (Kibor & Waiyaki, 1986). Mccp has
also been isolated from healthy sheep (Litamoi et al., 1990) and the role of sheep as a reservoir for the disease
has to be considered.
Recently CCPP was confirmed in wild ruminants kept in a wildlife preservation reserve in Qatar. The disease
affected wild goats (Capra aegagrus), Nubian Ibex (Capra ibex nubiana), Laristan mouflon (Ovis orientalis
laristanica) and Gerenuk (Litocranius walleri) with significant morbidity and mortality in these species (Arif et al.,
2005). Disease indistinguishable from naturally occurring CCPP has been experimentally reproduced with Mccp
by several groups of workers.
The diagnosis of outbreaks of respiratory disease in goats, and of CCPP in particular, is complicated, especially
where it is endemic. It must be differentiated from other similar clinico-pathological syndromes such as: peste des
petits ruminants, to which sheep are also susceptible; pasteurellosis, which can be differentiated on the basis of
distribution of gross lung lesions; and what has been called mastitis, arthritis, keratitis, pneumonia and
septicaemia syndrome or more often as contagious agalactia syndrome (Thiaucourt & Bolske, 1996). As the
longer name implies, the pneumonia is accompanied by prominent lesions in other organs. The disease caused
by Mccp is readily contagious and fatal to susceptible goats of all ages and both sexes, rarely affects sheep and
does not affect cattle.
1.
a)
Microscopy of lung exudates, impression smears or sections

Mccp is characterised histopathologically by an interstitial pneumonia with interstitial, intralobular oedema of
the lung (Kaliner & MacOwan, 1976; Nicholas, 2002). It shows a branching filamentous morphology in vivo
that can be observed by dark-field microscopy in exudates or tissue suspensions from lesions or pleural
fluid. Alternatively, smears made from cut lung lesions can be stained by the method of MayGrnwald
Giesma and examined by light microscopy. The other caprine mycoplasmas show a short filamentous or
coccobacillary morphology. Neither of these techniques provides a definitive diagnosis.
b)

Two polymerase chain reaction (PCR) assays for the specific identification of Mccp have now been
published. The first one (Bascunana et al., 1994) is based on the amplification of the 16S rRNA gene of the
mycoides cluster. The PCR product is then analysed by restriction enzyme cleavage for the identification of
the Mccp amplicon. The second one (Woubit et al., 2004) is based on a specific amplification. These PCR
techniques can be used directly on clinical materials such as lung tissue and pleural fluid (Blske et al.,
1996). Due to the difficulty in isolating Mccp, PCR is the technique of choice for the diagnosis of CCPP.
However, isolation of Mccp remains the confirmatory test. All mycoplasmas of the mycoides cluster can be
assigned a precise phylogenetic position by using a multilocus sequence typing approach which may be
used for identification purposes (Manso-Silvn et al., 2007).
c)
Gel precipitin tests to detect antigen in tissue specimens

Mccp releases an antigenic polysaccharide (Rurangirwa et al., 1987c) to which a specific monoclonal
antibody (MAb) (WM-25) has been produced (Rurangirwa et al., 1987d; 1992). This MAb
immunoprecipitates in agar gel with the polysaccharide produced by Mccp, and is used to identify the
causative agent in cases of CCPP, particularly when specimens are no longer suitable for culture because of
deterioration during transit.
996
d)
Isolation of mycoplasmas
i)
Selection of samples
The necropsy samples of choice are lung lesions, particularly from the interface between consolidated
and unconsolidated areas, pleural fluid, and mediastinal lymph nodes. If microbiological examination
cannot be performed immediately, samples or whole lungs can be stored at 20C for considerable
periods (months) with little apparent loss of mycoplasma viability. During transport, samples should
always be kept as cool as possible, as mycoplasma viability diminishes rapidly with increasing
temperature. Lung samples can be dispatched to other laboratories in frozen form.
ii)
Treatment of samples
Swabs are suspended in 23 ml of culture medium. Tissue samples are best minced using scissors,
and then shaken vigorously, or pulverised in medium1 using 1 g of tissue to 9 ml of medium. Tissues
should not be ground. The suspension is usually prepared with a mycoplasma medium, but if parallel
bacteriological examination is to be carried out, a high quality bacteriological medium, such as nutrient
broth, may be used to provide a suspension suitable for both forms of examination. Pleural fluid, or a
tissue suspension or swab, is serially diluted through at least three tenfold steps (to a nominal 104) in
the selected mycoplasma medium. Dilutions should also be plated on to solid medium.
iii)
Mycoplasma media
The medium used by MacOwan & Minette to culture Mccp organisms (MacOwan & Minette, 1976), is
termed viande foie goat (VFG), and includes goat-meat liver broth and goat serum. Alternative
suitable media are WJ (Jones & Wood, 1988), modified Hayflicks, and modified Newings tryptose
broth (Kibor & Waiyaki, 1986) Media enriched with 0.2% (or up to 0.8%) sodium pyruvate perform
considerably better, both for primary isolation and antigen production of Mccp (Miles & Wadher, 1990;
Mohan et al., 1990). Examples of suitable media are as follows (Blske et al., 1996; Thiaucourt &
Bolske, 1996; Thiaucourt et al., 1992):
CCPP medium
A. Autoclaved portion (121C for 15 minutes): Bacto PPLO (pleuropneumonia-like organisms) broth
without crystal violet (Difco) (21 g); deionised water (700 ml).
B. Membrane-filtered portion: Horse (alternatively pig or donkey) serum inactivated at 56C for
30 minutes (200 ml); fresh yeast extract (100 ml); glucose (sterile solution 0.5 g/ml) (2 ml); and sodium
pyruvate (sterile solution 0.5 g/ml) (8 ml).
Part B is added to A aseptically. Ampicillin (0.1 g/litre) and thallium acetate (250 mg/litre) can be added
to prevent contamination in primary isolations. The final pH of the medium should be 7.47.6.
Modified CCPP medium

A. Autoclaved portion (121C for 15 minutes): Bacto PPLO broth without crystal violet (Difco) (17.5 g);
glass distilled water (650 ml).
B. Membrane-filtered portion: Horse (alternatively pig or donkey) serum inactivated at 56C for
30 minutes (250 ml); fresh yeast extract (100 ml); 50% glucose (4 ml); 25% sodium pyruvate (8 ml); 5%
thallium acetate (4 ml); ampicillin (250 mg); and 0.5% phenol red (4 ml). The pH is adjusted to 7.8 with
sodium hydroxide or hydrochloric acid. Part B is added to A aseptically.
Modified Newings tryptose broth (Kibor & Waiyaki, 1986) and agar plates (Gourlays medium)
(Gourlay, 1964) are routinely used for isolation and maintenance of Mccp in Kenya.
iv)
Medium production, storage and quality control

Certain medium components, particularly serum, yeast extract and deionised water, should be
regularly monitored for growth-promoting capacity before incorporation into mycoplasma media. Lowpassage field isolates should be used for this screening purpose.
Broth media may be stored for at least 6 months at 25C before use, but penicillin or its analogues
should not be added until final dispensing. Broth media are dispensed into bijoux (1.8 ml or 2.7 ml) or
screw-capped tubes (4.5 ml), and stored for up to 3 weeks at 4C. Solid media are best made with
agarose (0.9% [w/v]), Noble agar (1.5% [w/v]), or purified agar (0.6% [w/v]). Plates, which are poured
to a depth of 68 mm, should be as fresh as possible when used, and should be stored for no more
than 2 weeks at 4C before use. All culture media should be subjected to quality control and must
For example, with the Stomacher 80, A.J. Seward, London, United Kingdom (UK).
997
support the growth of Mycoplasma spp. from small inocula. The reference stain should be cultured in
parallel with the suspicious samples to ensure that the tests are working correctly.
v)
Cultivation
Cultures are incubated at 37C. Plates are best incubated in a humid atmosphere of 5% CO2, 95% air
or N2, or in a candle jar with a moisture source.
Broth cultures are examined daily for evidence of growth colour change and the appearance of
floccular material. Gross turbidity indicates bacterial contamination; cultures showing this should be
passed through a 0.45-m membrane filter before subculture. Broth cultures are subcultured by
inoculation of fresh broth medium with one-tenth of their volume, or by streaking agar medium with a
loop.
Plate cultures are examined every 13 days using a stereo microscope (550 magnification) and
transmitted and incident light sources. If negative, the plates are discarded after 14 days. Subculture is
carried out by the transfer of excised agar blocks bearing isolated colonies to either agar (on which the
blocks are pushed, face down) or broth media. Alternatively, an agar plug bearing one colony is drawn
into a Pasteur pipette and discharged into fresh broth medium.
Cloning and purification of isolates is performed by repeated transfer of single colonies representing
each morphological type seen. Colony morphology varies with the medium used, the mycoplasma
species, its passage level and the age of the culture.
In early passage, many mycoplasma species, including M. capricolum subsp. capricolum (Mcc),
produce colonies of bizarre morphology, often small, centreless, and of irregular shape. This effect is
often associated with the use of marginally suitable medium. With passage, such isolates demonstrate
conventional fried egg colony morphology, except M. ovipneumoniae, which retains centreless
colonies. Colonies of M. mycoides subsp. mycoides Large Colony (MmmLC) and Mcc may be up to
3 mm in diameter.
Filtration of broth cultures through 0.45 m filters before subculture aids purification by excluding cell
aggregates.
Cultures suspected of being L-forms of bacteria should be examined for reversion to bacterial form by
three to five passages on solid mycoplasma medium from which antibiotics and thallium acetate have
been omitted.
Broth media used for primary isolation and which have shown no indication of growth by 7 days, should
be subcultured blind.
Cultures of each sample, including one blind subculture, should be examined for a minimum of
3 weeks before being discarded. Titrations in broths, if performed in full (to 1010), are also read at 3
4 weeks and are expressed as colour-changing units per transfer volume. Growth on plates is
expressed as colony-forming units (CFU) per ml.
e)
Identification of mycoplasmas
i)

Once the organism has been cultured, verification of Mccp can be achieved in 1 day by PCR. There
are now various PCR tests that can be applied for an identification of Mccp strains. The first test
(Bascunana et al., 1994) is based on amplification of a segment of the 16S rRNA gene. The amplified
fragment is common to the mycoides cluster. However, when the amplicon is digested with
endonuclease PstI, a unique cleavage pattern of three fragments for Mccp is observed when the
enzyme digests are analysed in agarose gel electrophoresis and stained with ethidium bromide (Blske
et al., 1996). The second test is based on a specific amplification and can allow confirmation of CCPP
within a few hours (Woubit et al., 2004).
Recently, PCR and sequencing has been used to establish the molecular epidemiology of CCPP.
These tests can be performed on dried samples, such as pleural fluid on filter papers. The sequencing
allows a precise identification of the species (the cleavage site for the 16S rRNA and a specific
detection for the locus H2) (Lorenzon et al., 2002; Pettersson et al., 1998).
Identification of Mccp strains by PCR (and sequencing) has now superseded all other techniques
because of its rapidity and reliability. However PCR reactions must be performed with great care to
prevent contaminations.
ii)
Biochemical tests
Wild strains should be passaged, and preferably cloned, three times before identification is attempted.
998
Biochemical tests cannot identify an isolate unequivocally, which at present can only be done by
serological or genetic means. Intraspecies variation in some biochemical reactions is often
considerable (Houshaymi et al., 2000), but some tests perform a useful function both as a preliminary
screening system and in providing supportive data for serological findings.
The tests most commonly used are glucose breakdown, arginine hydrolysis, film and spots formation,
reduction of tetrazolium chloride (aerobically and anaerobically), phosphatase activity, serum digestion
and digitonin sensitivity. The first three of these tests are performed routinely in isolation and cultivation
procedures. Glucose breakdown is indicated by acid (yellow) changes, and arginine hydrolysis by
alkaline (red) changes in broth media, using phenol red as indicator. Arginine use cannot be assessed
on conventional medium for isolation and culture as the media for testing the arginine deiminase
pathway should contain high concentrations of arginine and no glucose. Film and spots describes an
apparent wrinkling of the agar surface due to the deposition on it of an iridescent film of lipid, together
with the development of black spots within the medium in the vicinity of ageing colonies. This
phenomenon, produced by three mycoplasmas of small ruminants, is demonstrated on agar media
containing 20% or more serum, preferably of horse or pig origin. Supplementation of the medium with
10% egg yolk emulsion improves the sensitivity of the test.
The remaining biochemical tests require specific media or reagents. The test for tetrazolium reduction
provides corroborative evidence of the mycoplasmal nature of M. agalactiae isolates, as this organism is
neither glycolytic nor arginine-hydrolysing. Serum digestion (Freundt, 1983) distinguishes members of
ruminant mycoplasmas, and phosphatase production (Bradbury, 1983) separates Mcc from other members
of this cluster. Digitonin sensitivity distinguishes members of the order Mycoplasmatales from those of the
order Acholplasmatales (Freundt et al., 1973). A diagnostic medium is available that enables the specific
detection of Mccp growing on agar medium: colonies show a red coloration (Ozdemir et al., 2005).
iii)
Serological identification
Mycoplasmal antigens used in hyperimmune serum production are often contaminated with medium
constituents. The antibodies stimulated by these contaminants can cause false-positive reactions in
serological identification tests. This problem is avoided by absorption of the antiserum with the medium
used to produce the antigen (10 mg lyophilised medium per ml of antiserum), or by growing the
mycoplasmas to be used as antigens in medium containing homologous animal components, e.g.
growth in VFG medium to immunise goats.
Because of the close serological relationships between members of the M. mycoides cluster, isolates from
cases of CCPP should, preferably, be identified by at least two of the three tests described below.
Growth inhibition test
The growth inhibition test (GIT) is the simplest and most specific, but the least sensitive, of the tests
available. It depends on the direct inhibition of growth on solid medium by specific hyperimmune serum, and
detects primarily surface antigens (Dighero et al., 1970).
Mccp appears to be highly homogeneous serologically and wide zones of inhibition free of breakthrough
colonies are observed with antiserum to the type strain, regardless of the source of the test strain (Jones &
Wood, 1988). Mccp cross-reacts with Leachs bovine group 7 (PG50), M. equigenitalium and M. primatum in
the GIT when polyclonal antisera are used, but an MAb specific for Mccp in the GIT has been produced
(Rurangirwa et al., 1987d). The MAb reagent, WM25, has been reported to be specific for (Mccp) isolates by
the disc growth inhibition method, which will exclude M. agalactiae, Mcc and the other members of the
M. mycoides cluster associated with goats, but not bovine group 7 (not usually found in goats): the latter
can be excluded, however, by colony indirect fluorescence tests (Belton et al., 1994). A small proportion of
Mccp isolates also cross-react in the GIT with antiserum to Mcc. Group seven of Leach strains can
sometimes be found in goats although it is rare. Results should be interpreted carefully as some bovine
strains have been misidentified by the GIT using the specific antiserum.
Test procedure
i)
Broth culture in mid-to-late logarithmic phase is used at three tenfold dilutions, the selection of which is
related to the vigour of growth of the isolate on agar.
ii)
Agar plates are dried for 30 minutes at 37C.
iii)
Sterile paper disks of 67 mm in diameter are impregnated with a drop (1020 l) of undiluted
antiserum. Disks may be used wet, in which form they can be stored at 20C, or they can be
lyophilised (Dighero et al., 1970), which allows storage at 4C.
iv)
Using a separate plate for each dilution of culture, 1 ml or 2.5 ml volumes are pipetted on to 5 cm or
10 cm diameter plates, respectively. The inoculum is dispersed evenly over the plate, then the excess
is removed.
999
v)
The plates are dried at 2030C for 1520 minutes, preferably under a protective hood, until no visible
liquid is present on the surface. Sufficient residual moisture should remain to enable freeze-dried disks
to adhere to the agar surface.
vi)
Several disks, each impregnated with a different antiserum (selected on the basis of sample source
and the biochemical reactions and colony morphology of the isolate), are carefully placed on the agar
plates; isolates from CCPP cases should be screened with antisera against Mccp, MmmLC, Mcc,
M. mycoides subsp. capri (Mcc) and M. ovipneumoniae. A disk containing 1.5% digitonin should also
be included on the plates.
vii)
The plates are incubated at 37C for 26 days. Initial overnight incubation at 27C can increase the
sensitivity of the test. Inhibition by digitonin is generally readily apparent; however, inhibition by
antiserum may be more difficult to interpret, with suppression rather than total inhibition of growth,
depending on the species of mycoplasma, colony density and potency of the antiserum. Breakthrough
colonies are commonly observed within zones of inhibition. Circular precipitin bands are occasionally
seen around disks. Positive inhibition is regarded as a zone of 2 mm or more.
Growth precipitation test
The growth precipitation test detects soluble cytoplasmic and extramembranous antigens released by
growing cultures and allowed to diffuse through solid mycoplasma growth medium towards mycoplasma
antiserum during growth (Krogsgaard-Jensen, 1972). As with the gel precipitin test, there are strong crossreactions within the mycoides cluster. If growth inhibition is performed using MAb WM252, which is specific
for Mccp, both specific inhibition and a growth precipitin line are achieved simultaneously.
Indirect fluorescence antibody test
The direct and indirect fluorescent antibody tests are the most effective of the various serological methods
for identifying most mycoplasmas (Rosendal & Black, 1972). They are simple, rapid, and sensitive, yet
economical in the use of antiserum. Several forms have been described, the most commonly used and
perhaps best being the indirect fluorescent antibody (IFA) test applied to unfixed colonies on agar.
Antiserum against a single strain is sufficient to identify field isolates of that species, and antisera are diluted
before use. Cultures do not have to be cloned, but the test is usually applied only after several passages
have indicated whether the culture contains more than one species and the growth characteristics of the
organism(s) present.
Test procedure
i)
Two agar plates are predried at 37C for 30 minutes. Each one is flooded with a different dilution of test
broth culture, the dilutions being selected according to the vigour of growth of the strain on agar
medium. Alternatively, a drop of undiluted culture is spread over a single 5 cm plate using an L-shaped
glass rod.
ii)
The plates are incubated at 37C until the first evidence of growth is observed. If the IFA test cannot be
performed immediately, the plates can be stored at 4C for up to 4 weeks.
iii)
Several blocks of approximately 0.51 cm2 are excised from areas where colonies are numerous, but
not confluent. The blocks of each agar culture are cut to the same geometric shape to enable
recognition of origin, a different shape being used for each isolate. Several blocks of each isolate are
distributed (colony surface facing upwards) on to several different slides, each slide being used for a
different mycoplasma antiserum. The colony surface of each block is identified for future reference by
undercutting one corner.
iv)
Rabbit anti-mycoplasma (ra-m) serum or normal rabbit serum (NRS; as a control on a duplicate block)
at a suitable dilution in normal saline or phosphate buffered saline (PBS), pH 7.2, is gently pipetted on
to each agar block until the surface area is totally covered. The optimal dilution of ra-m is determined
by chequerboard titration against the fluorescein isothiocyanate (FITC)-conjugated anti-rabbit
immunoglobulin serum (a-r lg-FITC) used.
v)
The flooded blocks are incubated on their slides at room temperature for 30 minutes in a humid
chamber.
vi)
All blocks on one slide are tipped into a 10 ml tube containing approximately 7 ml of PBS.
vii)
The plugged tubes are rotated at 1830 rpm for 10 minutes. The PBS is then decanted and replaced
with fresh PBS, and the tubes are rotated again for 10 minutes.
viii) The PBS is decanted and the blocks are placed colony surface facing upwards on their respective
slides. Excess moisture is blotted off.
Available from Kenya Agricultural Research Institute (KARI), P.O. Box 57811, Nairobi, Kenya.
1000
ix)
All blocks are flooded with a-r lg-FITC at optimal dilution.
x)
The blocks are incubated again for 30 minutes at room temperature in a humid chamber, then tipped
into tubes containing fresh PBS, and washed twice by rotating, as before.
xi)
The blocks, replaced colony surface facing upwards on their respective slides, are examined by an epiimmunofluorescent microscope using the settings recommended by the manufacturer for FITC.
Notes on the indirect fluorescent antibody test
xii)
Working dilutions of ra-m and a-r lg-FITC should be kept at 4C, which limits their shelf life to
approximately 1 week.
xiii) Isolates from CCPP should be examined using antisera against Mccp, MmmLC, Mmc (M. mycoides
subsp. capri) and Mcc, positive control cultures should comprise their type strains, namely Mccp, Y
goat, PG3 and California kid, respectively.
xiv) A negative (NRS-treated) control should always be incorporated for each culture.
xv)
f)
Interpretation of the IFA test can be difficult. Autofluorescence is produced by some species,
particularly acholeplasmas. Even in pure cultures, a proportion of colonies may not stain positively with
the relevant antiserum; this is particularly true of Mcc. Otherwise, poor results are usually ascribable to
the use of an agar culture that has been allowed to grow for too long, or to the use of antiserum that
has deteriorated with dilution and age.
Other identification tests

Metabolism inhibition (Taylor-Robinson et al., 1966) and tetrazolium reduction inhibition (Senterfit & Jensen,
1996) are other tests sometimes used in the identification of caprine mycoplasmas. A gene probe, F38-12,
capable of distinguishing Mccp has been developed (Taylor et al., 1992).
A polysaccharide-specific antigen detection latex agglutination test has been developed to detect CCPP
antigen (March et al., 2000). In this test, latex beads are coated with polyclonal immunoglobulin IgG (rabbit)
directed against Mccp polysaccharide and used to detect the antigen in the serum of goats with CCPP. This
test is proposed to be inexpensive, easy to carry out in the field and useful for detecting CCPP in its earliest
stages.
2.
Serological tests
Serology has not been widely applied to identifying the cause of outbreaks of pleuropneumonia in goats and
sheep. Endemic infections with MmmLC and Mmc can produce a background of positive titres to these organisms
in a proportion of apparently healthy animals (Jones & Wood, 1988), and under experimental conditions
seroconversion to M. mycoides can occur in goats with no clinical signs of disease. Acute cases caused by Mccp
rarely show positive titres to the organism before death (MacOwan & Minette, 1977b; Muthomi & Rurangirwa,
1983; Thiaucourt & Bolske, 1996), perhaps because antibodies are eclipsed by circulating mycoplasma antigens
(Muthomi & Rurangirwa, 1983). Seroconversion to Mccp in experimentally infected animals is observed, by the
complement fixation (CF) test and indirect haemagglutination (IHA) test, to start 79 days after the appearance of
clinical signs, to peak between days 22 and 30, and to decline rapidly thereafter (Muthomi & Rurangirwa, 1983).
These various observations indicate that serology should be applied on a herd, not an individual basis, and that
whenever possible, paired serum samples collected 38 weeks apart, should be examined.
a)
Complement fixation test (the prescribed test for international trade) (MacOwan & Minette, 1976)
The CF test in various forms remains the most widely used serological test for diagnosis of contagious
bovine pleuropneumonia (Gourlay, 1983; Perreau et al., 1976). In CCPP, the CF test was used for detection
of Mccp infection (MacOwan & Minette, 1976) and it has been found to be more specific, though less
sensitive, than the IHA test (Muthomi & Rurangirwa, 1983). Its main disadvantage is the high level of
technical expertise required to perform the test (Gourlay, 1983).
One method of performing the test is as follows. To prepare the antigen, 2 litres of culture of titre higher than
109 CFU/ml is centrifuged at 40,000 g for 1 hour at 5C. The deposit is resuspended and washed three
times in physiological saline prior to storage in 0.51.0 ml volumes at 20C.
Sterile broth treated as above constitutes sediment antigen, and a freeze-dried broth reconstituted at
200 mg/ml constitutes a second control antigen. Prior to testing, the antigen is diluted 1/60 and
ultrasonicated for 3 minutes at low power in a container of iced water. The sonicate is centrifuged at 1250 g
for 30 minutes to remove any debris, and stored at 20C. If stored for more than 23 weeks the antigen
should be recentrifuged.
1001
Test procedure
Microtitre plate tests are performed using 0.025 ml volumes, two volumes containing three mean haemolytic
doses of complement, and a 1.5% (v/v) final concentration of sheep red blood cells (SRBCs) in U-bottomed
microtitre plates as follows:
i)
The following are mixed and incubated at 37C for 45 minutes:
25 l of doubling dilutions of test serum (heat inactivated at 56C for 30 minutes) starting with
dilution;
25 l of antigen (the dilution of the antigen must be determined in a chequerboard titration using a
known positive serum);
25 l of complement (3 haemolytic units).
ii)
25 l of sensitised SRBCs, at a final concentration of 1.5% (v/v), is mixed and the plates are incubated
at 37C for 45 minutes.
iii)
The plates are incubated at 4C for 1 hour to allow the unlysed cells to settle.
iv)
Reading the results: The titre will be the highest serum dilution that will fix 50% of the complement, i.e.
50% haemolysis.
Controls
In all CF tests a number of controls are required:
b)
i)
Indicator systems (RBCs + haemolysin) alone to ensure that RBCs do not lyse spontaneously.
ii)
Indicator system with complement only to show that enough complement is present to lyse the cells.
iii)
Indicator system with antigen only and no complement to show that antigen alone does not lyse the
cells.
iv)
Indicator system with serum alone and no complement to show that the serum alone does not lyse the
cells.
v)
Indicator system with complement and antigen to detect any anticomplementary activity of the antigen.
vi)
Indicator system with the complement and serum to detect any anticomplementary activity of the
serum.
Latex agglutination test

Latex beads sensitised with the polysaccharide produced by Mccp and present in culture supernatant have
been used in a slide agglutination test (Houshaymi et al., 2002; Rurangirwa et al., 1987a). This test is
presently used routinely in Kenya. It is a very useful test in an outbreak because it can be performed at the
penside using a drop of whole blood.
Both CF test and IHA test findings emphasise the difficulties inherent in the serological diagnosis of CCPP
when using whole cell or membrane preparations as antigen. The use of the more defined antigen, the
polysaccharide elaborated by Mccp, provides greater specificity, as there is no cross-reactivity with sera
against the other three principal caprine mycoplasmas.
c)

A competition ELISA has been developed (Thiaucourt et al., 1994) and proved both specific and sensitive.
However due to erratic and low demand the production of this test has been stopped until a new format is
developed to ensure longer stability of reagents.

The first experimental vaccine against Mccp was a live high passage Mccp (MacOwan & Minette, 1978). When
inoculated intratracheally, it proved innocuous and protected goats against experimental challenge. However,
more recent work has concentrated on inactivated forms of vaccine. The current form used in Kenya (where
inactivated Mccp, vaccines have been in use for several years) contains inactivated Mccp suspended in saponin,
has been described as having a shelf life of at least 14 months and the optimal dose of 0.15 mg provides
protection for over 1 year (Rurangirwa et al., 1987b).
1002
1.
Seed management
a)

The master seed was isolated from the lungs of a sick goat. In Kenya the strain used is called Yatta and it
has been confirmed to be Mccp by GIT and PCR, after 15 passages in culture. The master seed should also
be pure and free from other contaminants.
b)
Method of culture
The master seed is established and stored in a freeze-dried form in 1 ml ampoules. The working seed is
prepared by amplifying the master seed in modified Newings broth to make a bulk of 4 litres. Its growth is
arrested at the growth phase before filament formation.
This culture is tested for sterility before it is distributed in aliquots of 20 ml volumes and stored frozen at
20C.
c)
The master seed should be prepared from lung specimens or pleural fluids of a goat that dies of pneumonia,
showing all the clinical signs of CCPP. The isolate must be confirmed by GIT or by PCR to be Mccp. It must
be checked for sterility, safety, potency and extraneous agents.
2.
For vaccine production, a working seed is first established by amplifying an aliquot of freeze-dried master seed in
modified Newings broth (Kibor & Waiyaki, 1986), to make a bulk of 4 litres of culture. This culture is aliquoted in
20 ml volumes and frozen at 20C. The vaccine is grown in 5 litre pots containing 4 litres of modified Newings
broth. Each pot is sampled aseptically for sterility testing before 20 ml of working seed is inoculated into each
4 litres of medium. These pots are incubated at 37C for 46 days depending on how fast the mycoplasma grows.
After the filaments sediment, the antigen is harvested by centrifugation at 2600 g from pots that are not
contaminated. (The filaments are thin and white and sometimes unite to form a resemblance of an inverted pine
tree. This occurs on days 46 and it is at this point that they become heavy and sediment.) The pellet is
resuspended in sterile normal saline and centrifuged to remove the remnants of the growth medium. The pellet is
resuspended again in a small volume of sterile normal saline to make a viscous suspension.
Saponin is added to inactivate the mycoplasma at 3 mg saponin to 1 ml of antigen. It is left agitating overnight at
4C using a magnetic stirrer.
NOTE: Saponin also acts as an adjuvant. Three samples are taken aseptically from this suspension and tested
for sterility, for protein estimation and for the innocuity test.
3.
In-process control
During production, the following tests are carried out to ensure that the product remains pure and safe. These
tests are carried out by quality assurance staff. Each pot is sampled aseptically before inoculation to test for
sterility.
After maturity of culture, only pots that have not shown signs of contamination are pooled for centrifugation; the
contaminated pots are decontaminated and discarded after autoclaving.
After inactivation with saponin, a sample is taken aseptically for sterility, another one for innocuity and another
one for the protein estimation test.
a)
Sterility test
This test is aimed at verifying the absence of fungal and bacterial contaminants. Two tubes of thioglycollate
are inoculated with 1 ml of the sample each. The tubes contain about 15 ml of broth. These are incubated at
37C to eliminate aerobics, microaerophilics and anaerobics. The other medium used is soybean casein
digest broth. Four tubes are inoculated with 12 ml of sample each.
Two tubes are incubated at 37C and another two at room temperature (25C) to eliminate bacterial and
fungal contaminants. All media are incubated for 14 days with controls. Absence of any growth shows that
the sample is not contaminated.
1003
b)
Innocuity test
This test aims to demonstrate the absence of living mycoplasma in the vaccine: 0.3 ml of inactivated antigen
is added to 2.7 ml of modified Newings broth in a tube and tenfold dilutions are made from 101 to 109. A
positive control is set up using a viable culture of M. capripneumoniae and a negative control using three
uninoculated tubes of medium. All are incubated at 37C for 12 days. If there is no growth in the tubes
inoculated with test sample, the vaccine passes the test.
c)
Protein content estimation

The protein content is measured by the bicinchoninic acid method by comparison with a bovine albumin
standard.
4.
Batch control
a)
Sterility
The sterility test on the final batch is carried out as described in Section C.3.a except that four bottles of
each batch are pooled together and samples of the pool are used.
b)
Safety
Every batch of CCPP vaccine must be proven to be safe in laboratory animals. Two guinea-pigs are each
inoculated by the intramuscular route in the hind leg and another two guinea-pigs are inoculated by the
peritoneal route with 0.5 ml containing five doses of vaccine.
If the vaccine is safe, the guinea-pigs should not show any sign of disease for a 14-day observation period
and, on post-mortem, there should be no abscesses on the site of inoculation and in the peritoneal cavity,
respectively. If any vaccine-related deaths occur during the observation period, the vaccine fails. If on postmortem examination, abscesses are observed at the site of inoculation and the peritoneum, the vaccine also
fails.
c)
Potency
Research is in progress at the Kenya Agricultural Research Institute, Muguga, Kenya, to develop a test for
potency.
d)
The vaccine protects goats for 14 months. It is, however, recommended to boost immunity after 1 year.
e)
Stability
If stored at 4C, the vaccine has a shell life of 1 year. Before use the vaccine should be shaken thoroughly,
for even distribution of antigen.
f)
Preservatives
At present preservatives are not used in the vaccine.
g)
Precautions
Side-effects of the CCPP vaccine include development of a swelling at the site of inoculation and fever for
12 days following vaccination, accompanied with inappetance. The swelling may last between 1 and
14 days and is due to the saponin adjuvant. Accidental self-injection causes severe irritation.
5.
a)
Safety
Every batch of vaccine should be tested for safety in laboratory animals as described in C.4.b.
b)
Potency
Once the potency test has been developed, every batch of vaccine will be required to be tested for potency.
1004
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*
* *
NB: There is an OIE Reference Laboratory for Contagious caprine pleuropneumonia
diagnostic tests, reagents and vaccines for contagious caprine pleuropneumonia
1007
CHAPTER 2.7.7.
ENZOOTIC ABORTION OF EWES

(ovine chlamydiosis)
SUMMARY
Ovine chlamydiosis, also known as enzootic abortion of ewes (EAE) or ovine enzootic abortion
(OEA), is caused by the bacterium Chlamydophila abortus. Chlamydial abortion typically occurs in
the last 23 weeks of pregnancy with the appearance of stillborn lambs and inflamed placentas.
However, infection can also result in the delivery of full-term stillborn lambs or weak lambs that do
not survive longer than 48 hours. Infected ewes can also give birth to healthy lambs and it is not
uncommon to observe delivery of a dead and a weak or healthy lamb. There are rarely any
predictive signs that abortion is going to occur, although behavioural changes and a vulval
discharge can be observed in the last 48 hours of pregnancy.
Diagnosis of enzootic abortion depends on the isolation and identification of the causative agent or
detection of the agent or its nucleic acid in the products of abortion or vaginal excretions of freshly
aborted females. A humoral antibody response may be detected following abortion. Goats as well
as sheep and, less commonly, cattle, pigs, horses and deer, can be affected. Chlamydiosis of small
ruminants is a zoonosis and the organism must be handled with biosafety precautions. Pregnant
women are particularly at risk.
Identification of the agent: The basis for a positive diagnosis of infection with C. abortus depends
on a history of abortion in sheep or goats (often in late pregnancy), evidence of necrotic placentitis,
and the demonstration of large numbers of the organism in stained smears of affected placentae.
The still moist fleece of fetuses or vaginal swabs of females that have freshly aborted are also
useful. Care is needed to distinguish cotyledonary damage caused by Toxoplasma gondii and, in
stained smears, to be aware of the morphological similarities between C. abortus and Coxiella
burnetii, the agent of Q fever.
Chlamydial antigen can be detected by enzyme-linked immunosorbent assay, immunohistochemistry or the fluorescent antibody test, whereas chlamydial DNA can be detected by the
polymerase chain reaction or by microarray. Some of these methods are available in commercial kit
form.
Chlamydophila abortus can be isolated only in living cells; thus facilities for growth in chicken
embryos or cell cultures, with appropriate biohazard containment, are required.
Serological tests: A rise in antibody titre to C. abortus, detected by the complement fixation (CF)
test, is common after abortion or stillbirth, but this does not occur in every case. Chlamydophila
abortus shares common antigens with C. pecorum and some Gram-negative bacteria, so that the
CF test is not wholly specific, nor does it distinguish between responses to vaccination and to
infection. Low CF titres need to be interpreted with caution, particularly if these are encountered in
individual animals or in flocks with no history of abortion.
Alternative serological tests have been developed and some commercialised, but none has been
sufficiently appraised so far for field use. A delayed hypersensitivity reaction to chlamydial antigen
can be elicited in infected sheep, but the procedure is not amenable to routine use.
Requirements for vaccines: Inactivated and live vaccines are available that have been reported to
prevent abortion and to reduce excretion. They assist in control of the disease but will not eradicate
it. Serological screening during the period after parturition helps to identify infected flocks, to which
control measures can then be applied.
1008
Chapter 2.7.7. Enzootic abortion of ewes (ovine chlamydiosis)
A. INTRODUCTION
Ovine chlamydiosis (enzootic abortion of ewes [EAE] or ovine enzootic abortion [OEA]) is caused by the
bacterium Chlamydophila abortus. Chlamydial abortion in late pregnancy causes serious reproductive wastage in
many sheep-rearing areas of the world, particularly where flocks are closely congregated during the parturient
period (Aitken & Longbottom, 2007; Longbottom & Coulter, 2003). Abortion typically occurs in the last 23 weeks
of pregnancy with the appearance of stillborn lambs and grossly inflamed placentas. Infection can also result in
the delivery of full-term stillborn lambs and weak lambs that generally fail to survive beyond 48 hours. It is also not
uncommon in multiple births for an infected ewe to produce one dead lamb and one or more weak or healthy
lambs. Infection is generally established in a clean flock through the introduction of infected replacements and
results in a small number of abortions in the first year, which is followed by an abortion storm in the second year
that can affect up to around 30% of ewes.
Infected animals show no clinical illness prior to abortion, although behavioural changes and a vulval discharge
may be observed in ewes within the last 48 hours of pregnancy. Pathogenesis commences around day 90 of
gestation coincident with a phase of rapid fetal growth when chlamydial invasion of placentomes produces a
progressively diffuse inflammatory response, thrombotic vasculitis and tissue necrosis. Milder changes occur in
the fetal liver and lung and, in cases in which placental damage is severe, there may be evidence of hypoxic brain
damage (Buxton et al., 2002). Abortion probably results from a combination of impairment of materno-fetal
nutrient and gaseous exchange, disruption of hormonal regulation of pregnancy and induced cytokine aggression
(Entrican, 2002).
Chlamydial abortion also occurs in goats and, less frequently, cattle, pigs, horses and deer may be affected. In
sheep, abortion in late pregnancy with expulsion of necrotic fetal membranes are key diagnostic indicators, with
care being needed to distinguish the diffuse pattern of necrosis from that caused by Toxoplasma gondii
(cotyledons only). Distinction from other infectious causes of abortion such as brucellosis (see chapter 2.7.2),
coxiellosis (see chapter 2.1.12) or other bacterial pathogens (Campylobacter [see chapter 2.9.3], Listeria [see
chapter 2.9.7], Salmonella [see chapter 2.9.9]) can be achieved by microscopy and/or culture.
Taxonomically, the family Chlamydiaceae has been divided into two genera and nine species based on sequence
analysis of the 16s and 23s rRNA genes (Everett et al., 1999). The genus Chlamydia includes C. trachomatis
(humans), C. suis (swine) and C. muridarum (mouse and hamster). The genus Chlamydophila includes C. psittaci
(avian), C.felis (cat), C. abortus (sheep, goat and cattle), C. caviae (guinea-pig), the former species C. pecorum
(sheep and cattle) and C. pneumoniae (humans). A proposal has been mooted to recombine all the species in a
single genus (Chlamydia) but has not been adopted for the purposes of this chapter. The terms chlamydiosis
and chlamydia(e) are used to refer to members of the genus Chlamydia in general. However, a binomial of the
generic and specific names is used when referring to a particular chlamydial species. Infected females shed vast
numbers of infective C. abortus at the time of abortion or parturition, particularly in the placenta and uterine
discharges and at subsequent lambing (Papp et al., 1994), thus providing an infection source in the flock. Aborted
ewes do not usually abort again from C. abortus infection. Recent evidence suggests that the proportion of
infected ewes is reduced at the subsequent breeding season and only low levels of chlamydial DNA are detected
during the periovulation period and at lambing, so that this would not have significant impact on the epidemiology
(Livingstone et al., 2009; Gutierrez et al., 2011).
Zoonotic risk and biosafety requirements
Human infection may be acquired from infected products of abortion or parturition or from carelessly handled
laboratory cultures of the organism, with effects that range from subclinical infection to acute influenza-like illness.
Appropriate precautions should be taken and containment level 2 practices are recommended when handling
cultures and potentially infected tissues (see Chapter 1.1.3 Biosafety and biosecurity in the veterinary
microbiology laboratory and animal facilities). Authenticated cases of human placentitis and abortion caused by
C. abortus of ovine origin indicate that pregnant women are at special risk and should not be exposed to sources
of infection (Longbottom & Coulter, 2003; Sillis & Longbottom, 2011).
1.
a)
Smears
Where the clinical history of the flock and the character of lesions in aborted placentae suggest enzootic
abortion, a diagnosis can be attempted by microscopic examination of smears made from affected chorionic
villi or adjacent chorion. Several staining procedures are satisfactory, for example, modified Machiavello,
Giemsa, Brucella differential, or modified ZiehlNeelsen stains (Stamp et al., 1950). In positive cases
1009
stained by the latter method and examined under a high-power microscope, large numbers of small
(300 nm) coccoid elementary bodies are seen singly or in clumps stained red against the blue background of
cellular debris. Under dark-ground illumination, the elementary bodies are pale green. If placental material is
not available, smears may be made from vaginal swabs of females that have aborted within the previous
24 hours, or from the moist fleece of a freshly aborted or stillborn lamb that has not been cleaned by its
mother, or from the abomasal content of the aborted or stillborn lamb. In general, such preparations contain
fewer organisms than placental smears.
In terms of morphology and staining characteristics, C. abortus resembles the rickettsia Coxiella burnetii,
which, in some circumstances, may provoke abortion and which, in humans, causes Q fever. Care must be
taken to differentiate between these two organisms in cases lacking a good history or evidence of
chlamydial-induced placental pathology. Antigenic differences between C. abortus and Coxiella burnetii can
be detected serologically. Fluorescent antibody tests (FATs) using a specific antiserum or monoclonal
antibody may be used for identification of C. abortus in smears.
b)
Antigen detection
Several chlamydial genus-level antigen-detection tests are available commercially. A comparative
assessment of several such assays, on non-ovine material, indicated that those using enzyme-linked
immunosorbent assay (ELISA) methodology were more sensitive than kits employing a FAT (Wood &
Timms, 1992). Under the test conditions used, a kit that detects chlamydial lipopolysaccharide (LPS) was
judged to be the most sensitive of the rapid ELISA-based systems investigated. Though occasionally
yielding false-positive results, particularly with avian faecal samples, the kit also gave satisfactory results
with ovine placental samples (Wilsmore & Davidson, 1991) although it should be noted that it does not
differentiate between C. abortus and other chlamydial species that may contaminate the samples. In
histopathological sections, antigen detection can be performed using commercially available anti-Chlamydia
antibodies directed against LPS or MOMP (major outer membrane protein) (Borel et al., 2006).
c)
DNA
Amplification of chlamydial DNA by polymerase chain reaction (PCR) and real-time PCR provide alternative
approaches for verifying the presence of chlamydiae in biological samples without resorting to culture. PCR
is highly sensitive for this purpose, but has the attendant risk of cross-contamination between samples or
environmental contamination of samples in the field, so appropriate measures must be taken to avoid this
happening (see Guideline 3.2 Biotechnology in the diagnosis of infectious diseases). Another potential
problem is in the production of false negatives resulting from PCR-inhibitory substances in the samples.
Methods for discriminating between amplified DNA sequences originating from C. abortus and C. pecorum
have been described (DeGraves et al., 2003; Everett & Andersen, 1999; Jee et al., 2004; Laroucau et al.,
2001; Thiele et al., 1992). In the last few years, real-time PCR has become the preferred method in
diagnostic laboratories for its rapidity, high throughput and ease of standardisation (Sachse et al., 2009).
Recently, DNA microarray hybridisation assays using the ArrayTube platform have been developed and
hold much promise for the direct detection and identification of organisms from clinical samples (Borel et al.,
2008; Sachse et al., 2005). PCR assays in combination with restriction fragment length polymorphism
analysis have been developed with potential to differentiate naturally infected from vaccinated animals
(DIVA) (Laroucau et al., 2010; Wheelhouse et al., 2010).
d)
Tissue sections
Intracellular chlamydial inclusions can be demonstrated by Giemsa staining of thin (4 m) sections taken
from target tissues that have been suitably fixed in fluids such as Bouin or Carnoy. More striking results can
be obtained by immunological staining procedures. The direct immunoperoxidase method (Finlayson et al.,
1985) is rapid and simple, while the method with streptavidinbiotin is more complex (Szeredi & Bacsadi,
2002). Electron microscopy can also be performed using negative contrast, to differentiate chlamydiae from
Coxiella burnetii.
e)
Isolation of the agent

Chlamydophila abortus can be isolated in embryonated chicken eggs or in cell culture, the latter being the
method of choice for isolation of new strains. The causative agent of chlamydiosis is zoonotic and thus
isolation and identification procedures should be carried out under biosafety level 2 conditions.
Tissue samples, such as diseased cotyledons, placental membranes, fetal lung or liver, or vaginal swabs,
that may be subject to any delay before isolation procedures begin, should be maintained in a suitable
transport medium in the interim period. For optimal recovery such samples should be stored frozen,
preferably at 80C, or otherwise at 20C. The most satisfactory medium is sucrose/phosphate/glutamate
or SPG medium (sucrose [74.6 g/litre], KH2PO4 [0.512 g/litre], K2HPO4 [1.237 g/litre], L-glutamic acid
[0.721 g/litre]) supplemented with 10% fetal bovine serum, antibiotic (streptomycin and gentamycin are
1010
suitable, but not penicillin), and a fungal inhibitor (Spencer & Johnson, 1983). A tissue:medium ratio of 1:10
is commonly employed. Alternatively, approximately 1 g of tissue is ground with sterile sand in 8 ml of
transport medium.
Chicken embryos: Test samples are prepared as 10% suspensions in nutrient broth containing streptomycin
(not penicillin) (200 g/ml); 0.2 ml of suspension is inoculated into the yolk sac of 68-day old embryos,
which are then further incubated at 37C. Infected embryos die between 4 and 13 days after inoculation.
Smears prepared from their vascularised yolk sac membranes reveal large numbers of elementary bodies.
Cell cultures: Chlamydophila abortus of ovine origin can be isolated in a variety of cell types, but McCoy,
Buffalo Green Monkey (BGM) or baby hamster kidney (BHK) cells are most commonly used. For
confirmatory diagnosis, cultured cell monolayers are suspended in growth medium at a concentration of 2
105 cells/ml. Aliquots of 2 ml of the suspension are dispensed into flat-bottomed glass Universal bottles,
each containing a single 16 mm cover-slip. Confluent cover-slip monolayers are achieved after incubation for
24 hours at 37C. The growth medium is removed and replaced by 2 ml of test inoculum, which is then
centrifuged at 2500 g for 30 minutes on to the cover-slip monolayer to promote infection. After further
incubation for 23 days, the cover-slip monolayers are fixed in methanol and stained with Giemsa or
according to the method of Gimenez (Arens & Weingarten, 1981; Gimenez, 1964). After methanol fixation,
infected cultures contain basophilic (Giemsa) or eosinophilic (Gimenez) intracytoplasmic inclusions. Similar
procedures are used in culturing C. abortus for antigen preparation. FAT techniques can also be used and
are equally effective.
Chlamydial activity can be further enhanced by chemical treatment of cultured cells, before or during
infection, to favour chlamydial growth. Various substances that have been described for incorporation into
the infective inoculum to which cover-slip monolayers are exposed include: cycloheximide (0.5 g/ml) in the
maintenance medium, emetine (1 g/ml) for 5 minutes before infection, and 5-iodo-2-deoxyuridine
(80 g/ml) for 3 days prior to infection. Unless preconditioned cells are available, the latter isolation
procedure requires increased time for successful agent isolation.
2.
Serological tests
a)

Complement fixation (CF) is the most widely used procedure for detecting infection (sheep and goats are
generally tested within 3 months of abortion or parturition). The test will also detect evidence of vaccination.
Infection is evident principally during active placental infection in the last month of gestation and following
the bacteraemia that often accompanies abortion. Consequently, paired sera collected at the time of
abortion and again at least 3 weeks later may reveal a rising CF antibody titre that will provide a basis for a
retrospective diagnosis. Antigenic cross-reactivity between C. abortus and C. pecorum, as well as with some
Gram-negative bacteria (e.g. Acinetobacter), can give rise to low false-positive CF test results. Thus, titres
less than 1/32 in individual animals should be considered to be nonspecific for C. abortus, although they
could also be due to a low grade infection with C. abortus. Ambiguous results can be investigated further by
western blot analysis using purified elementary bodies (Jones et al., 1997).
Antigen is prepared from heavily infected yolk sac membranes obtained from chicken embryos that have
been inoculated in the same manner as those used to isolate the organism from field material. The
preparation of the antigen should be carried out in a biosafety cabinet with the appropriate biosecurity
precautions to prevent human infection (see chapter 1.1.3). Chopped and ground membranes are
suspended in phosphate buffer, pH 7.6, at the rate of 2 ml per g membrane. After removal of crude debris,
the supernatant fluid is centrifuged at 10,000 g for 1 hour at 4C, the deposit is resuspended in a small
volume of saline, and a smear of this is examined to ensure a high yield of chlamydiae. The suspension is
held in a boiling water bath for 20 minutes, or is autoclaved, and sodium azide (0.3%) is added as a
preservative. Antigen may also be prepared from cell cultures infected with C. abortus. Infected monolayers
are suspended in phosphate buffer, pH 7.6, and the cells are disrupted by homogenisation or
ultrasonication. Gross debris is removed and subsequent procedures are as for the preparation of antigen
from infected yolk sacs. In either case, CF tests with standardised complement and antisera will establish
the optimal working dilution for each batch of antigen.
b)
Other tests
The serological responses to C. abortus and C. pecorum can be resolved by indirect micro-immunofluorescence, but the procedure is too time-consuming for routine diagnostic purposes. ELISAs developed
independently by several research groups have not been adapted for general diagnostic work, partly
because of difficulties associated with the use of particulate antigens. However, a novel ELISA that
incorporates a stable, solubilised antigen has been used to test experimental and field samples, and has
given results that, though lacking species specificity, have a higher sensitivity than the CF test (Anderson et
1011
al., 1995; Jones et al., 1997). Other tests using monoclonal antibody technology in a competitive ELISA
(Salti-Montesanto et al., 1997) and recombinant antigen technology in indirect ELISAs (Longbottom et al.,
2002) have been developed and shown to be more sensitive and specific than the CF test in differentiating
animals infected with C. abortus from those infected with C. pecorum. However, these tests are currently
mainly used as research tools, and have not been developed commercially. A number of commercially
available serological tests have been evaluated and compared with these in-house tests with variable
results (Jones et al., 1997; Vretou et al., 2007, Wilson et al., 2009). None of the serological tests that is
available can differentiate vaccination titres from those acquired as a result of natural infection (Borel et al.,
2005).

1.
Background
a)

Currently, two types of vaccine (inactivated and attenuated live vaccines) are available commercially, to be
administered intramuscularly or subcutaneously at least 4 weeks before breeding to aid in the prevention of
abortion. A multi-component recombinant vaccine against C. abortus remains a future goal of chlamydial
vaccine research (Longbottom & Livingstone, 2006).
Inactivated vaccines can be prepared from infected yolk sacs or cell cultures (Jones et al., 1995) and
incorporate whole organisms or fractions of them (Tan et al., 1990) using the appropriate biosecurity
precautions to prevent human infection (see chapter 1.1.3). Operator care should be observed in handling
commercial inactivated vaccines that incorporate mineral oil-based adjuvants, as self-injection can result in
severe local inflammation and tissue necrosis. The commercial live, attenuated vaccine is a chemically
induced temperature-sensitive mutant strain of the organism that grows at 35C but not at 39.5C, the body
temperature of sheep (Rodolakis, 1986). This vaccine is supplied lyophilised and must be reconstituted in
diluent immediately before administration. Operator care should be observed in handling and administering
this live vaccine, particularly by immunocompromised individuals and pregnant women. Importantly, the live
vaccine must not be given to animals being treated with antibiotics, particularly tetracyclines. Inactivated
vaccines are safe for administration during pregnancy, whereas live vaccines cannot be used in pregnant
animals.
Both types of vaccine have a role to play in controlling disease, but neither confers absolute protection
against challenge or completely reduces the shedding of infective organisms. However, vaccinates exposed
to infection do experience significantly lower abortion rates and reduced excretion of chlamydiae for at least
two to three lambings after vaccination. It has been claimed that the live vaccine could be an aid to
eradication of disease (Nietfeld, 2001). In addition, the live vaccine strain 1B has been detected in the
placentas of vaccinated animals that have aborted as a result of OEA, suggesting a possible role for the
vaccine in causing disease (Wheelhouse et al., 2010), but despite this the use of live vaccine remains the
most effective method of protecting from the disease (Stuen & Longbottom, 2011).
Vaccine stored under refrigeration (53C) should remain stable for at least 1 year. No firm data are
available, but revaccination is recommended every 13 years, according to the exposure risk.
2.
a)

i)
One or more ovine abortion isolates that consistently grow productively in the chosen substrate are
suitable, and an early passage of the seed stock can be established. Alternatively, an isolate that has
been adapted to the chicken embryo by multiple passage (>100) can be used. This permits more of the
embryo to be used for vaccine production. Although adaptation to the embryo may diminish the
isolates virulence for sheep, there is no evidence that such change reduces its protective efficacy as
an inactivated vaccine.
ii)

Before inoculation of large numbers of embryos or cell cultures, the viability and freedom from
contamination of seed stock should be verified. It may be convenient to collect the total harvest in
separate manageable lots. In this case, the infectivity of an aliquot of each lot should be separately
titrated to ensure that each matches the requirements (see below). Store under refrigerated conditions.
1012
b)
i)
Procedure
For low passage isolates, the procedures described for the preparation of CF antigen are suitably
adapted and amplified for bulk production. Once the final harvest suspension is obtained, an aliquot is
removed for titration of its infectivity. The bulk is treated with formalin to a final concentration of 0.4%,
and stored until sterility tests confirm complete inactivation.
ii)

The inactivated harvest is centrifuged and resuspended in phosphate buffered saline containing 0.2%
formalin to a volume representing a preinactivation infectivity titre of approximately 108 infectious
units/ml. Usually, the aqueous suspension is blended with an oil adjuvant, either directly or after
precipitation by potassium alum (AlK[SO4]2.12 H2O). A preservative, such as 0.01% thiomersal, may
also be added.
iii)
In-process controls
The main requirements are to ensure adequate growth of C. abortus, avoidance of extraneous infection
of the culture substrate, completeness of inactivation and biohazard awareness by process workers.
iv)

Each separate batch of manufactured vaccine should be tested for sterility, safety and potency.
Safety
Subcutaneous inoculation into two or more seronegative sheep of twice the standard dose (usually
1.0 ml) of manufactured vaccine should elicit no systemic reaction, but oil-adjuvant vaccines can cause
a nonharmful swelling at the inoculation site.
Batch potency
At present, potency is judged by the occurrence of a serological response in previously unvaccinated
sheep given 1 ml of vaccine subcutaneously. Blood samples taken before and 28 days after
vaccination are compared. Ultimately, potency has to be judged against experimental challenge or field
performance, but no in-vitro correlation of protective efficacy has yet been established.
c)

i)
Safety requirements
See chapter 1.1.6 Principles of veterinary vaccine production.
ii)
See chapter 1.1.6 .
iii)
Stability
See chapter 1.1.6.
3.
a)

No biotechnology-based vaccines are currently in use for this disease.
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*
* *
NB: There are OIE Reference Laboratories for Enzootic abortion of ewes
diagnostic tests, reagents and vaccines for Enzootic abortion of ewes
1016
CHAPTER 2.7.8.
NAIROBI SHEEP DISEASE
See Chapter 2.9.1. Bunyaviral diseases of animals (excluding Rift Valley fever)
*
* *
1017
CHAPTER 2.7.9.
OVINE EPIDIDYMITIS
(Brucella ovis)
SUMMARY
Brucella ovis produces a clinical or subclinical disease in sheep that is characterised by genital
lesions in rams, and placentitis in ewes. Accordingly, the main consequences of the disease are
reduced fertility in rams, infrequent abortions in ewes, and an increased perinatal mortality. The
disease has been reported in Latin American, North American and European countries as well as
Australia, New Zealand and South Africa, but probably occurs in most sheep-raising countries.
Identification of the agent: The existence of clinical lesions (unilateral or, occasionally, bilateral
epididymitis) in rams may be indicative of the existence of infection, but laboratory examinations are
necessary to confirm the disease. Laboratory confirmation may be based on direct or indirect
methods. Direct diagnosis is made by means of bacteriological isolation of B. ovis from semen
samples or tissues of rams, or vaginal discharges and milk of ewes, on adequate selective media.
Molecular biological methods have being developed that could be used for complementary
identification based on specific genomic sequences. The polymerase chain reaction (PCR)
methods provide additional means of detection. However, indirect diagnosis based on serological
tests is preferred for routine diagnosis.
Serological tests: The complement fixation test (CFT), agar gel immunodiffusion (AGID) test and
indirect enzyme-Iinked immunosorbent assay (I-ELISA) using soluble surface antigens obtained
from B. ovis, can be used. Some I-ELISAs using recombinant proteins and monoclonal antibodies
are being tested in field trials. The sensitivities of the AGID test and ELISA are similar and
sometimes the I-ELISA has higher sensitivity than the CFT. A combination of the AGID test and IELISA seems to give the best results in terms of sensitivity. However, with regard to simplicity and
cost, the AGID test is the most practicable test for diagnosis of B. ovis. However, because of the
lack of standardised methods recognised at the international level for I-ELISA and AGID, the
prescribed test for international trade remains the CFT.
Requirements for vaccines and diagnostic biologicals: Seed cultures for antigen or vaccine
production should be obtained from internationally recognised laboratories. A single standard dose
(109 colony-forming units) of the live B. melitensis Rev.1 vaccine, administered subcutaneously or
conjunctivally, can be used safely and effectively in rams, for the prevention of B. ovis infection.
This vaccine strain should meet minimal quality standards: adequate concentration, absence of
dissociation, adequate residual virulence and immunogenicity and free of extraneous agents (see
Chapter 2.7.2 Caprine and ovine brucellosis [excluding B. ovis]).
A. INTRODUCTION
Brucella ovis causes a genital infection of ovine livestock manifested by epididymitis, infrequent abortions, and
increased lamb mortality. Passive venereal transmission via the ewe appears to be a frequent route of infection,
but ram-to-ram transmission is also common1 (Blasco, 1990). Infected ewes may excrete B. ovis in vaginal
1
Under the semi-extensive production systems (most common in European Mediterranean countries) rams are usually
housed together. Direct ram-to-ram transmission during non-breeding periods is quite frequent and has been suggested to take
place by several routes, including the rectal mucosa. Most ram-to-ram infections, however, are produced through the oral route.
Housed rams establish hierarchies (head-to-head combats), and it is frequent that dominated rams, after being mated by the
1018
Chapter 2.7.9. Ovine epididymitis (Brucella ovis)
discharges and milk and, accordingly, ewe-to-ram and lactating ewe-to-lamb transmission could also be
determinant mechanisms of infection. Accordingly, the ewes are as relevant as rams in the epidemiology of
infection, and control or eradication of B. ovis is feasible only if females are included in the corresponding
programme.
The demonstration of the existence of genital lesions (unilateral or, occasionally, bilateral epididymitis) by
palpating the testicles of rams may be indicative of the presence of this infection in a given flock. However, this
clinical diagnosis is not sensitive enough because only about 50% of rams infected with B. ovis present
epididymitis (Blasco, 1990). Moreover, the clinical diagnosis is extremely nonspecific due to the existence of
many other bacteria causing clinical epididymitis. The most frequently reported isolates causing epididymitis in
rams include Actinobacillus seminis, A. actinomycetemcomitans, Histophilus ovis, Haemophilus spp.,
Corynebacterium pseudotuberculosis ovis, B. melitensis and Chlamydophila abortus (formerly Chlamydia psittaci)
(Bulgin & Anderson, 1983; Burgess & McDowell, 1981; De Long et al., 1979; Ekdahl M.O., Money & Martin, 1968;
Livingstone & Hardy, 1964; Rodolakis & Bernard, 1977; Webb, 1983; Williamson & Nairn, 1980). It must be
emphasised that many palpable epididymal lesions in rams are sterile, trauma-induced spermatic granulomas.
Although cattle, goats and deer have been proved susceptible to B. ovis in artificial transmission experiments,
natural cases have been reported only in deer (Ridler, 2001). To date, no human cases have been reported, and
B. ovis is considered to be non-zoonotic. However, in areas where B. melitensis infection co-exists with B. ovis,
special care is required when handling samples, which should be transported to the laboratory in leak-proof
containers (for further details see Chapter 2.4.3 Bovine brucellosis).
biovars are given in the chapter 2.4.3.
1.
a)
The most valuable samples for the isolation of B. ovis from live animals are semen, vaginal swabs and milk.
For the collection of vaginal swabs and milk, see the instructions given in Chapter 2.7.2 Caprine and ovine
brucellosis (excluding B. ovis). Semen (genital fluids) can be collected easily in swabs taken from the
preputial cavity after electro-ejaculation. lf an electro-ejaculator is not available, swabs can be taken from the
vagina of brucellosis-free ewes immediately after natural mating.
For the isolation of B. ovis after necropsy, the preferred organs in terms of probability of isolation are the
epididymides, seminal vesicles, ampullae, and inguinal lymph nodes in rams, and the uterus, iliac and supramammary lymph nodes in ewes. However, to obtain maximum sensitivity, a complete search that includes
other organs and lymph nodes (spleen, cranial, scapular, prefemoral and testicular lymph nodes) should be
performed. Dead lambs and placentas should also be examined. The preferred culture sites in aborted or
stillborn lambs are abomasal content and lung.
Samples for culture should be refrigerated and transported to the laboratory to be cultured as soon as
possible after collection. The organism remains viable for at least 72 hours at room temperature and survival
is enhanced at 4C or, preferably, by freezing the tissue samples.
b)
Staining methods
Semen or vaginal smears can be examined following staining by Stamps method (Alton et al., 1988; Corbel
et al., 1978) (see chapter 2.7.2), and characteristic coccobacilli should be demonstrated in many infected
animals (Webb et al., 1980). Examination of Stamp-stained smears of suspect tissues (ram genital tract,
inguinal lymph nodes, placentas, and abomasal content and lung of fetuses) may also allow a rapid
presumptive diagnosis.
However, other bacteria with similar morphology or staining characteristics (B. melitensis, Coxiella burnetii,
and Chlamydophila abortus) can also be present in such samples, making the diagnosis difficult for
inexperienced personnel. Microscopy results should always be confirmed by culture of the microorganism.
dominant rams, lick the prepuce of these dominant rams as an act of submission. If these dominant rams are infected, the
probability of having B. ovis in the prepuce (excretion in the semen) is very high.
1019
c)
Culture
The best direct method of diagnosis is bacteriological isolation on adequate culture media. Semen, vaginal
swabs, or milk samples can be smeared directly on to plates with adequate culture media and incubated at
37C in an atmosphere of 510% CO2. Tissues should be macerated and ground in a small amount of
sterile saline or phosphate buffered saline (PBS) with a stomacher or blender, before plating.
Growth normally appears after 34 days, but cultures should not be discarded as negative until 7 days have
elapsed. Colonies of B. ovis become visible (0.52.5 mm) after 34 days of incubation, and are rough
phase, round, shiny and convex.
Brucella ovis can be isolated in nonselective media, such as blood agar base enriched with 10% sterile
ovine or bovine sera, or in blood agar medium with 510% sterile ovine blood. However, the inoculum
frequently contains other bacteria, which often overgrow B. ovis. Accordingly, the use of selective media
may be preferred. Various B. ovis selective media have been described. The modified ThayerMartins
medium (Brown et al., 1971; Marin et al., 1996) is recommended. Briefly, it can be prepared with GC
medium base (38 g/litre; Biolife Laboratories, Milan, Italy) supplemented with haemoglobin (10 g/litre; Difco)
and colistin methane-sulphonate (7.5 mg/litre), vancomycin (3 mg/litre), nitrofurantoin (10 mg/litre), nystatin
(100,000 International Units [IU]/litre = 17.7 mg) and amphotericin B (2.5 mg/litre) (all products from Sigma
Chemical, St Louis, United States of America [USA]). Working solutions are prepared as follows:
Solution A: Add 500 ml of distilled water to the GC medium base, heat the paste carefully while stirring
continuously and autoclave at 120C for 20 minutes.
Solution B: Suspend the haemoglobin in 500 ml of distilled water, adding the water slowly to avoid lumps.
Once dissolved, add a magnetic stirrer and autoclave at 120C for 20 minutes.
Antibiotic solution (prepared daily): colistin, nystatin and vancomycin are suspended in a mixture of
methanol/water (1/1); nitrofurantoin is suspended in 1 ml of a 0.1 M NaOH sterile solution. For amphotericin
B, it is recommended to prepare a stock solution of 10 mg/ml amphotericin B with 10 mg dissolved first in
1 ml sterile dimethyl sulphoxide (C2H6OS, for analysis; ACS) and then added to 9 ml of PBS (10 mM,
pH 7.2). Any stock solution remaining can be stored some days at 4C. All antibiotic solutions must be
filtrated through 0.22 m filters before addition to the culture medium.
Once autoclaved, stabilise the temperature (4550C) of both solutions A and B with continuous stirring. Mix
both solutions (adding A to B), avoiding bubble formation. Add the antibiotic solutions while stirring
continuously and carefully. Dispense into sterile plates.
Once prepared, the plates should not be stored for long periods, and freshly prepared medium is always
recommended. This medium is also suitable for the isolation of B. melitensis (see chapter 2.7.2).
All culture media should be subjected to quality control with the reference strain, to show that it supports
growth.
Another suitable, but less effective, antibiotic combination is: vancomycin (3 mg/litre); colistin (7.5 mg/litre);
nystatin (12,500 IU/litre); and nitrofurantoin (10 mg/litre).
The Farrells medium described for the culture of smooth brucellae is not appropriate for the culture of
B. ovis as it does not grow on this medium.
d)
Identification and typing

Brucella ovis colonies are not haemolytic. They are circular, convex, have unbroken edges, are always of
the rough type when examined by oblique illumination, and test positive in the acriflavine test (Alton et al.,
1988; Corbel et al., 1978). For growth, B. ovis needs an atmosphere of 510% CO2. It lacks urease activity,
fails to reduce nitrate to nitrite, is catalase positive and oxidase negative. It does not produce H2S and,
although it does not grow in the presence of methyl violet, it usually grows in the presence of standard
concentrations of basic fuchsin and thionin. The cultures are not lysed by Brucella-phages of the Tbilissi
(Tb), Weybridge (Wb) and Izatnagar (Iz) groups at the routine test dilution (RTD) or 104 RTD, while they are
lysed by phage R/C (Alton et al., 1988; 7). Most laboratories are not equipped for a complete identification,
and a practical schedule for presumptive identification is needed. Most B. ovis isolates can be correctly
identified on the basis of growth characteristics, direct observation using obliquely reflected light, Gram or
Stamps staining, catalase, oxidase, urease and acriflavine tests. However, definitive identification should be
carried out by reference laboratories with experience in identification and typing of Brucella.
1020
The polymerase chain reaction (PCR) and other recently developed molecular methods provide additional
means of detection and identification of Brucella sp. (see chapter 2.4.3).
2.
Serological tests
The most efficient and widely used tests are the complement fixation test (CFT), the double agar gel
immunodiffusion (AGID) test and the indirect enzyme-Iinked immunosorbent assay (I-ELISA). Several countries
have adopted various standard diagnostic techniques for B. ovis, but the only test prescribed by the OIE and the
European Union (EU) for international trade is the CFT. However, it has been demonstrated that the AGID test
shows similar sensitivity to the CFT, and it is a simpler test to perform. Although standardisation is lacking,
numerous independent studies have shown that the I-ELISA is more sensitive and specific than either the CFT or
AGID test, and with further validation and standardisation studies, the I-ELISA could become a suitable candidate
for future designation as a prescribed test for B. ovis diagnosis.
The International Standard anti-Brucella ovis Serum (International Standard 19852) is the one against which all
other standards are compared and calibrated. This reference standard is available to national reference
laboratories and should be used to establish secondary or national standards against which working standards
can be prepared and used in the diagnostic laboratory for daily routine use.
Antigens
When rough Brucella cells are heat-extracted with saline (hot-saline method, HS), they yield water-soluble
antigenic extracts, the major component of which precipitates with sera to rough Brucella (Diaz & Bosseray,
1973; Myers et al., 1972). For this reason, the HS extracts have been referred to as the rough-specific
antigen or, when obtained from B. ovis, as the B.-ovis-specific antigen. However, the chemical
characterisation of the HS extracts from B. ovis has shown that they are enriched in rough
lipopolysaccharide (R-LPS), group 3 outer membrane proteins and other outer membrane components
(Riezu-Boj et al., 1986). Thus, HS extracts contain LPS determinants specific for B. ovis, but also additional
antigenic components, some of them shared with rough and smooth B. melitensis and other Brucella
(Santos et al., 1984). Such components account for the cross-reactivity that is sometimes observed with the
HS method and sera of sheep infected with B. melitensis or vaccinated with Rev.1 (Riezu-Boj et al., 1986).
The HS extract, due to its water solubility and high content of relevant cell-surface epitopes, is the best
diagnostic antigen and has been widely used for the serological diagnosis of B. ovis infection.
Brucella ovis REO 198, a CO2- and serum-independent strain, is recommended as a source of the HS
antigens to be used in serological tests3. Solid media described in Section B.1.c. are satisfactory for the
growth of B ovis REO 198. HS antigen is prepared as follows:
i)
Exponentially grow a suitable strain of B ovis, preferably aerobic and nonserum dependent, e.g. REO
198, in one of the following ways: for 48 hours in trypticasesoy broth flasks in an orbital incubator at
37C and 150 rpm; or in Roux bottles of trypticasesoy agar, or other suitable medium, with 5% serum
added (not necessary when using the REO 198 strain); or in a batch-type fermenter as described for
B. abortus, but with the addition of 5% serum to the medium (not necessary when using the REO
198 strain).
ii)
Cells are resuspended in 0.85% saline or PBS, then washed twice in 0.85% saline (12 g of dried cells
or 30 g of wet packed cells in 150 ml).
iii)
The cell suspension is then autoclaved at 120C for 1530 minutes.
iv)
After cooling, the suspension is centrifuged (15,000 g, 4C, 15 minutes) and the supernatant fluid is
filtered and dialysed against distilled water using 100 times the volume of the suspension, at 4C; the
water should be changed three times over a minimum of 2 days.
v)
The dialysed fluid can be ultracentrifuged (100,000 g, 4C, 68 hours), and the sediment is
resuspended in a small amount of distilled water and freeze-dried. When produced to be used in the
CFT, the addition of control process serum replacement II (CPSRII) prior to freeze-drying may assist in
stability and anti-complementary activity.
HS is then resuspended either in distilled water (for use in the AGID test), veronal buffered saline (for use in
the CFT), or carbonate/bicarbonate buffer or PBS (for use in the I-ELISA) and titrated against a set of
adequate positive and negative sera.
2
3
Obtainable from the OIE Reference Laboratory for Brucellosis at AHVLA Weybridge, Addlestone, Surrey KT15 3NB,
United Kingdom.
Obtainable from the OIE Reference Laboratory for Brucellosis at Anses Maisons-Alfort, 94706, France.
1021
If it is to be used in the AGID test, the resuspended HS could be kept at 4C adding 0.5% phenol as
preservative. Freezing and thawing should be avoided (Diaz & Bosseray, 1973). The CFT antigen should be
standardised against the International anti-B. ovis Standard Serum to give 50% fixation at a 1/100 serum
dilution.
a)
Complement fixation test (the prescribed test for international trade)

There is no standardised method for the CFT, but the test is most conveniently carried out using the
microtitration method. Some evidence shows that cold fixation is more sensitive than warm fixation (Burgess
& Norris, 1982; Ris et al., 1984; Searson, 1982), but that it is less specific. Anticomplementary reactions,
common with sheep serum, are, however, more frequent with cold fixation.
Several methods have been proposed for the CFT using different concentrations of fresh sheep red blood
cells (SRBCs) (a 23% suspension is usually recommended) sensitised with an equal volume of rabbit antiSRBC serum diluted to contain several times (usually from two to five times) the minimum concentration
required to produce 100% lysis of SRBCs in the presence of a titrated solution of guinea-pig complement.
The latter is independently titrated (in the presence or absence of antigen according to the method) to
determine the amount of complement required to produce either 50% or 100% lysis of sensitised SRBCs in
a unit volume of a standardised suspension; these are defined as the 50% or 100% haemolytic unit of
complement (CH50 or CH100), respectively. It is generally recommended to titrate the complement before
each set of tests, a macromethod being preferred for an optimal determination of CH50. Usually, 1.25
2 CH100 or 56 CH50 are used in the test.
Barbital (veronal) buffered saline (VBS) is the standard diluent for the CFT. This is prepared from tablets
available commercially, otherwise it may be prepared according to the formula described elsewhere (see
Chapter 2.4.3 Bovine brucellosis). The test sera should be inactivated for 30 minutes in a water bath at 60
63C, and then diluted (doubling dilutions) in VBS. The stock solution of HS antigen (2.520 mg/ml in VBS)
is diluted in VBS as previously determined by titration (checkerboard titration). Usually, only one serum
dilution is tested (generally 1/10).
Using standard 96-well microtitre plates with round (U) bottom, the technique is usually performed as
follows:
i)
Volumes of 25 l of diluted inactivated test serum are placed in the well of the first and second rows.
Volumes of 25 l of CFT buffer are added to all wells except those of the first row. Serial doubling
dilutions are then made by transferring 25 l volumes of serum from the second row onwards.
ii)
Volumes of 25 l of antigen, diluted to working strength, are added to each well except wells in the first
row.
iii)
Volumes of 25 l of complement, diluted to the number of units required, are added to each well.
iv)
Control wells containing diluent only, complement + diluent, antigen + complement + diluent, are set up
to contain 75 l total volume in each case. A control serum that gives a minimum positive reaction
should be tested in each set of tests to verify the sensitivity of test conditions.
v)
The plates are incubated at 37C for 30 minutes or at 4C overnight, and a volume (25 or 50 l
according to the techniques) of sensitised SRBCs is added to each well. The plates are reincubated at
37C for 30 minutes.
vi)
The results are read after the plates have been centrifuged at 1000 g for 10 minutes at 4C or left to
stand at 4C for 23 hours to allow unlysed cells to settle. The degree of haemolysis is compared with
standards corresponding to 0, 25, 50, 75 and 100% lysis. The titre of the serum under test is the
highest dilution in which there is 50% or less haemolysis.
Standardisation of the results of the complement fixation test

There is a unit system that is based on the International Standard for anti-Brucella ovis Serum
(International Standard 1985 [see footnote 2]). This serum contains 1000 IU/ml. lf this serum is tested
in a given method and gives a titre of, for example 100, then the factor for an unknown serum tested by
that method can be found from the formula: 1000/100 titre of test serum = number of ICFTU
(International CFT units) of antibody in the test serum per ml. It is recommended that any country using
the CFT on a national scale should obtain agreement among the different laboratories performing the
test by the same method, to allow the same level of sensitivity and specificity to be obtained against an
adequate panel of sera from B. ovis culture positive and Brucella-free sheep. Results should always be
expressed in ICFTU, calculated in relation to those obtained in a parallel titration with a standard
serum, which itself may be calibrated against the International Standard.
1022
Interpretation of the results: sera giving a titre equivalent to 50 ICFTU/ml or more are considered to be
positive in the EU.
b)

The AGID test (Blasco, 1990) uses the following reagents: Good grade Noble agar or agarose, sodium
chloride (NaCI), and borate buffer (prepared with boric acid [12.4 g]; potassium chloride [14.5 g]; distilled
water [1600 ml]; adjusted to pH 8.3 with 0.2 M NaOH solution and made up to 2000 ml with distilled water).
To prepare the gels, dissolve 1 g of agarose (or Noble agar), 10 g of NaCI and 100 ml of borate buffer by
boiling while stirring continuously. On a flat surface, cover clean glass slides with the necessary amount of
molten gel to form a bed of 2.5 mm depth (3.5 ml approximately for standard microslides). After the gel has
solidified (1520 minutes), wells are cut in it using a gel puncher. The wells should be 3 mm in diameter and
3 mm apart, and should be arranged in a hexagonal pattern around a central well that is also 3 mm in
diameter. The test can be adapted to Petri dishes and other patterns.
Sera to be examined are placed in alternate wells separated by a control positive serum (infection proved by
bacteriology), with the antigen at its optimum concentration in the central well. The results are read after
incubation for 24 and 48 hours at room temperature in a humid chamber. A positive reaction is a clearly
defined precipitin line between the central well and the wells of the test sera that gives total or partial identity
with that of the positive controls. Precipitin lines not giving total identity may also appear and correspond to
minor antigenic components of HS extracts (antibodies to these components can also be common in
infections due to B. melitensis). These reactions should also be considered to be positive. Before a definitive
reading, it is important to wash the slides for 1 hour in a 5% sodium citrate water solution to clean unspecific
precipitin lines.
The HS (2.520 mg/ml) diluted in distilled water (optionally containing 0.5% phenol as a preservative) is the
antigen used in the AGID test (the preserved antigen can be stored at 4C for at least 1 month). Dilutions of
antigen are tested with a panel of 2030 sera from rams naturally infected with B. ovis and with a panel of
Brucella-free sheep. The optimum concentration of antigen is that giving the clearest precipitation line with
all the sera from B.-ovis-infected rams being negative with the sera from Brucella-free sheep.
c)
Enzyme-linked immunosorbent assay (the alternative test for international trade)

Several variations of this assay have been proposed. The assay described here is an indirect I-ELISA using
ABTS (2,2-azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]) as chromogen, but other procedures are
also suitable. Tests are performed on 96-weIl flat-bottomed ELISA plates. Reagent and serum dilutions are
made in PBS, pH 7.2, with the addition of 0.05% Tween 20 (PBST). Antigen dilutions are made in a
carbonate/bicarbonate buffer, pH 9.6, or, alternatively, in PBS, pH 7.2. Plates are washed after antigen
coating and between incubations, where appropriate, usually with PBST. The antigen (HS) and conjugate
are checkerboard titrated, and dilutions are selected to give the best discriminating ratio between negative
and positive standard sera. Secondary antibodies (anti-ovine IgG [H + L chains]) antibodies are usually
conjugated to horseradish peroxidase (HRPO), although other enzymes or conjugates (such as recombinant
protein G/HRPO) can be used. A monoclonal antibody to bovine IgG1HRPO conjugate has also been found
to be suitable for use in the I-ELISA (Vigliocco et al., 1997). If a peroxidase conjugate is used, the
chromogen, usually ABTS, is diluted in a substrate buffer (composed of citric acid trisodium and citric acid;
pH 4). The substrate, hydrogen peroxide (H2O2), is added to this, and the plates are incubated for 15
25 minutes at room temperature. The reaction may be stopped with 1 mM sodium azide, and the colour
change is read at 405414 nm (for further details see chapter 2.4.3).
The antigen used in the I-ELISA is the HS in stock solution at 1 mg/ml in coating buffer, titrated in a
checkerboard titration, with different dilutions of antigen, conjugate and substrate, against a standard serum
or against serial dilutions of a panel of sera from B. ovis culture positive and Brucella-free sheep to
determine the most sensitive and specific dilution (usually 510 g/ml).
i)
Microtitre plates of good quality polystyrene are coated by the addition of 100 l of a predetermined
antigen dilution in carbonate buffer, pH 9.6, to each well. Plates are incubated for 2 hours at 37C.
Alternatively, the coating can be made overnight at 4C with 100 l/well of the predetermined antigen
dilution in PBS, pH 7.2. Plates are then washed four times to remove unbound antigen and dried by
tapping firmly upside down on an absorbent paper. The coated plates can be used immediately or
dried and stored at 4C (the stability in these conditions is adequate for at least 1 month).
ii)
Sera: Dilute test and positive and negative control serum samples 1/200 by the addition of a minimum
of 10 l of serum to 2 ml PBST. This serum dilution is usually the optimal when using both polyclonal
and monoclonal conjugates. However, dilutions of 1/50 are the optimal when using the protein G-
1023
peroxidase conjugate (Marin et al., 1998) Add 100 l/well volumes of samples in duplicate to the
microtitre plates. The plates are covered, incubated at 37C for 1 hour, and washed three times with
the PBST washing buffer.
iii)
Conjugate: The titrated conjugate is diluted in PBST, added (100 l) to the wells, and the plate is
covered and incubated for 1 hour at 37C. After incubation, the plates are washed again three times
with PBST.
iv)
Substrate: The solution of ABTS in substrate buffer is added (100 l/well) and the plates are incubated
for 1560 minutes at room temperature with continuous shaking.
v)
Reading and interpreting the results: Absorbance is read automatically in a spectrophotometer at 405
414 nm. Absorbance values may be expressed as percentages of the mean absorbance of the positive
control or, preferably, transformed into I-ELISA units calculated either manually or by using a computer
and a curve-fitting program from a standard curve constructed with the series of positive control dilution
results.
The cut-off threshold should be properly established using the appropriate validation techniques (see
Chapter 1.1.5 Principles and methods of validation of diagnostic assays for infectious diseases). The
International Standard for anti-Brucella ovis Serum or the corresponding secondary or national standards
should be used to check or calibrate the particular test method in question.
Comparative studies have shown that the I-ELISA has better sensitivity than either the AGID test or the CFT
(Marin et al., 1989; Ris et al., 1984; Spencer & Burgess, 1984; Worthington et al., 1984; 1985). Due to the
existence of some I-ELISA-negative and AGID-positive sera, the combination of the AGID test and I-ELISA
gives optimal sensitivity (Marin et al., 1989). However, the combination of CF test and I-ELISA or CF and
AGID tests does not improve the sensitivity of I-ELISA alone (Marin et al., 1989). Moreover, the CFT has
other important disadvantages such as complexity, obligatory serum inactivation, anticomplementary activity
of some sera, the difficulty of performing it with haemolysed sera, and prozone phenomena. Because of its
sensitivity, simplicity and easy interpretation, the AGID test is very practicable for routine diagnosis in
nonspecialised laboratories.
Little is known about the existence of false positive results in B. ovis serological tests as a consequence of
infections due to bacteria showing cross-reacting epitopes with B. ovis. The foot rot agent (Dichelobacter
nodosus) has been described as showing cross-reactions with B. ovis (Whitington et al., 1996), but the
extent and practical consequences of this cross-reactivity in B. ovis diagnostic tests is unknown4.
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICAIS

Vaccination of both rams and ewes is probably the most economical and practical means for medium-term control
of B. ovis in areas with a high incidence of infection. For long-term control, consideration should be given to the
effect of vaccination on serological testing, and B.-ovis-free accreditation programmes have to be implemented.
The live B. melitensis strain Rev.1 (see chapter 2.7.2) is probably the best available vaccine for the prophylaxis of
B. ovis infection (Blasco, 1990). A single standard dose (109 colony-forming units) of Rev.1 administered
subcutaneously (in a 1 ml volume) or conjunctivally (in a 2530 l volume), to 35 month-old animals confers
adequate immunity against B. ovis. Conjunctival vaccination has the advantage of minimising the intense and
long-lasting serological response evoked by subcutaneous vaccination, thereby improving the specificity of
serological tests (Blasco, 1990). When used in both young and adult males, the safety of the Rev.1 vaccine is
adequate and side-effects appear to be very rare (Marin et al., 1990; Muoz et al., 2008). Therefore, in countries
with extensive management and high levels of incidence, it would be advisable to vaccinate both young and
healthy adult animals. In countries affected by B. ovis but free of B. melitensis, before using the B. melitensis
Rev.1 vaccine account should be taken of possible serological interferences and the conjunctival route should be
preferred to minimise this problem. The B. abortus RB51 live vaccine has not proven successful against B. ovis in
sheep (Jimenez et al., 1995) and no alternative vaccines are currently available.
REFERENCES
ALTON G.G., JONES L.M., ANGUS R.D. & VERGER J.M. (1988). Techniques for the Brucellosis Laboratory. INRA,
Paris, France.
Arcanobacterium pyogenes and Corynebacterium ovis, whose soluble extracts cross-react with B. ovis positive control
sera, have been recently isolated from several lymph nodes of rams giving strong positive responses in B. ovis AGID test
and I-ELISA (J.M. Blasco, unpublished results).
1024
BLASCO J.M. (1990). Brucella ovis. In: Animal Brucellosis, Nielsen K. & Duncan J.R., eds. CRC Press, Boca
Raton, Florida, USA, 351378.
BROWN G.M., RANGER C.R. & KELLEY D.J. (1971). Selective media for the isolation of Brucella ovis. Cornell Vet.,
61, 265280.
BULGIN M.S. & ANDERSON B.C. (1983). Association of sexual experience with isolation of various bacteria in cases
of ovine epididymitis. J. Am. Vet. Med. Assoc., 182, 372374.
BURGESS G.W. & MCDOWELL J.W. (1981). Escherichia coli epididymitis and seminal vesiculitis in a ram. Aust. Vet.
J., 57, 479480.
BURGESS G.W. & NORRIS M.J. (1982). Evaluation of the cold complement fixation test for diagnosis of ovine
brucellosis. Aust. Vet. J., 59, 2325.
CORBEL M.J., GILL K.P.W. & THOMAS E.L. (1978). Methods for the identification of Brucella. Ministry of Agriculture,
Fisheries and Food, UK, ADAS, RCV 22.
DE LONG W.J., WALDHALM D.G. & HALL R.F. (1979). Bacterial isolates associated with epididymitis in rams from
Idaho and eastern Oregon flocks. Am. J. Vet. Res., 40, 101102.
DIAZ R. & BOSSERAY N. (1973). Identification dun compos antignique spcifique de la phase rugueuse (R) des
Brucella. Ann. Rech. Vet., 4, 283292.
EKDAHL M.O., MONEY D.F. & MARTIN C.A. (1968). Some aspects of epididymitis of rams in New Zealand. N. Z. Vet.
J., 16, 8182.
JIMENEZ DE BAGUES M.P., BARBERAN M., MARIN C.M. & BLASCO J.M. (1995). The Brucella abortus RB51 vaccine
does not confer protection against Brucella ovis in rams. Vaccine, 13, 301304.
LIVINGSTONE C.W. & HARDY W.T. (1964). Isolation of Actinobacillus seminis from ovine epididymitis. Am. J. Vet.
Res., 25, 660663.
MARIN C.M., ALABART J.L. & BLASCO J.M. (1996). Effect of antibiotics contained in two Brucella selective media on
growth of B. abortus, B. melitensis and B. ovis. J. Clin. Microbiol., 34, 426428.
MARIN C.M., ALONSO-URMENETA B., MORIYON I., PEREZ S. & BLASCO J.M. (1998). Comparison of polyclonal,
monoclonal and protein G peroxidase conjugates in an enzyme-linked immunosorbent assay for the diagnosis of
Brucella ovis in sheep. Vet. Rec., 143, 390394.
MARIN C.M., BARBERAN M., JIMENEZ DE BAGUES M.P. & BLASCO J.M. (1990). Comparison of subcutaneous and
conjunctival routes of Rev. 1 vaccination for the prophylaxis of Brucella ovis infection in rams. Res. Vet. Sci., 48,
209215.
MARIN C.M., JIMENEZ DE BAGUES M.P., BLASCO J.M., GAMAZO C., MORIYON I. & DIAZ R. (1989). Comparison of three
serological tests for Brucella ovis infection of rams using different antigenic extracts. Vet. Rec., 125, 504508.
MUOZ P., DE MIGUEL M.J., GRILL M.J., MARN C.M., BARBERN M. & BLASCO J.M. (2008). Immunopathological
responses and kinetics of B. melitensis Rev 1 infection after subcutaneous or conjunctival vaccination in rams.
Vaccine, 26, 25622569.
MYERS D.M., JONES L.M. & VARELA-DIAZ V. (1972). Studies of antigens for complement fixation and gel diffusion
tests in the diagnosis of infections caused by Brucella ovis and other Brucella. Appl. Microbiol., 23, 894902.
RIDLER A. (2001) Brucella ovis infection in deer. Surveillance, 28 (3), 68.
RIEZU-BOJ J.I., MORIYON I., BLASCO J.M., MARIN C.M. & DIAZ R. (1986). Comparison of lipopolysaccharide and outer
membrane protein-lipopolysaccharide extracts in an enzyme-linked immunosorbent assay for the diagnosis of
Brucella ovis infection. J. Clin. Microbiol., 23, 938942.
RIS D.R., HAMEL K.L. & LONG D.L. (1984). Comparison of an enzyme-linked immunospecific assay (ELISA) with the
cold complement fixation test for the serodiagnosis of Brucella ovis infection. N.Z. Vet. J., 32, 1820.
1025
RODOLAKIS A. & BERNARD K. (1977). Isolement de Chlamydia des organes gnitaux de bliers atteints
dpididymite. Bull. Acad. Vet. Fr., 50, 6570.
SANTOS J.M., VERSTREATE D.R., PERERA V.Y. & WINTER A.J. (1984). Outer membrane proteins from rough strains of
four Brucella species. Infect. Immun., 46, 188194.
SEARSON J.E. (1982). Sensitivity and specificity of two microtitre complement fixation tests for the diagnosis of
Brucella ovis infection in rams. Aust. Vet. J., 58, 57.
SPENCER T.L. & BURGESS G.W. (1984). Enzyme-linked immunosorbent assay for Brucella ovis specific antibody in
ram sera. Res. Vet. Sci., 36, 194198.
VIGLIOCCO A.M., SILVA PAULO P.S., MESTRE J., BRIONES G.C., DRAGHI G., TOSSI M. & NIELSEN K. (1997).
Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis.
WEBB R.F. (1983). Clinical findings and pathological changes in Histophilus ovis infections of sheep. Res. Vet.
Sci., 35, 3034.
WEBB R.F., QUINN C.A., COCKRAM F.A. & HUSBAND A.J. (1980). Evaluation of procedures for the diagnosis of
Brucella ovis infection in rams. Aust. Vet. J., 56, 172175.
WHITINGTON R.J., SAUNDERS V.F. & EGERTON J.R. (1996). Antigenic cross-reactions between the causative agent of
ovine footrot, Dichelobacter nodosus, and other bacteria. Small Rumin. Res., 22, 5567.
WILLIAMSON P. & NAIRN M.E. (1980). Lesions caused by Corynebacterium pseudotuberculosis in the scrotum of
rams. Aust. Vet. J., 56, 496498.
WORTHINGTON R.W., STEVENSON B.J. & DE LISLE G.W. (1985). Serology and semen culture for the diagnosis of
Brucella ovis infection in chronically infected rams. N.Z. Vet. J., 33, 8486.
WORTHINGTON R.W., WEDDELL W. & PENROSE M.E. (1984). A comparison of three serological tests for the diagnosis
of B. ovis infection in rams. N.Z. Vet. J., 32, 5860.
*
* *
NB: There are OIE Reference Laboratories for Ovine epididymitis (Brucella ovis)
diagnostic tests, reagents and vaccines for ovine epididymitis (Brucella ovis)
1026
CHAPTER 2.7.10.
OVINE PULMONARY ADENOCARCINOMA

(adenomatosis)
SUMMARY
Ovine pulmonary adenocarcinoma (OPA), also known as ovine pulmonary adenomatosis and
jaagsiekte, is a contagious tumour of sheep and, rarely, of goats. It is a progressive respiratory
disease, principally affecting adult animals. The disease occurs in many regions of the world. A
beta-retrovirus (jaagsiekte sheep retrovirus: JSRV), distinct from the non-oncogenic ovine
lentiviruses, has been shown to cause the disease.
Identification of the agent: JSRV cannot yet be propagated in vitro, therefore routine diagnostic
methods, such as virus isolation, are not available for diagnosis. Diagnosis relies, at present, on
clinical history and examination, as well as on the findings at necropsy and by histopathology and
immunohistochemistry. Viral DNA or RNA can be detected in tumour, draining lymph nodes, and
peripheral blood mononuclear cells by polymerase chain reaction. Lambs become persistently
infected by JSRV at an early age, and, in an OPA-affected flock, most sheep are infected.
Serological tests: Antibodies to the retrovirus have not been detected in infected sheep and,
therefore, serological tests are not available for diagnosis.
Requirements for vaccines and diagnostic biologicals: There are no vaccines or diagnostic
biologicals available.
A. INTRODUCTION
Ovine pulmonary adenocarcinoma (OPA), also known as ovine pulmonary adenomatosis, jaagsiekte (Afrikaans =
driving sickness) and ovine pulmonary carcinoma (OPC), is a contagious lung tumour of sheep and, more rarely,
of goats. It is the most common pulmonary tumour of sheep and occurs in many countries around the world. It is
absent from Australia and New Zealand and has been eradicated from Iceland.
A number of different viruses have been linked aetiologically to OPA, including a herpesvirus and lentiviruses that
have been propagated from tumour tissue. However, the former does not have an aetiological role in OPA and
the latter exhibit characteristics of non-oncogenic lentiviruses. It has been demonstrated clearly that OPA is
caused by a beta-retrovirus that cannot yet be cultured in vitro, but the virus has been cloned and sequenced.
The term jaagsiekte sheep retrovirus (JSRV) is used in referring to this virus.
At present, diagnosis of OPA relies on clinical and pathological investigations, although polymerase chain
reaction (PCR) offers hope for ante-mortem diagnosis of OPA as a flock test. In flocks in which the disease is
suspected, its presence must be, at least once, confirmed by histopathological examination of affected lung
tissue. For such an examination, it is imperative to take specimens from several affected sites and, if possible,
from more than one animal. This is because secondary bacterial pneumonia, which might be the immediate cause
of death, often masks the lesions (both macroscopic and microscopic) of the primary disease. In the absence of
specific serological tests that can be used for the diagnosis of OPA in live animals, disease control relies on
regular flock inspections and prompt culling of suspected cases and, in the case of ewes, their offspring.
1.
Although ovine herpesvirus 1 (OvHV-1) had been isolated exclusively from OPA tumours, epidemiological studies
and experimental infections provide no evidence for a role in the aetiology of OPA. Ovine herpesvirus 2 (OvHV-2)
is the sheep-associated malignant catarrhal fever herpesvirus and has never been linked to OPA.
1027
Chapter 2.7.10. Ovine pulmonary adenomatosis (adenocarcinoma)
The association of retroviruses with OPA has been recognised for several years. Ovine lentiviruses have been
isolated on a number of occasions, but these viruses have no aetiological role in OPA.
For many years, the inability to culture JSRV and the lack of antibodies to the virus in affected sheep impeded the
confirmation of this virus as the aetiological agent. However, molecular biological techniques provided a key
advance, namely, the cloning and sequencing of the 7.5 kb JSRV genome following purification of virions from
lung washes of naturally affected sheep (York et al., 1992). JSRV has been designated as a beta-retrovirus
because of its genetic organisation and its structural proteins. Although cloned JSRV genes, used as hybridisation
probes, have revealed a range of homologous endogenous sequences in the genome of both healthy and OPAaffected sheep (Bai et al., 1996; Hecht et al., 1994; York et al., 1992), JSRV is clearly exogenous and associated
exclusively with OPA (Palmarini et al., 1996). JSRV is detected constantly in the lung fluid, tumour, peripheral
blood mononuclear cells, and lymphoid tissues of sheep affected by OPA or unaffected in-contact flockmates, and
never in sheep from unaffected flocks with no history of the tumour. Full-length proviral clones of JSRV have been
obtained from OPA tumour DNA and cells. JSRV virus particles, prepared from these clones by transient
transfection of a cell line, were used for intratracheal inoculation of neonatal lambs. OPA tumour was induced in
the lambs, thus demonstrating that JSRV is the causal agent of OPA (DeMartini et al., 2001; Palmarini et al.,
1999).
The sheep genome contains many copies of endogenous viral sequences that are highly related to JSRV.
Although they are not involved in the aetiology of OPA, their expression in the fetus may, by induction of
tolerance, account for the apparent lack of immune response of mature animals to exogenous JSRV (Palmarini et
al., 2004).
a)

Sequencing of JSRV and endogenous sequences in the sheep genome has led to the development of PCRs
that specifically detect JSRV (Bai et al., 1996; Palmarini et al., 1996). Using this sensitive procedure, JSRV
has been detected in peripheral blood mononuclear cells of unaffected in-contact sheep from flocks with
OPA, as well as experimentally infected lambs (De las Heras et al., 2005; Gonzalez et al., 2001; Holland et
al., 1999) and the bronchoalveolar lavage samples from unaffected in-contact sheep (Voigt et al., 2007).
Longitudinal studies in OPA-affected flocks have shown that lambs become infected at a very early age. A
high proportion of animals in these flocks are infected, yet only a minority develop OPA (Caporale et al.,
2005; Salvatori, 2005). JSRV has been found in colostrum and milk obtained from sheep in OPA-affected
flocks and JSRV can be detected within a few months in the blood of lambs fed artificially with colostrum and
milk (De las Heras et al., unpublished observations).
Control and treatment
b)
Animal inoculation
OPA cannot be transmitted to any laboratory animal and can be transmitted to sheep only with material that
contains JSRV, such as tumour homogenates, concentrated cell-free lung fluid from natural cases of OPA
and virus produced from molecular clones. Following the experimental inoculation of adult sheep, clinical
disease develops only after several months or years. In contrast, JSRV infection can be induced in 100% of
lambs aged 16 months at the time of inoculation and a high proportion of these animals develop clinical
signs (6290%) and lesions (87100%) of OPA (Salvatori et al., 2004).
At this time there is no practical animal inoculation method for the diagnosis of OPA.
c)
Virus isolation
There are no permissive cell culture systems for propagation of JSRV. Some cell cultures prepared from the
tumours occurring in young lambs can support virus replication for a short period (Jassim, 1988; Sharp et al.,
1985).
d)
Clinical signs and pathology

There is no reliable laboratory method for the ante-mortem diagnosis of OPA in individual animals at this
time, therefore flock history, clinical signs and post-mortem lesions are the primary method for the diagnosis
of the disease. As OPA has a long incubation period, clinical disease is encountered most commonly in
sheep over 2 years of age, with a peak occurrence at the age of 34 years. In exceptional cases, the
disease occurs in animals as young as 23 months of age. The cardinal signs are those of a progressive
respiratory embarrassment, particularly after exercise; the severity of the signs reflects the extent of tumour
development in the lungs. Accumulation of fluid within the respiratory tract is a prominent feature of OPA,
giving rise to moist rles that are readily detected by auscultation. Raising the hindquarters and lowering the
head of affected sheep may cause frothy mucoid fluid to run from the nostrils. Coughing and inappetance
are not common but, once clinical signs are evident, weight loss is progressive and the disease is terminal
1028
Chapter 2.7.10. Ovine pulmonary adenocarcinoma (adenomatosis)
within weeks or months. Death is often precipitated by a superimposed bacterial pneumonia, particularly that
due to Mannheimia (formerly Pasteurella) haemolytica. In clinically affected animals, a peripheral
lymphopenia characterised by a reduction in CD4+ T lymphocytes and a corresponding neutrophilia may
assist clinical diagnosis, but the changes are not pathognomonic and are not detected during early
experimental infection (Summers et al., 2002).
In some countries, another form of OPA (atypical OPA) occurs, which generally presents as an incidental
finding at necropsy or the abattoir (De las Heras et al., 2003).
e)
Necropsy
OPA lesions are in most cases confined to the lungs, although intra- and extrathoracic metastasis to lymph
nodes and other tissues can occur. In typical cases, affected lungs are considerably enlarged and heavier
than normal due to extensive nodular and coalescing firm grey lesions affecting much of the pulmonary
tissue. Usually lesions are present in both lungs, although the extent on either side does vary. Tumours are
solid, grey or light purple with a shiny translucent sheen and often separated from the adjacent normal lung
by a narrow emphysematous zone. The presence of frothy white fluid in the respiratory passages is a
prominent feature and is obvious even in lesions as small as a few millimetres. In advanced cases, this fluid
flows out of the trachea when it is cut or pendant. Samples should be taken at necropsy for histopathology,
immunohistochemistry or PCR for JSRV.
Pleurisy may be evident over the surface of the tumour and often abscesses are present in the
adenomatous tissue.
In atypical OPA, tumours comprise solitary or aggregated hard white nodules that have a dry cut surface and
show clear demarcation from surrounding tissues. The presence of excess fluid is not a prominent feature.
Adult sheep, which on post-mortem examination appear to have died from acute pasteurellosis, should have
their lungs examined carefully, as lesions of OPA may be masked by coexisting bronchopneumonia,
verminous pneumonia, chronic progressive pneumonia (maedi-visna) or combinations of these. Samples
should be taken at necropsy for histopathology.
f)
Histopathology
Histologically, the lesions are characterised by proliferation of mainly type II pneumocytes, a secretory
epithelial cell in the pulmonary alveoli. Nonciliated (Clara) and epithelial cells of the terminal bronchioli may
be involved. The cuboidal or columnar tumour cells replace the normal thin alveolar cells and sometimes
form papilliform growths that project into the alveoli. Intrabronchiolar proliferation may be present. In
advanced cases, extensive fibrosis may develop and, occasionally, nodules of loose connective tissue in a
mucopolysaccharide substance may be present.
A prominent feature is the accumulation of large numbers of alveolar macrophages in the alveoli adjacent to
the neoplastic lesions (Summers et al., 2005).
Where maedi-visna is concurrent, perivascular, peribronchiolar and interstitial lymphoid infilrates may be
prominent.
The histological appearance of atypical OPA is essentially the same as classical OPA, but with an
exaggerated inflammatory response (mostly lymphocytes and plasma cells) and fibrosis (De las Heras et al.,
2003).
For more detailed accounts of the clinical, post-mortem and histopathological aspects of OPA, the reader is
referred elsewhere (De las Heras et al., 2003; Sharp & DeMartini, 2003).
There appears to be a synergistic interaction between OPA and maedi-visna. Lateral transmission of maedivisna virus appears to be enhanced in sheep affected by OPA (Dawson et al., 1985; Gonzalez et al., 1993).
2.
Serological tests
At present, there are no laboratory tests to support a clinical diagnosis of OPA in the live animal. JSRV has been
associated exclusively with both typical and atypical forms of OPA, but antibodies to this virus have not been
detected in the sera of affected sheep, even with highly sensitive assays such as immunoblotting or enzymelinked immunosorbent assay (Ortin et al., 1997; Summers et al., 2002).
1029
Chapter 2.7.10. Ovine pulmonary adenomatosis (adenocarcinoma)

There are no vaccines or diagnostic biologicals available at the present time.
REFERENCES
BAI J., ZHU R.-Y., STEDMAN K., COUSENS C., CARLSON J.O., SHARP J.M. & DEMARTINI J.C. (1996). Unique long
terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine
pulmonary carcinoma from endogenous loci in the sheep genome. J. Virol., 70, 31593168.
CAPORALE M., CENTORAME P., GIOVANNINI A., SACCHINI F., DI VENTURA M., DE LAS HERAS M. & PALMARINI M. (2005).
Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of
naturally occurring JSRV infection during the commercial lifespan of sheep. Virology, 338, 144153.
DAWSON M., VENABLES C. & JENKINS C.E. (1985). Experimental infection of a natural case of sheep pulmonary
adenomatosis with maedi-visna virus. Vet. Rec. 116, 588589.
DE LAS HERAS M., GONZALEZ L.G. & SHARP J.M. (2003). Pathology of ovine pulmonary adenocarcinoma. Curr. Top.
Microbiol. Immunol., 275, 2554
DE LAS HERAS M., ORTN A., SALVATORI D., PREZ DE VILLAREAL M., COUSENS C., FERRER L.M., GARCA DE JALN J.A.,
GONZALEZ L. & SHARP J.M. (2005). A PCR technique for the detection of Jaagsiekte retrovirus in the blood suitable
for the screening of virus infection in sheep flocks. Res. Vet. Sci., 79, 259264.
DEMARTINI J.C., BISHOP J.V., ALLEN T.E., JASSIM F.A., SHARP J.M., DE LAS HERAS M., VOELKER D.R. & CARLSON J.O.
(2001). Jaagsiekte sheep retrovirus proviral clone JSRVJS7, derived from the JS7 lung tumor cell line, induces
ovine pulmonary carcinoma and is integrated into the surfactant protein A gene. J. Virol., 75, 42394246
GONZALEZ L., GARCIA-GOTI M., COUSENS C., DEWAR P., CORTABARRIA N., EXTRAMIANA B., ORTIN A., DE LAS HERAS M.
& SHARP J.M. (2001). Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the preclinical
period of sheep pulmonary adenomatosis. J. Gen. Virol., 82, 13551358.
GONZALEZ L., JUSTE R.A., CUERVO L.A., IDIGORAS I. & SAEZ DE OCARIZ C. (1993). Pathological and epidemiological
aspects of the coexistence of maedi-visna and sheep pulmonary adenomatosis. Res. Vet. Sci., 54, 140146.
HECHT S.J., CARLSON J.O. & DE MARTINI J.C. (1994). Analysis of a type D retroviral capsid gene expressed in ovine
pulmonary carcinoma and present in both affected and unaffected sheep genomes. Virology, 202, 480484.
HOLLAND M.J., PALMARINI M., GARCIA-GOTI M., GONZALEZ L., DE LAS HERAS M. & SHARP J.M. (1999). Jaagsiekte
retrovirus establishes a pantropic infection of lymphoid cells of sheep with naturally and experimentally acquired
pulmonary adenomatosis. J. Virol., 73, 40044008.
JASSIM F.A. (1988). Identification and characterisation of transformed cells in jaagsiekte, a contagious lung tumour
of sheep. PhD thesis. University of Edinburgh, UK.
ORTIN A., MINGUIJON E., DEWAR P., GARCIA M., FERRER L.M., PALMARINI M., GONZALEZ L., SHARP J.M. & DE LAS HERAS
M. (1997). Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep
retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis. Vet.
Immunol. Immunopathol., 61, 239237.
PALMARINI M., COUSENS C., DALZIEL R.G., BAI J., STEDMAN K, DEMARTINI J.C. & SHARP J.M. (1996). The exogenous
form of Jaagsiekte retrovirus (JSRV) is specifically associated with a contagious lung cancer of sheep. J. Virol.,
70, 16181623.
PALMARININ M., MURA M. & SPENCER T. (2004). Endogenous betaretroviruses of sheep: teaching new lessons in
retroviral interference and adaptation. J. Gen. Virol., 85, 113.
PALMARINI M., SHARP J.M., DE LAS HERAS M. & FAN H.Y. (1999). Jaagsiekte sheep retrovirus is necessary and
sufficient to induce a contagious lung cancer in sheep. J. Virol., 73, 69646972.
SALVATORI D. (2005). Studies on the pathogenesis and epidemiology of ovine pulmonary adenomatosis (OPA).
PhD thesis, University of Edinburgh, Scotland, UK.
1030
Chapter 2.7.10. Ovine pulmonary adenocarcinoma (adenomatosis)
SALVATORI D., COUSENS C., DEWAR P., ORTIN A., GONZALEZ L., DE LAS HERAS M., DALZIEL R.G. & SHARP J.M. (2004).
Effect of age at inoculation on the development of ovine pulmonary adenocarcinoma. J. Gen. Virol., 85, 3319
3324.
SHARP J.M. & DEMARTINI J.C. (2003). Natural history of JSRV in sheep. Curr. Top. Microbiol. Immunol., 275, 55
79.
SHARP J.M., HERRING A.J., ANGUS K.W., SCOTT F.M.M. & JASSIM F.A. (1985). Isolation and in vitro propagation of a
retrovirus from sheep pulmonary adenomatosis. In: Slow Virus Diseases in Sheep, Goats and Cattle. Sharp J.M.
& Hoff-Jorgensen R., eds. CEC Report EUR 8076 EN, Luxembourg, 345348.
SUMMERS C., NEILL W., DEWAR P., GONZALEZ L., VAN DER MOLEN R., NORVAL M. & SHARP J.M. (2002). Systemic
immune responses following infection with jaagsiekte sheep retrovirus and in the terminal stages of ovine
pulmonary adenocarcinoma. J. Gen. Virol., 83, 17531757.
SUMMERS C., NORVAL M., DE LAS HERAS M., GONZALEZ L., SHARP J.M. & WOODS G.M. (2005). An influx of
macrophages is the predominant local immune response in ovine pulmonary adenocarcinoma. Vet. Immunol.
Immunopathol., 106, 285294.
VOIGT K., BRGMANN M., HUBER K., DEWAR P., COUSENS C., HALL M., SHARP J.M. & GANTER M. (2007). PCR
examination of bronchoalveolar lavage samples is a useful tool in pre-clinical diagnosis of ovine pulmonary
adenocarcinoma (Jaagsiekte). Res. Vet. Sci., 83 (3), 419427.
YORK D.F., VIGNE R., VERWOERD D.W. & QUERAT G. (1992). Nucleotide sequence of the jaagsiekte retrovirus, an
exogenous and endogenous type D and B retrovirus of sheep and goats. J. Virol., 66, 49304939.
*
* *
1031
CHAPTER 2.7.11.
PESTE DES PETITS RUMINANTS
SUMMARY
Peste des petits ruminants (PPR), is an acute contagious disease caused by a Morbillivirus in the
family Paramyxoviridae. It affects mainly sheep and goats and occasionally wild small ruminants.
Based on the fact that PPR has been reported on a few occasions in camels, cattle and buffaloes,
those animal species are considered to be susceptible although their potential role in the circulation
of PPR virus (PPRV) has not been formally established. PPR occurs in Africa except Southern
Africa, in the Arabian Peninsula, throughout most of the Near East and Middle East, and in Central
and South-East Asia.
The clinical disease resembles rinderpest in cattle. It is usually acute and characterised by pyrexia,
serous ocular and nasal discharges, erosive lesions on different mucous membranes particularly in
the mouth, diarrhoea and pneumonia. At necropsy, erosions may be noted in the gastrointestinal
and urogenital tracts. The lungs may show interstitial bronchopneumonia and often secondary
bacterial pneumonia. PPR can also occur in subclinical form.
The disease must be differentiated from rinderpest, bluetongue, foot and mouth disease and other
exanthemous conditions.
Identification of the agent: The collection of specimens at the correct time is important to achieve
diagnosis by virus isolation and they should be obtained in the acute phase of the disease when
clinical signs are still apparent. The specimens from live animals can be swabs of conjunctival
discharges, nasal secretions, buccal and rectal mucosae, and anticoagulant-treated blood.
Rapid diagnosis is done by immunocapture enzyme-linked immunosorbent assay (ELISA), counter
immunoelectrophoresis and agar gel immunodiffusion. Polymerase chain reaction may also be
used.
Serological tests: The serological tests that are routinely used are the virus neutralisation and the
competitive ELISA.
Requirements for vaccines: In the past, control of PPR was ensured through vaccination with the
rinderpest tissue culture vaccine because of the existence of a strong antigenic relationship
between PPR and rinderpest viruses. The use of this heterologous vaccine has been abandoned in
favour of the live attenuated PPR virus vaccine, which is now widely commercially available.
A. INTRODUCTION
Peste des petits ruminants (PPR) is an acute viral disease of small ruminants characterised by fever, oculonasal
discharges, stomatitis, diarrhoea and pneumonia with foul offensive breath. Because of the respiratory signs, PPR
can be confused with contagious caprine pleuropneumonia (CCPP) or pasteurellosis. In many cases,
pasteurellosis is a secondary infection of PPR, a consequence of the immunosuppression that is induced by the
PPR virus (PPRV). PPRV is transmitted mainly by aerosols between animals living in close contact (Lefevre &
Diallo, 1990). Infected animals present clinical signs similar to those historically seen with rinderpest in cattle,
although the two diseases are caused by distinct virus species.
On the basis of its similarities to rinderpest, canine distemper and measles viruses, PPRV has been classified
within the genus Morbillivirus in the family Paramyxoviridae (Gibbs et al., 1979). Virus members of this group
have six structural proteins: the nucleocapsid protein (Np), which encapsulates the virus genomic RNA, the
phosphoprotein (P), which associates with the polymerase (L for large protein) protein, the matrix (M) protein, the
fusion (F) protein and the haemagglutinin (H) protein. The matrix protein, intimately associated with the internal
1032
Chapter 2.7.11. Peste des petits ruminants
face of the viral envelope, makes a link between the nucleocapsid and the virus external glycoproteins: H and F,
which are responsible, respectively, for the attachment and the penetration of the virus into the cell to be infected.
PPR was first described in Cte dIvoire (Gargadennec & Lalanne, 1942), but it occurs in most African countries
from North Africa to Tanzania (Kwiatek et al., 2011; Lefevre & Diallo, 1990; Swai, et al., 2009), and in nearly all
Middle Eastern countries to Turkey (Furley et al., 1987; Lefevre et al., 1991; Ozkul et al., 2002; Perl et al., 1994;
Taylor et al., 1990). PPR is also widespread in countries from a central Asia to South and South-East Asia
(Banyard et al., 2010; Shaila et al., 1989; Wang et al., 2009).
The natural disease affects mainly goats and sheep. It is generally admitted that cattle can only be infected
subclinically. However, in poor conditions it might be possible for cattle to develop lesions following PPRV
infection, clinical signs of which would be ascribed to rinderpest. Indeed, in the 1950s, disease and death were
recorded in calves experimentally infected with PPRV-infected tissue (Mornet et al., 1956). In addition, the virus
that was involved in what was thought to be a natural rinderpest outbreak in cattle, sheep and goats in 1971 in
Sudan was later shown to be PPRV (Kwiatek et al., 2011). Moreover, PPRV was isolated from an outbreak of
rinderpest-like disease in buffaloes in India in 1995 (Govindarajan et al., 1997). Antibodies to PPRV as well as
PPRV antigen and nucleic acid were detected in some samples from an epizootic disease that affected onehumped camels in Ethiopia and Sudan (Abraham et al., 2005; Ismail et al., 1992; Khalafalla et al. 2010; Kwiatek
et al., 2011; Roger et al., 2000; 2001). Cases of clinical disease have been reported in wildlife resulting in deaths
of wild small ruminants (Abubakar et al., 2011; Bao et al., 2011; Elzein et al., 2004; Furley et al., 1987; Kinne et
al., 2010). The American white-tailed deer (Odocoileus virginianus) can be infected experimentally with PPRV
(Hamdy & Dardiri, 1976). Dual infections can occur with other viruses such as pestivirus or goatpox virus.
The incubation period is typically 46 days, but may range between 3 and 10 days. The clinical disease is acute,
with a pyrexia up to 41C that can last for 35 days; the animals become depressed, anorexic and develop a dry
muzzle. Serous oculonasal discharges become progressively mucopurulent and, if death does not ensue, persist
for around 14 days. Within 4 days of the onset of fever, the gums become hyperaemic, and erosive lesions
develop in the oral cavity with excessive salivation. These lesions may become necrotic. A watery blood-stained
diarrhoea is common in the later stage. Pneumonia, coughing, pleural rales and abdominal breathing also occur.
The morbidity rate can be up to 100% with very high case fatality in severe cases. However, morbidity and
mortality may be much lower in milder outbreaks, and the disease may be overlooked. A tentative diagnosis of
PPR can be made on clinical signs, but this diagnosis is considered provisional until laboratory confirmation is
made for differential diagnosis with other diseases with similar signs.
At necropsy, the lesions are very similar to those observed in cattle affected with rinderpest, except that
prominent crusty scabs along the outer lips and severe interstitial pneumonia frequently occur with PPR. Erosive
lesions may extend from the mouth to the reticulorumen junction. Characteristic linear red areas of congestion or
haemorrhage may occur along the longitudinal mucosal folds of the large intestine and rectum (zebra stripes), but
they are not a consistent finding. Erosive or haemorrhagic enteritis is usually present and the ileocaecal junction
is commonly involved. Peyers patches may be necrotic. Lymph nodes are enlarged, and the spleen and liver may
show necrotic lesions.
There are no known health risks to humans working with PPRV as no report of human infection with the virus
exists.
1.
Samples for virus isolation must be kept chilled in transit to the laboratory. In live animals, swabs are made of the
conjunctival discharges and from the nasal and buccal mucosae. During the very early stage of the disease,
whole blood is also collected in anticoagulant for virus isolation, polymerase chain reaction (PCR) and
haematology. At necropsy, samples from two to three animals should be collected aseptically from lymph nodes,
especially the mesenteric and bronchial nodes, lungs, spleen and intestinal mucosae, chilled on ice and
transported under refrigeration. Samples of organs collected for histopathology are placed in 10% neutral buffered
formalin. It is good practice to collect blood for serological diagnosis at all stages, but particularly later in the
outbreak.
2.
a)

Agar gel immunodiffusion (AGID) is a very simple and inexpensive test that can be performed in any
laboratory and even in the field. Standard PPR viral antigen is prepared from infected mesenteric or
bronchial lymph nodes, spleen or lung material and ground up as 1/3 suspensions (w/v) in buffered saline
1033
(Durojaiye et al., 1983). These are centrifuged at 500 g for 1020 minutes, and the supernatant fluids are
stored in aliquots at 20C. The cotton material from the cotton bud used to collect eye or nasal swabs is
removed using a scalpel and inserted into a 1 ml syringe. With 0.2 ml of phosphate buffered saline (PBS),
the sample is extracted by repeatedly expelling and filling the 0.2 ml of PBS into an Eppendorf tube using
the syringe plunger. The resulting eye/nasal swab extracted sample, like the tissue ground material prepared
above, may be stored at 20C until used. They may be retained for 13 years. Negative control antigen is
prepared similarly from normal tissues. Standard antiserum is made by hyperimmunising sheep with 1 ml of
PPRV with a titre of 104 TCID50 (50% tissue culture infective dose) per ml given at weekly intervals for
4 weeks. The animals are bled 57 days after the last injection (Durojaiye, 1982).
i)
Dispense 1% agar in normal saline, containing thiomersal (0.4 g/litre) or sodium azide (1.25 g/litre) as a
bacteriostatic agent, into Petri dishes (6 ml/5 cm dish).
ii)
Six wells are punched in the agar following a hexagonal pattern with a central well. The wells are 5 mm
in diameter and 5 mm apart.
iii)
The central well is filled with positive antiserum, three peripheral wells with positive antigen, and one
well with negative antigen. The two remaining peripheral wells are filled with test antigen, such that the
test and negative control antigens alternate with the positive control antigens.
iv)
Usually, 13 precipitin lines will develop between the serum and antigens within 1824 hours at room
temperature (Durojaiye et al., 1983). These are intensified by washing the agar with 5% glacial acetic
acid for 5 minutes (this procedure should be carried out with all apparently negative tests before
recording a negative result). Positive reactions show lines of identity with the positive control antigen.
Results are obtained in one day, but the test is not sensitive enough to detect mild forms of PPR due to the
low quantity of viral antigen that is excreted.
b)
Counter immunoelectrophoresis
Counter immunoelectrophoresis (CIEP) is the most rapid test for viral antigen detection (Majiyagbe et al.,
1984). It is carried out on a horizontal surface using a suitable electrophoresis bath, which consists of two
compartments connected through a bridge. The apparatus is connected to a high-voltage source. Agar or
agarose (12%, [w/v]) dissolved in 0.025 M barbitone acetate buffer is dispensed on to microscope slides in
3-ml volumes. From six to nine pairs of wells are punched in the solidified agar. The reagents are the same
as those used for the AGID test. The electrophoresis bath is filled with 0.1 M barbitone acetate buffer. The
pairs of wells in the agar are filled with the reactants: sera in the anodal wells and antigen in the cathodal
wells. The slide is placed on the connecting bridge and the ends are connected to the buffer in the troughs
by wetted porous paper. The apparatus is covered, and a current of 1012 milliamps per slide is applied for
3060 minutes. The current is switched off and the slides are viewed by intense light: the presence of 1
3 precipitation lines between pairs of wells is a positive reaction. There should be no reactions between
wells containing the negative controls.
c)
Immunocapture enzyme-linked immunosorbent assay

Advice on the use and applicability of ELISA methods is available from the OIE Reference Laboratories for
PPR. Both the methods described are available as commercial kits.
The immunocapture enzyme-linked immunosorbent assay (ELISA) (Libeau et al., 1994) using two
monoclonal antibodies (MAb) raised to the N protein, allows a rapid identification of PPRV. The instructions
provided by kit supplier should be followed, but the following shows a typical procedure for the test.
i)
Microtitre ELISA plates (such as Nunc Maxisorp) are coated with 100 l of a capture MAb solution
(diluted according to the instructions of the kit supplier). Coating may be overnight at 4C or for 1 hour
at 37C.
ii)
After washing, 50 l of the sample suspension is added to two wells, and control wells are filled with
buffer.
iii)
Immediately, add 25 l of a detection biotinylated MAb for PPR and 25 l of streptavidin/peroxidase to

two wells.
iv)
The plates are incubated at 37C for 1 hour with constant agitation.
v)
After three vigorous washes, 100 l of ortho-phenylenediamine (OPD) in hydrogen peroxide is added,
and the plates are incubated for 10 minutes at room temperature.
vi)
The reaction is stopped by the addition of 100 l of 1 N sulphuric acid, and the absorbance is
measured at 492 nm on a spectrophotometer/ELISA reader.
1034
The cut-off above which samples are considered to be positive is calculated from the blank as three times
the mean absorbance values.
The test is very specific and sensitive (it can detect 100.6 TCID50/well of PPRV). The results are obtained in
2 hours.
A sandwich form of the immunocapture ELISA is widely used in India (Singh et al., 2004a): the sample is
first allowed to react with the detection MAb and the immunocomplex is then captured by the MAb or
polyclonal antibody adsorbed on to the ELISA plate. The assay shows high correlation to the cell infectivity
assay (TCID50) with a minimum detection limit of 103 TCID50/ml (Saravanan et al., 2008).
d)

Reverse transcription PCR (RT-PCR) techniques based on the amplification of parts of the N and F protein
genes has been developed for the specific diagnosis of PPR (Couacy-Hymann et al., 2002; Forsyth &
Barrett, 1995). This technique is 1000 times more sensitive than classical virus titration on Vero cells
(Couacy-Hymann et al., 2002) with the advantage that results are obtained in 5 hours, including the RNA
extraction, instead of 1012 days for virus isolation. A multiplex RT-PCR, based on the amplification of
fragments of N and M protein genes, has been reported (George et al., 2006). Another format of the N genebased RT-PCR has also been described (Saravanan et al., 2004; Kumar et al., 2007). Instead of analysing
the amplified product the amplicon by agarose gel electrophoresis, it is detected on a plate by ELISA
through the use of a labelled probe. This RT-PCR-ELISA is ten times more sensitive than the classical RTPCR. In recent years, nucleic acid amplification methods for PPR diagnosis have been significantly
improved with quantitative real-time RT-PCR (Adombi et al., 2011; Balamurugan et al., 2010; Bao et al.,
2008; Batten et al., 2011; Kwiatek et al., 2010). This method is also ten times more sensitive than the
conventional RT-PCR, as well as minimising the risk of contamination. The application of nucleic acid
isothermal amplification to PPR diagnosis has also been described (Li et al., 2010). The sensitivity of this
assay seems to be similar to that of the real-time RT-PCR. This assay is simple to implement, rapid and the
result can be read by naked eye.
Because this is a rapidly developing field, users are advised to contact the OIE and FAO1 Reference
Laboratories for PPR (see Table given in Part 4 of this Terrestrial Manual) for advice on the most
appropriate techniques.
e)
Culture and isolation methods

Even when diagnosis has been carried out by rapid techniques, the virus should always be isolated from
field samples in tissue cultures for further studies (Durojaiye et al., 1983; Lefevre & Diallo, 1990).
PPRV may be isolated in primary lamb kidney/lung cells and some cell lines (Vero, B95a). Unfortunately,
PPRV isolation using such cells is not always successful on first passage and may require multiple blind
passages. Recently derivatives of cell lines (Vero, CV1) expressing the morbillivirus receptor, the signalling
lymphocyte activation molecule (SLAM or CD150), have been developed that can enable isolation of field
viruses from pathological specimens in less than 1 week, without requirement for blind passages. These
include a derivative of the monkey cell line CV1 expressing goat SLAM (Adombi et al., 2011) and derivatives
of Vero cells expressing dog SLAM. Monolayer cultures are inoculated with suspect material (swab material,
buffy coat or 10% tissue suspensions) and examined daily for evidence of cytopathic effect (CPE). The CPE
produced by PPRV can develop within 5 days and consists of cell rounding and aggregation culminating in
syncytia formation in lamb kidney cells and cell lines expressing SLAM. In Vero cells, it is sometimes difficult
to see the syncytia. If they exist, they are very small. However, small syncytia are always seen in infected
Vero cells stained with haematoxylin and eosin. Syncytia are recognised by a circular arrangement of nuclei
giving a clock face appearance. Cover-slip cultures may give a CPE earlier than day 5. Some cells may
contain intracytoplasmic and intranuclear inclusions, others may be vacuolated. Similar cellular changes
may be seen in stained histopathological sections of infected tissues. After 56 days, blind passages should
always be carried out as CPE may take time to appear.
3.
Serological tests
The demonstration of antibodies in PPRV infected goats and sheep can be used to support a diagnosis by the
antigen-detection tests. Tests that are routinely used are the virus neutralisation (VN) test and the competitive
ELISA.
Food and Agriculture Organization of the United Nations.
1035
a)
Virus neutralisation (the prescribed test for international trade)

This test is sensitive and specific, but it is time-consuming. The standard neutralisation test is now usually
carried out in 96-well microtitre plates (Rossiter et al., 1985) although roller-tube cultures may also be used.
Vero cells are preferred, but primary lamb kidney cells may also be used.
This test requires the following materials: cell suspensions at 600,000/ml; 96-well cell culture plates; sera to
be titrated (inactivated by heating to 56C for 30 minutes); complete cell culture medium; PPRV diluted to
give 1000, 100, 10 and 1 TCID50/ml.
b)
i)
Dilute the sera at 1/5, and then make twofold serial dilutions in cell culture medium.
ii)
Mix 100 l of virus at 1000 TCID50/ml (to give 100 TCID50 in each well) and 100 l of a given dilution of
serum (using six wells per dilution) in the wells of the cell culture plate.
iii)
Arrange a series of control wells for virus and uninfected cells as follows: six wells with 100 TCID50
(100 l) per well; six wells with 10 TCID50 (100 l) per well; six wells with 1 TCID50 (100 l) per well; six
wells with 0.1 TCID50 (100 l) per well; and six wells with 200 l of virus-free culture (control cells) per
well.
iv)
Make the wells containing the virus controls up to 100 l with complete culture medium, and incubate
the plates for 1 hour at 37C.
v)
Add 50 l of cell suspension to each well. Incubate the plates at 37C in the presence of CO2.
vi)
Read the plates after 1 and 2 weeks of incubation. The results should be as follows: 100% CPE in virus
control wells of 100 and 10 TCID50, 50% CPE for the 1 TCID50 dilution, no CPE for the 0.1 TCID50
dilution, no CPE in wells where the virus had been neutralised by serum during the test, and CPE in
wells where the virus had not been neutralised by serum during the test.

Several competitive ELISAs (C-ELISA) have been described, based on the use of MAbs that recognise virus
proteins. They are of two types: those where the MAb recognises the N protein and use recombinant N
protein produced in baculovirus as the antigen (Choi et al., 2005; Libeau et al., 1995); and those with a viral
attachment protein (H) specific MAb and antigen consisting of purified or part-purified PPRV (vaccine strain)
(Anderson & McKay, 1994; Saliki et al., 1993; Singh et al., 2004b). All the assays work on the principle that
antibodies to PPRV in test sera can block the binding of the MAb to the antigen.
Advice on the use and applicability of ELISA methods is available from the OIE Reference Laboratories for
PPR. Some methods are available as commercial kits. An example protocol for one method (Libeau et al.,
1995) is given below.
i)
Coat microtitre plates (such as Nunc Maxisorp) with 50 l of a predetermined dilution of N-PPR protein
(produced by a recombinant baculovirus) for 1 hour at 37C with constant agitation.
ii)
Wash the plates three times and blot dry.
iii)
Distribute 45 l of blocking buffer (PBS + 0.05% Tween 20 + 0.5% fetal calf serum) to all wells, and
then add 5 l of test sera to test wells in duplicate (at a final dilution of 1/20) and 5 l of the different
control sera (strong positive, weak positive and negative serum) to control wells.
iv)
Add 50 l of MAb diluted to working strength as advised by the supplier, and incubate at 37C for
1 hour.
v)
Wash the plates three times and blot dry.
vi)
Add 50 l of anti-mouse conjugate diluted 1/1000, and incubate at 37C for 1 hour.
vii)
Wash the plates three times.
viii) Prepare OPD in hydrogen peroxide solution. Add 50 l of substrate/conjugate mixture to each well.
Stop the reaction after 10 minutes with 50 l of 1 M sulphuric acid.
ix)
Read on an ELISA reader at 492 nm.

The absorbance is converted to percentage inhibition (PI) using the formula:
PI = 1 (absorbance of the test wells/absorbance of the MAb control wells) 100
Sera showing PI greater than 50% in both duplicate wells are positive.
1036

1.
Background
a)

Sheep and goats that recover from PPR develop an active life-long immunity against the disease (Durojaiye,
1982). Several homologous PPR vaccines are available, being cell culture-attenuated strains of natural
PPRV (Sen et al., 2010). In 1998, the OIE International Committee endorsed the use of such a vaccine in
countries that have decided to follow the OIE pathway for epidemiological surveillance for rinderpest in
order to avoid confusion when serological surveys are performed. There have also been three published
reports on the preliminary results from recombinant capripox-based PPR vaccines that are able to protect
against both capripox and PPR (Berhe et al., 2003; Diallo et al., 2002, Chen et al., 2010). The production
and validation of vaccine from the commercially available attenuated PPRV is described here.
2.
a)

The PPRV vaccine Nigeria 75/1 strain is a live vaccine cultured in Vero cells. The original strain of the virus
was isolated in Nigeria in 1975 (Taylor & Abegunde, 1979). It has been attenuated by serial passages in
Vero cell cultures (Diallo et al., 1989b). The strain provided for vaccine production is the 70th passage in
Vero cells (PPRV 75/1 I.K6 BK2 Vero 70). It is stored in freeze-dried form at 20C and may be obtained
from Reference Laboratories (see Table given in Part 4 of this Terrestrial Manual). Tests of vaccine activity
show that it retained the ability to protect (at a dose of 103 TCID50) up to the 120th passage in Vero cells, the
latest passage tested so far.
b)
Method of culture
Cells
PPR vaccine is produced in Vero cells, which must be free from all bacterial, fungal and viral contamination.
Culture medium
The culture medium consists of minimal essential medium (MEM) supplemented with antibiotics (for
example penicillin + streptomycin at final concentrations of 100 IU [International Units]/ml and 100 g/ml,
respectively), and an antifungal agent (nystatin [Mycostatin] at a final concentration of 50 g/ml). The
medium is enriched with 10% fetal calf serum (complete medium) for cell growth. This proportion of serum is
reduced to 2% for maintenance medium when the cell monolayer is complete.
Primary seed batch of vaccine virus
This consists of virus in its 70th passage in Vero cells (PPRV 75/1 LK6 BK2 Vero 70). The freeze-dried
contents of a flask from the seed bank are reconstituted with 2 ml of sterile water (or cell culture medium
without serum). This liquid is mixed with Vero cells suspended in complete culture medium to provide at
least 0.001 TCID50 per cell. Cell culture dishes are filled with this virus/cell mixture (around 2 107 Vero
cells in a 175 cm2 dish), and are incubated at 37C. The cultured cells are examined regularly to detect a
CPE. The medium is renewed every 2 days, reducing the proportion of serum to 2% once the cell monolayer
is complete. Virus is first harvested when there is 4050% CPE. This viral suspension is stored at 70C.
Successive harvesting is made every 2 days until the CPE reaches 7080%, which is the time for final
freezing of the culture dishes (in general, at least two further harvestings can be made before final freezing
of the culture dishes). All suspensions of virus collected are submitted to two freezethaw cycles, then
added to form a single batch, which serves as the primary seed batch. This is divided into small volumes in
bottles and stored at 70C. The contents of five flasks are thawed and titrated (minimum titre required:
105 TCID50/ml). It is best to freeze-dry this seed in order to store it at 20C. In this case it will be necessary
to titrate the freeze-dried virus (five bottles). A batch made up in this way must pass all tests for sterility.
When preparing seed batches, it is important to avoid infecting the cells with high doses of virus (high
multiplicity of infection), as this will lead to accumulation of defective particles in the viral suspension
produced, which will diminish the titre of subsequent products. On the other hand, very weak multiplicity of
infection (e.g. 0.0001) will prolong the culture time.
1037
Preparation of the working seed batch
This is done under the same conditions as for the primary seed batch. A large stock of virus is formed, from
which the final vaccine will be produced. This batch is distributed into receptacles and stored at 70C. It
must satisfy tests for sterility. Five samples are titrated (minimum titre required: 106 TCID50/ml).
c)
It is necessary to confirm or rule out the presence of PPRV in the product under test. For this purpose, antiPPR serum is used to neutralise the virus in cell culture.
Test procedure
i)
Mix the contents of two vaccine bottles with sterile double-distilled water to provide a volume equal to
the volume before freeze-drying.
ii)
Make tenfold dilutions of the reconstituted vaccine in serum-free culture medium (0.5 ml of viral
suspension + 4.5 ml of medium).
iii)
Make two series of mixtures for virus dilutions from each bottle on a 96-well plate as follows:
Series 1:
Dilutions of viral suspension:

Viral suspension (in l)
Culture medium (in l)
1
50
50
2
50
50
3
50
50
4
50
50
Series 2:
Dilutions of viral suspension:

Viral suspension (in l)
PPR antiserum (in l)
1
50
50
2
50
50
3
50
50
4
50
50
(Note: PPR antiserum used for this purpose is prepared in goats and freeze-dried. It is reconstituted
with 1 ml of sterile double-distilled water in a dilution of 1/10.)
iv)
Incubate the mixtures at 37C for 1 hour
v)
Add to each well 100 l of cells suspended in complete culture medium (30,000 cells/well).
vi)
Incubate the microplate at 37C in the presence of CO2.
vii)
Read the plate after 1015 hours of incubation.
Normally a CPE is present only in the wells containing cells infected with the mixture of virus and culture
medium. If it is detected in the wells of Series 2, it will be necessary to identify PPRV by
immunofluorescence, using a PPR MAb, or by immunocapture (specific PPR MAb, and the immunocapture
test kit are available from the OIE Reference Laboratory for PPR in France [see Table given in Part 4 of this
Terrestrial Manual]). If this identification confirms the presence of PPRV, the PPR antiserum used must have
been too weak, or the batch must be changed. If immunofluorescence or immunocapture is negative, a viral
contaminant must be present, and the material under test must be destroyed.
2.
a)
Vaccine production
This operation is performed on a larger scale. Cells can be infected with virus at a multiplicity of infection as
before or with high doses, e.g. up to 0.01. Products of the various harvests, after two freezethaw cycles,
are brought together (to form the final product) and stored at 70C pending the results of titration and tests
for sterility. If these results are satisfactory, the vaccine is freeze-dried.
b)
Freeze-drying
The freeze-drying medium (Weybridge medium) is composed of 2.5% (w/v) lactalbumin, 5% (w/v) sucrose
and 1% (w/v) sodium glutamate, pH 7.2.
This medium is added to an equal volume of viral suspension for freeze-drying (which may have been
diluted beforehand to provide the desired number of vaccine doses per bottle). The resulting mixture is kept
cool, homogenised, then distributed into bottles and freeze-dried. At the end of a freeze-drying cycle, the
probe is adjusted and kept at 35C for 4 hours. Once this operation has been completed, the bottles are
capped under vacuum. Randomly selected samples (e.g. 5%) of this final batch are submitted to tests for
innocuousness, efficacy and sterility, and residual moisture is estimated by the Karl Fisher method (optimum
3.5%). If the tests give unsatisfactory results, the entire batch is destroyed.
1038
3.
In-process control
Cells used in cultures must be checked for normal appearance and shown to be free from contaminating viruses,
especially bovine viral diarrhoea virus. A virus titration must be undertaken on the seed lot: using MEM (serumfree) medium, a series of tenfold dilutions is made (0.5 ml virus + 4.5 ml diluent) down to 106 of the product to be
titrated. Vero cells from one flask are trypsinised and suspended in complete culture medium at 300,000/ml. They
are distributed on a 96-well plate (30,000 cells per well, equivalent to 100 l of cell suspension). Then, 100 l of
virus diluted tenfold is added to the cells (dilutions ranging from 102 to 106). One row of wells serves as a
control for uninfected cells to which virus-free culture medium (100 l) is added. The plate is incubated at 37C in
the presence of CO2. The plates are read (by examining for CPE) 1015 days after infection.
Virus titre is determined by the SpearmanKrber method. The minimum titre per dose is 102.5.
4.
Batch control
a)
Identity
The contents of one container from each filling lot must be checked for identity by culture after neutralisation
with specific antiserum.
b)
Sterility
This consists of testing for viral, bacterial or fungal contaminants. It is done on cells and sera before their
use in vaccine production, and on the seed stock and the vaccine before and after freeze-drying. Any
product that fails this test for sterility is destroyed.
c)
Safety
This test is done in rodents in order to detect any nonspecific toxicity associated with the product. The test
requires reconstituted vaccine in solvent (mixed contents of five bottles), six guinea-pigs, each weighing
200250 g; ten unweaned mice (1722 g, Swiss line or similar).
Vaccine, 0.5 ml, is injected intramuscularly into a hind limb of two guinea-pigs, 0.5 ml into the peritoneal
cavity of two guinea-pigs, and 0.1 ml into the peritoneal cavity of six mice. Two guinea-pigs and four mice
are kept as uninoculated controls. The animals are observed for 3 weeks. If one guinea-pig or two mice die,
the test must be repeated. Dead animals undergo post-mortem examination to ascertain the cause of death.
At the end of 3 weeks of observation, all animals are killed for post-mortem examination. All the results are
recorded. The vaccine is considered to be satisfactory if, during the first or second test, at least 80% of
animals remain in good health during the period of observation, and no significant post-mortem lesion is
found.
d)
Potency and efficacy in small ruminants

This test requires the following: vaccine reconstituted with normal saline (the mixed contents of five bottles)
to provide 100 doses and 0.1 dose/ml; six goats and six sheep, all approximately 1-year old and free from
antibodies to PPR; sterile syringes and needles; and pathogenic PPRV previously titrated in goats, diluted
with sterile normal saline to provide 103 of the 50% lethal dose for goats (LD50).
Vaccinate two goats and two sheep subcutaneously with 100 doses per animal; vaccinate two goats and two
sheep subcutaneously with 0.1 dose per animal; keep the remaining animals as in-contact controls. The
animals are observed and temperature measurements are recorded daily for 3 weeks. At the end of this
period, blood is taken from all animals for the preparation of sera. All animals are challenged by
subcutaneous injection of a 1 ml suspension of pathogenic PPRV (103 LD50 per animal). The animals are
observed and their body temperature measurements are recorded daily for 2 weeks.
The vaccine is considered to be satisfactory if all vaccinated animals resist the challenge infection, while at
least half of the in-contact controls develop signs of PPR. The serum neutralisation test must be positive for
PPR antibody (in serum diluted at least 1/10) in vaccinated animals only in samples taken 3 weeks after
vaccination. If any of the controls are also positive, the experiment must be repeated using another batch of
pathogenic PPRV. The batch of vaccine is destroyed if vaccinated animals react to the virulent challenge.
1039
Titration of neutralising PPR antibody
This test requires the following: cell suspensions at 600,000/ml; 96-well cell culture plates; sera to be titrated
(inactivated by heating to 56C for 30 minutes); complete cell culture medium; PPRV diluted to give 1000,
100, 10 and 1 TCID50/ml.
Dilute the sera at 1/5, then make a twofold serial dilutions in cell culture medium. Mix 100 l of virus at
1000 TCID50/ml (to give 100 TCID50 in each well) and 100 l of a given dilution of serum (using six wells per
dilution) in the wells of the cell culture plate. Arrange a series of control wells for virus and uninfected cells
as follows: six wells with 100 TCID50 (100 l) per well; six wells with 10 TCID50 (100 l) per well; six wells
with 1 TCID50 (100 l) per well; six wells with 0.1 TCID50 (100 l) per well; and six wells with 200 l of virusfree culture (control cells) per well.
Make the wells containing the virus controls up to 100 l with complete culture medium, and incubate the
plates for 1 hour at 37C. Add 50 l of cell suspension to each well. Incubate the plates at 37C in the
presence of CO2. Read the plates after 1 and 2 weeks of incubation. The results should be as follows: 100%
CPE in virus control wells of 100 and 10 TCID50, 50% CPE for the 1 TCID50 dilution, no CPE for the
0.1 TCID50 dilution, no CPE in wells where the virus had been neutralised by serum during the test, and
CPE in wells where the virus had not been neutralised by serum during the test.
e)
Duration of immunity is at least 3 years.
f)
Stability
Freeze-dried vaccine can be kept for at least 2 years at 28C (although storage at 20C is better),
provided it is stored under vacuum and protected from light. Recently, it has been demonstrated that this
vaccine, suspended in medium containing trehalose and submitted to the ultra-rapid method of dehydration,
can resist at 45C for a period of 14 days with minimal loss of potency (Worrwall et al., 2001).
5.
a)
Safety
See Section C.4.c.
b)
Potency
See Section C.4.d.
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*
* *
NB: There are OIE Reference Laboratories for Peste des petits ruminants
diagnostic tests, reagents and vaccines for Peste des petits ruminants
1043
CHAPTER 2.7.12.
SALMONELLOSIS
(S. abortusovis)
See Chapter 2.9.9. Salmonellosis
*
* *
1044
CHAPTER 2.7.13.
SCRAPIE
SUMMARY
Scrapie is a naturally occurring, infectious, neurodegenerative disease of sheep and goats
characterised by vacuolar or spongy changes in the central nervous system (CNS). It has been
recognised as a clinical disorder for more than 250 years, and is now classified as a transmissible
spongiform encephalopathy (TSE), or prion disease, defined by the accumulation of an abnormal
form (referred to as PrPSc) of a host membrane glycoprotein (prion protein or PrP), in the CNS. In
some animals, PrPSc accumulation is also detectable in lymphoreticular tissues. Polymorphisms of
the sheep PrP gene are associated with susceptibility to scrapie. PrP genotyping has been used as
a tool in the control of classical scrapie, but no genotype appears to be completely resistant to
infection, and the recently identified variant atypical scrapie has been reported in sheep of PrP
genotypes that are apparently resistant to classical scrapie. Classical scrapie is endemic in many
parts of the world, where it has often been introduced by importation. Australia and New Zealand
have maintained freedom by use of strict restrictions on imports and other measures. The infection
in sheep may be passed from ewe to lamb in the period from parturition to weaning. Infection can
also pass horizontally to unrelated sheep or goats, especially when parturition occurs in confined
areas. Fetal membranes are known to be a source of infection, and sheep milk from clinically
affected animals has been demonstrated to transmit disease. The incubation time between primary
infection and clinical disease is nearly always longer than 1 year and may sometimes exceed the
commercial lifespan of the sheep. The majority of cases occur in sheep between 2 and 5 years of
age. Clinical disease only develops if the infection enters the CNS. Atypical scrapie, when it
presents clinically, appears to affect older animals predominantly, and occurs with a geographical
distribution suggestive of a spontaneous disease, although it can be transmitted experimentally.
Identification of the agent: The disease is recognised by the clinical signs, which are variable, but
generally start insidiously with behavioural abnormalities then progress to more obvious
neurological signs, including pruritus, incoordination and poor bodily condition. Clinical diagnosis is
confirmed by the presence of gray matter vacuolation of target areas within the brain tissue and/or
the immuno-detection of disease-specific accumulations of PrPSc in the brain or lymphoreticular
tissues. Immunochemical detection of the protein in brain samples forms the basis of rapid tests
used in active surveillance programmes. In experimental studies of the disease in sheep and goats,
detectable PrPSc accumulation in the brain does not start until several months after challenge, so it
must be remembered that a negative result does not necessarily equate to an unexposed animal.
Detection of PrPSc in lymphoreticular tissues during the incubation period of scrapie in some
animals offers a means of preclinical diagnosis of scrapie infection and may be particularly useful
for surveillance purposes. It can also be performed in vivo using biopsied tissue. It is not, however,
appropriate for atypical cases, or a proportion of classical cases, so can only be used to confirm
presence of infection and cannot be used to prove absence of disease.
Most, but not all, currently recognised forms of scrapie can be transmitted to laboratory rodents by
injecting them with infected brain tissue, but the variable efficiency of transmission coupled with
long incubation times preclude this as a practical diagnostic procedure.
Serological tests: Scrapie infection is not known to elicit any specific immune response and there
is no basis for establishing a diagnosis by detecting specific antibodies.
available.
1045
Chapter 2.7.13. Scrapie
A. INTRODUCTION
Scrapie (also known as la tremblante; Traberkrankheit; Gnubberkrankheit) is a naturally occurring progressive,
fatal, infectious, neurodegenerative disease of sheep and goats that has been recognised for at least 250 years,
and has been reported in Europe, North America, Asia and Africa. Scrapie and other related diseases in humans
and animals were later classified as transmissible spongiform encephalopathies (TSE), although they are now
known as prion diseases. The occurrence of scrapie preceded recognition of other prion diseases of mammals
and so, in retrospect, it is the archetype of prion disorders (Hrnlimann, 2006). Prion diseases are defined by the
accumulation of an abnormal isoform (PrPSc) of the host-encoded protein (PrPC) in the central nervous system
(CNS) in all cases. The abnormal protein may also be detected in other tissues such as in the lymphoreticular
system (LRS) and variably in other tissues/body fluids.
An atypical form of scrapie was first described in Norway in 1998 (Benestad et al., 2008). Subsequently, active
surveillance using rapid immunochemical methods has provided evidence for the widespread occurrence of this
atypical form of scrapie (also known as Nor98), throughout Europe, with reports of cases also in the Falkland
Islands (Epstein et al., 2005) and North America (Loiacono et al., 2009). Although the epidemiology is not
suggestive of transmission in the field (Benestad et al., 2008) this type of scrapie can be transmitted
experimentally (Simmons et al., 2007) Retrospective studies have now identified British cases from the late
1980s, predating active surveillance (EFSA, 2005b). Atypical scrapie has been identified in sheep of genotypes
considered to be resistant to classical scrapie, and in goats (Arsac et al., 2007; Seuberlich et al., 2007).
The interaction of agent variables (particularly strain) and host variables determines the disease phenotype. In
sheep, different PrP genotypes are associated with relative susceptibility to TSEs (Goldmann, 2008).
Polymorphisms at codon 171 are of particular significance in determining overall susceptibility of sheep to
classical scrapie, while variations at 141 and 154 affect susceptibility of sheep to the more recently identified
atypical form of disease (Benestad et al., 2008; Moum et al., 2005; Saunders et al., 2006).
Characterisation of different strains of scrapie isolates has, historically, relied upon transmission to rodents
(biological strain typing) (Bruce et al., 2004), principally using inbred (wild-type) mice, but increasingly also a
number of different transgenic mouse constructs (Groschup & Buschmann, 2008). Molecular TSE-typing uses
differential epitope binding of PrPSc in immuno-histochemistry (IHC) or Western immunoblotting (Jeffrey et al.,
2001). The ability to distinguish scrapie from bovine spongiform encephalopathy (BSE) is of particular importance
in small ruminants because of the zoonotic nature of the latter and the potential for past exposure of small
ruminants to the feed-borne BSE agent. However, the mechanisms by which strain and host parameters influence
disease phenotype are still incompletely understood.
Because of the known inadequacies of baseline (passive) surveillance and the absence of active surveillance
components, the true scrapie status of many countries is unknown, and it is probably impossible to establish
freedom from infection in a national flock without recourse to disproportionate levels of active surveillance. Some
countries have never recorded the disease against a background of general and/or targeted surveillance, while
others have maintained freedom for various periods through rigorous preventative policies and monitoring. The
disease usually occurs in sheep 25 years of age. Rarely are cases present in sheep less than 1 year of age. In
atypical scrapie, significant numbers of cases have been reported in sheep over 5 years of age. In some
instances, the commercial lifespan of the sheep may be too short or exposure has occurred too late in life to allow
the clinical disease to develop. Classical scrapie has also been described in goats, and captive moufflon (Ovis
musimon), a primitive type of sheep. Most breeds of sheep are affected. The infection in sheep may be passed
from ewe to lamb in the period from parturition to weaning. Infection can also pass horizontally to unrelated sheep
or goats, especially when parturition occurs in confined areas. Fetal membranes are known to be a source of
infection, and milk has recently been shown to be infectious to lambs (Konold et al., 2008). Pasture previously
grazed by, or buildings previously inhabited by, infected sheep may also represent a risk. Animals incubating the
disease, and even animals that never develop clinical signs, may still be a source of infection to others.
The biohazard for humans from scrapie diagnostic testing appears to be limited, but appropriate precautions
should be taken. Creutzfeldt-jakob disease (CJD) has been found to occur at no greater frequency in those with
occupations providing closest contact with the agent, but the extreme chemical and physical resistance of the
scrapie agent and the fact that it is experimentally transmissible by injection to a wide spectrum of mammalian
species suggest the prudence of preventing human exposure.
1.
The specific causative agent has never been isolated or described for any prion disease. Characterisation is
therefore based on the identification of phenotypic parameters, such as clinical signs, through histopathological
profiles, to increasingly complex immunochemical and biological parameters.
1046
The clinical signs of scrapie (Ulvund, 2006) usually start insidiously, often with behavioural changes that are
evident only from repeated inspections. These subtle presenting features, which may include apparent confusion,
separation from the flock and a staring gaze, progress to a more definite neurological illness, frequently
characterised by signs of pruritus and ataxia or incoordination of gait. Either the pruritus or the ataxia usually
emerges to dominate the clinical course. Death may occur after a protracted period of only vague neurological
signs or may even occur without premonitory signs. These clinical signs, individually, are not disease-specific and
clinical suspicion of disease should be confirmed by further testing.
Pruritus is recognised principally by compulsive rubbing or scraping against fixed objects, nibbling at the skin and
scratching with a hind foot or horns. This results in extensive loss of wool, particularly over the lateral thorax,
flanks, hindquarters and tail head. The persistence of pruritus often results in localised self-inflicted skin lesions.
These may occur in areas of wool loss and on the poll, face, ears and limbs. A characteristic nibble reflex can
often be elicited by scratching the back, and may also be evoked by the sheeps own scraping movements. Some
sheep or goats with scrapie, however, may not present with evident signs of pruritus.
Ataxia or incoordination of gait may first become apparent as difficulty in positioning the hind limbs on turning,
swaying of the hindquarters and a high stepping or trotting gait of the forelimbs. Stumbling and falling occur, but
the sheep is generally able to quickly regain a standing posture. These signs progress to weakness and
recumbency. Information on the phenotypic variants of scrapie, termed atypical scrapie, or Nor98 suggests
clinical features being dominated by ataxia in the absence of pruritus; circling has also been observed (Benestad
et al., 2003; Konold et al., 2007; Moum et al., 2005; Simmons et al., 2009; Ulvund, 2006). Other signs of scrapie
may include teeth grinding (bruxism), low head carriage, a fine head or body tremor and, rarely, seizures or visual
impairment. In most cases, there is also a loss of bodily condition or weight.
Progress of the clinical disease is very variable, lasting for a week or up to several months, with an inevitably fatal
outcome. There is also variation in the clinical signs among individual animals and in different breeds of sheep.
These variations may be due to the influence of host genotype and strain of agent. Environmental factors may
also have an influence on the disease course. The clinical diagnosis of individual cases of scrapie can therefore
be difficult. The clinical signs may, especially in the early phase of the disease, resemble those of some other
conditions of adult sheep, including ectoparasitism, pseudorabies (Aujeszkys disease), rabies, cerebral listeriosis,
ovine progressive pneumonia (maedi-visna), pregnancy toxaemia (ketosis), hypomagnesaemia and chemical and
plant intoxications.
Video-clips illustrating the clinical signs of scrapie can be viewed on the website of the OIE Reference Laboratory
at Animal Health and Veterinary Laboratories Agency (AHVLA), which is also the European Commission TSE
Community Reference Laboratory1. Other historical videotape footage of classical scrapie signs can also be
sourced (Wells & Hawkins, 2004). Video-clips of atypical scrapie signs are also available (Konold et al., 2007).
Full narratives describing the clinical signs of scrapie are available in the literature (Benestad et al., 2008; Konold
et al., 2007; Ulvund, 2006).
According to the prion hypothesis, demonstration of PrPSc would constitute identification of the agent, but, by
definition, transmission from infected tissues, usually to laboratory rodents by injection, is the only available
means of detection of infectivity. Long incubation periods and the failure of some natural scrapie sources to
transmit to specific mice strains mean that it is impractical to use the criterion of transmissibility for diagnosis.
However, biological characterisation on transmission is an important experimental component of the definition of
any emerging new phenotypic variants of scrapie and for discriminatory approaches to distinguish cases of
scrapie from BSE in sheep or goats (see footnote 1). Given the absence of any specific gross pathological
changes, the laboratory diagnosis of classical scrapie (Gavier-Widen et al., 2005) has, in the past, been reliant on
the demonstration of histopathological changes, notably vacuolation, in the CNS. Prior to the routine use of
immunochemical detection of PrPSc, morphological demonstration of PrPSc in the form of scrapie-associated
fibrils (SAF) was employed as an adjunct to histopathological diagnosis. SAF are visualised in unfixed brain
extracts using negative-stain electron microscopy and have also been recovered from formaldehyde-fixed and
autolysed brain tissue (Chaplin et al., 1998; Stack et al., 1996).
The histopathological diagnosis was historically based on examination of a single section of medulla oblongata
taken at the level of the obex, considered the predilection site for morphological vacuolar changes (Wood et al.,
1997). This approach is still valid for the confirmation of classical scrapie, but it will not detect atypical scrapie.
However, PrPSc detection using IHC examination and/or Western immunoblotting techniques, performed on the
medulla oblongata, have increased the efficiency of the detection methods, and this has now extended to the
active surveillance of large populations using rapid PrP detection tests (see below). Detectable PrPSc precedes
vacuolation and clinical signs, making the immuno-based tests a more sensitive option (Hamir et al., 2001). While
clinically suspect cases of scrapie should, where suitable samples are available, continue to be investigated
European Commission: TSE Community Reference Laboratory Web Resources:

http://www.vla.gov.uk/science/sci_tse_rl_video.htm
1047
initially by histopathological examination for morphological changes, diagnostic criteria must now include the
demonstration of PrPSc in the CNS. The medulla remains the most consistent and appropriate diagnostic level of
the CNS for classical scrapie, however the accumulation pattern of PrPSc in atypical scrapie is different (Benestad
et al., 2008; Moore et al., 2008). In atypical scrapie, the medulla shows only minimal change, while much more
consistent and overt lesions can usually be identified in the cerebellum, thalamus and basal ganglia. Therefore,
taking practical and logistical sampling considerations into account the medulla and cerebellum should both be
examined as a minimum for robust diagnosis. This approach assists in the characterisation of the prion protein
that is present, and contributes to discrimination of disease phenotypes, particularly between classical and
atypical scrapie, and indeed is also sufficient for the discrimination of scrapie and BSE (Baron et al., 2006).
Some commercially available rapid methods for the detection of PrPSc, introduced originally for the diagnosis of
BSE, are also approved for scrapie diagnosis (European Food Safety Authority [EFSA] (2005a). These rapid tests
take the form of Western immunoblot, luminescence (LIA)- or enzyme-linked immunosorbent assay (ELISA)based methods, and provide preliminary screening from which positive or inconclusive results are subject to
examination by confirmatory IHC or Western blot methods. All these methods have been shown to be able to
detect classical scrapie in the appropriate samples (EFSA, 2005a), but some have subsequently been shown to
detect atypical scrapie to a varying degree or not at all. The analytical sensitivity of these kits is kept under review
by the European Commission, and links to information on the performance of currently approved tests can be
found on the Reference Laboratory webpages (see footnote 1).
Failure to observe characteristic histological changes or to detect disease-specific PrP/SAF does not confirm the
absence of the disease; agreement between the results of multiple diagnostic approaches provides the best
assurance of accuracy. Clearly, in surveillance situations where monitoring is for the purpose of obtaining
evidence of freedom from scrapie in small ruminant species, it may be necessary to apply multiple diagnostic
criteria and to use at least two laboratory methods (histopathological and IHC, or immunoblotting) on accurately
sampled CNS tissue (minimum medulla and cerebellum) to maintain a high degree of confidence in negative
results.
Passive surveillance of scrapie, comprising the examination of CNS material from clinically suspect cases, has, in
recent years, been complemented in many countries by active surveillance, targeting healthy adult culls and fallen
stock (diseased or dead animals, also called risk animals) screened at post-mortem using the rapid test methods.
In scrapie, the opportunity also exists for screening approaches that do not rely solely on examination of the CNS
tissue from dead animals to detect exposed animals, but uses the widespread presence of PrPSc in
lymphoreticular tissue in many animals to enable demonstration of infected animals by biopsy of palatine tonsil,
nictitating membrane, superficial lymph nodes or, most recently, rectal mucosa lymphoreticular tissue (Gonzalez
et al., 2006). Any of these tissues can usefully be included in the screening of cull populations too, to maximise
disease detection. It must be noted that not all animals with classical scrapie have detectable lymphoreticular
involvement (Andreoletti et al., 2000; Gonzalez et al., 2006), and no PrPSc has yet been detected in the
lymphoreticular tissues of cases of sheep or goats with atypical scrapie (Benestad et al., 2003). However, the
testing of lymphoreticular tissue offers the opportunity to detect some animals infected with classical scrapie at
relatively early stages of incubation, before the CNS is positive.
Whereas large-scale testing, to determine freedom from scrapie, should include targeted examination of
peripheral tissues as well as the CNS of younger animals, surveillance for prevalence of disease could potentially
limit tissue examination to the CNS of adult sheep and goats for the reasons given above. However, testing to
estimate disease prevalence needs to take into account a number of factors, including the stratification of the
sheep-farming industry, dose or level of infection within particular flocks, frequency of disease and relative
involvement of the LRS in different genotypes, and the effect of genotype/agent strain combination on incubation
period.
The need to distinguish between cases of scrapie and possible BSE in sheep and goats has required the
development of diagnostic methods with the potential to discriminate between the agents causing these
infections. Studies suggest that the conformation of disease-specific PrP produced in BSE-infected sheep is
different from that of disease-specific PrP found in natural sheep scrapie (EFSA, 2005b; Jeffrey et al., 2001; Stack
et al., 2006). These conformational differences may be detected by immunoblotting or IHC techniques using
epitope-specific antibodies. Within the European Union, the strategy for this distinction comprises examination of
source CNS material after initial detection through active or passive surveillance (initial screening), in a primary,
secondary and tertiary phase procedure (see footnote 1), involving a Western immunoblot method capable of
such discrimination, followed by peer review and further investigation by biochemical and IHC methods, and
finally, if necessary, mouse transmission to a standard panel of wild-type mice (see Chapter 2.4.6 Bovine
spongiform encephalopathy). Interpretation of the in-vitro methods (Western immunoblot or ELISA) is reliant on
differences between BSE and scrapie in the N-terminal cleavage site for Proteinase-K digestion of PrPSc. The insitu IHC approach relies on distribution and epitope-specific labelling patterns of PrPSc in brain and
lymphoreticular tissues. Increasingly, for biological characterisation of agent strain, appropriate transgenic mouse
constructs are being used, although comprehensive data comparing models are still sparse.
1048
Quality control (QC), quality assurance (QA) and appropriate positive and negative control samples are essential
parts of testing procedures and advice and control materials can be requested from the OIE Reference
Laboratories.
a)
Sample preparation
Concerns regarding BSE in small ruminant populations and, the recognition of atypical scrapie, have
influenced the strategies for sampling and diagnosis. Although comprehensive sampling and multiple testing
methods would provide the most robust contingencies for these and possible future uncertainties in the
diagnosis of prion diseases of small ruminants, operational factors also determine what is practically and
economically possible. The relative implementation of passive and active surveillance programmes, and the
diagnostic methods applied, further influence sampling strategy. Selection and recommendation of methods
is therefore under constant review.
For routine diagnosis, the sampled CNS material is either stored fresh or frozen for subsequent biochemical
tests or is fixed for histological preparations. Where programmes are in place to identify possible infections
with BSE in small ruminant populations, all sampling should be conducted aseptically, using new sterile
disposable instruments, or instruments sterilised under conditions specified for the decontamination of prions
(see chapter 2.4.6). Cross contamination at necropsy/sampling should be avoided. Thus, in the following
procedures where fresh tissue is sampled for biochemical methods, an aliquot should, if required, be
reserved for transmission studies. Although in many instances disease can be confirmed on autolysed or
suboptimally preserved material, such samples can only provide limited evidence of the absence of scrapie.
Sheep in which clinical disease is suspected (passive surveillance) should be killed by intravenous
injection of barbiturate and the whole brain removed by standard necropsy procedures as soon after death
as possible. Whole brain removal is advisable to allow pathological examinations for differentiation between
possible different TSE phenotypes and differential diagnosis of non-TSE brain disorders. Methods of
subdividing the brain tissue, for application of PrP-detection techniques requiring fresh tissue and for
histological techniques, are dependent on the optimum sensitivities of each of the tests for different brain
areas and the compromise that precisely the same area cannot be used for both fresh/frozen and fixed
tissue approaches. The following protocol is suggested but may be subject to modification to satisfy the
particular portfolio of tests. Further information can be obtained from OIE Reference Laboratories (see
footnote 1 or Table in Part 4 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date
list).
Initially, a coronal block of medulla oblongata inclusive of the obex (see chapter 2.4.6, Figure 1) is taken for
fixation into at least 10 times its volume of 10% formol saline and held for 35 days prior to trimming and
histological processing to paraffin wax for histopathological and IHC examinations. Care should be taken to
ensure that this sample is not frozen. For the detection of PrP (or SAF), fresh tissue samples are taken and
stored frozen (20C or below) prior to extraction of protein. Samples should, if possible, provide 5 g of
tissue. This should be taken initially from the caudal medulla and, if necessary supplemented with brainstem
immediately rostral to the medulla obex sample. Subdivision of this tissue to accommodate multiple
biochemical methods can be achieved by hemisecting in the median plane or by transverse sectioning.
Possible variation in sampling for rapid test requirements at the level of the obex is dealt with below in the
discussion on active surveillance approaches. Where the whole brain is available additional fresh samples
are advocated to minimise false-negative diagnoses, taking into account the possibility that there may be
strain-specific targeting of other parts of the brain. For example, in atypical scrapie, cerebellum, thalamus
and basal ganglia regions provide the optimum sites for testing (Benestad et al., 2008; Moore et al., 2008).
The remaining brain tissue is fixed in approximately 10 times its volume of 10% formol saline for at least
1 week and then cut transversely as required to obtain blocks for histological processing to paraffin wax. The
initial sampling of the single block of the medulla may well be sufficient for IHC and the morphological
diagnosis (see chapter 2.4.6, Fig. 1). Requirements for pathological characterisation or differential diagnosis
can be fulfilled by taking additional areas of the brain stem and, as necessary, representative blocks of all
major brain regions. Sections 5 m in thickness, are stained with haematoxylin and eosin and examined
initially for the morphological changes and, as required, for IHC detection of PrPSc, as outlined below.
For active surveillance programmes, methods for removal of the brainstem via the foramen magnum
using proprietary spoon-shaped instruments, similar to those employed in cattle for sampling for BSE
diagnosis (see chapter 2.4.6, Figure 2) have been devised for sheep. Although not advisable, the approach
can also be used for clinical suspect cases. The minimum sampling is the brainstem at the level of the obex
and, if it is required to also diagnose atypical scrapie, the cerebellum. The brainstem portion is either
hemisectioned in the median plane to provide half (fresh/frozen) for a rapid test and half (fixed) for
histopathology. Alternatively a complete coronal slice inclusive of the obex is fixed and a similar adjacent
caudal medulla slice taken for the rapid test. The complete coronal slice has been recommended in the past,
to establish the symmetry of morphological changes, but with the use of rapid molecular techniques there is
1049
competition between tests for the optimal diagnostic sites at the obex. Some rapid test kits use a core
sampling approach to obtain an appropriate mass of material from the obex region. While hemisectioning of
the obex region of the brainstem will result in loss of the ability to assess vacuolar lesion symmetry, the
greater specificity provided by IHC to detect PrPSc largely offsets this disadvantage. However if this, or a
core sampling approach, is adopted, it becomes critical to ensure that the contralateral target site is not
compromised. The dorsal nucleus of the vagus nerve (the optimal target area for cases of classical scrapie)
is a narrow column and lies close to the midline (see chapter 2.4.6, Figure 3). The options are also
dependent on the specific sampling instruments provided by the test kit manufacturer. For all sampling
methods it is vital that operators are trained and that the training includes instruction in the gross and crosssectional neuroanatomy of the brainstem and the precise location of the target areas for disease-specific
PrPSc accumulation.
For differentiation of classical and atypical cases, portions of cerebellum are required fixed and fresh/frozen.
b)
Histological examination
Morphological changes in the CNS are those of a spongiform encephalopathy comprising principally
vacuolation of neuronal cell bodies and the surrounding neuropil, accompanied by a variable and usually
less conspicuous gliosis (particularly an astrocytic reaction). Typically, the lesions have a bilaterally
symmetrical distribution. There is considerable variation in the distribution pattern of vacuolation and other
changes, but lesions, at least in classical scrapie, are usually most apparent in the brain stem and frequently
affect the dorsal nucleus of the vagus nerve. Care must be taken if interpreting histopathology alone, as
some incidental vacuolation of neurons may also be present in the brains of apparently healthy sheep, albeit
at a low frequency (Wells et al., 2006). There is no direct correlation between the severity of clinical signs
and pathological changes (Begara-McGorum et al., 2000; Fraser, 1976). A clinical diagnosis of suspected
scrapie cannot be refuted by a failure to find significant vacuolar changes in the brain. The absence of
lesions is therefore not evidence of the absence of scrapie infection, as this can exist without either clinical
signs or detectable morphological changes. This is particularly relevant for atypical scrapie, in which there is
no vacuolation in the brainstem. In these cases, vacuolation, if it occurs at all, is generally restricted to the
molecular layer of the cerebellar cortex, the cerebral cortex and the basal ganglion.
Despite such reservations, the histological examination of sections of medulla oblongata at the obex may be
sufficient to confirm a diagnosis, in most cases, of clinically suspect classical scrapie (Gavier-Widen et al.,
2005; Wood et al., 1997). Clearly, the absence of lesions can be established with greatest confidence by
examining a number of areas representative of the whole brain.
c)
Detection of disease-specific forms of PrP

Methods for the demonstration of accumulation of disease-specific forms of PrP in specified target areas
now provide the principal approach to the diagnosis of natural scrapie (Gavier-Widen et al., 2005). In
suspect clinical cases the combined use of IHC and Western immunoblotting is advocated to confirm the
diagnosis. IHC on tissue sections to demonstrate accumulation of PrPSc should be carried out in parallel
with routine histology in suspected cases. Combined use of IHC and Western immunoblotting is also
recommended where histological lesions are mild in severity and considered equivocal. In active
surveillance programmes, the primary diagnosis will usually be accomplished using rapid test methods and,
in the case of positive or inconclusive results, confirmatory methods should also be applied. Test methods
are detailed on the web sites of OIE Reference Laboratories (see chapter 2.4.6). A wide range of antisera
and monoclonal antibodies for PrP detection by immunochemical methods are now in use and some are
Immunohistochemical methods
Disease-specific PrPSc in scrapie-affected brain is demonstrated by IHC on routinely formalin-fixed

material by the application of a variety of epitope demasking techniques and the use of appropriate
antibodies to PrP (see footnote 1 or Table in Part 4 of this Terrestrial Manual or consult the OIE Web
site for the most up-to-date list). Recognition of morphological disease-specific immunolabelling
configurations, their cellular associations and neuroanatomical distribution patterns provide the basis
for a confirmatory diagnosis in classical (Ryder et al., 2001) and in atypical (Benestad et al., 2008)
scrapie. The method used at the European TSE Community Reference Laboratory, AHVLA, is provided
in the following link http://www.defra.gov.uk/vla/science/docs/sci_tse_rl_prp_ihc.pdf. In recognition of the
distribution of generic skills in national reference laboratories, and the power of the IHC approach, variation
in methodology is possible from laboratory to laboratory, subject to appropriate proficiency testing and
quality assurance.
If histopathological examination and IHC results cannot be achieved, e.g. because of autolysis (owing to the
poor state of the sample, i.e. severely autolysed cases), then immunoblotting, the ELISA methods and SAF
detection are the remaining test options available. Similarly, these methods can also be applied in
1050
circumstances when, sometimes in error at necropsy, CNS material intended for fixation and histological
examination has been frozen. IHC methods can still be applied to such samples if they are subsequently
fixed, but the ability to identify anatomical sites may be compromised, meaning that any negative result
must be qualified. With modification, the method for SAF detection may also be applied successfully to
formalin-fixed tissue (Chaplin et al., 1998). Because of its lower diagnostic sensitivity, SAF detection should
not be used as a sole test if an immunochemical method is possible.
Western immunoblot methods
All Western immunoblotting techniques rely on detergent extraction followed by treatment with proteinase K
enzyme to digest any normal host protein (PrPc). This leaves only PrPres (the truncated, partially proteaseresistant form of the abnormal prion protein [PrPSc]) to be bound by a specific antibody, which provides a detection
system in positive brain samples (see also chapter 2.4.6). A diagnosis based on the detection of PrPres by
Western immunoblotting for classical scrapie cases requires that a wide region of three immunostained bands
corresponding to proteins of molecular mass 19 kDa (unglycosylated PrPres) to 32 kD (diglycosylated PrPres) be
present in the proteinase-K-treated scrapie sample lanes only, and that control sample lanes provide appropriate
comparisons. Several sensitive Western immunoblotting methods for the detection of ovine classical scrapie
PrPres have been published (Arsac et al., 2007; Baron et al., 2006; Stack, 2004).
For atypical scrapie cases four protein bands are visualised by Western immunoblotting, ranging from
19 kDa (unglycosylated PrPres) to 27 kDa (diglycosylated PrPres) with a fourth band below 15 kDa. The
techniques used to detect atypical scrapie employ a reduced concentration of proteinase K enzyme in the
procedure (Arsac et al., 2007; Baron et al., 2006; Benestad et al., 2008).
The original technique used for diagnosis of BSE, which has been referred to as the OIE Western
immunoblotting technique (see footnote 1; Stack, 2004; Stack et al., 2006) relies on detergent extraction of
large amounts of fresh brain material (nominally 4 g) followed by ultracentrifugation to concentrate the PrP
and finally the proteinase K treatment is applied. This technique will also detect classical and atypical
scrapie samples.
Rapid test methods
Rapid immunodiagnostic test formats for the detection of PrPSc in small ruminant brain tissue have been
developed and have been evaluated for diagnostic use (EFSA, 2005a), and these are all commercially
available. Reference should be made to instructions provided by commercial manufacturers, which will have
been subject to approval before use, and subsequent quality assurance. Deviation from test methods
provided by commercial manufacturers is not normally permitted, and not recommended.
The rapid tests rely on the optimisation of the reagents used for extraction and digestion and specific
antibodies for detection, which negates the need for lengthy ultracentrifugation steps. The tests require fresh
medulla taken at the obex or just caudal to the obex. Most rapid tests use less that 0.5 g of material and
many sampling tools are designed to sub-sample precise amounts. However, to allow for possible additional
testing at least 1 g of initial sample is advised. Some laboratories use the OIE Western immunoblotting
technique (if enough tissue is available) to confirm any weak-positive samples that are initially detected
using a rapid test (see footnote 1). The increased amount or concentration of PrPres extracted by
ultracentrifugation from the larger aliquot of brain tissue can give improved sensitivity. Rapid test methods
have been used to identify atypical scrapie cases (EFSA, 2005b) and detection is optimised if the
appropriate brain sample (such as cerebellum) is tested.
Prospects for more sensitive diagnostic tests for scrapie and other TSEs are mainly directed at present on the
refinement of existing methods and the development of new approaches to the detection of disease-specific
forms of PrP. Achievement of the consistent performance of rapid test methods for the primary diagnostic
approach is paramount, particularly with regard to the capacity of tests to recognise both classical and atypical
scrapie phenotypes. Overall diagnostic sensitivity is strongly influenced by the accuracy of sampling.
d)
Other diagnostic tests

As for BSE of cattle (see chapter 2.4.6) tests that can be applied effectively to the live animal to detect
scrapie cases in the early stage of incubation remain elusive, despite several avenues of research. The
pursuit of non-prion protein biomarkers, including possibly through metabolomic or proteomic approaches,
may offer prospects but there are constraints, including that the key tissue is accessible and that specificity
is demonstrable.
2.
Serological tests
A serological immune response to the scrapie agent has not been detected.
1051
3.
Genetic screening
Scrapie control and elimination strategies based on genetic selection for resistance to classical scrapie have been
deployed successfully in some countries. Selection is made on determination of the common polymorphisms of
the sheep PrP gene. As an aid to the control of classical scrapie: breeding stock, particularly rams, of appropriate
PrP genotype can be selected to produce progeny with reduced risk of developing disease (Dawson et al., 1998).
Such genotyping services are available on a commercial basis in North America and in several countries in
Europe. The test is performed using DNA extracted from white blood cells obtained from ethylene diamine tetraacetic acid (EDTA)-treated blood samples. (Other tissue such as skin [e.g. ear punches] can also be used, as can
other tissues such as brain, for screening cull population samples.) Selection of breeding stock can be based on
the most scrapie-resistant animals, homozygous for arginine at codon 171, thereby reducing the incidence of
classical scrapie in individual flocks. However, these animals are not always common in flocks, and in some
breeds the genotype is actually absent.
A strategic approach to eliminating scrapie infection from national sheep flocks or geographical regions by
adopting a national genetic breeding programme is possibly premature. The shortage of sheep that are
homozygous for arginine at codon 171 is one factor. The lack of data on the effects of a high prevalence of such a
genotype on productivity, resistance to diseases other than scrapie and viability in general is another factor.
Furthermore, there is, as yet, insufficient knowledge concerning the prevalence and epidemiology of atypical
scrapie, although early indications are that an alternative breeding strategy would be required for this form of the
disease. Decisions on the appropriateness of such programmes must take account of a thorough evaluation of
the current national/regional/local scrapie situation, the availability of replacement resistant sheep, the sheep
importation policy, availability of testing facilities and the desirability and support of the sheep industry; especially
the willingness of sheep breeders to commit themselves to the programme for a long period of time.

There are no biological products available.
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scrapie by the detection of fibrils and the abnormal protein isoform. In: Methods in Molecular Medicine: Prion
Diseases, Ridley R.M. & Baker H.F., eds. Humana Press Inc., Totowa, New Jersey, USA, 85103.
ULVUND M.J. (2006). Clinical findings in scrapie. In: Prions in Humans and Animals, Hrnlimann B., Riesner D. &
Kretzschmar H., eds. de Gruyter, Berlin, Germany, 398407.
WELLS G.A.H. & HAWKINS S.A.C. (2004). Animal models of transmissible spongiform encephalopathies:
experimental infection, observation and tissue collection. In: Techniques in Prion Research. Lehmann S. & Grassi
J., eds. Birkhuser Verlag, Switzerland, 3771.
WELLS G.A.H., RYDER S.J. & HADLOW W.J. (2006). The pathology of prion diseases in animals. In: Prions in
Humans and Animals. Hrnlimann B., Riesner D. & Kretzschmar H., eds. de Gruyter, Berlin, Germany, 315327.
WOOD J.L.N., MCGILL I.S., DONE S.H. & BRADLEY R. (1997). Neuropathology of scrapie: a study of the distribution
patterns of brain lesions in 222 cases of natural scrapie in sheep, 19821991. Vet. Rec., 140, 167174.
*
* *
NB: There are OIE Reference Laboratories for Scrapie
diagnostic tests and reagents for scrapie
1054
CHAPTER 2.7.14.
SHEEP POX AND GOAT POX
SUMMARY
Sheep pox and goat pox are viral diseases of sheep and goats characterised by fever, generalised
papules or nodules, vesicles (rarely), internal lesions (particularly in the lungs), and death. Both
diseases are caused by strains of capripoxvirus, all of which can infect sheep and goats. Although
most of the strains examined cause more severe clinical disease in either sheep or goats, some
strains have been isolated that are equally pathogenic in both species.
Capripox is endemic in Africa north of the Equator, the Middle East, Turkey, Iran, Iraq, Afghanistan,
Pakistan, India, Nepal, parts of the Peoples Republic of China and Bangladesh. The most recent
outbreaks occurred in Mongolia in 2008 and 2009, an isolated outbreak in Greece in 2008, and in
Kazakhstan and Azerbaijan in 2009. Goat pox has been established in Vietnam since 2005. The
first goat pox outbreak in Chinese Taipei occurred in 2008 and in 2010 the disease reoccurred and
was declared endemic.
Identification of the agent: Laboratory confirmation of capripox is most rapid using the
polymerase chain reaction (PCR) method or by the identification of typical capripox virions using
the transmission electron microscope in combination with a clinical history consistent with
generalised capripox infection. The capripox virion is distinct from that of the other poxvirus
commonly infecting sheep and goats a parapoxvirus that causes orf or contagious pustular
dermatitis. A precipitating antigen can be identified by an agar gel immunodiffusion test (AGID)
using biopsy material, however due to low sensitivity and cross reactivity with parapoxvirus, this is
no longer recommended. Capripoxvirus will grow on tissue culture of ovine, caprine or bovine
origin, although field isolates may require up to 14 days to grow or require one or more additional
tissue culture passage(s). The virus causes intracytoplasmic inclusions, clearly seen using
haematoxylin and eosin staining. The antigen can also be detected in tissue culture using specific
sera and immunoperoxidase or immunofluorescence techniques. Capripoxvirus antigen and
inclusion bodies may be seen in stained cryostat or paraffin sections of biopsy or post-mortem
lesion material.
An antigen-detection enzyme-linked immunosorbent assay (ELISA) using a polyclonal detection
serum raised against a recombinant immunodominant antigen of capripoxvirus has been
developed. Genome detection by a conventional or a real-time polymerase chain reaction (PCR)
method using capripoxvirus-specific primers has also been reported.
Serological tests: The virus neutralisation test is the most specific serological test, but because
immunity to capripox infection is predominantly cell mediated, the test is not sufficiently sensitive to
identify animals that have had contact with the virus and developed only low levels of neutralising
antibody. The AGID and indirect immunofluorescence tests are less specific due to cross-reactions
with antibody to other poxviruses. Western blotting using the reaction between the P32 antigen of
capripoxvirus with test sera is both sensitive and specific, but is expensive and difficult to carry out.
The use of this antigen or some other appropriate antigens expressed by a suitable vector, in an
ELISA offers the prospect of an acceptable and standardised serological test.
Requirements for vaccines: Live and inactivated vaccines have been used for the control of
capripox. All strains of capripoxvirus so far examined share a major neutralisation site and will cross
protect. Inactivated vaccines give, at best, only short-term immunity.
1055
Chapter 2.7.14 Sheep pox and goat pox
A. INTRODUCTION
Sheep pox and goat pox (capripox) are endemic in central and North Africa, the Middle East, India, China
(Peoples Rep. of), Vietnam and Chinese Taipei. Capripox is caused by strains of capripoxvirus and produces a
characteristic clinical disease in fully susceptible breeds of sheep and goats and would usually be difficult to
confuse with any other disease. In indigenous animals, generalised disease and mortality are less common,
although they are seen where disease has been absent from an area or village for a period of time, when
intensive husbandry methods are introduced, or in association with other disease agents, such as peste des petits
ruminants virus or foot and mouth disease virus.
Capripox is a major constraint to the introduction of exotic breeds of sheep and goats, and to the development of
intensive livestock production. Strains of capripoxvirus that cause lumpy skin disease (such as Neethling), are
also found in cattle, but there is no evidence that these strains will naturally cause disease in sheep and goats.
The geographical distribution of lumpy skin disease partially differs from that of sheep pox and goat pox.
Strains of capripoxvirus do pass between sheep and goats, although most cause more severe clinical disease in
only one species; recombination also occurs between these strains, producing a spectrum showing intermediate
host preferences and a range of virulence. Some strains are equally pathogenic in both sheep and goats.
Capripox has the potential to spread and become established in countries outside its normal distribution. In 1983
it spread into Italy, in 1985 and 1989 into Cyprus, and in 1988 and numerous subsequent occasions into Greece,
but did not become established in these countries. In 1984, however, it spread into Bangladesh where it has
persisted. In 2005, an outbreak in goats in Vietnam indicated that capripox has a wider distribution than previously
recognised.
The incubation period is between 8 and 13 days following contact between an infected and susceptible animals. It
may be as short as 4 days following experimental infection by intradermal inoculation or mechanical transmission
by insects. Some breeds of European sheep, such as Soay, may die of acute infection before the development of
skin lesions. In other breeds there is an initial rise in rectal temperature to above 40C, followed in 25 days by
the development of, at first, macules small circumscribed areas of hyperaemia, which are most obvious on
unpigmented skin and then of papules hard swellings of between 0.5 and 1 cm in diameter which may cover
the body or be restricted to the groin, axilla and perineum. Papules may be covered by fluid-filled vesicles, but this
is rare. A flat haemorrhagic form of capripox has been observed in some breeds of European goat, in which all
the papules appear to coalesce over the body; this form is always fatal.
Within 24 hours of the appearance of generalised papules, affected animals develop rhinitis, conjunctivitis and
enlargement of all the superficial lymph nodes, in particular the prescapular lymph nodes. Papules on the eyelids
cause blepharitis of varying severity. As the papules on the mucous membranes of the eyes and nose ulcerate,
so the discharge becomes mucopurulent, and the mucosae of the mouth, anus, and prepuce or vagina become
necrotic. Breathing may become laboured and noisy due to pressure on the upper respiratory tract from the
swollen retropharyngeal lymph nodes, due to the developing lung lesions.
If the affected animal does not die in this acute phase of the disease, the papules start to become necrotic from
ischaemic necrosis following thrombi formation in the blood vessels at the base of the papule. In the following
510 days the papules form scabs, which persist for up to 6 weeks, leaving small scars. The skin lesions are
susceptible to fly strike, and secondary pneumonia is common. Anorexia is not usual unless the mouth lesions
physically interfere with feeding. Abortion is rare.
On post-mortem examination of the acutely infected animal, the skin lesions are often less obvious than on the
live animal. The mucous membranes appear necrotic and all the body lymph nodes are enlarged and
oedematous. Papules, which may be ulcerated, can usually be found on the abomasal mucosa, and sometimes
on the wall of the rumen and large intestine, on the tongue, hard and soft palate, trachea and oesophagus. Pale
areas of approximately 2 cm in diameter may occasionally be seen on the surface of the kidney and liver, and
have been reported to be present in the testicles. Numerous hard lesions of up to 5 cm in diameter are commonly
observed throughout the lungs, but particularly in the diaphragmatic lobes.
The clinical signs and post-mortem lesions vary considerably with breed of host and strain of capripoxvirus.
Indigenous breeds are less susceptible and frequently show only a few lesions, which could be confused with
insect bites or contagious pustular dermatitis. However, lambs that have lost their maternally derived immunity,
animals that have been kept isolated and animals brought into endemic areas from isolated villages, particularly if
they have been subjected to the stress of moving long distances and mixing with other sheep and goats, and their
pathogens, can often be seen with generalised and sometimes fatal capripox. Invariably there is high mortality in
unprotected imported breeds of sheep and goats following capripoxvirus infection. Capripox is not infectious to
humans.
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1.
Sample collection, submission and preparation
Material for virus isolation and antigen detection should be collected by biopsy or at post-mortem from skin
papules, lung lesions or lymph nodes. Samples for virus isolation and antigen-detection enzyme-linked
immunosorbent assay (ELISA) should be collected within the first week of the occurrence of clinical signs, before
the development of neutralising antibodies. Samples for genome detection by polymerase chain reaction (PCR)
may be collected when neutralising antibody is present. Buffy coat from blood collected into EDTA (ethylene
diamine tetra-acetic acid) during the viraemic stage of capripox (before generalisation of lesions or within 4 days
of generalisation), can also be used for virus isolation. Samples for histology should include tissue from the
surrounding area and should be placed immediately following collection into ten times the sample volume of 10%
formalin.
Tissues in formalin have no special transportation requirements. Blood samples, for virus isolation from the buffy
coat, should be collected in tubes containing anticoagulant, placed immediately on ice and processed as soon as
possible. In practice, the blood samples may be kept at 4C for up to 2 days prior to processing, but should not be
frozen or kept at ambient temperatures. Tissues and dry scabs for virus isolation, antigen detection and genome
detection should preferably be kept at 4C, on ice or at 20C. If it is necessary to transport samples over long
distances without refrigeration, the medium should contain 10% glycerol; the samples should be of sufficient size
that the transport medium does not penetrate the central part of the biopsy, which should be used for virus
isolation/detection.
Material for histology should be prepared by standard techniques and stained with haematoxylin and eosin (H&E).
Lesion material for virus isolation and antigen detection are homogenised. The following is an example of one
technique for homogenisation: The tissue is minced using sterile scissors and forceps, and then ground with a
sterile pestle in a mortar with sterile sand and an equal volume of sterile phosphate buffered saline (PBS)
containing sodium penicillin (1000 international units [IU]/ml), streptomycin sulphate (1 mg/ml), mycostatin
(100 IU/ml) or fungizone (2.5 g/ml) and neomycin (200 IU/ml). The homogenised suspension is freezethawed
three times and then partially clarified by centrifugation using a bench centrifuge at 600 g for 10 minutes. In cases
where bacterial contamination of the sample is expected (such as when virus is isolated from skin samples), the
supernatant can be filtered through a 0.45 m pore size filter after the centrifugation step. Buffy coats may be
prepared from 58 ml unclotted blood by centrifugation at 600 g for 15 minutes; the buffy coat is carefully
removed into 5 ml of cold double-distilled water using a sterile Pasteur pipette. After 30 seconds, 5 ml of cold
double-strength growth medium is added and mixed. The mixture is centrifuged at 600 g for 15 minutes, the
supernatant is discarded and the cell pellet is suspended in 5 ml of growth medium, such as Glasgows modified
Eagles medium (GMEM). After centrifugation at 600 g for a further 15 minutes, the resulting pellet is suspended
in 5 ml of fresh GMEM. Alternatively, the buffy coat may be separated from a heparinised sample using a Ficoll
gradient.
a)
Culture
Capripoxvirus will grow in tissue culture of bovine, ovine or caprine origin, although primary or secondary
cultures of lamb testis (LT) or lamb kidney (LK) cells are considered to be the most susceptible, particularly
those derived from a wool sheep breed. The following is an example of an isolation technique; Either 1 ml of
buffy coat cell suspension or 1 ml of clarified biopsy preparation supernatant is inoculated on to a 25 cm2
tissue culture flask of 90% confluent LT or LK cells, and supernatant is allowed to absorb for 1 hour at 37C.
The culture is then washed with warm PBS and covered with 10 ml of a suitable medium, such as GMEM,
containing antibiotics and 2% fetal calf serum. If available, tissue culture tubes containing LT or LK cells and
a flying cover-slip, or tissue culture microscope slides, are also infected.
The flasks should be examined daily for 714 days for evidence of cytopathic effect (CPE). Contaminated
flasks should be discarded. Infected cells develop a characteristic CPE consisting of retraction of the cell
membrane from surrounding cells, and eventually rounding of cells and margination of the nuclear
chromatin. At first only small areas of CPE can be seen, sometimes as soon as 4 days after infection; over
the following 46 days these expand to involve the whole cell sheet. If no CPE is apparent by day 7, the
culture should be freezethawed three times, and clarified supernatant inoculated on to fresh LT or LK
culture. At the first sign of CPE in the flasks, or earlier if a number of infected cover-slips are being used, a
cover-slip should be removed, fixed in acetone and stained using H&E. Eosinophilic intracytoplasmic
inclusion bodies, which are variable in size but up to half the size of the nucleus and surrounded by a clear
halo, are indicative of poxvirus infection. Syncytia formation is not a feature of capripoxvirus infection. If the
CPE is due to capripoxvirus infection of the cell culture, it can be prevented or delayed by inclusion of
specific anti-capripoxvirus serum in the medium; this provides a presumptive identification of the agent.
1057
Some strains of capripoxvirus have been adapted to grow on African green monkey kidney (Vero) cells, but
these are not recommended for primary isolation.
Electron microscopy
Material from the original suspension is prepared for transmission electron microscope examination, prior to
centrifugation, by floating a 400-mesh hexagon electron microscope grid, with pileoform-carbon substrate
activated by glow discharge in pentylamine vapour, on to a drop of the suspension placed on parafilm or a
wax plate. After 1 minute, the grid is transferred to a drop of Tris/EDTA buffer, pH 7.8, for 20 seconds and
then to a drop of 1% phosphotungstic acid, pH 7.2, for 10 seconds. The grid is drained using filter paper, airdried and placed in the electron microscope. The capripox virion is brick shaped, covered in short tubular
elements and measures approximately 290 270 nm. A host-cell-derived membrane may surround some of
the virions, and as many as possible should be examined to confirm their appearance (Kitching & Smale,
1986).
The virions of capripoxvirus are indistinguishable from those of orthopoxvirus, but, apart from vaccinia virus,
no orthopoxvirus causes lesions in sheep and goats. However, capripoxvirus is distinguishable from the
virions of parapoxvirus, that cause contagious pustular dermatitis, as they are smaller, oval in shape, and
each is covered in a single continuous tubular element, which appears as striations over the virion.
Histology
Following preparation, staining with H&E, and mounting of the formalin-fixed biopsy material, a number of
sections should be examined by light microscopy. On histological examination, the most striking aspects of
acute-stage skin lesions are a massive cellular infiltrate, vasculitis and oedema. Early lesions are
characterised by marked perivascular cuffing. Initially infiltration is by macrophages, neutrophils and
occasionally eosinophils, and as the lesion progresses, by more macrophages, lymphocytes and plasma
cells. A characteristic feature of all capripoxvirus infections is the presence of variable numbers of sheep
pox cells in the dermis. These sheep pox cells can also occur in other organs where microscopic lesions of
sheep and goat pox are present. These cells are large, stellate cells with eosinophilic, poorly defined
intracytoplasmic inclusions and vacuolated nuclei. Vasculitis is accompanied by thrombosis and infarction,
causing oedema and necrosis. Epidermal changes consist of acanthosis, parakeratosis and hyperkeratosis.
Changes in other organs are similar, with a predominant cellular infiltration and vasculitis. Lesions in the
upper respiratory tract are characterised by ulceration.
Animal inoculation
Clarified biopsy preparation supernatant (see Section B.1.a Culture) may also be used for intradermal
inoculation into susceptible lambs. These lambs should be examined daily for evidence of typical skin
lesions of capripox.
b)
Fluorescent antibody tests
Capripoxvirus antigen can also be identified on infected cover-slips or tissue culture slides using fluorescent
antibody tests. Cover-slips or slides should be washed and air-dried and fixed in cold acetone for
10 minutes. The indirect test using immune sheep or goat sera is subject to high background colour and
nonspecific reactions. However, a direct conjugate can be prepared from sera from convalescent sheep or
goats or from rabbits hyperimmunised with purified capripoxvirus. Uninfected tissue culture should be
included as a negative control because cross-reactions, due to antibodies to cell culture antigens, can cause
problems. The fluorescent antibody tissue section technique has also been used on cryostat-prepared
slides.
An agar gel immunodiffusion (AGID) test has been used for detecting the precipitating antigen of
capripoxvirus, but has the disadvantage that this antigen is shared by parapoxvirus. Agarose (1%) is
prepared in borate buffer, pH 8.6, dissolved by heating, and 2 ml is poured on to a glass microscope slide
(76 26 mm). When the agar has solidified, wells are cut to give a six-well rosette around a central well.
Each well is 5 mm in diameter, with a distance of 7 mm between the middle of the central well and the
middle of each peripheral well. The wells are filled as follows: 18 l of the lesion suspension is added to
three of the peripheral wells, alternately with positive control antigen, and 18 l of positive capripoxvirus
control serum is added to the central well. The slides are placed in a humidified chamber at room
temperature for 48 hours, and examined for visible precipitation lines using a light box. The test material is
positive if a precipitation line develops with the control serum that is confluent with that produced by the
positive control antigen. This test will not, however, distinguish between capripox infection and contagious
pustular dermatitis (orf).
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To prepare antigen for the AGID, one of two 125 cm2 flasks of LT or LK cells is infected with capripoxvirus,
and harvested when there is 90% CPE (812 days). The flask is freezethawed twice, and the cells are
shaken free of the flask. The contents are centrifuged at 4000 g for 15 minutes, most of the supernatant is
decanted and stored, and the pellet is resuspended in the remaining supernatant. The cells should be lysed
using an ultrasonic probe for approximately 60 seconds. This homogenate is then centrifuged as before, the
resulting supernatant being pooled with that already collected. The pooled supernatant is added to an equal
volume of saturated ammonium sulphate at pH 7.4 and left at 4C for 1 hour. This solution is centrifuged at
4000 g for 15 minutes, and the precipitate is collected and resuspended in a small volume of 0.8% saline for
use in the AGID test. The uninfected flask is processed in an identical manner throughout, to produce a
tissue culture control antigen (Kitching et al., 1986a).
Following the cloning of the highly antigenic capripoxvirus structural protein P32, it is possible to use
expressed recombinant antigen for the production of diagnostic reagents, including the raising of P32
monospecific polyclonal antiserum and the production of monoclonal antibodies (MAbs) (Carn, 1995). Using
hyperimmune rabbit antiserum raised by inoculation of rabbits with purified capripoxvirus, capripox antigen
from biopsy suspensions or tissue culture supernatant can be trapped on an ELISA plate. The presence of
the trapped antigen can then be detected using guinea-pig serum raised against the group-specific structural
protein P32, commercial horseradish-peroxidase-conjugated rabbit anti-guinea-pig immunoglobulin and a
chromogen/substrate solution.
c)

A conventional or a real-time PCR method can be used to detect the capripoxvirus genome in biopsy or
tissue culture samples. Primers for the viral attachment protein gene and the viral fusion protein gene are
specific for all the strains within the genus Capripoxvirus (Heine et al., 1999; Ireland & Binepal, 1998). It is
not possible to distinguish between strains of capripoxvirus from cattle, sheep or goats using serological
techniques. Strains of virus can be identified using sequence and phylogenetic analysis (Le Goff et al.,
2009). Strains can also be characterised by comparing the genome fragments generated by HindIII digestion
of their purified DNA (Black et al., 1986; Kitching et al., 1986). Using this techniques and by sequencing of
the genome, differences between isolates from the different species have been identified (Hosamani et al.,
2004), but these are not consistent and there is evidence for the movement of strains between species and
recombination between strains in the field (Gershon et al., 1989).
The conventional gel-based PCR method described in Chapter 2.4.14 Lumpy skin disease can be used for
the detection of sheep pox and goat pox viral DNA. Recently described real-time PCR methods are reported
to be faster and have higher sensitivity (Balinsky et al., 2008; Bowden et al., 2008).
2.
Serological tests
a)
A test serum can either be titrated against a constant titre of capripoxvirus (100 TCID50 [50% tissue culture
infective dose]) or a standard virus strain can be titrated against a constant dilution of test serum in order to
calculate a neutralisation index. Because of the variable sensitivity of tissue culture to capripoxvirus, and the
consequent difficulty of ensuring the use of 100 TCID50, the neutralisation index is the preferred method,
although it does require a larger volume of test sera. The test is described using 96-well flat-bottomed tissue
culture grade microtitre plates, but it can be performed equally well in tissue culture tubes with the
appropriate changes to the volumes used, although it is more difficult to read an end-point in tubes. The use
of Vero cells in the virus neutralisation test has been reported to give more consistent results (Kitching &
Taylor, 1985).
Test procedure
i)
Test sera including a negative and a positive control are diluted 1/5 in Eagles/HEPES (N-2hydroxyethylpiperazine, N-2-ethanesulphonic acid) and inactivated at 56C for 30 minutes.
ii)
Next, 50 l of the first inactivated serum is added to columns 1 and 2, rows A to H of the microtitre
plate. The second serum is placed in columns 3 and 4, the third in columns 5 and 6, the positive control
serum is placed in columns 7 and 8, the negative control serum is placed in columns 9 and 10, and
50 l of Eagles/HEPES without serum is placed in columns 11 and 12 and to all wells of row H.
iii)
A reference strain of capripoxvirus, usually a vaccine strain known to grow well in tissue culture, with a
titre of over log10 6 TCID50 per ml is diluted in Eagles/HEPES in bijoux bottles to give a log dilution
series of log10 5.0; 4.0; 3.5; 3.0; 2.5; 2.0; 1.5 TCID50 per ml (equivalent to log10 3.7; 2.7; 2.2; 1.7; 1.2;
0.7; 0.2 TCID50 per 50 l).
1059
iv)
Starting with row G and the most diluted virus preparation, 50 l of virus is added to each well in that
row. This is repeated with each virus dilution, the highest titre virus dilution being placed in row A.
v)
The plates are covered and incubated for 1 hour at 37C.
vi)
LT cells are prepared from pregrown monolayers as a suspension of 105 cells/ml in Eagles medium
containing antibiotics and 2% fetal calf serum. Following incubation of the microtitre plates, 100 l of
cell suspension is added to all the wells, except wells H11 and H12, which serve as control wells for
the medium. The remaining wells of row H are cell and serum toxicity controls.
vii)
The microtitre plates are covered and incubated at 37C for 9 days.
viii) Using an inverted microscope, the monolayers are examined daily starting at day 4 for evidence of
CPE. There should be no CPE in the cells of row H. Using the 0240 KSGP vaccine strain of
capripoxvirus, the final reading is taken on day 9, and the titre of virus in each duplicate titration is
calculated according to the Krber method. If left longer, there is invariably a breakthrough of virus in
which virus that was at first neutralised appears to disassociate from the antibody.
ix)
Interpretation of the results: The neutralisation index is the log titre difference between the titre of the
virus in the negative serum and in the test serum. An index of 1.5 is positive. The test can be made
more sensitive if serum from the same animal is examined before and after infection. Because
immunity to capripox is predominantly cell mediated, a negative result, particularly following vaccination
in which the response is necessarily mild, does not imply that the animal from which the serum was
taken is not protected.
A constant-virus/varying-serum method has been described using serum dilutions in the range 1/5 to
1/500 and fetal calf muscle cells. Because these cells have a lower sensitivity to capripoxvirus than LT
cells, the problem of virus breakthrough is overcome.
b)

The AGID test cannot be recommended as a serological test for the diagnosis of capripox because of the
cross-reaction with antibody to contagious pustular dermatitis virus, which is the main differential diagnosis.
A consequence of this cross-reaction is many false-positive results.
c)

Capripoxvirus-infected tissue culture grown on flying cover-slips or tissue culture microscope slides can be
used for the indirect fluorescent antibody test. Uninfected tissue culture control, and positive and negative
control sera, should be included in the test. The infected and control cultures are fixed in acetone at 20C
for 10 minutes and stored at 4C. Dilutions of test sera are made in PBS, starting at 1/5, and positives are
identified using an anti-sheep gamma-globulin conjugated with fluorescein isothiocyanate (Davies & Otema,
1978). Cross-reactions can occur with orf, bovine papular stomatitis virus and perhaps other poxviruses.
d)
Western blot analysis

Western blotting of test sera against capripoxvirus-infected cell lysate provides a sensitive and specific
system for the detection of antibody to capripoxvirus structural proteins, although the test is expensive and
difficult to carry out (Chand et al., 1994).
Capripoxvirus-infected LT cells should be harvested when 90% CPE is seen, freezethawed three times,
and the cellular debris pelleted by centrifugation. The supernatant should be decanted, and the proteins
should then be separated by SDS/PAGE (sodium dodecyl sulphate/polyacrylamide gel electrophoresis). A
vertical discontinuous gel system, using a stacking gel made up of acrylamide (5%) in Tris (125 mM),
pH 6.8, and SDS (0.1%), and a resolving gel made up of acrylamide (1012.5%) in Tris (560 mM), pH 8.7,
and SDS (0.1%), is recommended for use with a glycine running buffer containing Tris (250 mM), glycine
(2 M), and SDS (0.1%). Samples of supernatant should be prepared by boiling for 5 minutes with an
appropriate lysis buffer prior to loading. As an alternative to tissue culture antigen, it is possible to use
purified virus or expressed recombinant P32 (Carn et al., 1994; Heine et al., 1999).
Molecular weight markers should be run concurrently with the protein samples. The separated proteins in
the SDS/PAGE gel should then be transferred by electroblotting to a nitrocellulose membrane (NCM). After
blotting, the NCM is rinsed thoroughly in PBS and blocked in 3% bovine serum albumin (BSA) in PBS, or 5%
skimmed milk powder in PBS, on a rotating shaker at 4C overnight. The NCM can then be separated into
strips by employing a commercial apparatus to allow the concurrent testing of multiple serum samples, or
may be cut into strips and each strip can be incubated separately thereafter. The NCM is washed thoroughly
with five changes of PBS for 5 minutes on a rotating shaker, and then incubated at room temperature on the
shaker for 1.5 hours, with the appropriate serum at a dilution of 1/50 in blocking buffer (3% BSA and 0.05%
Tween 20 in PBS; or 5% milk powder and 0.05% Tween 20 in PBS). The membrane is again thoroughly
washed and incubated (in blocking buffer) with anti-species immunoglobulin horseradish-peroxidase-
1060
conjugated immunoglobulins at a dilution predetermined by titration. After further incubation at room

temperature for 1.5 hours, the membrane is washed and a solution of diaminobenzidine tetrahydrochloride
(10 mg in 50 ml of 50 mM Tris-HCl, pH 7.5, and 20 l of 30% [v/v] hydrogen peroxide) is added. This is then
incubated for approximately 37 minutes at room temperature on a shaker with constant observation, and
the reaction is stopped by washing in PBS before excessive background colour is seen. A positive and
negative control serum should be used on each occasion.
Positive test samples and the positive control will produce a pattern consistent with reaction to proteins of
molecular weights 67, 32, 26, 19, and 17 kDa the major structural proteins of capripoxvirus whereas
negative serum samples will not react with this pattern. Hyperimmune serum prepared against parapoxvirus
(orf virus) will react with some of the capripoxvirus proteins, but not the 32 kDa protein that is specific for
capripoxvirus.
e)

ELISAs for the detection of antibodies against capripox virus have been developed using the expressed
structural P32 protein (Carn et al., 1994; Heine et al., 1999). More recently ELISA based on recombinant
capripox antigens (Bowden et al., 2009) and on inactivated, purified whole sheep pox virus (Babiuk et al.,
2009) have been described.

1.
Background
a)

A variety of attenuated live and inactivated capripoxvirus vaccines has been used to provide protection to
sheep and goats against capripox (see refs Carn, 1993 and Kitching, 1983 for reviews). All strains of
capripoxvirus of ovine, caprine or bovine origin examined so far share a major neutralising site, so that
animals recovered from infection with one strain are resistant to infection with any other strain (Capstick,
1961). Consequently, it is possible to use a single strain of capripoxvirus to protect both sheep and goats
against all field strains of virus, regardless of whether their origin was in Asia or Africa (Kitching et al., 1986b;
Kitching & Taylor, 1985).
There are two antigenic forms of capripoxvirus, the intact virion covered in short tubular elements, and the
intact virion additionally covered in a host-cell-derived membrane. The latter is the form usually produced by
the infected animal, whereas the former is that seen when virus is produced by freezethawing infected
tissue culture. Dead vaccines produced from tissue culture are almost entirely naked virions, and when used
as a vaccine do not stimulate immunity to the membrane-bound virion. This in part explains the poor
success of inactivated vaccines. An additional factor is that inactivated vaccines are less effective than live,
replicating vaccine virus in stimulating the cell-mediated immune response, which is the predominant
protective response to poxvirus infection. Dead capripox vaccines provide, at best, only temporary
protection. A number of strains of capripoxvirus have had widespread use as live vaccines (Davies &
Mbugwa, 1985), for example the 0240 Kenya sheep and goat pox strain used in sheep and goats, the
Romanian and RM-65 strains used mainly in sheep, and the Mysore and Gorgan strains used in goats. The
protective dose depends on the vaccine strain used. Immunity in sheep and goats against capripox following
vaccination with the 0240 strain lasts over a year, and will probably provide lifelong protection against lethal
challenge. Similarly the Romanian strain gave protection for at least 30 months. The 0240 strain should not
be used in Bos taurus breeds of cattle.
2.
General requirements set for the facilities used for the production of vaccines and for the documentation and
record keeping throughout the whole manufacturing process are described in Chapter 1.1.6 Principles of
veterinary vaccine production. The documentation should include the standard operating procedures (SOP) for
the method of manufacture and each step for the testing of cells and reagents used in the process, each batches
and the final product.
a)

i)
A strain of capripoxvirus used for vaccine production must be accompanied by a history describing its
origin and tissue culture or animal passage. It must be safe to use in all breeds of sheep and goats for
which it is intended, including pregnant and young animals. It must be non-transmissible, remain
1061
attenuated after further tissue culture passage, and provide complete protection against challenge with
virulent field strains for a minimum of 1 year. A quantity of master seed vaccine virus should be
prepared and stored in order to provide a consistent working seed for regular vaccine production.
ii)

Each master seed must be tested to ensure its identity and shown to be free from adventitious viruses,
in particular pestiviruses, such as border disease and bovine viral diarrhoea virus, and free from
contamination with bacteria, fungi and/or mycoplasmas. The general procedures for sterility/purity tests
are described in chapter 1.1.7. The master seed must also be safe and produce no clinical reaction in
all breeds of sheep or goats when given by the recommended route and stimulate complete immunity
to capripox in all breeds of sheep and goats for at least 1 year. The necessary safety and potency tests
are described in paragraph C.2.b.iv Final product batch tests.
b)
The method of manufacture should be documented as the Outline of Production.
i)
Procedure
Vaccine seed should be lyophilised and stored in 2 ml vials at 20C. It may be stored wet at 20C,
but when wet, is more stable at 70C or lower. The virus should be cultured in primary or secondary
LT or LK cells of wool sheep origin for maximum yield. Vero cells may also be used with suitably
adapted strains.
Vaccine batches are produced on fresh monolayers of secondary LT or primary LK cells. A vial of seed
virus is reconstituted with GMEM or another appropriate medium and inoculated on to an LT or LK
monolayer that has been previously washed with warm PBS, and allowed to adsorb for 15 minutes at
37C before being overlaid with additional GMEM. After 46 days, there will be extensive (5070%)
CPE. The culture should be examined for any evidence of nonspecific CPE, medium cloudiness or
change in medium pH. The culture is freezethawed three times, the suspension removed and
centrifuged at 600 g for 20 minutes. A second passage may be required to produce sufficient virus for
a production batch. Live vaccine may be produced on roller bottles.
The procedure is repeated and the harvests from individually numbered flasks are each mixed
separately with an equal volume of sterile and chilled 5% lactalbumin hydrolysate and 10% sucrose,
and transferred to individually numbered bottles for storage at 20C. Prior to storage, 0.2 ml is
removed from each bottle for sterility control. An additional 0.2 ml is removed for virus titration; 2 ml
pools composed of 0.2 ml samples taken from ten bottles are used. A written record of all the
procedures must be kept for all vaccine batches.
Inactivated vaccines are produced, usually from unattenuated field strains of capripoxvirus, grown in
tissue culture as described above, inactivated with 0.03% formaldehyde, and mixed with an equal
volume of alhydrogel as adjuvant. Formaldehyde is no longer considered to be a suitable inactivant for
certain other viral vaccines because its mode of action cannot be guaranteed to be totally effective in
inactivating all the live virus. This has not been fully investigated for capripoxvirus.
ii)
Requirements for substrate and media

The specification and source of all ingredients used in the manufacturing procedure should be
documented and the freedom from extraneous agents: bacteria, fungi, mycoplasma and viruses should
be tested. The detailed testing procedure is described in the chapter 1.1.7. The use of antibiotics must
meet the requirements of the licensing authority.
iii)
In-process controls
Cells: Cells should be obtained from the testis or kidney of a healthy young lamb from a scrapie-free
flock of a wool sheep breed. During cultivation, cells must be observed for any evidence of CPE, and
for normal morphology (predominantly fibroblastic). They can usually be passaged successfully up to
ten times. When used for vaccine production, uninfected control cultures should be grown in parallel
and maintained for at least three additional passages for further observation. They should be checked
for the presence of noncytopathic strains of bovine virus diarrhoea or border disease viruses by
immunofluorescence or immunoperoxidase techniques. If possible, cells should be prepared and
screened prior to vaccine production, and a stock stored in sterile DMSO (dimethyl sulphoxide) in liquid
nitrogen (12 ml aliquots containing 20 106 cells/ml).
Serum: Bovine serum used in the growth or maintenance medium must be tested free from antibody to
capripoxvirus and contamination with pestivirus or any other viruses, extraneous bacteria, mycoplasma
or fungi.
Medium must be tested free from contamination with pestivirus or any other viruses, extraneous
bacteria, mycoplasma or fungi.
Virus: Seed virus and final vaccine must be titrated in tissue culture tubes or microtitre plates. Vaccine
samples must be examined for the presence of adventitious viruses including cytopathic and
1062
noncytopathic strains of pestivirus, and should be mixed with a high-titre capripoxvirus-immune serum
that has tested negative for antibody to pestivirus to prevent the vaccine virus itself interfering with the
test. The vaccine bulk can be held at 20C or below until all sterility tests and titrations have been
completed, at which time it should be freeze-dried in 1 ml aliquots in vials sufficient for 100 doses. The
vaccine harvest diluted with lactalbumin hydrolysate and sucrose should have a minimum titre
log10 4.5 TCID50 per ml after freeze-drying, equivalent to a field dose of log10 2.5 TCID50. A further
titration is carried out on five randomly chosen vials of the freeze-dried preparation to confirm the titre.
iv)

Sterility/purity
Safety and efficacy
The efficacy and safety studies should be demonstrated by statistically valid vaccination-challenge
studies using seronegative young sheep and goats of known susceptibility to capripox virus. The
procedure described is suitable for vaccine strains such as 0240 that are equally immunogenic in both
sheep and goats. The choice of target animal should be adapted for strains with a more restricted host
preference, such as Romanian and RM-65 strains for sheep or Mysore and Gorgan strains for goats.
Four sheep and four goats are placed in a high containment level animal unit and serum samples are
collected. Five randomly chosen vials of the freeze-dried vaccine are reconstituted in 1 ml of sterile
PBS each, and pooled. Overdose studies: one sheep and one goat are inoculated intradermally or
subcutaneously with 0.2 ml of the 10 concentrated vaccine. The remaining vaccine is diluted with
sterile PBS and two sheep and two goats are inoculated intradermally or subcutaneously with 0.2 ml of
the recommended field dose. The remaining sheep and goat are control animals. The animals are
clinically examined daily and rectal temperatures are recorded. On day 21 after vaccination, the eight
animals are again serum sampled and challenged with a known virulent capripoxvirus strain by
intradermal inoculation. (The challenge virus solution should also be tested free from extraneous
viruses that can contaminate the vaccine.) The clinical response is recorded during the following
14 days. Control animals should develop the typical clinical signs of capripox, whereas there should be
no local or systemic reaction in the vaccinates other than a delayed-type hypersensitivity reaction,
which will disappear within 4 days. Serum samples are again collected on day 30 after vaccination. The
day 21 serum samples are examined for seroconversion to selected viral diseases that could have
contaminated the vaccine, and the day 0 and 30 samples are compared to confirm the absence of
antibody to pestivirus.
The fully reconstituted vaccine is also tested in mice and guinea-pigs. Two guinea-pigs are inoculated
intramuscularly with 0.5 ml into the hind leg, and two guinea-pigs and six mice are inoculated
intraperitoneally with 0.5 ml and 0.1 ml, respectively. Two guinea-pigs and four mice are kept as
uninoculated controls. The animals are observed for 3 weeks, humanely killed and a post-mortem
examination is carried out. There should be no evidence of pathology due to the vaccine.
Batch potency
Potency tests must be undertaken if the minimum immunising dose of the virus strain is not known.
This is usually carried out by comparing the titre of a virulent challenge virus on the flanks of
vaccinated and control animals. Following vaccination, the flanks of at least three animals and three
controls are shaved of wool or hair. Log10 dilutions of the challenge virus are prepared in sterile PBS
and six dilutions are inoculated intradermally (0.1 ml per inoculum) along the length of the flank; four
replicates of each dilution are inoculated down the flank. An oedematous swelling will develop at
possibly all 24 inoculation sites on the control animals, although preferably there will be little or no
reaction at the four sites of the most dilute inocula. The vaccinated animals should develop an initial
hypersensitivity reaction at sites of inoculation within 24 hours, which should quickly subside. Small
areas of necrosis may develop at the inoculation site of the most concentrated challenge virus. The
macule/papule is measured at between 8 and 10 days post-challenge. The titre of the challenge virus
is calculated for the vaccinated and control animals; a difference of log10 titre > 2.5 is taken as
evidence of protection.
c)

i)
Safety requirements
The vaccine must be safe to use in all breeds of sheep and goats for which it is intended, including
young and pregnant animals. It must also be non-transmissible, remain attenuated after further tissue
culture passage.
Safety tests should be carried out on the final product of each batch as described in paragraph C.2.b.iv
Final product batch tests.
1063
The safety of the vaccine in non-target animals must have been demonstrated using mice and guineapigs as described in paragraph C.2.b.iv Final product batch tests. There should be no evidence of
pathology caused by the vaccine.
The selected final vaccine should not revert to virulence during a further passages in target animals.
Attenuated vaccine should not be able to perpetuate autonomously in a cattle, sheep or goat
populations. Vaccines using the 0240 strain should not be used in Bos taurus breeds. Strains of
capripoxvirus are not a hazard to human health. There are no precautions other than those described
above for sterility and freedom from adventitious agents.
ii)
For animal production
The efficacy of the vaccine must be demonstrated in vaccination challenge experiment under
laboratory conditions. As described in Section C.2.b.iv Final product batch tests.
Once the potency of the particular strain being used for vaccine production has been determined in
terms of minimum dose required to provide immunity, it is not necessary to repeat this on the final
product of each batch, provided the titre of virus present has been ascertained.
For control and eradication
Vaccination is the only effective way to control the sheep pox and goat pox outbreaks in endemic
countries. Unfortunately, currently no marker vaccines allowing the differentiation of infected from
vaccinated animals are available.
Immunity to virulent field virus following vaccination of sheep or goats with the 0240 strain lasts over
1 year, and protection against generalised infection following intradermal challenge lasts at least
3 years and is effective lifelong. The duration of immunity produced by other vaccine strains should be
ascertained in both sheep and goats by undertaking controlled trials in an environment in which there is
no possibility of field strains of capripoxvirus confusing the results. The inactivated vaccines provide
immunity for less than 1 year, and for the reasons given at the beginning of this section, may not give
immunity to the form of capripoxvirus usually associated with natural transmission.
ii)
Stability
All vaccines are initially given a shelf-life of 24 months before expiry. Real-time stability studies are
then conducted to confirm the appropriateness of the expiry date. Multiple batches of the vaccine
should be re-titrated periodically throughout the shelf-life to determine the vaccine variability.
Properly freeze-dried preparations of capripox vaccine, particularly those that include a protectant,
such as sucrose and lactalbumin hydrolysate, are stable for over 25 years when stored at 20C and
for 24 years when stored at 4C. There is evidence that they are stable at higher temperatures, but no
long-term controlled experiments have been reported. The inactivated vaccines must be stored at 4C,
and their shelf- life is usually given as 1 year.
No preservatives other than a protectant, such as sucrose and lactalbumin hydrolysate, are required
for the freeze-dried preparation.
3.
a)

Currently, no recombinant vaccines for capripoxviruses are commercially available. However, a new
generation of capripox vaccines is being developed that uses the capripoxvirus genome as a vector for the
genes of other ruminant pathogens, for instance genes of rinderpest and peste des petits ruminants (PPR)
viruses. The recombinant vaccine will provide protection against capripox, rinderpest and PPR in a single
vaccination (Berhe et al., 2003).
b)
Special requirements for biotechological vaccines, if any

Not applicable.
1064
REFERENCES
BABIUK S., WALLACE D.B., SMITH S.J., BOWDEN, T.R., DALMAN B., PARKYN G., COPPS J. & BOYLE D.B. (2009).
Detection of antibodies against capripoxviruses using an inactivated sheeppox virus ELISA. Transbound. Emerg.
Dis., 56, 132141.
BALINSKY C.A, DELHON G, SMOLIGA G, PRARAT M, FRENCH R.A, GEARY S.J, ROCK D.L & RODRIGUEZ L.L. (2008). Rapid
preclinical detection of sheep pox virus by a real-time PCR assay. J. Clin. Microbiol., 46, 438442.
BERHE G., MINET C., LE GOFF C., BARRETT T., NGANGNOU A., GRILLET C., LIBEAU G., FLEMING M., BLACK D.N. & DIALLO
A. (2003). Development of a dual recombinant vaccine to protect small ruminants against peste-des-petitsruminants virus and capripoxvirus infections. J. Virol., 77, 15711577.
BLACK D.N., HAMMOND J.M. & KITCHING R.P. (1986). Genomic relationship between capripoxviruses. Virus Res., 5,
277292.
BOWDEN T.R, BABIUK S.L, PARKYN G.R., COPPS J.S. & BOYLE D.B. (2008). Capripox virus tissue tropism and
shedding: A quantitative study in experimentally infected sheep and goats. Virology , 371, 380393.
BOWDEN T.R., COUPAR B.E., BABIUK S.L., WHITE J.R., BOYD V., DUCH C.J., SHIELLL B.J., UEDA N., PARKYN G.R.,
COPPS J.S. & BOYLE D.B. (2009). Detection of antibodies specific for sheeppox and goatpox viruses using
recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay. J. Virol. Methods, 161 (1),
1929.
CAPSTICK P.B. (1961). Annual Report. Kenya Veterinary Department, Kenya, 4547.
CARN V.M. (1993). Control of capripoxvirus infections. Vaccine, 11, 12751279.
CARN V.M. (1995). An antigen trapping ELISA for the detection of capripoxvirus in tissue culture supernatant and
biopsy samples. J. Virol. Methods, 51, 95102.
CARN V.M., KITCHING R.P., HAMMOND J.M., CHAND P., ANDERSON J. & BLACK D.N. (1994). Use of a recombinant
antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus. J. Virol. Methods, 49, 285294.
CHAND P., KITCHING R.P. & BLACK D.N. (1994). Western blot analysis of virus-specific antibody responses to
capripoxvirus and contagious pustular dermatitis infections in sheep. Epidemiol. Infect., 113, 377385.
DAVIES F.G. & MBUGWA G. (1985). The alterations in pathogenicity and immunogenicity of a Kenya sheep and goat
pox virus on serial passage in bovine foetal muscle cell cultures. J. Comp. Pathol., 95, 565576.
DAVIES F.G. & OTEMA C. (1978). The antibody response in sheep infected with a Kenyan sheep and goat pox
virus. J. Comp. Pathol., 88, 205210.
GERSHON P.D., KITCHING R.P., HAMMOND J.M. & BLACK D.N. (1989). Poxvirus genetic recombination during natural
virus transmission. J. Gen. Virol., 70, 485489.
HEINE H.G., STEVENS M.P., FOORD A.J. & BOYLE D.B. (1999). A capripoxvirus detection PCR and antibody ELISA
based on the major antigen P32, the homolog of the vaccinia virus H3L gene. J. Immunol. Methods, 227, 187
196.
HOSAMANI M., MONDAL B., TEMBHURNE P.A., BANDYOPADHYAY S.K., SINGH R.K. & RASOOL T.J. (2004). Differentiation
of sheep pox and goat poxviruses by sequence analysis and PCR-RFLP of P32 gene. Virus Genes, 29, 7380.
IRELAND D.C. & BINEPAL Y.S. (1998). Improved detection of capripoxvirus in biopsy samples by PCR. J. Virol.
Methods, 74, 17.
KITCHING R.P. (1983). Progress towards sheep and goat pox vaccines. Vaccine, 1, 49.
KITCHING R.P., BHAT P.P. & BLACK D.N. (1989). The characterization of African strains of capripoxvirus. Epidemiol.
Infect., 102, 335343.
KITCHING R.P., HAMMOND J.M. & BLACK D.N. (1986a). Studies on the major precipitating antigen of capripoxvirus. J.
Gen. Virol., 67, 139148.
1065
KITCHING R.P., HAMMOND J.M. & TAYLOR W.P. (1986b). A single vaccine for the control of capripox infection in
sheep and goats. Res. Vet. Sci., 42, 5360.
KITCHING R.P. & SMALE C. (1986). Comparison of the external dimensions of capripoxvirus isolates. Res. Vet. Sci.,
41, 425427.
KITCHING R.P. & TAYLOR W.P. (1985). Clinical and antigenic relationship between isolates of sheep and goat pox
viruses. Trop. Anim. Health Prod., 17, 6474.
LE GOFF C., LAMIEN C.E., FAKHFAFH E., CHADEYRAS A., ABU-ADULUGBAD E., LIBEAU G., TUPPURAINEN E., WALLACE D.,
ADAM T., SILBER R., GULYAZ V., MADANI H., CAUFOUR P., HAMAMMI S., DIALLO A. & ALBINA E. (2009). Capripoxvirus Gprotein-coupled chemokine receptor, a host-range gene suitable for virus-animal origin discrimination. J. Gen.
Virol., 90, 6777.
*
* *
NB: There are OIE Reference Laboratories for Sheep pox and goat pox
diagnostic tests, reagents and vaccines for sheep pox and goat pox
1066
SECTION 2.8.
SUIDAE
CHAPTER 2.8.1.
AFRICAN SWINE FEVER
SUMMARY
African swine fever (ASF) is an infectious disease of domestic and wild pigs of all breeds and ages,
caused by a virus that produces a range of syndromes. Acute disease is characterised by high
fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. Soft ticks of the
Ornithodoros genus, especially O. moubata and O. erraticus, have been shown to be both
reservoirs and transmission vectors of ASF virus (ASFV).
ASFV is the only member of the Asfarviridae family, genus Asfivirus.
Laboratory diagnostic procedures for ASF fall into two groups: the first contains the tests for virus
isolation and the detection of virus antigens and genomic DNA, while the second contains the tests
for antibody detection. The selection of the tests to be carried out depends on the disease situation
and laboratory diagnostic capacity in the area or country.
Identification of the agent: Laboratory diagnosis must be directed towards isolation of the virus by
simultaneously carrying out the inoculation of pig leukocyte or bone marrow cultures, the detection
of antigen in smears or cryostat sections of tissues by fluorescent antibody test (FAT) and the
detection of genomic DNA by the polymerase chain reaction (PCR). The PCR is an excellent, highly
sensitive, and rapid technique for ASFV detection and it is very useful under a wide range of
circumstances. It is especially useful if the tissues are unsuitable for virus isolation and antigen
detection.
In doubtful cases, the material is passaged in leukocytes cell cultures and the procedures described
above are repeated.
Serological tests: Pigs that survive natural infection usually develop antibodies against ASFV from
710 days post-infection and these antibodies persist for long periods of time. Where the disease is
endemic, or where a primary outbreak is caused by a strain of low virulence, the investigation of
new outbreaks should include the detection of specific antibodies in serum or extracts of the tissues
submitted. A variety of methods such as indirect fluorescent antibody (IFA), the enzyme-linked
immunosorbent assay (ELISA) and the immunoblotting test is available for antibody detection.
Requirements for vaccines and diagnostic biologicals: At present, there is no vaccine for ASF.
A. INTRODUCTION
African swine fever virus (ASFV) is a complex large icosahedral enveloped DNA virus that exhibits many features
common to both iridovirus and poxvirus families (Arias & Snchez-Vizcano, 2002a; Vinuela, 1985). This virus is
currently classified as the only member of a family called Asfarviridae (Dixon et al., 2005). At least 28 structural
proteins have been identified in intracellular virus particles (200 nm) (Snchez-Vizcano, 2006).More than a
hundred infectious proteins have been identified in infected porcine macrophages, and at least 50 of them react
with sera from infected or recovered pigs.The virus genome comprises between 170 and 192 kilobases (kb), with
a conserved central region of about 125 kb and variable ends. These variable regions encode five multigene
families that are directly involved with the variability of the virus genome (Blasco et al., 1989).The complete
1067
Chapter 2.8.1. African swine fever
analysis of the sequence of several ASFV strains has been accomplished (Chapman et al., 2008; De Villier et al.,
2010).Different strains of ASFV vary in their ability to cause disease, but at present there is only one recognised
serotype of the virus detectable by antibody tests.
The molecular epidemiology of the disease is investigated by sequencing of the C- terminal end of VP72 gene,
which differentiates up to 22 distinct genotypes (Boshoff et al., 2007; Lubisi et al., 2005). Full genome sequence
of the p54-gene has been confirmed as a valuable additional genotyping method for molecular epidemiological
studies (Gallardo et al., 2009). Enhanced discrimination is obtained by analysis of the central variable region
(CVR) within the B602L-gene, described as the most variable locus to distinguish between closely related isolates
and identify virus subgroups within several of the 22 genotypes (Gallardo et al., 2009).
ASF viruses produce a range of syndromes varying from peracute, acute to chronic disease and apparently
healthy virus carriers. Pigs are the only domestic animal species that is naturally infected by ASFV. European wild
boars and feral pigs are also susceptible to the disease, exhibiting clinical signs and mortality rates similar to
those observed in domestic pigs. In contrast African wild pigs such as warthogs (Phacochoerus aethiopicus),
bush pigs (Potamochoerus porcus) and giant forest hogs (Hylochoerus meinertzhageni) are resistant to the
disease and show few or no clinical signs. These species of wild pig act as reservoir hosts of ASFV in Africa
(Snchez-Vizcano, 2006).
The incubation period in nature is usually 419 days. The more virulent strains produce peracute or acute
haemorrhagic disease characterised by high fever, loss of appetite, haemorrhages in the skin and internal organs,
and death in 410 days, sometimes even before the first clinical signs are observed. Mortality rates may be as
high as 100%. Less virulent strains produce mild clinical signs slight fever, reduced appetite and depression
which can be readily confused with many other conditions in pigs and may not lead to suspicion of ASF. Low
virulent, non-haemadsorbing strains occasionally produce mainly subclinical non-haemorrhagic infection and
seroconversion, but some animals may develop discrete lesions in the lungs or on the skin in areas over bony
protrusions and other areas subject to trauma. Animals which have recovered from either acute or chronic
infections may become persistently infected, acting as virus carriers. The biological base for the persistence of
ASFV is still not well understood (Carrillo et al., 1994). Recovered ASFV carrier pigs and persistently infected wild
pigs constitute the biggest problems in controllingthe disease. The serological recognition of carrier pigs has been
vital for the success of eradication programmes (Arias & Snchez-Vizcano, 2002b).
ASF cannot be differentiated from classical swine fever (hog cholera; CSF) by either clinical or post-mortem
examination, and both diseases should be considered in the differential diagnosis of any acute febrile
haemorrhagic syndrome of pigs. Bacterial septicaemias may also be confused with ASF and CSF. Laboratory
tests are essential to distinguish between these diseases.
In countries free from ASF but suspecting its presence, the laboratory diagnosis must be directed towards
isolation of the virus by simultaneously carrying out the inoculation of pig leukocyte or bone marrow cultures, the
detection of antigen in smears or cryostat sections of tissues by the fluorescent antibody test (FAT) and, by the
detection of genomic DNA by the polymerase chain reaction (PCR), which is the most sensitive technique for
detecting the presence of the agent in persistently infected animals and is particularly useful if samples submitted
are unsuitable for virus isolation and antigen detection because they have undergone putrefaction. ASFV can be
detected by PCR from a very early stage of infection in tissues, ethylene diamine tetra-acetic acid (EDTA)-blood
and serum samples. Pigs recovered from acute or chronic infections usually exhibit a viraemia for several weeks
making the PCR test a very useful tool for the detection of ASFV DNA in pigs infected with low or moderately
virulent strains.
As no vaccine is available, the presence of ASFV antibodies is indicative of previous infection and,as antibodies
are produced from the first week of infection and persist for long periods, they are a good marker for the diagnosis
of the disease.The early appearance (from 7 to 10 days post-infection) and subsequent long-termpersistence of
antibodies make antibody detection techniques,such as ELISA, immunoblotting or IFA test, very useful in
diagnosing the subacute and chronic forms of disease.
ASF epidemiology is complex with different epidemiological patterns of infection occurring in Africa and Europe.
ASF occurs through transmission cycles involving domestic pigs, wild boars, wild African suids, and soft ticks
(Snchez-Vizcano et al., 2009).In regions where Ornithodorus soft-bodied ticks are present, the detection of
ASFV in these reservoirs of infection contributes to a better understanding of the epidemiology of the disease.
This is of major importance in establishing effective control and eradication programmes (Basto et al., 2006).
ASF is not a zoonotic disease and does not affect public health (Snchez-Vizcano et al., 2009).
ASFV should be handled in laboratories that have been approved by their Competent Authorities following OIE
guidelines for the biocontainment of Group 3 and 4 pathogens.
1068
1.
Where ASF is suspected, the following samples should be sent to the laboratory: blood in anticoagulant (EDTA),
spleen, lymph nodes, tonsil and kidney. These should be kept as cold as possible, without freezing, during transit.
After the samples arrive at the laboratory, they should be stored at 70C if processing is going to be delayed. As
maintaining a cold chain is not always possible, samples can be submitted in glycerosaline; this may slightly
decrease the likelihood of virus isolation, but it may facilitate the submission of samples to the laboratory so that
an outbreak can be confirmed.
a)
Sample preparation for haemadsorption test

i)
Prepare suspensions of tissues by grinding 0.51.0 g pieces with a pestle and mortar containing sterile
sand, and then add 510 ml of a buffered salt solution or tissue culture medium containing antibiotics.
ii)
Clarify the suspensions by centrifugation at 1000 g for 5 minutes. Use the supernatant for cell culture/
haemadsorption (Section B.1.a below).
Haemadsorption test
The haemadsorption (HAD) test (Malmquist & Hay, 1960) is based on the fact that pig erythrocytes will
adhere to the surface of pig monocyte or macrophage cells infected with ASFV and that most virus isolates
produce this phenomenon of haemadsorption. A positive result in the HAD test is definitive for ASF
diagnosis. A very small number of non-haemadsorbing viruses have been isolated, most of which are a
virulent, but some do produce typical acute ASF. The test is carried out by inoculating blood or tissue
suspensions from suspect pigs into primary leukocyte cultures, (Procedure 1 below) or into alveolar
macrophages cell cultures, and also by preparing leukocyte cultures from the blood of pigs inoculated at the
laboratory or from the blood of suspect pigs collected in the field (Procedure 2 below). Up to 300 cultures in
tubes can be prepared from each 100 ml of defibrinated blood collected. It is essential to carry out all
procedures in such a way as to prevent contamination of the cultures.
Procedure 1: Haemadsorption test in primary leukocyte cultures
i)
Collect the required volume of fresh defibrinated pig blood.
ii)
Centrifuge at 700 g for 30 minutes and collect the buffy coat cells. Add three volumes of 0.83%
ammonium chloride to the leukocytes obtained. Mix and incubate at room temperature for 15 minutes.
Centrifuge at 650 g for 15 minutes and carefully remove the supernatant. Wash pellet in medium or
phosphate buffered saline (PBS).
iii)
Resuspend the cells at a concentration of 106107 cells/ml in tissue culture medium containing 10
30% pig serum and antibiotics. In order to prevent nonspecific haemadsorption, the medium should
contain serum or plasma from the same pig from which the leukocytes were obtained. If a large volume
of samples is to be tested, the homologous serum can be replaced by serum that has been identified
by pre-screening as capable of preventing the nonspecific auto-rosette formation.
iv)
Dispense the cell suspension in 96-well plates with 200 l per well (300,000 cells/well) and incubate at
37C in a humidified 5%CO2 incubator. This procedure can also be performed in aliquots of 1.5 ml in
160 16 mm tubes and incubate in a sloping position (510 from the horizontal) at 37C.
Note: For routine diagnosis, only 24-day-old cultures are sufficiently sensitive.
v)
After 3 days inoculate three tubes or well plates by adding 0.2 ml/tube or 0.02 ml (1/10 final
dilution)/well of prepared samples. It is advisable to inoculate ten-fold and hundred-fold dilutions into
cultures, and this is especially important when the field material submitted is in poor condition.
vi)
Inoculate positive control cultures with haemadsorbing virus. Uninoculated negative controls are
essential to monitor the possibility of nonspecific haemadsorption.
vii)
Add 0.2 ml of a fresh preparation of 1% pig erythrocytes in buffered saline to each tube. In the case of
the 96-well plates, add 0.02 ml of 1% pig erytrocytes per well.
viii) Examine the cultures daily for 710 days under a microscope for cytopathic effect (CPE) and
haemadsorption.
ix)
Reading the results: Haemadsorption consists of the attachment of large numbers of pig erythrocytes
to the surface of infected cells. A CPE consisting of a reduction in the number of adherent cells in the
absence of haemadsorption may be due to the cytotoxicity of the inoculum, Aujeszkys disease virus or
non-haemadsorbing ASFV, which can be detected by the FAT on the cell sediment or by use of PCR
(see below). If no change is observed, or if the results of the immunofluorescence and PCR tests are
1069
negative, subinoculate the supernatant up to three times into fresh leukocyte cultures. All isolations
should be confirmed by PCR and sequencing.
Procedure 2: Haemadsorption autorosette test with peripheral blood leukocytes from infected pigs
This procedure is quicker than the preparation and inoculation of primary pig leukocyte cultures (described in
Procedure 1 above) and will give more rapid results in positive cases. It can be performed in laboratories
that are not equipped for routine virological examinations; the minimum requirements are slides and coverslips, a microscope and sterile medium, tubes or bottles and pipettes. Blood from suspect pigs in the field, or
those inoculated in the laboratory, is collected in heparin and leukocyte cultures are prepared for direct
examination for haemadsorption. However, the results of the test are difficult to evaluate and it is now being
replaced by the PCR.
b)
i)
Collect 20 ml of whole blood in a syringe containing 2000 IU heparin in 2 ml of saline, mix and transfer
to a glass tube or narrow bottle.
ii)
Place the tube/bottle vertically in an incubator or water bath at 37C, and allow the cells to settle.
Sedimentation is improved by the addition of 2 ml of a plasma volume expander, such as
Dextravan150 which is a solution of Dextran 150 in 0.9% NaCl for injection (Fisons, United Kingdom).
iii)
Incubate the cultures for 68 hours at 37C, and then examine the cultures at 23-hour intervals by
transferring small aliquots of the white-cell-rich supernatant, together with some erythrocytes, on to a
glass slide.
iv)
Reading the results: The presence of haemadsorbing cells identified under a microscope indicates the
presence of ASFV. Haemadsorption consists of the attachment of large numbers of pig erythrocytes to
the surface of infected cells. Any evidence of haemadsorption would be enough to consider repeating
the assay or confirming the presence of ASFV by another test such as PCR.
Antigen detection by fluorescent antibody test

The FAT (Bool et al., 1969) can be used as an additional method to detect antigen in tissues of suspect pigs
in the field or those inoculated at the laboratory. Positive FAT plus clinical signs and appropriate lesions can
provide a presumptive diagnosis of ASF. It can also be used to detect ASFV antigen in leukocyte cultures in
which no HAD is observed and can thus identify non-haemadsorbing strains of virus. It also distinguishes
between the CPE produced by ASFV and that produced by other viruses, such as Aujeszkys disease virus
or a cytotoxic inoculum. However, it is important to note that in subacute and chronic disease, FAT has a
significantly decreased sensitivity. This reduction in sensitivity may be related to the formation of antigenantibody complexes in the tissues of infected pigs which block the interaction between the ASFV antigen
and ASF conjugate (Snchez-Vizcano, 2006).
c)
Test procedure
i)
Prepare cryostat sections or impression smears of test tissues, or spreads of cell sediment from
inoculated leukocyte cultures on slides, air dry and fix with acetone for 10 minutes at room
temperature.
ii)
Stain with fluorescein isothiocyanate (FITC)-conjugated anti-ASFV

recommended or pretitrated dilution for 1 hour at 37C in a humid chamber.
iii)
Fix and stain positive and negative control preparations similarly.
iv)
Wash by immersing four times in fresh clean PBS, mount stained tissues in PBS/glycerol, and examine
under an ultraviolet light microscope with suitable barrier and exciter filters.
v)
Reading the results: Tissues are positive if specific granular cytoplasmic fluorescence is observed in
paracortical tissue of lymphoid organs or in fixed macrophages in other organs, or in inoculated
leukocyte cultures.
immunoglobulin
at
the
Detection of virus genome by the polymerase chain reaction

PCR techniques have been developed, using primers from a highly conserved region of the genome, to
detect and identify a wide range of isolates belonging to all the known virus genotypes, including both nonhaemadsorbing viruses and isolates of low virulence. The PCR techniques are particularly useful for
identifying virus DNA in pig tissues that are unsuitable for virus isolation or antigen detection because they
have undergone putrefaction, or when there is good reason to believe that virus may have been inactivated
before samples are received in the laboratory.
Two validated PCR procedures are described and consist of a sample preparation followed by the test
procedure. These procedures serve as a general guideline and a starting point for the PCR protocol. Optimal
reaction conditions (incubation times and temperatures, models and suppliers of equipment, concentrations
1070
of assay reagents such as the primers and dNTPs) may vary so the described conditions should be
evaluated first.
Sample preparation procedure (Agero et al., 2003; 2004)
A sensitive extraction procedure using the commercial High Pure PCR Template Preparation Kit1 is
described below. A number of other DNA extraction kits are commercially available for the preparation of
template suitable for PCR depending on the sample submitted for analysis and may be appropriate for use.
Different samples can be used in this procedure such as cell culture supernatants, EDTA-blood, serum and
tissue homogenates, even if the latter have been kept in warm conditions and undergone a degree of
putrefaction. This procedure has the advantage in that it can be used for both the extraction of ASFV DNA
and CSFV RNA which enables the simultaneous detection of both viruses in a multiplex PCR assay (Agero
et al., 2004).
The High Pure PCR Template Preparation Kit1 includes the following reagents: Binding Buffer, Proteinase K,
Inhibitor Removal Buffer, Wash Buffer, and High Pure Filter Tubes and collection tubes.
For organ and tissue samples, first prepare a 1/10 homogenate of the material in PBS, then centrifuge to
clarify at 12,000 g for 5 minutes. Extract DNA/RNA from the resultant supernatant fluid. Sometimes it is
recommended to process a 1/10 dilution of the supernatant in parallel.
Extraction for control samples: at least one positive and one negative control should be included in each
nucleic acid extraction run. Positive control samples should be 200 l of ASFV-positive serum, EDTA-blood,
or 1/10 tissue homogenates (same kind of tissue as samples to be analysed): the negative control should be
200 l of water or ASFV-negative EDTA-blood, or tissue homogenate. Controls should be processed with
the test samples.
Preparation of working solutions
Lyophilised proteinase K: dissolve proteinase K in 4.5 ml sterile distilled water, and aliquot the solution in
500 l vials. Store at 20C until use.
Inhibitor Removal Buffer: add 20 ml absolute ethanol to the original vial. Label and date bottle accordingly.
Wash buffer: add 80 ml absolute ethanol to the original vial. Label and date bottle accordingly.
i)
Pipette 200 l of sample into a 1.5 ml microcentrifuge tube.
ii)
Add 200 l of binding buffer and 40 l of proteinase K. Mix immediately. Incubate for 10 minutes at
72C.
iii)
Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
iv)
Add 100 l of isopropanol to the sample tube.
v)
Place the High Pure filter tube in a collection tube and pipette the sample in the upper reservoir.
Centrifuge for 1 minute at 8000 rpm. (With blood samples, repeat the centrifugation step if sample
remains in the filter tube.)
v)
Discard the collection tube and place the filter tube into a clean collection tube.
vii)
Add 500 l of Inhibitor Removal Buffer to the upper reservoir and centrifuge for 1 minute at 8000 rpm.
viii) Discard the collection tube and place the filter tube into a clean collection tube.
ix)
Add 450 l of wash buffer to the upper reservoir and centrifuge for 1 minute at 8000 rpm.
x)
Discard the collection tube and repeat the washing step.
xi)
Discard the collection tube and place the filter tube into a clean collection tube. Centrifuge for
10 seconds at 13000 rpm to remove residual wash buffer.
xii)
Discard the collection tube and place the filter tube in a clean 1.5 ml microcentrifuge tube.
xiii) For the elution of nucleic acids, add 50 l of prewarmed (70C) sterile water to the upper reservoir (be
careful not to use the Elution Buffer included in the kit for CSFV RNA). Centrifuge for 1 minute at
8000 rpm.
xiv) Use immediately or store at 20C for future use.
Roche Diagnostics.
1071
PCR amplification by conventional PCR (Agero et al., 2003)
The ASFV primer set described in this procedure can be combined with a specific primer set for CSFV in a
multiplex RT-PCR method that allows the simultaneous and differential detection of both virus genomes in a
single reaction (Agero et al., 2004).
Stock solutions
Nuclease-free sterile water.
Hot Start Taq Gold DNA polymerase, 10 PCR Buffer II, and magnesium chloride are commercially
available.
PCR nucleotide mix containing 10 mM of each dNTP is commercially available.
Primers at a concentration of 20 pmol/l: Primer PPA-1 sequence 5-AGT-TAT-GGG-AAA-CCC-GACCC-3 (forward primer); primer PPA-2 sequence 5-CCC-TGA-ATC-GGA-GCA-TCC-T-3 (reverse
primer).
10 Loading buffer: 0.2% xylene cyanol, 0.2% bromophenol blue, 30% glycerol.
TAE buffer (50) for agarose gel: Tris base (242 g); glacial acetic acid (57.1 ml); 0.5 M EDTA, pH 8.0
(100 ml); distilled water (to 1 litre).
Marker DNA: 100 base-pair ladder is commercially available.
i)
In a sterile 1.5 ml microcentrifuge tube prepare the PCR reaction mixture described below for each
sample. Prepare the reaction mixture in bulk for the number of samples to be assayed allowing for at
least one extra sample.
ii)
Nuclease-free or sterile distilled water (17.375 l), 10 PCR Buffer II (2.5 l), magnesium chloride
25 mM (2 l), dNTP mix 10mM (0.5 l), primer PPA-1, 20 pmol/l (0.25 l), primer PPA-2, 20 pmol/l
(0.25 l), Taq Gold DNA polymerase 5 U/l (0.125 l).
iii)
Add 23 l of the PCR reaction mix to the required number of 0.2 ml PCR tubes.
iv)
Add 2l of extracted sample template to each PCR tube. Include a positive reaction control (2 l of
ASFV DNA) and a negative reaction control (2 l of distilled water) for each PCR run.
v)
Place all the tubes in an automated thermal cycler and run the following programme:
One cycle at 95C for 10 minutes.
40 cycles at 95C for 15 seconds, 62C for 30 seconds and 72C for 30 seconds.
Hold at 4C.
vi)
At the end of the programme, remove PCR tubes and add 2.5 l of 10 loading buffer to each tube.
vii)
Load all the samples in a 2% agarose gel in TAE buffer containing ethidium bromide at a final
concentration of 0.5 g/ml.
viii) Add marker DNA to one lane on each side of the gel.
ix)
Run the gel at a constant voltage of 150-200 volts for about 30 minutes.
x)
Reading the results: Examine the gel over a UV light source. In a positive sample, a discrete band will
be present that should co-migrate with the PCR product of the positive control. Calculate the size of the
PCR products in the test samples and the positive control by reference to the standard markers. The
PCR product of the positive control has a size of 257 base pairs. No bands should be seen in the
negative control.
xi)
Optional: An additional confirmatory assay could be performed by BsmA I restriction endonuclease

digestion of the amplified products. For this assay, incubate for 2.5 hours at 55C a total of 5 l of
amplified DNA product in a final volume of 20 l digestion mix: 2 l of 10 buffer, 1 l of BsmA I
(5 U/l) and 12 l of sterile distilled water. Then, run the samples in a 3% agarose gel as described
above. The restriction pattern should include two fragments of 173177 and 8480 base pairs in the
positive samples.
PCR Procedure: TaqMan PCR protocol (King et al., 2003)
Sample preparation
A number of DNA extraction kits are commercially available for the preparation of template suitable for PCR
depending on the sample submitted for analysis and may be appropriate for use in Reference Laboratories.
1072
The QIAamp Viral RNA Mini Kit (QIAGEN) procedure (spin protocol) is described below. This kit can be
used for blood from suspected swine fever animals. Blood from infected swine should be taken in EDTA.
The detection of ASFV can be performed in parallel to that for CSF virus (see Chapter 2.8.3 Classical swine
fever for CSFV molecular detection methods).
i)
Pipette 560 l of the supplied buffer AVL into a 1.5 ml microcentrifuge tube.
ii)
Add 140 l of test or control sample and mix by pulse-vortexing for about 15 seconds. Negative ASF
control samples consisting of spleen homogenates from uninfected pigs and uninfected porcine bone
marrow (PBM) and peripheral blood mononuclear (PBL) cells should be processed alongside the test
samples. Additional extraction negative controls can also be prepared for each test sample and
uninfected negative control by running parallel extractions of nuclease-free water (all controls should
subsequently be assayed by the PCR procedure along with the test samples).
iii)
Incubate at room temperature for at least 10 minutes.
iv)
Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
v)
Add 560 l ethanol to the sample, pulse-vortex for approximately 15 seconds and briefly centrifuge to
remove drops from the inside of the lid.
vi)
Add 630 l of the solution from v) to a QIAamp spin column (in a 2-ml collection tube) without wetting
the rim. Close the cap and centrifuge for 1 minute at 6000 g. Place the spin column into a clean 2 ml
collection tube and discard the tube containing the filtrate.
vii)
Carefully open the QIAamp spin column and repeat step vi.
viii) Carefully open the QIAamp spin column and add 500 l of Buffer AW1. Close the cap and centrifuge
for 1 minute at 6000 g. Place the spin column into a clean 2-ml collection tube and discard the tube
containing the filtrate.
ix)
Carefully open the QIAamp spin column and add 500 l of Buffer AW2. Close the cap and centrifuge
for 3 minutes at 20,000 g.
x)
Place the QIAamp spin column in a new 1.5 ml microcentrifuge tube. Discard the old collection tube
containing the filtrate. Carefully open the QIAamp spin column and add 60 l of Buffer AVE. Close the
cap and incubate at room temperature for 1 minute. Centrifuge for 1 minute at 6000 g.
xi)
Discard the QIAamp spin column. Store the extracted DNA (60 l) at 20C until required for PCR
amplification procedure.
Stock solutions
i)
Nuclease-free or another appropriate sterile water and TaqMan PCR reaction master mix (2).
ii)
Primers at a concentration of 50 pmol/l: Primer 1 sequence 5-CTGCT-CATGG-TATCA-ATCTTATCGA-3 (positive strand); Primer 2 sequence 5-GATAC-CACAA-GATC(AG)-GCCGT-3 (negative
strand).
iii)
TaqMan probe at a concentration of 5 pmol/l: (5-[6-carboxy-fluorescein (FAM)]-CCACG-GGAGGAATAC-CAACC-CAGTG-3-[6-carboxy-tetramethyl-rhodamine (TAMRA)]).
PCR amplification by TaqMan assay (Fernndez-Pinero et al., 2010)
i)
In a sterile 1.5 ml microcentrifuge tube prepare the PCR reaction mixture described below for each
sample. Prepare the reaction mixture in bulk for the number of samples to be assayed but allowing for
one extra sample.
Nuclease-free or sterile water (7.5 l); (2 conc.) TaqMan PCR reaction master mix (12.5 l); primer
1, 50 pmol (1.0 l); primer 2, 50 pmol (1.0 l); TaqMan probe, 5 pmol (1 l).
ii)
Add 22 l PCR reaction mix to a well of a MicroAmp optical reaction plate for each sample to be
assayed.
iii)
Add 3 l of extracted sample template or blank extraction control and securely cover each well with a
cap.
iv)
Spin the plate for 1 minute in a suitable centrifuge to mix the contents of each well.
v)
Place the plate in a TaqMan Sequence Detection System for PCR amplification and run the following
programme:
One cycle at 50C for 2 minutes,
Forty cycles at 95C for 15 seconds, 58C for 1 minute.
1073
Note: If a TaqMan thermal cycler is not available, an ordinary thermal cycler can be used and the
PCR products analysed by end-point fluorescence readers or alternatively by electrophoresis on a
1.5% agarose gel. A product of 250 bp is expected.
vi)
Reading the results: Assign a threshold cycle (CT) value to each PCR reaction from a scan of all
amplification plots (a plot of the fluorescence signal versus cycle number). Negative test samples,
uninfected negative or extraction blank controls should have a CT value >40.0. Positive test samples
and controls should have a CT value < 40.0 (strongly positive samples have a CT value <30.0).
Modifications of this protocol using a range of commercial amplification kits can provide even higher PCR
yields, however these amplification kits should be fully validated prior to use.
2.
Serological tests
Antibodies persist in recovered pigs for long periods after infection, sometimes for life, and a number of tests are
available for detecting these antibodies, although only a few of them have been developed for routine use in
diagnostic laboratories (Escribano et al., 1990; Pastor et al., 1990; Snchez-Vizcano, 1987). The most commonly
used is the ELISA (Snchez-Vizcano et al., 1983; Wardley et al., 1979), which is suitable for examining either
serum or fluid from the tissues. Confirmatory testing of ELISA-positive samples should be carried out in critical
cases using an alternative test, such as the IFA test, immunoperoxidase staining or immunoblotting (Escribano et
al., 1990; Pastor et al., 1989). Antibody is usually not detected in pigs infected with virulent ASFV as they die
before it is produced. Antibodies are produced in pigs infected with low or moderately virulent ASF viruses, but
these are not fully neutralising antibodies.
Recently, an extensive study carried out to assess the specificity and sensitivity of ASF serological tests in the
different epidemiological scenarios of Africa and Europe, with currently circulating ASFV isolates (including the
Caucasian genotype II and the eastern African isolates exhibiting more variability), has shown that the OIE
prescribed tests are able to detect with accuracy and high sensitivity the presence of ASF antibodies in all the
epidemiological situations evaluated (Gallardo et al., 2010).
Where ASF is endemic, confirmation of suspected cases of disease is best done using a standard serological test
(ELISA), combined with an alternative serological test (IFA) or an antigen-detection test. In some countries, over
95% of positive cases have been identified using a combination of IFA tests and FAT (Snchez-Vizcano, 2006).
It should be noted that when pigs have been infected with avirulent isolates or those of low virulence, serological
tests may be the only way of detecting infected animals.
a)
Enzyme-linked immunosorbent assay (the prescribed test for international trade)

The ELISA (Pastor et al., 1990) is a direct test that can detect antibodies to ASFV in pigs that have been
infected by viruses of low or moderate virulence. A highly sensitive and specific commercial ELISA kit based
on a competition format that has been validated for use under different epidemiological situations is
available. A cheaper alternative is to prepare a soluble antigen for use in an indirect ELISA, and the
procedure using this soluble antigen is described below.
ELISAs show a decreased sensitivity when the serum samples to be tested are poorly preserved. To solve
this problem, several new ELISAs based on the use of new ASFV recombinant proteins are now being
validated (Gallardo et al., 2006).
Carrying out a second confirmatory test such as the immunoblotting test, IFA test or Immunoperoxidase test
described below is recommended in the case of a doubtful result or a positive result when sera are
suspected to be poorly preserved.
Antigen preparation for ELISA
The ELISA antigen is prepared from infected cells grown in the presence of pig serum (Escribano et al.,
1989).
i)
Infect MS (monkey stable) cells at multiplicity of infection of 10 with adapted virus, and incubate in
medium containing 2% pig serum.
ii)
Harvest the cells at 3648 hours post-infection, when the CPE is extensive. Wash in PBS, sediment at
650 g for 5 minutes, wash the cell pellet in 0.34 M sucrose in 5 mM Tris/HCl, pH 8.0, and centrifuge to
pellet cells.
Carry out steps (iii) to (v) on ice:
1074
iii)
Resuspend the cell pellet in 67 mM sucrose in 5 mM Tris/HCl, pH 8.0 (1.8 ml per 175 cm2 flask), and
leave for 10 minutes with agitation after 5 minutes.
iv)
Add nonionic detergent Nonidet P-40 to a final concentration of 1% (w/v), and leave for 10 minutes
(with agitation after 5 minutes) to lyse the cells.
v)
Add sucrose to a final concentration of 64% (w/w) in 0.4 M Tris/HCl, pH 8.0, and centrifuge at 1000 g
for 10 minutes to pellet nuclei.
vi)
Collect the supernatant and add EDTA (2 mM final concentration), beta-mercaptoethanol (50 mM final
concentration) and NaCl (0.5 M final concentration) in 0.25 mM Tris/HCl, pH 8.0, and incubate for
15 minutes at 25C.
vii)
Centrifuge at 100,000 g for 1 hour at 4C over a layer of 20% (w/w) sucrose in 50 mMTris/HCl, pH 8.0.
Remove the band immediately above the sucrose layer and use as the ELISA antigen. Store at 20C.
Indirect ELISA procedure (Pastor et al., 1990)
i)
Coat ELISA polysorp microtitre plate(s) with antigen by adding 100 l of the recommended or
pretitrated dilution of antigen in 0.05 M carbonate/bicarbonate buffer, pH 9.6, to each well.
ii)
Incubate at 4C for 16 hours (overnight) and then wash five times with 0.05% Tween 20 in PBS,
pH 7.2.
iii)
Dilute the test sera and positive and negative control sera 1/30 in 0.05% Tween 20 in PBS, pH 7.2, and
add 100 l of each diluted serum to duplicate wells of the antigen-coated plate(s).
If four pairs of each positive and negative control serum are added to wells in different parts of the
plate, 40 sera can be tested in duplicate on one plate, as shown on the plate plan below.
1
10 11 12
iv)
Incubate plates at 37C for 1 hour (optionally on a plate shaker), and then wash five times with 0.05%
Tween 20 in PBS.
v)
To each well add 100 l of protein-A/horseradish-peroxidase conjugate (Pierce) at the recommended

or pretitrated dilution in 0.05% Tween 20 in PBS.
vi)
Incubate the plates at 37C for 1 hour, and then wash five times with 0.05% Tween 20 in PBS.
vii)
Substrate: Add hydrogen peroxide to the substrate solution (0.04% orthophenylenediamine (OPD) in
phosphate/citrate buffer, pH 5.0) at the rate of 10 l/25 ml, and add 100 l of substrate to each well.
Alternatively, DMAB/MBTH substrate solution can be used instead of OPD: Add 200 l of substrate to
each well (10 ml DMAB 80.6 mM Solution + 10 ml of MBTH1.56mM solution + 5 l H2O2).
DMAB/MBTH substrate preparation:
DMAB 3-Dimethylaminobenzoic acid (SIGMAD-07871643); MBTH 3-Methyl-2-benzothiazolinone
hydrazone hydrochloride monohidrate (SIGMA M-8006).
DAMB 80,6 mM Solution: Dissolve 13.315 g of DAMB acid in 1000 ml of 0.1 M phosphate buffer, pH 7
(5.3 g KH2PO4, 8.65 g Na2HPO4 made up to 1000 ml in distilled water) by continuous agitation for
1 hour at room temperature, adjusting the pH to 7 with NaOH (5 M). Filter through a funnel.
MBTH 1,56 mM solution: Dissolve 0.3646 g of MBTH in 1000 ml of 0.1 M phosphate buffer, pH 7 (5.3 g
KH2PO4, 8,65 g Na2HPO4 made up to 1000 ml in distilled water) by continuous agitation for 1 hour,
adjusting the pH to 6,25 with concentrated Hydrochloric acid. Filter through a funnel.
1075
The volume required per plate is: 10 ml DMAB + 10 ml MBTH + 5 l H2O2 30%
Substrate can be prepared as stock solutions, aliquoted and kept at 20C. Mix the DAMB and the
MBTH solutions (1:1) just before use and add the required quantity of 30% H2O2.
viii) Incubate at room temperature for approximately 610 minutes (before the negative control begins to be
coloured). The time necessary for the colour to develop will depend on both the temperature of the
substrate when added to the wells, and the room temperature.
ix)
Stop the reaction by adding 100 l of 3 N sulphuric acid to each well.
x)
Reading the results: Positive sera have a clear colour (yellow in case of OPD substrate, blue in case of
DMAB/MBTH substrate) and can be read by eye, but to ensure that all positive sera are identified, it is
necessary to read the absorbance in each well spectrophotometrically, at 492 nm (OPD substrate) or
at 600620 nm (in case of DMAB/MBTH), in an ELISA reader. Using OPD substrate any serum is
considered to be positive if it has an absorbance value of more than twice the mean absorbance value
of the control negative sera on that plate. Using DMAB/MBTH substrate, the test is validated when the
mean absorbance value of the positive control is more than four times the mean of absorbance of the
negative control.
To correctly interpret the results it is necessary to calculate a cut-off point which enables the
differentiation of negative, inconclusive and positive results. The cut-off point is established by the
following equation:
The cut-off point = optical density negative serum 1 + optical density positive serum 0.2.
b)
Sera with an optical density below the cut-off point 0.1 can be considered negative.
Sera with an optical density higher than cut-off point + 0.1 can be considered positive.
Sera with an optical density between cut-off point 0.1 can be considered inconclusive and the
result needs to be confirmed by the IB technique.

This test (Pan et al., 1974) should be used as a confirmatory test for sera from areas that are free from ASF
and are positive in the ELISA, and for sera from endemic areas that give an inconclusive result in the ELISA.
c)
Test procedure
i)
Prepare a suspension of ASFV-infected pig kidney or monkey cells at a concentration of 5

105 cells/ml, spread small drops on glass slides, air dry and fix with acetone at room temperature for
10 minutes. Note that slides can be stored at 20C until ready for use.
ii)
Heat inactivate test sera at 56C for 30 minutes.
iii)
Add appropriate dilutions of test sera and positive and negative control sera in buffered saline to slides
of both infected and uninfected control cells, and incubate for 1 hour at 37C in a humid chamber.
iv)
Wash the slides by immersing four times in fresh clean PBS and then distilled water.
v)
Add predetermined or recommended dilutions of anti-pig immunoglobulin/FITC or protein-A/FITC

conjugate to all slides, and incubate for 1 hour at 37C in a humid chamber.
vi)
Wash the slides by immersing four times in fresh clean PBS and then distilled water, mount in
PBS/glycerol, and examine under an ultraviolet light microscope with suitable barrier and exciter filters.
vii)
Reading the results: The control positive serum on infected cells must be positive and all other controls
must be negative before the test can be read. Sera are positive if infected cultures show specific
fluorescence.
Immunoblotting test (Escribano et al., 1990; Pastor et al., 1989)

This test should be used as an alternative to the IFA test to confirm equivocal results with individual sera.
The immunoblotting test is very specific and enables easier and more objective interpretation of the results
and a better recognition of weak-positive samples.
Viral proteins that induce specific antibodies in pigs have been determined. These polypeptides have been
placed on antigen strips and have been shown in the immunoblotting test to react with specific antibodies
from 9 days post-infection.
Preparation of antigen strips
i)
Prepare cytoplasmic soluble virus proteins as described for the preparation of ELISA antigen in Section
B.2.a.
1076
ii)
Electrophorese through 17% acryl-amide/N,N-diallyltartardiamide (DATD) gels with appropriate

molecular weight standards.
iii)
Transfer the proteins on to a 14 14 cm2 nitrocellulose membrane by electrophoresis at a constant

current of 5 mA/cm in transfer buffer (20% methanol in 196 mM glycine, 25 mMTris/HCl, pH 8.3).
iv)
Dry the membrane and label the side on to which the proteins were electrophoresed.
v)
Cut one strip from the edge of the filter and carry out the immunoblotting procedure described below.
Identify the region containing proteins of 2335 kDa by comparison with the molecular weight
standards run in parallel, and cut this region into 0.5 cm wide strips. Label each strip on the side on to
which the proteins were electrophoresed.
These strips (approximately 4 cm long) constitute the antigen strips used for immunoblotting and contain
proteins with which antibodies in both acute and convalescent pig sera will react. These antibodies persist
for life in some pigs.
Preparation of chloranaphthol substrate solution
This solution must be prepared immediately before use.

i)
Dissolve 6 mg of 4-chloro-1-naphthol in 2 ml of methanol and add this solution slowly to 10 ml of PBS

while it is being stirred.
ii)
Remove the white precipitate that is formed by filtration through Whatman No.1 filter paper (optional).
iii)
Add 4 l of 30% hydrogen peroxide.
Test procedure
The antigen strips must be kept with the labelled side uppermost during the immunoreaction procedure.
i)
Incubate the antigen strips in blocking buffer (2% non-fat dried milk in PBS) at 37C for 30 minutes with
continuous agitation.
ii)
Prepare 1/40 dilutions of test sera and positive and negative control sera in blocking buffer.
iii)
Incubate the antigen strips in the appropriate serum at 37C for 45 minutes with continuous agitation.
Incubate one antigen strip in positive control serum and one in negative control serum. These two
strips are controls. Wash four times in blocking buffer; the final wash should be for 5 minutes with
continuous agitation.
iv)
Add protein-A/horseradish-peroxidase conjugate at the recommended or pretitrated dilution (usually at

1/1000 dilution) in blocking buffer to all antigen strips. Incubate at 37C for 45 minutes with continuous
agitation. Wash four times in blocking buffer; the final wash should be for 5 minutes with continuous
agitation.
v)
Prepare the substrate solution, add to the antigen strips, and incubate at room temperature for 5
15 minutes with continuous agitation.
vi)
Stop the reaction with distilled water when the protein bands are suitably dark.
vii)
Reading the results: Positive sera react with more than one virus protein in the antigen strip; they must
give a similar protein pattern and have the same intensity of colour as the antigen strips stained with
positive control serum.

At present there is no vaccine for ASF.
REFERENCES
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AGERO M., FERNNDEZ J., ROMERO L., ZAMORA M.J., SANCHEZ C., BELK S., ARIAS M. & SNCHEZ-VIZCANO J.M.
(2004). A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential
diagnosis of African swine fever and Classical swine fever. Vet. Res., 35, 113.
1077
ARIAS M. & SNCHEZ-VIZCANO J.M. (2002a). African swine fever. In: Trends in Emerging Viral Infections of Swine,
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BASTO A.P., PORTUGAL R.S., NIX R.J., CARTAXEIRO C., BOINAS F., DIXON L.K., LEITAO A. & MARTINS C. (2006).
Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in
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BLASCO R., AGERO M., ALMENDRAL J.M. & VIUELA E. (1989). Variable and constant region in African swine fever
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BOOL P.H., ORDAS A. & SANCHEZ BOTIJA C. (1969). El diagnostico de la peste porcina africana por
inmunofluorescencia. (The diagnosis of African swine fever by immunofluorescence). Bull. OIE, 72, 819839.
BOSHOFF C.I., BASTOS A.D., GERBER L.J. &VOSLOO W. (2007).Genetic characterisation of African swine fever
viruses from outbreaks in southern Africa (19731999).Vet.Microbiol.,121 (12), 4555.
CARRILLO C., BORCA M.V., AFONSO C.L., ONISK D.V. & ROCK D.L. (1994). Long-term persistent infection of swine
monocytes/macrophages with African swine fever virus.J. Virol., 68, 580583.
CHAPMAN D.A., TCHEREPANOV V., UPTON C. & DIXON L.K (2008).Comparison of the genome sequences of nonpathogenic and pathogenic African swine fever virus isolates. J.Gen. Virol., 89, 397408.
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analysis of 11 complete African swine fever virus genome sequences.Virol., 400 (1), 128136.
DIXON L.K., ESCRIBANO J.M., MARTINS C., ROCK D.L., SALAS M.L. & WILKINSON P.J. (2005). In: Virus Taxonomy, VIIIth
Report of the ICTV, Fauquet C.M., Mayo M.A., Maniloff J, Desselberger U.& Ball L.A., eds. Elsevier/Academic
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ESCRIBANO J.M., PASTOR M.J., ARIAS M. & SANCHEZ VIZCAINO J.M. (1990). Confirmacin de sueros positivos a
ELISA-peste porcina africana, mediante la tcnica de Immunoblotting. Utilizacin de las protenas inducidas por
el virus con pesos moleculares comprendidos entre 23 y 35 kilodaltons, en el desarrollo de un kit de diagnstico.
(Confirmation of sera positive by ASF ELISA with the immunoblotting technique. Use of virus-induced proteins of
2325 kDa in the development of a diagnostic kit.) Med. Vet., 7, 135141.
ESCRIBANO J.M., PASTOR M.J. & SANCHEZ VIZCAINO J.M. (1989). Antibodies to bovine serum albumin in swine sera:
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1122.
FERNNDEZ-PINERO J., GALLARDO C., ELIZALDE M., RASMUSSEN T.B., STAHL K., LOEFFEN W., BLOME S., BATTEN C.,
CROOKE H., LE POTIER M.F.,UTTENTHAL ., LEBLANC N., ALBINA E., KOWALCZYK A., MARKOWSKA-DANIEL I., TIGNON M.,
DE MIA G.M., GIAMMARIOLI M., ARIAS M. & HOFFMANN B. (2010). EPIZONE ring trial on ASFV real-time PCR. Annual
Meeting of National African Swine Fever Laboratories, 18 May 2010, Pulawy, Poland.
GALLARDO C., BLANCO E., RODRGUEZ M.J., CARRASCOSA A. & SANCHEZ-VIZCAINO J.M. (2006). Antigenic properties
and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells. J. Clin. Microbiol., 44
(3), 14891495.
GALLARDO C., MWAENGO D.M., MACHARIA J.M., ARIAS M., TARACHA E.A., SOLER A., OKOTH E., MARTN E., KASITI J.&
BISHOP RP.(2009). Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of
the p54, p72, and pB602L (CVR) genes. Virus Genes, 38 (1), 8595.
GALLARDO C., SOLER A., SIMN A., MARTN E., MARTN R., PELAYO V.,OKOTH E.,BISHOP R., SNCHEZ M.A., DE MIA
G.,FASINA F.O., SNCHEZ-VIZCANO, J.M. & ARIAS M.(2010). African Swine Fever Threat: Evaluating diagnostic tools
with ASFV circulating strains. Annual Meeting of National African Swine Fever Laboratories, 18 May 2010,
Pulawy, Poland.
KING D.P., REID S.M., HUTCHINGS G.H., GRIERSON S.S., WILKINSON P.J., DIXON L.K., BASTOS A.D.S. & DREW T.W.
(2003). Development of a TaqMan PCR assay with internal amplification control for the detection of African
swine fever virus. J. Virol. Methods, 107, 5361.
1078
LUBISI B.A., BASTOS A.D., DWARKA R.M. & VOSLOO W.(2005).Molecular epidemiology of African swine fever in East
Africa. Arch Virol., 150 (12), 24392452.
MALMQUIST W.A. & HAY D. (1960). Haemadsorption and cytopathic effect produced by African swine fever virus in
swine bone marrow and buffy coat cultures. Am. J. Vet. Res., 21, 104108.
PAN I.C., TRAUTMAN R., HESS W.R., DE BOER C.J., TESSLER J., ORDAS A., SANCHEZ BOTIJA C., OVEJERO J. & SANCHEZ
M.C. (1974). African swine fever: comparison of four sero tests on porcine serums in Spain. Am. J. Vet. Res., 35,
787790.
PASTOR M.J., ARIAS M. & ESCRIBANO J.M. (1990). Comparison of two antigens for use in an enzyme-linked
immunosorbent assay to detect African swine fever antibody. Am. J. Vet. Res.,51, 15401543.
PASTOR M.J., LAVIADA M.D., SANCHEZ VIZCAINO J.M. & ESCRIBANO J.M. (1989). Detection of African swine fever virus
antibodies by immunoblotting assay. Can. J. Vet. Res., 53, 105107.
SNCHEZ-VIZCANO J.M. (1987). African swine fever diagnosis. In: African Swine Fever, Becker Y., ed. Martinus
Nijhoff, Boston, USA, 6371.
SNCHEZ-VIZCANO J.M. (2006). African swine fever. In: Diseases of Swine,Ninth Edition,Straw B.,DAllaire S.,
Mengeling W., Taylor D., eds. Iowa State University,USA, pp. 291298.
SNCHEZ-VIZCANO J.M., CROWTHER J.R. & WARDLEY R.C. (1983). A collaborative study on the use of the ELISA in
the diagnosis of African swine fever. In: African Swine Fever. (CEC/FAO Research Seminar, Sardinia, Sept.
1981). Wilkinson P.J., ed. Commission of the European Communities Publication EUR 8466 EN, 297325.
SNCHEZ-VIZCANOJ.M., MARTINEZ-LPEZ B., MARTINEZ-AVILS M., MARTINS C., BOINAS B., VIAL L., MICHAUD V., JORI
F., ETTER E., ALBINA E. & ROGER F. (2009). Scientific Review on African Swine Fever. CFP/EFSA/AHAW/2007/02,
pp: 1141.
VINUELA E. (1985). African swine fever. Curr. Top. Microbiol. Immunol., 116, 151170.
WARDLEY R.C., ABU ELZEIN E.M.E., CROWTHER J.R. & WILKINSON P.J. (1979). A solid-phase enzyme-linked
immunosorbent assay for the detection of African swine fever antigen and antibody. J. Hyg., 83, 363369.
*
* *
NB: There are OIE Reference Laboratories for African swine fever
diagnostic tests and reagents for African swine fever
1079
CHAPTER 2.8.2.
ATROPHIC RHINITIS OF SWINE
SUMMARY
Definition of the disease: Atrophic rhinitis is an infectious disease of swine characterised by
serous to mucopurulent nasal discharge, shortening or twisting of the snout, atrophy of the turbinate
(conchal) bones and reduced productivity. It may occur enzootically or more sporadically,
depending on a variety of factors including herd immunity. The most severe progressive form is
caused by infection with toxigenic strains of Pasteurella multocida alone or in combination with
Bordetella bronchiseptica. Infections with B. bronchiseptica alone can cause a mild to moderate
form with nonprogressive turbinate bone atrophy. Turbinate atrophy may only be obvious at
slaughter or may be detected in the live animal by use of radiography or tomography.
Environmental and management factors also contribute to the severity and incidence of this
disease. A large proportion of apparently normal pig herds may be infected with B. bronchiseptica
or nontoxigenic P. multocida and show a mild degree or low prevalence of turbinate atrophy.
Identification of the agents: The diagnosis of atrophic rhinitis depends on clinical and postmortem observations in affected swine assisted by the recovery and characterisation of
P. multocida and B. bronchiseptica. The isolation of both organisms is often complicated by the
more profuse growth of other bacteria. Isolation rates are improved by preservation of the nasal or
tonsillar swab at 48C in a non-nutritive transport medium and by using a selective culture
medium.
Pasteurella multocida and B. bronchiseptica can be identified by traditional biochemical tests.
Pasteurella multocida isolates may be further characterised by their capsular and somatic antigens.
Capsular type D is most prevalent in many areas of the world, but in some regions type A
predominates. Capsular antigens may be distinguished serologically by indirect haemagglutination
or immunofluorescence, chemically by flocculation in acriflavine, or by susceptibility to
hyaluronidase. Somatic antigen types can be distinguished by a gel diffusion precipitation test, with
type 3 found most frequently in swine. Toxigenicity of P. multocida isolates can be demonstrated by
testing for cytotoxicity in cultured cells. A commercially available toxin-specific enzyme-linked
immunosorbent assay (ELISA) is now widely used in some areas of the world to differentiate
toxigenic from nontoxigenic isolates. In addition, it is suitable for detection of toxin production by
bacteria from primary culture plates without the need for prior isolation and identification of
individual colonies.
Assays based on the use of DNA probes or polymerase chain reaction (PCR) provide rapid,
sensitive and highly specific detection of B. bronchiseptica and both toxigenic and nontoxigenic
P. multocida for those laboratories with the capability to perform them. A multiplex PCR useful for
capsular typing of P. multocida has also been described.
Serological tests: Detection of antibodies to P. multocida and B. bronchiseptica is of little value as
nontoxigenic strains of P. multocida share cross-reactive antigens with toxigenic strains and
B. bronchiseptica can be isolated from many swine herds. A test based on detection of antibodies
to the P. multocida toxin is commercially available but its usefulness is limited as not all infected
swine develop such antibodies. Widespread vaccination with P. multocida toxoid induces antibodies
of vaccinal origin, complicating interpretation of results.
Requirements for vaccines and diagnostic biologicals: Several vaccines are available
commercially that contain bacterins of B. bronchiseptica and a mixture of toxigenic and/or
nontoxigenic strains of P. multocida, or a toxoid derived from P. multocida or from a recombinant
Escherichia coli.
1080
Chapter 2.8.2. Atrophic rhinitis of swine
A. INTRODUCTION
Definition of the disease: Atrophic rhinitis is an infectious disease of swine characterised by stunted
development or deformation of the nasal turbinate (conchal bone) and septum.
Causal pathogens: Two forms of atrophic rhinitis have been recognised, depending on the causal agent(s) (De
Jong, 2006):
a)
A severe progressive form caused by toxigenic isolates of Pasteurella multocida, most commonly capsular
types D or A, alone or in combination with Bordetella bronchiseptica.
b)
A less severe, nonprogressive form with mild to moderate turbinate atrophy, often without significant snout
changes, caused by B. bronchiseptica.
Description of the disease: Initial clinical signs include sneezing, snuffling and eye discharge with resultant dark
tear-staining and subsequent nasal discharge, which can vary from serous to mucopurulent; in some cases pigs
may show epistaxis. Atrophy of the nasal turbinate and septal deviation may lead to shortening or twisting of the
snout and, in severe cases, difficulty in eating. Increased severity is associated with overstocking and poor
management, housing and environmental conditions. Reduced productivity is generally associated with moderate
to severe atrophic rhinitis, although the precise relationship between infection with these bacteria and reduced
weight gains has not been thoroughly elucidated.
Bordetella bronchiseptica or toxigenic P. multocida may be present in a herd without clinical evidence of disease,
especially when other respiratory pathogens are absent and environmental and management conditions are
optimal. Such carrier herds pose a risk of transmitting these agents to other herds in which progression to severe
disease may occur. Bordetella bronchiseptica and toxigenic P. multocida are commonly found in many
domesticated and wild animal species that could potentially transmit the bacteria to swine herds.
Zoonotic risk and biosafety requirements: Infection of humans by B. bronchiseptica is rare and occurs most
often in immunocompromised persons exposed to infected or vaccinated pets; transmission from swine to
humans has not been reported. Pasteurella multocida can be a serious human pathoen but most zoonotic
infections are associated with exposure to pets or wild animals. Pasteurella multocida has also been frequently
isolated from healthy human carriers working in swine production sites or living nearby and has additionally been
associated with chronic or acute respiratory disease in such individuals (Donnio et al., 1999; Marois et al., 2009).
Transmission occurs primarily through bites or scratches and wound contamination from infected material, but
may also result from inhalation of aerosols. Appropriate precautions should be observed by persons having
contact with swine infected with P. multocida, particularly those who may be immunocompromised. Containment
level 2 practices are recommended for handling clinical specimens and cultures of B. bronchiseptica and
P. multocida.
Differential diagnosis: Porcine cytomegalovirus also causes rhinitis in young pigs but does not progress to
turbinate atrophy or nasal distortion. Asymmetric bone development resulting from habitual biting or chewing of
stalls or drinkers may lead to a noticeable misalignment of the jaw in some pigs. Careful inspection can
distinguish this condition from shortness or deviation of the snout characteristic of atrophic rhinitis.
The diagnosis of atrophic rhinitis depends on clinical, pathological and microbiological investigations, with the
latter being particularly important for herds infected subclinically. It is generally accepted that a herd in which
toxigenic P. multocida is present be defined as affected with progressive atrophic rhinitis, whether or not clinical
signs of the disease are evident (Pedersen et al., 1988). Control in many countries has, therefore, centred on
detection of infection, even in subclinically infected animals considered to be potential carriers.
1.
Pathological diagnostic criteria
Turbinate atrophy may only be seen at slaughter when snout sections at the level of the first/second premolar
tooth are examined. Subjective assessment of turbinate atrophy is convenient and often useful for monitoring
herds (De Jong, 2006), but objective scales of measurement (Gatlin et al., 1996) are best suited for studies
requiring data analysis. Radiography (Done, 1976) and tomography (Magyar et al., 2003) can provide objective
observations from live animals; tomography reveals not only severe lesions but more subtle changes that may not
be apparent by radiography. However, these techniques are of limited use due to the equipment and expertise
required. Diagnosis is assisted by detection of characteristic histopathological features including fibrous
replacement of the bony plates of the ventral conchae with varying degrees of inflammatory and reparative
changes.
1081
2.
Identification of the agents
a)
In vitro culture
As P. multocida preferentially colonises the tonsil, tonsillar swabs or biopsies will provide the highest
isolation rates (Ackermann et al., 1994). Nasal swabs are preferred for isolation of B. bronchiseptica. When
sampling the tonsil is not practical, nasal swabs suffice for isolation of both organisms. Swabs with flexible
shafts should be used; sample collection in young pigs is facilitated by the use of mini-tipped swabs. A single
swab is used to sample both sides of the nasal cavity and should then be placed in a non-nutritive transport
medium (e.g. phosphate buffered saline) and kept at 48C during transit to avoid overgrowth by other
faster-growing bacteria. Transit time should not exceed 24 hours.
Although both P. multocida and B. bronchiseptica grow readily on blood agar, a selective medium is
preferred as overgrowth of other bacteria that are present in higher numbers often interferes with their
detection. An additional difficulty related to B. bronchiseptica is that this organism grows more slowly than
most other bacteria present in clinical samples. Various formulations of media containing antibiotics have
been used for isolation of P. multocida, but comparison of studies in the literature indicates that the highest
isolation rates are obtained with modified Knight medium (bovine blood agar containing 5 g/ml clindamycin,
0.75 g/ml gentamicin) (Lariviere et al., 1993) or KPMD (bovine blood agar containing 3.75 U/ml bacitracin,
5 g/ml clindamycin, 0.75 g/ml gentamicin, and 2.25 g/ml amphotericin B) (Ackermann et al., 1994).
MacConkey agar with 1% glucose and 20 g/ml furaltadone is used by many laboratories for selective
growth of B. bronchiseptica from nasal swabs, but a modified Smith-Baskerville medium (a peptone agar
formulation containing 20 g/ml penicillin, 20 g/ml furaltadone, and 0.5 g/ml gentamicin) appears superior,
especially when the number of B. bronchiseptica present is low (Lariviere et al., 1993; Smith & Baskerville,
1979). A further improvement in isolation rate was reported using blood agar containing 40 g/ml cephalexin
(Lariviere et al., 1993). A selective blood agar medium for simultaneous isolation of both P. multocida and
B. bronchiseptica, containing 5 mg/litre clindamycin-HCl, 0.75 mg/litre gentamicin sulphate, 2.5 mg/litre Ktellurite, 5 mg/litre amphotericin-B and 15 mg/litre bacitracin, has also been described (De Jong & Borst,
1985). However, it should be noted that K-tellurite has sometimes been found to be inhibitory to the growth
of type D P. multocida (Lariviere et al., 1993).
b)
Biochemical characteristics
P. multocida is a Gram-negative, bipolar, pleomorphic rod and forms nonhaemolytic, greyish colonies on
blood agar with a characteristic, sweetish odour. It fails to grow on MacConkey agar but yields positive
oxidase and catalase reactions and produces indole.
B. bronchiseptica is also a Gram-negative rod, forming convex colonies 12 mm in size, usually haemolytic,
on blood agar or Bordet-Gengou medium after 48 hours of growth. It is nonfermentative, but positive for
oxidase, catalase, citrate and urea and grows in 6.5% NaCl.
Agglutination tests using specific antisera have been described for confirming the identity of presumptive
B. bronchiseptica isolates but appropriate sera are not widely available for use.
Capsular typing of P. multocida
Capsular typing of P. multocida is useful for epidemiological purposes, as P. multocida often has a mucoid
capsule. Serotyping by indirect haemagglutination has traditionally been used (Carter, 1955) but only a few
laboratories throughout the world make and maintain the antisera required. However, simpler chemical
methods can usually distinguish most swine isolates. Those producing a type D capsule form a heavy
flocculate in 1/1000 aqueous acriflavine (Carter & Subronto, 1973), while capsular type A strains can be
identified by inhibition of growth in the presence of hyaluronidase (Carter & Rundell, 1975). A small
proportion of swine isolates are noncapsulated.
Acriflavine test procedure for capsular type D P. multocida
i)
Inoculate a tube containing 3 ml of brainheart infusion broth, using a freshly grown bovine blood agar
culture, for each P. multocida isolate to be tested. Include a known type D strain and a known type A
strain as positive and negative controls.
ii)
Incubate inoculated tubes at 37C for 1824 hours.
iii)
Pellet bacteria by centrifugation and remove 2.5 ml of the supernatant.
iv)
Add 0.5 ml of a 1/1000 aqueous solution of acriflavine neutral. Acriflavine solution should be freshly
prepared each week and stored at 4C, protected from light.
v)
Mix to resuspend the bacterial pellet and incubate the tube at room temperature, without shaking.
vi)
Observe at 5 minutes for the presence of a heavy flocculent precipitate.
1082
Hyaluronidase test procedure for capsular type A P. multocida
i)
Prepare fresh bovine blood agar cultures of the isolates to be tested. Include a known type A strain and
a known type D strain as positive and negative controls.
ii)
Inoculate each strain to be examined on a separate trypticase soy blood agar plate with 5% sheep
blood or 6% bovine blood by streaking several parallel lines of growth, approximately 35 mm apart,
across the diameter of the plate. For maximum production of hyaluronic acid it is important that the
plates be fresh and not dehydrated.
iii)
Heavily streak a hyaluronidase-producing strain of Staphylococcus aureus at right angles to the lines of
P. multocida growth.
iv)
Incubate the plates at 37C, in a humidified atmosphere, and observe periodically for up to 24 hours.
Type A strains will exhibit a marked inhibition of growth in the region adjacent to the growth lines of
S. aureus.
Somatic antigen typing of P. multocida
Differences in the cell wall lipopolysaccharide among P. multocida strains provide the basis for somatic
antigen typing. Sixteen types can be distinguished by a gel diffusion precipitation test (Heddleston et al.,
1972), with type 3 found most frequently in swine. Although the required antisera are not widely available,
many reference laboratories and some diagnostic laboratories offer somatic antigen typing.
Detection of the P. multocida toxin
Diagnosis of progressive atrophic rhinitis depends upon characterisation of P. multocida isolates as

toxigenic. The heat-labile toxin of P. multocida produces dermonecrosis in guinea pigs and is lethal in mice
following intraperitoneal injection. Toxigenicity can also be demonstrated in vitro by testing for cytopathic
effects on monolayers of embryonic bovine lung (EBL) cells (Rutter & Luther, 1984), African green monkey
kidney (Vero) cells (Pennings & Storm, 1984) or bovine turbinate cells (Eamens et al., 1988). The bacteria
are grown in brainheart infusion broth incubated at 37C for 24 hours and then pelleted by centrifugation.
The supernatant is sterilised by filtration and titrated in monolayer cultures prepared in microtitre plates.
Following incubation at 37C for 23 days, the monolayers are stained with crystal violet and examined
microscopically to detect cytopathic effects. A rapid cell culture test, in which the suspect colonies are grown
on an agar overlay of EBL cells (Chanter et al., 1986), permits more efficient analysis of large numbers of
isolates.
An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies can detect the toxin in
mixtures of bacteria recovered from primary isolation media (Foged, 1992). This is an important advantage
as swine may be colonised simultaneously with a mixture of toxigenic and nontoxigenic strains (Ackermann
et al., 1994; De Jong, 2006). Cell culture methods would require every colony of P. multocida in the sample
to be tested, which is clearly impractical, to achieve the same level of sensitivity as the ELISA.
This ELISA is commercially available throughout Europe and in a few other selected regions of the world1
(though not in the United States of America) and has been widely adopted in many areas as the preferred
test for identification of carriers and for control of progressive atrophic rhinitis. Though highly specific, a
positive result without previous history of disease or suspicious signs should be thoroughly investigated to
recover toxigenic isolates from the animals sampled.
c)
Molecular methods
Colony morphology and biochemical testing remain the basis for identification of toxigenic P. multocida and
B. bronchiseptica in many laboratories. However, a number of assays based on the use of DNA probes
(Kamps et al., 1990; Register et al., 1998) or polymerase chain reaction (PCR) (Kamp et al., 1996;
Lichtensteiger et al., 1996; Nagai et al., 1994; Register & DeJong, 2006) for detection of toxigenic
P. multocida and/or B. bronchiseptica from swine provide faster, more specific and more sensitive diagnostic
tools. Diagnostic laboratories are increasingly using PCR for identification of these agents as the equipment
and expertise become more readily available. Proper in-house validation with known controls as well as
standardised, ongoing quality control measures are essential (see Chapter 1.1.5 Principles and methods of
validation of diagnostic assays for infectious diseases).
A multiplex PCR assay for capsular typing of P. multocida (Townsend et al., 2001) appears to provide more
reliable results than phenotypic methods and is frequently used in suitably equipped diagnostic laboratories.
For availability/purchase information contact DakoCytomation Denmark A/S, Produktionsvej 42, DK-2600 Gloistrup,
Denmark; http://www.dakocytomation.com.
1083
Various DNA fingerprinting techniques, including restriction endonuclease analysis (REA), ribotyping,
pulsed-field gel electrophoresis, and PCR-based methods have been evaluated by numerous groups for the
purpose of differentiating P. multocida isolates. Few direct comparisons between methods have been carried
out using strains from pigs with atrophic rhinitis, but REA currently appears to be the method of choice for
epidemiologic investigations as it provides a high level of discrimination without the need for specialized
equipment or reagents (Djordjevic et al., 1998; Gardner et al., 1994; Harel et al., 1990)
3.
Serological tests
At present there are no satisfactory serological tests that can be relied on to detect those animals infected with
toxigenic P. multocida and capable of developing or spreading the disease. Detection of antibodies to
P. multocida is not helpful, as nontoxigenic strains share many cross-reacting antigens with toxigenic strains. An
ELISA for detection of antibodies to the P. multocida toxin has been described (Foged, 1992) that is commercially
available in Europe and a few other areas of the world (see footnote 1). However, many animals infected with
toxigenic P. multocida fail to produce antibodies to the toxin, and widespread use of toxoid-containing vaccines
limits the diagnostic value of this ELISA to herds with no history of vaccination or to detection of vaccine response
in vaccinated herds.
Infection with B. bronchiseptica can be detected serologically by agglutination testing with formalin-treated
bacteria or with a more sensitive ELISA (Venier et al., 1984). Unless monitoring the status of a negative herd,
B. bronchiseptica serology may be of little value as the organism is present in many apparently healthy pig herds.

1.
Background
There are several commercially available vaccines that contain whole-cell bacterins of B. bronchiseptica
combined with a toxigenic P. multocida bacterin and/or a P. multocida toxoid. The toxigenic P. multocida bacterin
component is most often capsular type D but some vaccines additionally include a type A strain, which may be
toxigenic or nontoxigenic. Live, attenuated B. bronchiseptica vaccines are also available. Vaccines containing
only B. bronchiseptica are not suitable for control of progressive atrophic rhinitis, but may be of benefit in herds
with the nonprogressive form. Pasteurella multocida and B. bronchiseptica vaccines appear to reduce the level of
colonisation by these bacteria, but do not eliminate them or prevent infection. Most commercially available
vaccines contain either an oil adjuvant or aluminium hydroxide gel.
The P. multocida toxin is the single most important protective antigen with respect to progressive atrophic rhinitis.
Vaccines based on a P. multocida toxoid offer specific protection against the action of the toxin, which, by itself,
can be used to reproduce all of the major signs of this disease (see ref. 14 for review). The level of toxin produced
by P. multocida is relatively low and the toxin-specific antibody response induced by bacterin-only vaccines may
not be optimal. Purified toxoid (inactivated by formaldehyde) is more immunogenic than crude toxoid and the
immunogenicity of the inactivated form is not affected by mixture with a B. bronchiseptica bacterin. However, the
difficulty and expense of large-scale purification from cultures of P. multocida prevent routine incorporation of
native, chemically inactivated toxoid into vaccines. Recombinant vaccines containing subunit toxin proteins or
genetically detoxified derivatives of the full-length toxin are highly efficacious and less costly to produce (Hsuan et
al., 2009; Pejsak et al., 1994). A DNA vaccine encoding a full-length but enzymatically inactive toxoid was shown
to be highly immunogenic in pigs but has so far not been evaluated for efficacy against challenge (Register et al.,
2007).
Bordetella bronchiseptica produces a variety of toxins and adhesins that are potential virulence factors in swine.
Only one, the outer membrane protein pertactin, has been shown to protect against disease in pigs (Kobisch &
Novotny, 1990). Despite this fact, a dermonecrotic toxin produced by B. bronchiseptica, unique from the toxin
produced by P. multocida, has traditionally been regarded as the primary virulence factor and protective
immunogen in swine (De Jong, 2006). Several studies strongly implicate the toxin as a virulence factor and it
undoubtedly plays a role in pathogenesis and, perhaps, in protection. However, the role of pertactin and several
additional virulence factors in protective immunity is most likely equal to, or perhaps exceeds, that of the toxin.
Bordetella bronchiseptica is subject to phenotypic variation under certain growth conditions (e.g. temperatures
below 37C or the presence of chemical modulators such as MgSO4 or nicotinic acid), in which production of most
virulence factors is reversibly turned off. Spontaneously occurring mutants, permanently unable to produce most
virulence factors, also arise with a low frequency during culture. Careful attention to colony morphology, on an
area of the plate with well-separated colonies, is essential to retain cultures in the phase I (also known as Bvg+),
or virulent, mode. Phase I colonies are small (12 mm in diameter), domed, and haemolytic on blood agar. Loss
of haemolysis and the appearance of larger, flat colonies indicate conversion to the avirulent form. Whenever
1084
possible, cultures should be propagated using single haemolytic colonies to minimise the slow accumulation of
avirulent clones within the culture.
2.
a)

i)
The seed-lot system should be employed for the bacterial strains used to prepare whole-cell bacterins,
as well as for the strains from which purified antigens are derived.
In the case of whole-cell bacterins, the origin and history of both the P. multocida and B. bronchiseptica
strains should be described and the full characterisation of the master seeds should be laid down in a
master seed batch protocol. Bordetella bronchiseptica used for vaccine production should be a phase I
virulent culture and the P. multocida isolates used should be toxigenic.
Working seeds used for vaccine production should be derived from the master seed and checked for
all relevant properties as described in the master seed batch protocol.
ii)
Quality criteria
Both the master seed and the working seed must be pure cultures, free from bacterial, mycotic,
mycoplasmal and viral contamination.
Identity of the bacterial species and the production of relevant antigens should be confirmed.
b)
i)
Procedure
Precise details of standards for the production of effective commercial vaccines are not available, but
they are known to contain 1010 cells of formalin-killed B. bronchiseptica and 10 g of P. multocida
toxoid per dose. Bordetella bronchiseptica should be confirmed to be a phase I culture and, for
P. multocida, it should be confirmed that the culture contains sufficient levels of toxin. A defined
number of passages should be used to give the production culture. Bordetella bronchiseptica cells, and
either P. multocida cells and/or toxin, are inactivated, detoxified and formulated with an adjuvant. As
the toxin of P. multocida has an intracellular location and is released on cell lysis during the stationary
phase, the culture supernatant should be harvested approximately 48 hours after the end of the
exponential phase of growth.
ii)
Requirements for media

All bacterial strains should be propagated in media that support efficient growth and allow optimal
expression of the antigens that are relevant for the induction of protective antibodies.
iii)
In-process controls
Seed and production cultures are inoculated on blood agar plates and incubated. No nonspecific
colonies should grow on these plates.
Cultures are inactivated with formaldehyde. Tests are performed to check the effectiveness of the
inactivation process and to test for residual formaldehyde.
Quantification of antigens is carried out by performing a total cell count using a bacterial counting
chamber for enumerating whole cells or an antigenic mass determination for defined antigens, e.g.
P. multocida toxin, by quantitative enzyme immunoassay.
iv)

Every batch of vaccine should be tested for sterility according to standard methods (see Chapter 1.1.7
Tests for sterility and freedom from contamination of biological materials) described in the European
Pharmacopoeia or the United States Code of Federal Regulations.
Every batch of vaccine should be tested for safety in the target animal, by giving a double dose by the
recommended route of vaccination, and a second, single dose 2 weeks later. No abnormal local or
systemic reactions should occur. When a preservative is used, the concentration should be measured
for each batch. It must not exceed the maximum permitted level.
Every batch of vaccine should be tested for potency using a validated serological test that correlates
with the protection obtained in the efficacy experiment, as described under Section C.2.c.ii. The
potency test is not necessarily carried out in the target animal mice or rabbits can be used. In these
latter cases, correlation has to be shown with protective antibody levels in the target animal.
1085
c)

i)
Safety requirements
Although inactivation of the bacterial cultures by a validated method is a standard procedure, both
bacterial species produce dermonecrotic toxins; detoxification of these toxins should be confirmed
when toxoids are used as vaccine components. Standard safety tests for inactivated vaccines should
be carried out (see Chapter 1.1.6 Principles of veterinary vaccine production).
When an oil emulsion is used as the adjuvant, accidental injection of the operator can cause a severe
local reaction. Medical attention should be sought immediately, treating the wound as a grease-gun
injury.
ii)
Normally the vaccine is applied during the late stage of pregnancy, so that progeny will be protected by
the uptake of colostral antibodies. The efficacy of a trial vaccine should be measured by vaccinating
groups of pregnant sows. Their progeny should be challenged by virulent cultures of B. bronchiseptica
and toxin-producing P. multocida. Significant protection should be obtained against the clinical signs of
the progressive form of atrophic rhinitis, i.e. turbinate atrophy. The clinical signs induced in the controls
and vaccinates may be compared according to the scoring system of Done (1976).
When the vaccine may be applied irrespective of the stage of pregnancy, duration of immunity should
be at least 6 months, so that booster vaccinations twice a year should maintain effective antibody
levels.
iii)
Stability
Every batch of vaccine should be subjected to an accelerated shelf-life test, which has been correlated
with real-time shelf-life testing.
3.
A number of vaccines incorporating enzymatically inert P. multocida toxin subunits or whole toxin rendered
inactive through mutation have been described and evaluated under experimental conditions. The recombinant
proteins are generally expressed in Escherichia coli and purified prior to use. At present, only a few vaccines that
include recombinant toxin or toxin subunits are commercially available; they may or may not include a
P. multocida bacterin. Evidence so far suggests such vaccines elicit levels of toxin-specific antibody equal to or
greater than those obtained with vaccines containing native, chemically inactivated toxoid.
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by toxigenic and nontoxigenic strains of Pasteurella multocida in conventionally raised swine. J. Vet. Diagn.
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CARTER G.R. (1955). Studies on Pasteurella multocida. I. A hemagglutination test for the identification of
serological types. Am. J. Vet. Res., 16, 481484.
CARTER G.R. & RUNDELL S.W. (1975). Identification of type A strains of P. multocida using staphylococcal
hyaluronidase. Vet. Rec., 96, 343.
CARTER G.R. & SUBRONTO P. (1973). Identification of type D strains of Pasteurella multocida with acriflavine. Am.
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CHANTER N., RUTTER J.M. & LUTHER P.D. (1986). Rapid detection of toxigenic Pasteurella multocida by an agar
overlay method. Vet. Rec., 119, 629630.
DE JONG M.F. (2006). In: Diseases of Swine, Ninth Edition, Straw B.E., Zimmerman J.J., dAllaire S..& Taylor D.J.,
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DE JONG M.F. & BORST G.H. (1985). Selective medium for the isolation of P. multocida and B. bronchiseptica. Vet.
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progressive atrophic rhinitis. J. Med. Microbiol., 47, 679688.
DONE J.T. (1976). Porcine atrophic rhinitis: snout radiography as an aid to diagnosis and detection of the disease.
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toxin-producing strains of Pasteurella multocida subsp. multocida isolated from man and swine. J Med. Microbiol.,
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EAMENS G.J., KIRKLAND P.D., TURNER M.J., GARDNER I.A., WHITE M.P. & HORNITZKY C.L. (1988). Identification of
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FOGED N.T. (1992). Pasteurella multocida toxin. The characterisation of the toxin and its significance in the
diagnosis and prevention of progressive atrophic rhinitis in pigs. APMIS Suppl., 25, 156.
GARDNER I.A., KASTEN R., EAMENS G.J., SNIPES K.P. & ANDERSON R.J. (1994). Molecular fingerprinting of
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measure the severity of induced atrophic rhinitis. Can. J. Vet. Res., 60, 121126.
HAREL J., CT S. & JACQUES M. (1990). Restriction endonuclease analysis of porcine Pasteurella multocida
isolates from Quebec. Can. J. Vet. Res., 54, 422426.
HEDDLESTON K.L., GALLAGHER J.E. & REBERS P.A. (1972). Fowl cholera: gel diffusion precipitation test for
serotyping Pasteurella multocida from avian species. Avian Dis., 16, 925936.
HSUAN S.-L., LIAO C.-M., HUANG C., WINTON J.R., CHEN Z.-W., LEE W.-C., LIAO J.-W., CHEN T.-H., CHIOU C.-J., YEH
K.-S. & CHIEN M.-S. (2009). Efficacy of a novel Pasteurella multocida vaccine against progressive atrophic rhinitis
of swine. Vaccine, 27, 29232929.
KAMP E.M., BOKKEN G.C., VERMEULEN T.M., DE JONG M.F., BUYS H.E., REEK F.H. & SMITS M.A. (1996). A specific
and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar
swabs specimens of pigs. J. Vet. Diagn. Invest., 8, 304309.
KAMPS A.M., BUYS W.E., KAMP E.M. & SMITS M.A. (1990). Specificity of DNA probes for the detection of toxigenic
Pasteurella multocida subsp. multocida strains. J. Clin. Microbiol., 28, 18581861.
KOBISCH M. & NOVOTNY P. (1990). Identification of a 68 kilodalton outer membrane protein as the major protective
antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets. Infect. Immun., 58, 352357.
LARIVIERE S., LEBLANC L., MITTAL K.R. & MARTINEAU G.P. (1993). Comparison of isolation methods for the recovery
of Bordetella bronchiseptica and Pasteurella multocida from the nasal cavities of piglets. J. Clin. Microbiol., 31,
364367.
LICHTENSTEIGER C.A., STEENBERGEN S.M., LEE R.M., POLSON D.D. & VIMR E.R. (1996). Direct PCR analysis for
toxigenic Pasteurella multocida. J. Clin. Microbiol., 34, 30353039.
MAGYAR T., KOVCS F., DONK T., BR H., ROMVRI R., KOVCS M. & REPA I. (2003). Turbinate atrophy evaluation in
pigs by computed tomography. Acta Vet. Hung., 51, 485491.
MAROIS C., FABLET C., GAILLOT O., MORVAN H., MADEC F. & KOBISCH M. (2009). Molecular diversity of porcine and
human isolates of Pasteurella multocida. J. Appl. Microbiol., 107, 18301836.
NAGAI S., SOMENO S. & YAGIHASHI T. (1994). Differentiation of toxigenic from nontoxigenic isolates of Pasteurella
multocida by PCR. J. Clin. Microbiol., 32, 10041010.
PEDERSEN K.B., NIELSEN J.P, FOGED N.T., ELLING F., NIELSEN N.C. & WILLEBERG P. (1988). Atrophic rhinitis in pigs:
proposal for a revised definition. Vet. Rec., 122, 190191.
PEJSAK Z., WASISKA B., MARKOWSKA-DANIEL I. & HOGG A. (1994). Field evaluation of thirteen regimens for the
control of progressive atrophic rhinitis. Comp. Immunol. Microbiol. Infect. Dis., 17, 125132.
PENNINGS A.M. & STORM P.K. (1984). A test in vero cell monolayers for toxin production by strains of Pasteurella
multocida isolated from pigs suspected of having atrophic rhinitis. Vet. Microbiol., 9, 503508.
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REGISTER K.B. & DEJONG K.D. (2006). Analytical verification of a multiplex PCR for identification of Bordetella
bronchiseptica and Pasteurella multocida from swine. Vet. Microbiol., 117, 201210.
REGISTER K.B., LEE R.M. & THOMSON C. (1998). Two-color hybridization assay for simultaneous detection of
Bordetella bronchiseptica and toxigenic Pasteurella multocida from swine. J. Clin. Microbiol., 36, 33423346.
REGISTER K.B., SACCO R.E. & BROCKMEIER S.L. (2007). Immune response in mice and swine to DNA vaccines
derived from the Pasteurella multocida toxin gene. Vaccine, 25, 61186128.
RUTTER J.M. & LUTHER P.D. (1984). Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of
pigs. Vet. Rec., 114, 393396.
SMITH I.M. & BASKERVILLE A.J. (1979). A selective medium facilitating the isolation and recognition of Bordetella
bronchiseptica in pigs. Res. Vet. Sci., 27, 187192.
TOWNSEND K.M., BOYCE J.D., CHUNG J.Y., FROST A.J. & ADLER B. (2001). Genetic organization of Pasteurella
multocida cap loci and development of a multiplex capsular PCR typing system. J. Clin. Microbiol., 39, 924929.
VENIER L., ROTHSCHILD M.F. & WARNER C.M. (1984). Measurement of serum antibody in swine vaccinated with
Bordetella bronchiseptica: comparison of agglutination and enzyme-linked immunosorbent assay methods. Am. J.
Vet. Res., 45, 26342636.
*
* *
1088
CHAPTER 2.8.3.
CLASSICAL SWINE FEVER

(hog cholera)
SUMMARY
Classical swine fever (CSF), also known as hog cholera, is a contagious viral disease of pigs. The
causative virus is a member of the genus Pestivirus of the family Flaviviridae, and is closely related
to the viruses of bovine viral diarrhoea and Border disease. There is only one serotype of CSF virus
(CSFV).
The disease may run an acute, subacute, chronic, late onset, or inapparent course, depending on a
variety of viral and host factors of which the age of the animals, the virulence of the virus and the
time of infection (pre- or post-natal) are of greatest importance. Adult pigs usually display less
severe signs of disease than young animals and stand a better chance of survival. In pregnant
sows, the virus may cross the placental barrier and reach the fetuses. In-utero infection with strains
of the virus of moderate or low virulence can result in what is referred to as the carrier sow
syndrome followed by prenatal or early post-natal death, the birth of diseased piglets or an
apparently healthy but persistently infected litter. An outbreak of CSF has serious consequences
for trade in pigs and pig products.
The highly variable clinical picture of CSF often precludes a diagnosis on clinical and pathological
grounds alone. Laboratory methods are therefore essential for an unambiguous diagnosis.
Detection of virus or viral nucleic acid in whole blood and of antibodies in serum are the methods of
choice for diagnosing CSF in live pigs, whereas detection of virus, viral nucleic acid or antigen in
organ samples is most suitable when the pig is dead.
Identification of the agent: The direct fluorescent antibody test (FAT) on cryostat sections of
organs from affected pigs can be used for the detection of CSF antigen. A panel of monoclonal
antibodies (MAbs) is used to determine whether the fluorescence is due to CSF or non-CSF
Pestivirus antigens. For the detection of CSF genome, polymerase chain reaction (PCR) is
commonly used. The isolation of CSFV should be attempted in the pig kidney (PK-15) cell line, or
other suitable cell lines. The cultures are examined for virus growth by immunofluorescence or
immunoperoxidase staining; positive isolates are further characterised by the use of MAbs and by
partial genetic sequencing. Polymerase chain reaction protocols for the identification of CSFV
nucleic acid have now gained international acceptance and are being used in several laboratories,
both for detection of the agent and differentiation from ruminant pestiviruses. Antigen-capture
enzyme-linked immunosorbent assays (ELISAs) are also useful for herd screening, but should not
be used on a single animal basis.
Serological tests: Detection of virus-specific antibodies is particularly useful in herds suspected of
being infected at least 21 days previously with CSFV. Serological methods are also valuable for
monitoring and for prevalence studies, and are essential if a country wishes to be internationally
recognised as being free from the disease in the absence of vaccination.
As CSFV cross-reactive antibodies against ruminant Pestivirus are occasionally observed in
breeding pigs, screening tests have to be followed by confirmatory tests that are CSFV-specific.
Certain ELISAs are relatively CSFV-specific, but the definitive method for differentiation is the
comparative neutralisation test, which compares the level of antibodies to different Pestivirus
species.
Requirements for vaccines and diagnostic biologicals: Vaccines against CSF are based on live
virus that has been attenuated by passage through cell cultures or through a suitable host species
1089
Chapter 2.8.3. Classical swine fever (hog cholera)
that is not of the family Suidae. The production of these modified live virus (MLV) vaccines is based
on a seed-lot system that has been validated with respect to virus identity, sterility, purity, safety,
nontransmissibility, stability and immunogenicity. If CSFV is used in the production of vaccine or in
challenge studies, the facility should meet the OIE requirements for Containment Group 4
pathogens.
Effective inactivated, conventional whole virus vaccines are not available. Subunit marker vaccines
are now available, which, in contrast to MLV vaccines, induce antibodies that can be distinguished
from antibodies induced by field virus using an accompanying diagnostic test. The presently
registered marker vaccines are based on the major envelope glycoprotein (E2-subunit) of CSFV,
and are produced in insect cells using recombinant DNA technology.
A. INTRODUCTION
The viruses that cause classical swine fever (CSF), bovine viral diarrhoea (BVD) and Border disease (BD) are
members of the family Flaviviridae, genus Pestivirus, and are closely related, both antigenically and structurally.
Clinical signs and lesions seen at post-mortem in pigs affected with CSF are highly variable due to both viral and
host factors. Furthermore, congenital infections with ruminant pestiviruses in pigs can give rise to a clinical
disease that is indistinguishable from CSF (Terpstra & Wensvoort, 1988b; Vannier & Carnero, 1985; Wensvoort &
Terpstra, 1988).
Spread of disease in all age groups, accompanied by pyrexia, huddling, inappetance, dullness, weakness,
conjunctivitis, constipation followed by diarrhoea, and an unsteady gait are the prevailing signs. Several days after
the onset of clinical signs, the ears, abdomen and inner thighs may show a purple discoloration. Animals with
acute disease die within 13 weeks. Sudden death in the absence of clinical illness is not symptomatic of CSF.
Under certain circumstances related to the animals age and condition, as well as to the virus strain involved,
subacute or chronic clinical illness may develop, which can be protracted for 24 weeks or even months. Chronic
illness leads to a stunting of growth, anorexia, intermittent pyrexia and diarrhoea. Congenital persistent infections
may go undetected for months and may be confined to only a few piglets in the herd or may affect larger
numbers. The clinical signs are nonspecific: wasting in the absence of pyrexia. Chronic, persistent infections
always lead to the death of the animal. Herd mortality rates may be slightly above the expected level. CSF affects
the immune system, a main characteristic being generalised leukopenia, which can often be detected before the
onset of fever. Immunosuppression may lead to concurrent infections.
In acute cases, gross pathological lesions might be inconspicuous or absent. In typical cases, the lymph nodes
are swollen and marbled red, and haemorrhages occur on serosal and mucosal membranes of the intestinal
organs. Spleenic infarctions may occur. In subacute and chronic cases, necrotic or button ulcers may be
observed in the mucosa of the gastrointestinal tract, epiglottis and larynx, in addition to the above lesions.
Histopathological findings are not pathognomonic. Lesions may include parenchymatous degeneration of
lymphatic tissue, cellular proliferation of vascular interstitial tissue, and a nonsuppurative meningoencephalomyelitis, with or without vascular cuffing.
A useful critique of diagnostics and vaccination for CSF, from an authoritative source, has recently been
published (Blome et al., 2006), which, as well as general guidance, also provides sources of information on
validation and scientific opinion on the applicability of certain commercial products in these areas.
The variability of the clinical signs and post-mortem lesions do not provide firm evidence for unequivocal
diagnosis. Other viral diseases, such as African swine fever, porcine dermatitis and nephropathy syndrome
(PDNS), and post-weaning multisystemic wasting syndrome (PMWS), thrombocytopenic purpura and various
septicaemic conditions including salmonellosis (especially due to Salmonella choleraesuis), erysipelas,
pasteurellosis, actinobacillosis (due to Actinobacillus suis) and Haemophilus parasuis infections may be confused
with acute CSF. In fact, these bacteria often cause concurrent infections, and isolating these pathogens may
obscure the real cause of disease, the CSF virus (CSFV). Similarly concurrent PDNS can lead to oversight of an
underlying CSF infection.
A tentative diagnosis based on clinical signs and post-mortem lesions must therefore be confirmed by laboratory
investigations. As pyrexia is one of the first signs of CSF and is accompanied by a viraemia (Depner et al., 1994),
1090
detection of virus or viral nucleic acid in whole blood, collected in heparin or ethylene diamine tetra-acetic acid
(EDTA), or in tissues, collected from a few febrile animals, is the method of choice for detecting infected herds at
an early stage. This is all the more necessary in view of the serious consequences of an outbreak of CSF for
trade in pigs and pig products.
Laboratory methods for diagnosis of CSF are aimed at detection of the virus, viral nucleic acid or viral antigens, or
detection of specific antibodies. For a correct interpretation of the test results the inspecting veterinarian should
pay particular attention to the simultaneous and clustered occurrence of two or more of the prevailing signs of
disease listed above. Random sampling is unsuitable for CSF diagnosis. Additionally, whole blood samples for
virus detection and reverse-transcription polymerase chain reaction (RT-PCR) analyses can be collected from a
larger group of pigs.
CSF is subject to official control and the virus has a high risk of spread from the laboratory: consequently, a risk
analysis should be carried out to determine the level of biosecurity needed for the diagnosis and characterisation
of the virus. The facility should meet the requirements for the appropriate Containment Group as determined by
the risk assessment and as outlined in Chapter 1.1.3 Biosafety and biosecurity in the veterinary microbiology
laboratory and animal facilities. Countries lacking access to such a specialised national or regional laboratory
should send specimens to an OIE Reference Laboratory.
Antibodies develop in the third week of illness and persist in the surviving animal for life. Samples for antibody
detection are collected in ordinary (nonheparinised) tubes from convalescent pigs and from contact herds when
more than 3 weeks have elapsed since the suspected contact with a confirmed outbreak took place.
1.
a)
The fluorescent antibody test (FAT) is a rapid test that can be used to detect CSFV antigen in cryostat
sections of tonsils, spleen, kidney, lymph nodes or distal portions of the ileum. Tissues should be collected
from several (febrile and/or diseased) animals (Bouma et al., 2001) and transported without preservatives
under cool conditions, but not frozen. Cryostat sections are stained directly with anti-CSF immunoglobulin
conjugated to fluorescein isothiocyanate (FITC) or indirectly using a secondary FITC conjugate and
examined by fluorescence microscopy. During the first stage of the infection, tonsillar tissue is the most
suitable, as this is the first to become affected by the virus irrespective of the route of infection (Ressang,
1973). In subacute and chronic cases, the ileum is frequently positive and occasionally may be the only
tissue to display fluorescence. A negative FAT result does not completely rule out CSF infection. When
suspicion of CSF continues, further samples should be obtained or attempts made at virus isolation in cell
culture (e.g. pig kidney [PK-15]) or another cell line of pig origin that is as sensitive and known to be free
from Pestivirus contamination.
There is a relatively high risk of false (positive and negative) results when FAT is used by laboratories not
thoroughly acquainted with the method. Thus FAT should only be used by laboratories that have experience
of using the technique, perform the technique on a routine basis and have had training in interpreting the
fluorescence.
Test procedure
Include positive and negative control sections in each series of organ samples to be examined.
i)
Cut out a piece of tonsil, spleen, kidney and ileum of approximately 1 1 0.5 cm, and mount it with a
cryo-embedding compound or distilled water on a cryostat table.
ii)
Freeze the piece of organ on to the cryostat table.
iii)
Cut sections not more than 48 m thick and mount these on to 10 32 mm grease-free cover-slips
with one corner cut-off. All sections are mounted with this corner in the same position (e.g. top right).
iv)
After drying, fix the mounted sections for 10 minutes at room temperature in acetone (analytical grade)
or air-dry for 20 minutes at 37C.
v)
Immerse the sections briefly in phosphate buffered saline (PBS), remove excess fluid with tissue paper
and place them (cut off corner top right) on a frame in an incubation chamber humidified with a small
volume of water placed in the bottom of the chamber.
vi)
Dispense the anti-CSF immunoglobulin at working dilution on to the entire section and incubate in the
closed chamber for 30 minutes at 37C. If a secondary FITC conjugate is required, wash the section
1091
five times for 2 minutes each in PBS at room temperature, then add the FITC conjugate at working
dilution and incubate as previously described.
vii)
Wash the sections five times for 2 minutes each in PBS at room temperature.
viii) Remove the remaining PBS by touching the cover-slip against tissue paper and mount the cover-slip
(with the section between cover-slip and slide) with mounting buffer on to a microscope slide.
ix)
Remove excess mounting fluid with tissue paper and examine the sections for fluorescence using a UV
microscope. A CSF-positive section shows brilliant green fluorescing cells. In the tonsils, fluorescence
in the epithelial lining of the crypts is particularly evident. In kidney sections, fluorescence is most
abundant in the proximal and distal tubules of the renal cortex and the collecting ducts in the medulla.
In the ileum, fluorescence is most prominent in the epithelial cells of the Lieberkhn glands, whereas in
the spleen reactivity is more diffuse, with concentrations of lymphoid cells in the periarterial lymphoid
sheath (PALS).
The FAT involves the use of an anti-CSF immunoglobulin prepared from a polyclonal antibody to CSFV that
will not distinguish between the antigens of different pestiviruses. Conjugates used for the FAT on cryostat
sections or inoculated cell cultures should be prepared from anti-CSFV gamma-globulins raised in specific
pathogen free pigs. The working dilution of the conjugates (at least 1/30) should combine a maximum
brilliance with a minimum of background. The test should only be performed on samples from freshly dead
animals, as autolysis and bacterial contamination can often result in high background staining.
Strains of modified live virus (MLV) vaccine multiply mainly in the regional lymph nodes and in the crypt
epithelium of the tonsils. Pigs vaccinated with MLV strains may yield a positive FAT for 2 weeks after
vaccination (Ogawa et al., 1973; Terpstra, 1978). Rabbit inoculation is used to differentiate between
lapinised and field strains of CSFV. In contrast to field strains, lapinised strains given intravenously cause a
febrile reaction and induce an immune response in rabbits. As nucleic acid sequencing has become
available and more reliable, animal inoculation is no longer necessary to differentiate between field strains
and vaccine strains of CSFV.
Pigs infected with ruminant pestiviruses can give false-positive FAT reactions. Congenital infections with
ruminant pestiviruses can cause clinical signs and pathological lesions indistinguishable from those in
chronic CSF (Terpstra & Wensvoort, 1988b; Vannier & Carnero, 1985; Wensvoort & Terpstra, 1988).
Infections by CSFV or ruminant pestiviruses can be differentiated by testing sera from the dam and litter
mates, or from other contacts of an FAT-positive piglet, for neutralising antibodies to each virus. If the virus
was isolated, or viral nucleic acid can be detected, using RT-PCR, subsequent sequencing provides a rapid
and accurate tool to distinguish ruminant pestiviruses from CSFV. Another method of differentiating these
viruses is by the inoculation of seronegative piglets with a suspension of suspect material, followed at least
4 weeks by virus neutralisation (VN) tests on their sera for the respective antibodies. However, VN tests may
take several days, and animal inoculation methods take several weeks.
Immunoperoxidase procedure for differentiation of pestiviruses by monoclonal antibodies
The use of a panel of three monoclonal antibodies (MAbs), either horseradish peroxidase (HRPO) or FITCconjugated, or used in conjunction with an anti-mouse conjugate and specifically detecting all field strains of
CSFV, vaccine strains of CSFV and ruminant pestiviruses, respectively, would allow an unambiguous
differentiation between field and vaccine strains of CSFV on the one hand, and between CSFV and other
pestiviruses on the other (Edwards et al., 1991; Wensvoort et al., 1986; 1989b). A prerequisite is that the
MAb against CSFV recognises all field strains and that the anti-vaccine MAb recognises all vaccine strains
used in the country. No single MAb selectively reacts with all ruminant pestiviruses (Edwards et al., 1991).
The use of an MAb to differentiate a CSF vaccine strain can be omitted in nonvaccination areas. A
polyclonal anti-CSF immunoglobulin conjugated to HRPO serves as a positive control. Caution should be
exercised when using evidence of a single MAb as sole confirmation of an isolate as CSF.
Test procedure
i)
Cut eight or more cryostat sections (48 m) of the FAT-positive tonsil, or another positive organ if the
tonsil is not available.
ii)
Fix the sections on to flying cover-slips for 10 minutes in acetone (analytical grade) and allow to air dry.
iii)
Prepare working dilutions of the respective MAb-peroxidase conjugates in PBS + 0.01% Tween 80 +
5% horse serum, pH 7.6. (FITCMAb can also be used, as well as unconjugated MAb provided that a
secondary conjugate is used.)
iv)
After rinsing with PBS, overlay two sections with the working dilution of the respective monoclonal
conjugates, and two sections with the working dilution of the polyclonal conjugate (controls).
v)
Incubate for 1 hour at 37C in a humid chamber.
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vi)
Wash the sections six times for 10 seconds each in PBS.
vii)
Stain the sections with freshly prepared chromogensubstrate solution* for 515 minutes at room
temperature.
viii) Rinse the sections in 0.05 M sodium acetate, pH 5.0, in distilled water and mount them on microscope
slides.
ix)
Examine sections with a light microscope. Dark red staining of the cytoplasm of the epithelial cells
lining the tonsillar crypts indicates recognition of the virus isolate by the respective conjugate, and is
considered to be positive.
x) Interpretation of the test:

Polyclonal antibody
Monoclonal antibody specific for
Interpretation
CSF strain
CSF vaccine strain
BVD/BD strain
CSF field strain
CSF vaccine strain
BVD/BD strain
Other non-CSF Pestivirus
The existence of novel strains of CSF should always be considered and any isolate from cases where CSF is still
suspected should be sent to an OIE Reference Laboratory.
Antigen-capture assay
For rapid diagnosis of CSF in live pigs, antigen-capture enzyme-linked immunosorbent assays (ELISAs)
have been developed for screening herds suspected of having been recently infected. The ELISAs are of the
double-antibody sandwich type, using monoclonal and/or polyclonal antibodies against a variety of viral
proteins in either serum, the blood leukocyte fraction or anticoagulated whole blood; in addition, some test
kits can be used to test clarified tissue homogenates (Depner et al., 1995). The technique is relatively simple
to perform, does not require tissue culture facilities, is suitable for automation and can provide results within
half a day. The disadvantage of being less sensitive than virus isolation, especially in adult pigs and mild or
subclinical cases, may be compensated by testing all pigs of the suspect herd showing pyrexia. However,
the lowered specificity of these tests should also be taken into consideration. The test is not suitable for the
diagnosis of CSF in a single animal.
b)
Isolation of virus
Isolation of virus in cell cultures is a more sensitive but slower method for diagnosis of CSF than
immunofluorescence on frozen sections. Isolation is best performed in rapidly dividing PK-15 cells seeded
on to cover-slips simultaneously with a 2% suspension of the tonsil in growth medium. Other pig cell lines
may be used, but should be demonstrably at least as sensitive as PK-15 cells for isolation of CSFV. The
cultures are examined for fluorescent foci by FAT after 2472 hours or after 45 days incubation are fixed
for immunoperoxidase staining.
The tonsil is the most suitable organ for virus isolation from pigs that died or were killed for diagnostic
purposes. Alternatively, spleen, kidney, ileum or lymph nodes can also be used.
A detailed procedure for virus isolation is as follows:
i)
Prepare a 100-fold strength glutamineantibiotic stock solution: dissolve glutamine (2.92 g) in 50 ml

distilled water (solution A) and sterilise by filtration. Dissolve each of the following antibiotics in 510 ml
sterile distilled water: penicillin (106 International Units [IU]); streptomycin (1 g); mycostatin (5 105 U);
polymixin B (15 104 U); and kanamycin (1 g). Pool these solutions (solution B). Mix aseptically
*
A.
Chromogensubstrate solution
Stock solution of chromogen: 0.4% 3-amino-9-ethyl carbazole; N,N-dimethyl-formamide (1 ml).
Caution TOXIC compound. Both chemicals are carcinogens and irritants to eyes, skin and respiratory tract.
B.
0.05 M sodium acetate, pH 5.0; 19 ml (sterile filtered through a membrane).
C.
Stock solution of substrate (30% hydrogen peroxide).
Keep stock solutions A and C at 4C in the dark and solution B at room temperature. Stock solution A can be kept at 4C for at
least 6 months and solution C for 1 year. Immediately before use, dilute 1 ml of solution A in 19 ml of solution B. Then add 10 l
of stock solution C. Mix well and stain the sections.
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solutions A and B, make up to 100 ml with sterile distilled water, and store in 5 ml aliquots at 20C.
Exact antibiotic constitution is not critical, provided sterility is achieved and cells are not affected.
ii)
Cut 12 g of tissue into small pieces and, using a mortar and pestle or other device, grind in a small
amount of cell culture medium with sterile sand into a homogeneous paste. Alternatively, use an
appropriate crushing machine at 4C.
iii)
Make a 20% (w/v) suspension by adding Hanks balanced salts solution (BSS) or Hanks minimal
essential medium (MEM); 1 ml of the glutamineantibiotic stock is added for each 10 ml of suspension.
This mixture is held at room temperature for up to 1 hour.
iv)
Centrifuge at 1000 g for 15 minutes.
v)
A PK-15 monolayer is trypsinised, the cell suspension is centrifuged at 160 g for 10 minutes, and
resuspended to contain approximately 2 106 cells/ml in growth medium (Eagles MEM with Earles
salts; 5% fetal bovine serum free from ruminant pestiviruses and pestivirus antibodies ; and 0.2 ml of
the glutamineantibiotic stock solution per 10 ml cell suspension). As a guide, one 75 cm2 flask will
give approximately 50 ml of cell suspension at the appropriate concentration.
vi)
Either:
Suspension inoculation: mix nine parts of cell suspension (from step v) and one part of supernatant
fluid (from step iv) and inoculate 1.01.5 ml into 68 Leighton tubes with cover-slips or other
appropriate cell culture flasks. Three tubes are inoculated with 1.01.5 ml of cell suspension alone as
controls. After completion of the sample inoculations, three tubes are inoculated with CSFV as positive
controls. Careful precautions must be taken to avoid cross-contamination with this known positive virus
suspension. Negative cultures should also be prepared. Incubate at 37C.
Or:
Pre-formed monolayer inoculation: for each tissue, inoculate 1.01.5 ml of cell suspension (prepared
as in step v) into 68 Leighton tubes with cover-slips or other appropriate cell culture flasks. Incubate at
37C for a minimum of 4 hours and a maximum of 36 hours. Then drain the medium and inoculate
0.2 ml of supernatant fluid (from step iv), incubate for 1 hour at 37C, rinse and overlay with 1 ml of
growth medium and incubate at 37C.
vii)
At 1, 2 and 3 days after inoculation, two cultures, together with a positive and negative control culture
are washed twice for 5 minutes each in Hanks BSS, Hanks MEM or PBS, fixed with cold acetone
(analytical grade) for 10 minutes, and stained with a direct anti-CSFV conjugate at its appropriate
working dilution or indirectly, as described in Section B.1.a.
If the 2% tonsil suspension proves to be toxic for the cells, then the test should be repeated using a
higher dilution or another organ. Use of the method employing pre-formed monolayers (above) will help
to avoid such.
viii) After washing in PBS three times for 5 minutes each, the cover-slip cultures are mounted in 90%
carbonate/bicarbonate buffered glycerol, pH>8.0, and examined for fluorescent foci.
Instead of Leighton tubes, 6-well plates with cover-slips can be used. Alternatively, cultures growing on flatbottomed microtitre plates or M24-plates can also be used for virus isolation. In such case, plates are fixed
and stained as described later for the neutralising peroxidase-linked assay (NPLA).
Whole blood (heparin or EDTA treated) from clinically diseased pigs is a suitable sample for early CSF
diagnosis. The leukocyte fraction or other components may be used, but for reasons of sensitivity and
simplicity whole blood is preferred (De Smit et al., 1994). The procedure is as follows:
i)
Freeze a sample of whole blood at 20C and thaw in a waterbath at 37C.
ii)
Inoculate 300 l haemolysed blood on to a PK-15 monolayer grown to approximately 75% confluence*
in an M24-plate, and allow adsorption for 1 hour at 37C.
ii)
Remove inoculum, wash the monolayer once with Hanks BSS or Hanks MEM, and add growth
medium.
iv)
After a further incubation period of 34 days, the plates are washed, fixed and stained, as described
later for the NPLA, using in each step a volume of 300 l to compensate for the larger cell surface.
Note: this method is less sensitive than conventional virus isolation for the detection of acute CSF.
Simultaneous inoculation, though slightly more sensitive, is less suitable as the anticoagulant may interfere with the
adhesion of cells on to the surface.
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Reverse-transcription polymerase chain reaction
Many methods for RT-PCR have been described and are still being developed (McGoldrick et al., 1998).
This internationally accepted method is rapid and more sensitive than antigen-capture ELISAs or virus
isolation making it particularly suited to preclinical diagnosis. Several conventional and real-time PCR
protocols have been described (Hoffmann et al., 2005; McGoldrick et al., 1998; Paton et al., 2000b; Risatti et
al., 2003; 2005) and a suitable protocol may be obtained from the literature or from the OIE Reference
Laboratories for CSF (see Table given in Part 4 of this Terrestrial Manual). Due to its speed and sensitivity,
RT-PCR is a suitable approach to screening suspect cases of disease and is now accepted by a number of
countries and the European Union (EU) (European Commission, 2002). However, it has to be kept in mind
that false positive results due to laboratory contamination can occur as well as false negative results due to
inhibiters contained in the sample. Any positive results from primary outbreaks should always be confirmed
by other tests. It is mandatory to include an adequate number of positive and negative controls in each run; it
is also recommended that internal controls be included. See Chapter 1.1.5 Principles and methods of
validation of diagnostic assays for infectious diseases, for further details on PCR techniques. The test can
be applied to individual or pooled blood samples as well as solid organs and has been used successfully to
control outbreaks.
The molecular epidemiology of CSF is based on the comparison of genetic differences between virus
isolates. RT-PCR amplification of CSFV RNA followed by nucleotide sequencing is the simplest way of
obtaining the sequence data to make these comparisons. A number of different regions of the CSFV
genome may be targeted for molecular epidemiological studies (Paton et al., 2000a). Two regions have
been extensively studied and provide large sets of sequence data with which new isolates can be compared.
One of these regions lies within the 5-noncoding region (5NCR) of the genome (150 nucleotides) and the
other lies within the E2 major glycoprotein gene (190 nucleotides). In brief, the method used is to extract
virus RNA from infected PK-15 cell cultures, perform RT-PCR to amplify one or both targets within the
5NCR or the E2 gene, and then determine the nucleotide sequence of the products and compare with
stored sequence information held in the databases. A database of these sequences is available from the
OIE Reference Laboratory for CSF (Hanover, Germany). Recent findings on analysing ruminant pestivirus
sequences highlight the need for analysis of multiple regions in order to accurately type strains by this
method (Hurtado et al., 2003). CSFV isolates from primary outbreaks should be sent to an OIE Reference
Laboratory for investigation of molecular epidemiology. An import permit should be obtained prior to
dispatch.
2.
Serological tests
Detection of virus-specific antibodies is particularly useful on premises suspected of having infections with CSF
strains of low virulence. Due to the immunosuppressive effect of CSFV, antibodies cannot be detected with
certainty until 21 days post-infection. Serological investigations aimed at detecting residual foci of infection,
especially in breeding herds, may also be useful in a terminal phase of CSF eradication.
As the incidence of infection with ruminant pestiviruses may be high in breeding stock, only tests that will
discriminate between CSF and BVD/BD antibodies are useful. VN and the ELISA using MAbs satisfy the
requirements for sensitivity, but positive results should be confirmed by comparative VN testing.
Neutralisation tests are performed in cell cultures using a constant-virus/varying-serum method. As CSFV is
noncytopathic, any non-neutralised virus must be detected, after multiplication, by an indicator system. The NPLA
(Terpstra et al., 1984) and the fluorescent antibody virus neutralisation (FAVN) test (Liess & Prager, 1976) are the
most commonly used techniques. Both tests can be carried out in microtitre plates. The NPLA system is now
favoured, being easier to read and having the advantage that the results can be determined by use of an inverted
light microscope, though a crude assessment of titre can be made with the naked eye.
a)
Neutralising peroxidase-linked assay (a prescribed test for international trade)

The NPLA is carried out in flat-bottomed microtitre plates. Sera are first inactivated for 30 minutes at 56C.
For international trade purposes, it is best to test with an initial serum dilution of 1/5 (1/10 final dilution). For
surveillance schemes within a country, a screening dilution of 1/10 may suffice. Appropriate controls to
ensure specificity and sensitivity of reactions are incorporated into each test.
Test procedure
i)
Dispense dilutions of serum in growth medium (Eagles MEM, 5% fetal bovine serum and antibiotics) in
50 l volumes into duplicate wells of a microtitre plate. The fetal bovine serum must be free from both
BVDV and antibodies to it. A third well may be included for each sample. This well contains serum and
not virus and is used as a serum control (for cytotoxicity and/or nonspecific staining).
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ii)
Add 50 l of virus suspension to the wells, diluted in growth medium to contain approximately
100 TCID50/50 l, and mix the contents on a microplate shaker for 20 seconds.
iii)
Incubate the plates in a CO2 incubator for 1 hour at 37C.
iv)
Add to all wells 50 l of growth medium containing 2 105 cells/ml.
v)
Allow the cells to grow at 37C in 5% CO2 to become confluent, usually within 34 days.
vi)
Discard the growth medium and rinse the plates once in 0.15 M NaCl.
vii)
Drain the plates by blotting on a towel.
viii) The cell monolayers may be fixed in one of several ways:
The plates are incubated for 45 minutes at 37C, and then for at least a further 45 minutes at
20C. The plates are removed from the freezer, the wells are filled with 100 l 4%
paraformaldehyde in PBS and reincubated for 510 minutes at room temperature. The
paraformaldehyde is discarded and the plates are rinsed with 0.15 M NaCl; or
The plates are incubated at 7080C for 12 hours; or
The plates are fixed with 80% acetone and incubating at 7080C for 1 hour; or
The plates are fixed in 20% acetone in PBS for 10 minutes followed by thorough drying at 25
30C for 4 hours. (This can be done quickly with the aid of a hair-dryer after 35 minutes
complete dryness is obtained as observed by the whitish colour of the cell monolayer.)
ix)
Add to each well 50 l of a hyperimmune porcine CSF antiserum or monoclonal antibody, diluted in
0.5 M NaCl containing 1% Tween 80 + 0.1% sodium azide, pH 7.6. Incubate at 37C for 15 minutes.
The working dilution of the antiserum should be determined by prior titration: i.e. a serum with an NPLA
titre of 1/30,000 could be used at 1/100.
x)
Wash the plates five times in 0.15 M NaCl containing 1% Tween 80, pH 7.6.
xi)
Add to each well 50 l of an anti-porcine or anti-murine (as appropriate) IgG-HRPO conjugate, diluted
to its working concentration in 0.5 M NaCl with 1% Tween 80, pH 7.6, and then incubate for 10 minutes
at 37C.
xii)
Wash the plates five times in 0.15 M NaCl containing 1% Tween 80, pH 7.6.
xiii) Add 50 l of chromogensubstrate solution to each well and stain for 1530 minutes at room
temperature. This solution is described in Section B.1.a Immunoperoxidase procedure for
differentiation of pestiviruses by monoclonal antibodies.
xiv) The test is read visually. Infected cell sheets are completely or partially stained reddish brown. The
monolayer should be examined by low-power microscopy to determine the end-point of the titration.
The cytoplasm of infected cells is stained dark red.
xv)
The following controls are included in the test: cell control, positive serum and back titration of test
virus. The back-titration should confirm that virus has been used at a concentration of between 30 and
300 TCID50/50 l.
NOTE: The incubation times given above are for guidance only. Longer incubation times, with reagent
dilutions optimised to such times, may be used, in order to conserve reagents.
b)
Fluorescent antibody virus neutralisation test (a prescribed test for international trade)
Leighton tube method:
i)
Seed a suspension of PK-15 cells at a concentration of 2 105 cells/ml into Leighton tubes with a
cover-slip.
ii)
Incubate the cultures for 12 days at 37C until they reach 7080% confluency.
iii)
Inactivate the sera for 30 minutes at 56C. For international trade purposes, it is best to test with an
initial serum dilution of 1/5 (1/10 final dilution).
iv)
Incubate equal volumes of diluted serum and virus suspension containing 200 TCID50 (50% tissue
culture infective dose) per 0.1 ml for 12 hours at 37C. Thus a constant amount of CSFV of
100 TCID50 is used for each reaction well.
v)
Remove the cover-slips from the Leighton tubes, wash briefly in serum-free medium, overlay the cell
sheet with the serum/virus mixture (from step iv) and incubate for 1 hour at 37C in a humid
atmosphere.
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vi)
Place the cover-slip in a clean Leighton tube and incubate the cultures in maintenance medium for
2 more days.
vii)
Remove the cover-slips from the Leighton tubes, wash the monolayers twice for 5 minutes each in
PBS, pH 7.2, fix in pure acetone for 10 minutes and stain with the working dilution of the conjugate for
30 minutes at 37C before washing.
viii) Mount the cover-slips on grease-free microscope slides with 90% carbonate/bicarbonate buffered
glycerol, pH>8.0, and examine for fluorescence.
When the FAVN test is performed in microtitre plates, the procedure for the NPLA (see below) can be
followed up to step viii. The plates are then stained with the working dilution of the conjugate for 30 minutes
at 37C and examined for fluorescence. Note: When detecting fluorescence, microplates are best examined
from above, using a long focal-length objective.
Occasionally, sera from pigs infected with BVDV or BDV react in the FAVN or NPLA at low dilution as if they
were infected with CSFV. The extent of cross-reactivity depends on the strain of ruminant pestivirus involved
and the interval between infection and time of sampling (Wensvoort et al., 1989a). The usually high antibody
levels reached after exposure to CSF infection, including strains of low virulence, allow the use of
comparatively high initial dilutions in NPLA tests for CSF antibody, thus avoiding most, but not all, crossreactions (Terpstra et al., 1984; Terpstra & Wensvoort, 1988a). In case of continued doubt, comparative
tests using a strain of CSFV, a strain of BVDV and a strain of BDV, that are representative for the country or
region, have proved useful. Comparative neutralisation tests are end-point titrations in which the same
series of twofold dilutions of the suspected serum sample is tested in duplicate against 100 TCID50 of each
selected virus strain. The comparative tests are performed according to the protocols described for the
FAVN or NPLA; the cell lines used must be suitable for BVDV and BDV. Neutralisation titres are expressed
as the reciprocal of the highest serum dilution that prevents virus growth in 50% of two replicate wells. A
three-fold difference or more between end-points of two titrations should be considered decisive for an
infection by the virus species yielding the highest titre. It may be necessary to use different strains of the
same genotype, and/or to test several pigs from an infected herd to obtain a definitive result.
c)

Competitive, blocking and indirect techniques may be used on any suitable support and a number have
been described (e.g. Colijn et al., 1997; Have, 1987; Leforban et al., 1990; Moser et al., 1996; Wensvoort et
al., 1988). The tests used should minimise cross-reactions with BVDV and other pestiviruses. However, the
test system must ensure identification of all CSF infections, and at all stages of the immune response to
infection.
Antigen: The antigen should be derived from or correspond to viral proteins of one of the recommended
CSFV strains. Cells used to prepare antigen must be free from any other Pestivirus infection.
Antisera: Polyclonal antisera for competitive or blocking assays should be raised in pigs or rabbits by
infection with one of the recommended CSFV strains or with the lapinised C strain. MAbs should be directed
against or correspond to an immunodominant viral protein of CSFV. Indirect assays should use an antiporcine immunoglobulin reagent that detects both IgG and IgM.
The sensitivity of the ELISA should be high enough to score positive any serum from convalescent animals,
i.e. at least 21 days postinoculation that reacts in the neutralisation test. The ELISA may only be used with
serum or plasma samples derived from individual pigs. If the ELISA procedure used is not CSF-specific,
then positive samples should be further examined by differential tests to distinguish between CSF and other
pestiviruses.
The complex-trapping blocking ELISA (Colijn et al., 1997) is a one-step method and is suitable for use in
automated ELISA systems e.g. robots. The sera are tested undiluted. The test is fast and easy to perform,
and detects antibodies against low virulence strains of CSFV at an early stage after infection. As the MAbs
are specific for CSFV, the complex-trapping blocking ELISA will only rarely detect antibodies against BVDV,
although BD antibodies can be more problematic. Positive sera are retested for confirmation by the NPLA.
Recently, a novel ELISA has been described that uses fused protein derived from viral peptides (Lin et al.,
2005). The test claims to provide greater sensitivity and earlier detection of antibody than is obtained by
conventional ELISAs, but, at this time, its reactivity with antibody induced by diverse strains of CSF is not
known.
More information on commercial kits for diagnosis can be obtained from the OIE Reference Laboratories.
1097

Effective inactivated whole virus vaccines against CSF are not available.
C1. Modified live virus vaccines

MLV vaccines are produced from CSFV strains that have been attenuated by passage either in cell cultures or in
a suitable host species not belonging to the family Suidae. Production is carried out in cell cultures, or in nonSuidae, based on a seed-lot system. This must be validated with respect to identity, sterility, purity, safety,
nontransmissibility, stability and immunogenicity.
The vaccine production facility should operate under the appropriate biosecurity procedures and practices. If
CSFV is used for vaccine production or for vaccinechallenge studies, that part of the facility where this work is
done should meet the requirements for Containment Group 4 pathogens as outlined in chapter 1.1.3.
To produce a seed lot and a final vaccine of high quality, the optimal conditions for virus yield must be
determined. For vaccines produced in cell cultures, growth curve experiments must be done to study the effect of
composition of the medium, regulation of pH and atmospheric CO2 content, starting concentration of seeded cells,
ratio between cell sheet surface and medium volume, phase of the cell growth at the time of viral infection,
stationary or rolling conditions during viral replication, etc. For vaccines produced in animals, their age, breed,
weight, the size of inoculum (number of animal ID50 [50% infectious dose]), pathogenesis of the infection, and the
clinical signs are factors that must be investigated to determine the peak of virus growth and the tissues to be
harvested.
Regardless of the production method, the substrate should be harvested under aseptic conditions and be
subjected to a freezethaw cycle to release cell-associated virus. Coarse cell or tissue elements are removed by
filtration or low-speed centrifugation. A stabiliser is added, such as lactose at a final concentration of 5%. The
vaccine is homogenised before lyophilisation to ensure a uniform batch.
The vaccine virus in the final product should not differ by more than five passages from the material used for
validating the seed lot. The commercial vaccine should be produced in batches in lyophilised form as a
homogeneous product.
1.
Seed management
a)

To validate a seed lot for an MLV CSF vaccine, samples of the seed lot must first pass pilot experiments.
Except for tests to confirm identity, sterility, purity and stability of attenuation, pilot experiments may also be
performed with representative samples of the final commercial product. These samples must originate from
the same seed lot as tested above.
Except where otherwise specified, all pigs used in pilot tests are 68 weeks of age, healthy, free from
antibodies against CSFV and BVDV, of the same breed and origin, grouped at random if necessary, and
kept under the same conditions. Pregnant sows must be of equal parity.
The seed virus must be sterile and induce specific neutralising antibodies against a virulent strain of CSFV in
pigs.
b)
Method of culture
Production is performed in cell cultures or in a suitable host of a species not of the family Suidae.
c)
i)
Purity
Vaccine must be virologically pure.
Each of three seronegative pigs is inoculated intramuscularly with an amount of seed-lot virus
equivalent to tenfold the amount of virus contained in one dose of vaccine. This is repeated 3 weeks
later using the same dose and route of administration. Serum samples are collected 2 weeks after the
1098
last inoculation, and tested by the most sensitive method for freedom from antibodies to the viruses of
African swine fever, Aujeszkys disease, BVD, foot and mouth disease (all types), transmissible
gastroenteritis, swine vesicular disease, porcine reproductive and respiratory syndrome, and porcine
influenza (types H1N1 and H3N2); and for porcine adenoviruses, porcine teschoviruses (types 1 and
2), porcine parvovirus and porcine circoviruses (types 1 and 2).
ii)
Safety
Vaccines should be tested for any pathogenic effects on healthy pigs, and also on pigs that might be
immunosuppressed due to the presence of concurrent infection or medication, as well as testing to
ensure that the vaccine does not cross the placenta of pregnant sows.
For tests of safety in conventional pigs, each of ten seronegative pigs is inoculated intramuscularly with
ten vaccine doses. Ten other pigs serve as controls. All pigs are observed for 3 weeks thereafter. Body
temperatures are recorded and blood samples are collected daily, with an anticoagulant, for the first
week. Body weights are recorded at inoculation and 2 weeks later. No animal should die or show signs
of illness caused by the vaccine (seed-lot) virus. A daily group average body temperature should not
reach 40.5C or more throughout the trial period. The average weight gain should not fall significantly
(p <0.05) below that of the controls. Leukopenia (white blood cell [WBC] count <7 106 cells/ml) may
be disregarded if it is only in one pig for 1 day.
To ensure attenuation, even in immunosuppressed pigs, each of ten pigs is immunosuppressed by
daily injections, each of 2 mg prednisolone/kg body weight, for 5 consecutive days. On day 3, each
animal is inoculated with the equivalent of one dose of vaccine, and kept under observation for
3 weeks thereafter. No animal should die or become ill due to the vaccine virus.
To ensure safety in pregnant animals, each of ten sows, 2535 days pregnant, is inoculated
intramuscularly with the equivalent of one dose of vaccine. A further ten animals of the same parity and
gestation serve as controls. The vaccination should not interfere with normal gestation to term, and the
number of live piglets born from the test group should not be significantly fewer (p <0.05) than that for
the control pigs.
For field trials, a minimum of 200 pigs is used, farrowed and reared by at least 20 dams, and
seronegative for CSF and BVD. The litters are equally distributed over at least two farms. Half of the
piglets in each litter are inoculated intramuscularly at 714 days of age with the equivalent of one dose
of vaccine. The uninoculated littermates are controls. All piglets are weighed at inoculation and
2 weeks later, and are kept under observation for 3 weeks. A mortality rate that exceeds 5% due to
causes other than vaccination invalidates the trial. No animal should die or show signs of disease due
to the vaccine virus. The average weight gain of the inoculated pigs in the litters should not be more
than 20% below that of the controls during the 2 weeks post-inoculation.
iii)
Nontransmissibility
To confirm nontransmissibility, 24 seronegative pigs are divided into four groups of equal size. Five
pigs in each group are inoculated intramuscularly with the equivalent of one dose of vaccine. The
remaining pigs represent in-contact controls. All pigs are challenged 6 weeks later with at least
105 PID50 (50% pig infectious dose) of a virulent strain of CSFV. All in-contact animals should be
serologically negative at the time of challenge, and then die within 3 weeks. All vaccinated pigs should
remain healthy and survive.
iv)
Stability of attenuation
To confirm stability of virus attenuation, two pigs are each inoculated intramuscularly with an amount of
seed-lot virus equivalent to 100 doses of vaccine, and then killed 67 days later. The tonsils of both
pigs are pooled and made into a 10% suspension in PBS, pH 7.2. This is used to inoculate two further
pigs intramuscularly with 2 ml, and these are then killed 67 days later. This protocol is repeated five
times. During these passages, the tonsillar tissue may be stored at 4C, if storage is to be under
24 hours, or at 70C for longer periods. At the same time, the presence of CSF antigen is confirmed
at each passage by the direct FAT in cryostat sections of the tonsils, or by virus isolation in a suitable
substrate. If CSFV or antigen cannot be demonstrated after a certain passage, a second series of
passages is performed to show infection, commencing with the last two pigs of the previous series.
Five pigs are inoculated intramuscularly with the sixth pig passage of the seed-lot virus, equivalent to
one vaccine dose or, if this passage has not been reached, the highest passage of the two series
where virus or viral antigen was detected. Five further pigs are similarly inoculated with one dose of the
seed-lot virus, equivalent to one vaccine dose. All pigs are weighed at the time of inoculation and again
2 weeks later. Blood is collected daily into anticoagulant during the first week, and all pigs are kept
under observation for 3 weeks. No animal should die or become ill from the vaccine virus. The average
weight gain of the two groups during the first 2 weeks should not differ significantly (p <0.05).
Leukopenia (WBC count of <7 106/ml) is permitted, at the most, in one pig of either group for 1 day.
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v)
Immunogenicity
To demonstrate adequate immunogenicity, ten pigs are each inoculated with an amount of virus
equivalent to one dose of vaccine, and two other pigs are housed separately as uninoculated controls.
All pigs are challenged 7 days later with 105 PID50 of a virulent strain of CSFV. Only the controls
should die.
In a test for duration of immunity, ten pigs are each inoculated with one dose, and two others are
maintained separately as controls. Six months later, the sera of the inoculated pigs are tested for CSF
antibodies; at least eight pigs should be positive. All pigs are then challenged with at least 105 PID50 of
a virulent strain of CSFV, and observed for 3 weeks. Only the controls should die.
To test protection of the development of the carrier sow syndrome, 20 pregnant sows at the same
stage of gestation are randomly divided into two groups. The sows of one group are vaccinated once or
twice with an amount of virus equivalent to one dose of vaccine, and intranasally challenged 4 weeks
after the last vaccination with a field strain of low virulence, together with the unvaccinated control
sows. All sows are killed 4 weeks after challenge and the fetuses are examined for the presence of
CSFV or viral antigen. Vaccination should significantly reduce transplacental transmission of the virus.
Under the storage conditions prescribed by the manufacturer for the final product, a volume of virus
equivalent to one dose of vaccine must maintain its immunogenicity at least until the end of the stated
shelf life.
2.
Each batch of MLV CSF vaccine must be derived from the same seed lot that has been used for the pilot tests.
Also, each batch must be prepared according to the production protocol and under the conditions laid down for
the registration of the final product. The properties of each batch and those of the seed lot must be verified as
uniform.
3.
In-process control
The protocol for production will depend on the vaccine strain, the production system (animals or cell cultures), and
available facilities. The norms for cell culture vaccines may vary according to the production system, namely,
primary cultures, cell lines, monolayers or suspension cultures.
4.
Batch control
All pigs used in batch control tests must be 68 weeks of age and free from antibodies to CSFV and BVDV. They
must be uniform in origin, breed, husbandry, and randomly distributed into any groups where necessary.
a)
Identity
The vaccine must induce specific neutralising antibodies against a virulent strain of CSFV.
b)
Sterility
Tests for sterility and freedom from contamination of biological material may be found in chapter 1.1.7.
c)
Safety
Each of three pigs is inoculated intramuscularly with ten doses of the reconstituted vaccine as a single
injection. The pigs are observed for 3 weeks thereafter and body temperatures are taken daily for the first
week. No pig should die or show signs of disease attributable to the vaccine, the average daily body
temperature must at no time reach 40.5C or more, and the pigs should grow normally.
d)
Purity
The batch must be virologically pure. To test for this, three pigs are each inoculated intramuscularly with ten
vaccine doses. Serum samples are collected at the time of inoculation and again 5 weeks later. These are
tested for antibodies to BVD (neutralisation for 1 hour at 37C) and porcine parvovirus (haemagglutination
inhibition using four haemagglutinating units). All three pigs must remain disease free. Tests for virological
purity need not be carried out when using vaccines produced in rabbits.
1100
e)
Potency
Potency is expressed as the number of 50% protective doses (PD50) contained in one vaccine dose. One
vaccine dose is at least 100 PD50.
Two groups of five 68-week-old piglets are inoculated intramuscularly with a 1/40 and a 1/160 dilution of
the reconstituted vaccine, respectively, using buffered salt solution, pH 7.2. The vaccinated pigs together
with two controls are challenged intramuscularly with 105 PID50 of a virulent strain of CSFV 2 weeks later.
The pigs are observed for 2 weeks thereafter, during which time the controls should die. From the pigs that
survive without showing any signs of CSF, the number of PD50 contained in the vaccine is calculated using
the usual statistical methods.
This potency test may be replaced by an infectivity assay, provided that the manufacturer can show that
there is a distinct and reproducible relationship between the virus content of the vaccine and the protection it
will confer on pigs against challenge.
f)
Stability
The period of validity of a batch of lyophilised CSF vaccine should not be under 1 year.
C2. Marker vaccines

Despite the existence of safe and effective MLV vaccines against CSF, their use has been discouraged in the EU
and some other CSF-free or near free countries, because antibodies provoked by such vaccines cannot be
distinguished from antibodies induced by the wild-type virus. A marker vaccine, which allows discrimination of
infected from vaccinated animals (DIVA), does not have this disadvantage: it can elicit a protective immune
response that can be distinguished from the immune response induced by field virus. An additional prerequisite of
any DIVA strategy is the availability of a companion serological test that is highly discriminatory, for demonstrating
absence of infection and in tracing residual infections.
The minimum demands for CSF marker vaccines and the companion discriminatory tests have been formulated
as follows (European Commission, 1997):
a)
Vaccine
The vaccine should provide protection against any natural-contact challenge, i.e. it should prevent clinical
signs and re-excretion of the virus. The efficacy of vaccination should be shown experimentally by studies in
which transmission of wild-type virus in vaccinated groups of pigs is studied. The protective effect of
vaccination should be achieved within the shortest possible period and ideally less than 2 weeks. A fast and
reliable protection should preferably be obtained after one single application. Furthermore, it should be
ensured that infection of vaccinated pregnant sows does not lead to transplacental infection and the birth of
litters congenitally infected with CSFV. Duration of immunity should be at least 6 months.
Many different marker vaccines for CSF are under development, and two have been registered in the EU.
Both are subunit vaccines that employ the E2 glycoprotein of CSFV as an immunogen and have been
subject to independent assessment (Depner et al., 2001; uttenthal et al., 2001). The E2 subunit is produced
by insect cells that are infected by genetically modified baculovirus, which contains the E2 gene of CSFV.
The vaccines, therefore, do not contain any CSFV, while the baculo (vector) virus is chemically inactivated.
The final preparations are adjuvanted with mineral oils to form a double (water/oil/water), or a single (water
in oil) emulsion. Several studies of the ability of these E2 DIVA vaccines to prevent horizontal and vertical
transmission have given conflicting results (Blome et al., 2006).
b)
Companion discriminatory test

The companion discriminatory serological test should be very sensitive because vaccination will reduce the
prevalence of the disease. It should ideally provide discrimination within the same time-frame as for
development of antibody to the immunising protein and should be used primarily as a herd test. If a high
sensitivity reduces the specificity of the test, already compromised by the presence of antibodies to other
pestiviruses, good and fast confirmatory assays should be available to discriminate positive from falsepositive results.
The existing accompanying DIVA tests for E2 subunit vaccines are ELISAs that rely on the detection of
antibody to the Erns protein (Floegel-Niesmann, 2001; Langedijk et al., 2001). Such tests have recently
been approved by the European Commission (2003), for use in determining whether herds vaccinated with
an E2 subunit vaccine may also have been exposed to field virus. An assessment of their performance
1101
(Floegel-Niesmann, 2001) has revealed that neither discriminatory ELISA consistently detected individual
marker-vaccinated, CSF-challenged weaner pigs, hence the recommendation only to employ such a
strategy at the herd level.
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COLIJN E.O., BLOEMRAAD M. & WENSVOORT G. (1997). An improved ELISA for the detection of serum antibodies
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swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows. Vet. Microbiol., 83, 107120.
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Blood Components for Early Diagnosis of CSF. Rep. Meeting Nat. Swine Fever Lab. Brussels 2425 November.
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monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses. Vet. Microbiol., 29, 101
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EUROPEAN COMMISSION (1997). The Use of Marker Vaccines in the Control of Infectious Diseases in Particular,
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confirmation of classical swine fever (2002/106/EC). Official Journal of the European Union, L39/71.
EUROPEAN COMMISSION (2003). Commission decision of 5 December 2003 amending Decision 2002/106/EC as
regards the establishment of a classical swine fever discriminatory test. (2003/859/EC). Official Journal of the
European Union, L324/55.
FLOEGEL-NIESMANN G. (2001). Classical swine fever (CSF) marker vaccine. Trial III. Evaluation of discriminatory
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HAVE P. (1987). Use of enzyme-linked immunosorbent assays in diagnosis of viral diseases in domestic livestock.
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HOFFMANN B., BEER M., SCHELP C., SCHIRRMEIER H. & DEPNER K. (2005). Validation of a real-time RT-PCR assay for
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HURTADO A., GARCIA-PEREZ A.L., ADURIZ G. & JUSTE R.A. (2003). Genetic diversity of ruminant pestiviruses from
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LANGEDIJK J.P., MIDDEL W.G., MELOEN R.H., KRAMPS J.A. & DE SMIT J.A. (2001). Enzyme-linked immunosorbent
assay using a virus type-specific peptide based on a subdomain of envelope protein E(rns) for serologic diagnosis
of pestivirus infections in swine. J. Clin. Microbiol., 39, 906912.
1102
LEFORBAN Y., EDWARDS S., IBATA G. & VANNIER P. (1990). A blocking ELISA to differentiate hog cholera virus
antibodies in pig sera from those due to other pestiviruses. Ann. Rech. Vet., 21, 119129.
LIESS B. & PRAGER D. (1976). Detection of neutralising antibodies (NIF) test: use of new technical equipment for
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LIN M., TROTTIER E. & MALLORY M. (2005). Enzyme-linked immunosorbent assay based on a chimeric antigen
bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus
infection. Clin. Diagn. Lab. Immunol., 12, 877881.
MCGOLDRICK A., LOWINGS J.P., IBATA G., SANDS J.J., BELAK S. & PATON D.J. (1998). A novel approach to the
detection of classical swine fever virus by RT-PCR with a fluorogenic probe (Taq Man). J. Virol. Methods, 72,
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MOSER C., RUGGLI N., TRATSCHIN J.D. & HOFMANN M.A. (1996). Detection of antibodies against classical swine
fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2. Vet. Microbiol., 51, 41
53.
OGAWA N., NAKAGAWA H., YAMAMOTO H., SAWADA M., HANAKI T. & SAZAWA H. (1973). Viral detection in pigs
inoculated with the GPE-strain of hog cholera attenuated virus. Ann. Rep. Nat. Vet. Assay Lab. (Japan), 10, 15
19.
PATON D.J., MCGOLDRICK A., GREISER-WILKE I., PARCHARIYANON S., SONG J.-Y., LIOU P.P., STADEJEK T., LOWINGS
J.P., BJORKLUND H. & BELAK S. (2000a). Genetic typing of classical swine fever. Vet. Microbiol., 73, 137157.
PATON D.J., MCGOLDRICK A., BENSAUDE E., BELAK S., MITTELHOLZER C., KOENEN F., VANDERHALLEN H., GREISERWILKE I., SCHEIBNER H., STADEJEK T., HOFMANN M. & THUER B. (2000b). Classical swine fever virus: a second ring
test to evaluate RT-PCR detection methods. Vet. Microbiol., 77, 7181.
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RISATTI G.R., CALLAHAN J.D., NELSON W.M. & BORCA M.V. (2003). Rapid detection of classical swine fever virus by
a portable real-time reverse transcriptase PCR assay. J. Clin. Microbiol., 41 (1), 500505.
RISATTI G., HOLINKA L., LU Z., KUTISH G., CALLAHAN J.D., NELSON W.M., BREA TIO E. & BORCA M.V. (2005).
Diagnostic evaluation of a real-time reverse transcriptase PCR assay for detection of classical swine fever virus.
J. Clin. Microbiol., 43 (1), 468471.
TERPSTRA C. (1978). Detection of C-strain virus in pigs following vaccination against swine fever. Tijdschr.
Diergeneeskd, 103, 678684.
TERPSTRA C., BLOEMRAAD M. & GIELKENS A.J.L. (1984). The neutralising peroxidase-linked assay for detection of
antibody against swine fever virus. Vet. Microbiol., 9, 113120.
TERPSTRA C. & WENSVOORT G. (1988a). The protective value of vaccine-induced neutralising antibody titres in
swine fever. Vet. Microbiol., 16, 123128.
TERPSTRA C. & WENSVOORT G. (1988b). Natural infections of pigs with bovine viral diarrhoea virus associated with
signs resembling swine fever. Res. Vet. Sci., 45, 137142.
UTTENTHAL A., LE POTIER M.F., ROMERO L., DE MIA G.M. & FLOEGEL-NIESMANN G. (2001). Classical swine fever
(CSF) marker vaccine. Trial I. Challenge studies in weaner pigs. Vet. Microbiol., 83, 85106.
VANNIER P. & CARNERO R. (1985). Effets pour le porc dun virus propag par un vaccin contre la maladie
dAujeszky. Point Vet., 17, 325331.
WENSVOORT G., BLOEMRAAD M. & TERPSTRA C. (1988). An enzyme immunoassay employing monoclonal antibodies
and detecting specifically antibodies to classical swine fever virus. Vet. Microbiol., 17, 129140.
WENSVOORT G. & TERPSTRA C. (1988). Bovine viral diarrhoea infections in piglets born from sows vaccinated
against swine fever with contaminated vaccine. Res. Vet. Sci., 45, 143148.
1103
WENSVOORT G., TERPSTRA C., BOONSTRA J., BLOEMRAAD M. & VAN ZAANE D. (1986). Production of monoclonal
antibodies against swine fever virus and their use in laboratory diagnosis. Vet. Microbiol., 12, 101108.
WENSVOORT G., TERPSTRA C., DE KLUYVER E.P (1989a). Characterization of porcine and some ruminant
pestiviruses by cross-neutralisation. Vet. Microbiol., 20, 291306.
WENSVOORT G., TERPSTRA C., DE KLUYVER E.P., KRAGHTEN C. & WARNAAR J.C. (1989b). Antigenic differentiation of
pestivirus strains with monoclonal antibodies against hog cholera virus. Vet. Microbiol., 21, 920.
*
* *
NB: There are OIE Reference Laboratories for Classical swine fever
diagnostic tests, reagents and vaccines for classical swine fever
1104
CHAPTER 2.8.4.
NIPAH VIRUS ENCEPHALITIS
See Chapter 2.9.6. Hendra and Nipah virus diseases.
*
* *
1105
CHAPTER 2.8.5.
PORCINE BRUCELLOSIS
SUMMARY
Brucellosis in pigs is caused by Brucella suis, a bacterial infection that, after an initial bacteraemia,
causes chronic inflammatory lesions in the reproductive organs of both sexes, with occasional
localisation and lesions in other tissues. The species Brucella suis consists of five biovars, but the
infection in pigs is caused by B. suis biovars 1, 2 or 3. The disease caused by biovars 1 and 3 is
similar, while that caused by biovar 2 differs from 1 and 3 in its host range, its limited geographical
distribution and its pathology. Biovar 2 is rarely pathogenic for humans, whereas biovars 1 and 3
are highly pathogenic causing severe disease. Porcine brucellosis is of widespread occurrence;
generally, however, the prevalence is low, with the exception of South America and South-East
Asia where the prevalence is higher. In some areas, B. suis infection has become established in
wild or feral pigs diagnostic methods recommended for wild and feral pigs are the same as for
domestic pigs. Various biovars of B. suis cause infections in animals other than pigs, such as
reindeer, caribou, hares and various murine species, and occasionally in cattle and dogs. Brucella
suis infections in animals other than pigs are dealt with in an Appendix at the end of this chapter.
Signs of disease in sows include infertility and abortion at any stage of gestation, and birth of dead
or weak piglets. In boars, the most prominent sign is orchitis, and the secondary sex organs may be
affected. Brucella suis may be present in the semen, sometimes in the absence of clinical signs.
Transmission during copulation is more common than is the case with brucellosis in ruminants. In
both sexes, bones and especially joints and tendon sheaths may be affected, causing lameness
and sometimes paralysis. Pigs are susceptible to artificial infection with B. abortus and
B. melitensis, but reports of natural disease in pigs being caused by either of these organisms are
rare. In humans, the infection is usually confined to those who are occupationally exposed to pigs,
and to laboratory workers. The capability of B. suis to colonise the bovine udder with subsequent
shedding in milk, has the potential to be a serious human health risk.
Identification of the agent: Brucella suis is readily isolated from live pigs by culture of birth
products, and from carcasses by culture of lymph nodes and organs. Selective media are available
for culture of contaminated samples. In nature, B. suis occurs invariably in the smooth phase the
appearance on solid medium is typical of smooth brucellae. Biovars of porcine origin agglutinate
with monospecific A antiserum, and not with M antiserum. Definite identification of species and
biovars may be effected by phage typing, molecular and biochemical tests, preferably carried out in
specialised laboratories.
Serological tests: To date, none of the serological tests has been shown to be reliable in routine
diagnosis in individual pigs. The indirect and competitive enzyme-linked immunosorbent assays
(ELISAs), as well as the Rose Bengal test (RBT), complement fixation test (CFT) and fluorescence
polarisation assay (FPA) are the prescribed tests for international trade purposes. The allergic skin
test and the buffered plate agglutination test (BPAT) are also useful for identifying infected herds.
The procedures for all the tests are the same as those described in Chapter 2.4.3 Bovine
brucellosis.
Requirements for vaccines and diagnostic biologicals: Brucella suis strain 2 vaccine has been
used for immunising pigs in China (Peoples Rep. of). Confirmation of the results obtained in China
is required before strain 2 vaccine can be recommended for general use. In other countries,
experimental work has shown that B. melitensis Rev.1 vaccine is superior to B. suis strain 2 in
protecting sheep against B. melitensis. Sufficient data is not available to conclude if B. abortus
strain RB51 vaccine is efficacious in protecting swine against exposure to B. suis. In practice, no
1106
Chapter 2.8.5. Porcine brucellosis
product has yet found general acceptance. Preparation, testing and use of an established allergen,
brucellin is described.
A. INTRODUCTION
Porcine brucellosis is an infection caused by biovar 1, 2 or 3 of Brucella suis. It occurs in many countries where
pigs are raised. Generally, the prevalence is low, but in some areas, such as South America and South-East Asia,
the prevalence is much higher. Porcine brucellosis may be a serious, but presently unrecognised, problem in
some countries. Brucella suis biovar 1 infections have been reported from feral pigs in some of the southern
States of the United States of America (USA), and in Queensland, Australia. In both countries, a number of
human infections have been reported from people who hunt and handle material taken from feral pigs (Robson et
al., 1993; Starnes et al., 2004).
The disease is generally transmitted by consumption of feed contaminated by birth and/or abortion products and
uterine discharges. Pigs will readily eat aborted fetuses and placental membranes. Transmission during
copulation also occurs frequently, and this has implications for those practising artificial insemination.
In pigs, as in ruminants, after the initial bacteraemia, B. suis colonises cells of the reproductive tract of either sex.
In females, placentas and fetuses are invaded, while in males, invasion occurs in one or more of the following:
testis, prostate, epididymis, seminal vesicles, and/or bulbo-urethral glands. In males the lesions, which are most
often unilateral, start with a hyperplasia that may progress to abscess formation; the final stage is characterised
by sclerosis and atrophy. Arthritis may occur in various joints, and sometimes spondylitis occurs.
The most common manifestation of brucellosis in female pigs is abortion, occurring very early or at any time
during gestation. Vaginal discharge is not often evident, and, in chronically infected herds, infertility rather than
abortion is the most relevant clinical sign of the disease. In males, brucellosis is more likely to be persistent, with
lesions in the genital tract often leading to interference with sexual activity, which can be temporary or permanent.
The boar may excrete brucellae in the semen without any apparent abnormality in the sex organs or interference
with sexual activity.
In both sexes, there may be swollen joints and tendon sheaths, lameness and, occasionally, posterior paralysis. A
significant proportion of both male and female pigs will recover from the infection, often within 6 months, but many
will remain permanently infected.
Brucellosis caused by B. suis biovar 2 differs from infection caused by biovars 1 and 3 in its host range, its
distribution, and in its pathology. In general, the geographical distribution of biovar 2 has historically been in a
broad range between Scandinavia and the Balkans (Alton, 1990). The prevalence in wild boars appears to be
high throughout continental Europe (Al Dahouk et al., 2005; Cvetnic et al., 2003; Garin-Bastuji et al., 2000;
Godfroid & Kasbohrer, 2002; Godfroid et al., 1996). In recent outbreaks in Europe, wild boars have been
implicated as the source of transmission of biovar 2 to outdoor reared pigs (Garin-Bastuji et al., 2000). In addition
to wild boars, the European hare (Lepus capensis) is also considered as a reservoir for B. suis biovar 2 and has
been implicated as a possible source of transmission to domestic livestock (Alton, 1990; Godfroid & Kasbohrer,
2002; Szulowski et al., 1999). Brucella suis biovar 2 causes miliary lesions, particularly reproductive tissues, that
often become purulent. To date, biovar 2 has rarely been reported as the cause of human brucellosis. However,
biovar 2 infection has been reported in two immuno-compromised hunters, who had been extensively exposed
through gutting or skinning boars or hares (Garin-Bastuji et al., 2006).
The most common B. suis biovars (1 and 3) are serious human pathogens and adequate precautions are needed
when handling and disposing of potentially infective material. This is especially so in the laboratory after culture
has greatly increased the number of organisms present. Laboratory manipulation of the cultures or contaminated
material from infected animals must be done under strict biosafety conditions to safely handle this dangerous
zoonotic agent. Biosafety containment level 3 is recommended (see Chapter 1.1.3 Biosafety and biosecurity in the
veterinary microbiology laboratory and animal facilities).
biovars are given in the Chapter 2.4.3 Bovine brucellosis.
As far as biovars 1 and 3 are concerned, culture methods are at least as sensitive as serology (Ferris et al.,
1995). Biovar 2 appears to be highly sensitive to selective media and could be more difficult to isolate (GarinBastuji & Blasco, unpublished data). As the product of almost all pig-raising enterprises passes through abattoirs,
1107
surveillance methods (serology and culture) can be applied effectively at this point. In many areas, traditional
village pig breeding is now accompanied by the development of larger commercial units, thereby increasing the
use of artificial insemination. Whereas artificial insemination using brucellosis-free boars can be a valuable aid in
the control of porcine brucellosis, the inadvertent use of infected semen could, obviously, cause incalculable
damage.
1.
Optimal samples for bacteriologic culture and methods for processing of samples are similar to those described
for bovine brucellosis in chapter 2.4.3. Standard and selective media used for other species of brucellae are
suitable for B. suis (see chapter 2.4.3). The addition of serum is not essential, but basal medium containing 5%
serum is a satisfactory medium, both for isolation, maintenance of cultures and typing. The addition of CO2 to the
atmosphere is not required.
In nature, B. suis invariably occurs in the smooth form and colonies are morphologically indistinguishable from
other smooth brucellae, described in chapter 2.4.3.
Biovars 1, 2 and 3 of B. suis are all A surface antigen dominant, and growth may be presumptively identified by
slide agglutination with the monospecific A antiserum. Confirmatory identification of species and biovar should be
performed in a specialised reference laboratory. The OIE Reference Laboratories for brucellosis are listed in the
Table given in Part 4 of this Terrestrial Manual.
Confirmation of species and biovar depends on phage tests, production of H2S (only biovar 1 produces H2S), and
growth in the presence of dyes. However, some strains of B. suis biovar 1 are atypical in that they grow on media
containing 20 g/ml of basic fuchsin. Most strains of B. suis are inhibited by safranin O at a concentration of
1/10,000, whereas B. suis usually reacts more rapidly in the urease test than either B. abortus or B. melitensis.
Oxidative metabolic tests are supplemental tests that can be used for distinguishing B. suis from other smooth
Brucella species.
Molecular genetic techniques using the polymerase chain reaction (PCR) and specific primers are available that
can identify B. suis and other species of Brucella (see chapter 2.4.3). Some of these techniques can distinguish
biovars of B. suis (Ferro-Beck et al., 2006; Fretin et al., 2008).
2.
Serological tests
None of the serological tests used for the diagnosis of porcine brucellosis are reliable for diagnosis in individual pigs.
The major antigen involved in the serological tests currently available is the smooth lipopolysaccharide (LPS). The
OPS moiety of this molecule contains epitopes that cross-react with those existing in the corresponding LPS from
Yersinia enterocolitica serotype O:9 (Jungersen et al., 2006). Therefore, available serological tests are unable to
distinguish between antibodies raised to these two infections. Y. enterocolitica O:9 infection in pigs is not
uncommon in some areas (Al Dahouk et al., 2005; Wrathall et al., 1983). Studies have suggested that the
sensitivities and specificities of the Rose Bengal test (RBT), the indirect and competitive enzyme-linked
immunosorbent assay (I- and C-ELISAs), and the fluorescent polarisation assay (FPA) are similar (Paulo et al.,
2000). With the exception of acute stages of infection (<9 weeks) (Jungersen et al., 2006), the use of the FPA
(Nielsen et al., 1999) or C-ELISA has been reported to eliminate cross-reactivity with Y. enterocolitica but this
should be confirmed in additional field studies performed in various epidemiological situations. Swine serum may
sometimes also contain nonspecific antibody, thought to be of the IgM isotype, further reducing the specificity of
conventional tests, especially the serum agglutination test (SAT). Also, swine complement interacts with guineapig complement to produce a pro-complementary activity that reduces the sensitivity of the complement fixation
test (CFT). Sensitivity levels may be low for the CFT; therefore caution should be taken when interpreting test
results from individual animals. For international and other trade, e.g. purchasing boars, the disease status of the
herd and of the area in which the herd is situated are of more importance than tests on individual animals.
Reference sera
The OIE reference standards are those against which all other standards are compared and calibrated. For
the RBT and CFT, please refer to chapter 2.4.3 for antigen standardisation and test protocols. A porcine
reference standard for ELISAs and FPA has been developed and will be available to national laboratories in
the near future1.
a)
Enzyme-linked immunosorbent assay (prescribed tests for international trade)

Indirect and competitive ELISAs have been developed for the diagnosis of brucellosis in individual pigs and
for screening large numbers of sera. These techniques promise to be more efficient than any of the tests
Obtainable from the Canadian Food Inspection Agencies, 3851 Fallowfield Road, Nepean, Ontario K2H 8P9, Canada.
1108
mentioned above. At this time the indirect ELISA and the reference sera for this test are not commercially
available.
Indirect ELISA
A method for the I-ELISA is described in detail in Chapter 2.4.3 Bovine brucellosis, however, antibody
specific for porcine IgG conjugated with horseradish peroxidase (HRPO) has to be used. The I-ELISA may
be used as a screening test. Before being used for international trade, the cut-off of I-ELISA should be
properly established using the appropriate validation techniques (see Chapter 1.1.5 Principles and methods
of validation of diagnostic assays for infectious diseases) and the test should be standardised against the
above-mentioned Standard.
Competitive ELISA
C-ELISA procedures for detection of porcine antibody to Brucella sp. (Nielsen et al., 1999) are identical to
the procedures used for bovine antibody to B. abortus described in chapter 2.4.3. This assay is capable of
eliminating some reactions due to Y. enterocolitica O:9 and in some situations other cross-reacting antibody,
such as IgM, will not compete well. Before being used for international trade, the cut-off of C-ELISA should
be properly established using the appropriate validation techniques (see chapter 1.1.5) and the test should
be standardised against the above-mentioned Standard
b)
Fluorescence polarisation assay (a prescribed test for international trade)

The FPA for detection of porcine antibody to Brucella sp. is essentially the same as that described for cattle
(for more details see chapter 2.4.3); an example serum dilution used is 1/25 for the tube test and 1/10 for the
plate test (Nielsen et al., 1999). It is a simple technique for measuring antigen/antibody interaction and may
be performed in the laboratory or in the field. Lyophilised porcine sera tend to increase background activity in
this assay. The FPA may be used as a screening and/or confirmatory test. Before being used for
international trade, the cut-off of FPA should have been properly established using the appropriate validation
techniques (see chapter 1.1.5) and the test should be standardised against the above-mentioned Standard.
c)
Rose Bengal test (a prescribed test for international trade)

The preparation and standardisation of RBT antigen and the method of performing the test are described in
chapter 2.4.3. The B. abortus antigens are appropriate for testing swine sera in RBT and will identify
antibody against all three biovars of B. suis. The RBT may be used as a screening test, bearing in mind its
lack of specificity for discriminating reactions caused by brucellosis from those caused by infections by
Y. enterocolitica O:9 and other cross-reacting bacteria.
d)

The preparation and standardisation of CFT antigen and the method of performing the test are described in
chapter 2.4.3. The B. abortus antigens are appropriate for testing swine sera in CFT and will identify
antibody against all three biovars of B. suis. The CFT has a reduced sensitivity for diagnosing B. suis
infection and is not capable of eliminating all reactions caused by Y. enterocolitica O:9. It may be used as a
complementary test.
3.
Other Tests
a)
Allergic (hypersensitivity) tests

Brucelin-INRA is an sLPS free cytosolic extract from rough B. melitensis B115. This preparation does not
stimulate the formation of antibodies that would be reactive in BBAT, CFT or ELISAs. The product has been
developed for use in ruminants, but is also effective for confirming the disease at the herd level in pigs. A
rough strain is used in its preparation, thereby avoiding the presence of sLPS. The preparation,
standardisation and testing, of Brucellin-INRA is described in detail in chapter 2.4.3. As a diagnostic agent in
pigs 0.1 ml of the allergen is injected intradermally into the skin at the base of the ear or preferably next to
the base of the tail. The latter appears more practical and less hazardous. The reaction is read after
48 hours. A positive reaction shows erythema of non-pigmented skin and an oedematous swelling. In severe
reactions, there may also be some necrosis.

Numerous attempts have been made to develop a vaccine to immunise pigs against B. suis, but none has been
found fully effective. Only one immunogen B. suis strain 2 (S2) vaccine has been reported suitable after
1109
extensive field use in south China (Peoples Rep. of) (Liang Xingxian, 1991; Xin, 1986)2 but experiments under
strictly controlled conditions are not available. To date, it does not appear to have been used elsewhere in pigs,
probably because it has been shown that this vaccine does not confer adequate protection in sheep against
B. melitensis (Verger et al., 1995). Similarly, the B. abortus strain RB51 vaccine has been reported as
inefficacious in protecting swine against exposure to B. suis (Stoffregen et al., 2006).
APPENDIX: BRUCELLA SUIS INFECTIONS IN ANIMALS OTHER THAN PIGS

1.
Rangiferine brucellosis
Brucella suis biovar 4 causes serious disease in reindeer or caribou (Rangifer tarandus and its various
subspecies) throughout the Arctic region, including Siberia, Canada and Alaska (Jungersen et al., 2006; Orlow,
1963). Some of these animals are domesticated, others are wild and migratory. Rangifer tarandus is very
susceptible to B. suis infection, which causes fever, depression and various local signs, such as abortion, retained
placentas, metritis, sometimes with blood-stained discharge, mastitis, bursitis and orchitits. In the Arctic region,
B. suis biovar 4 constitutes a serious zoonosis (Forbes, 1991). Transmission to humans may be by direct contact
or through consumption of milk and other inadequately heated products from reindeer. Bone marrow, which is
considered to be a special delicacy in this region, is also a source of human infection.
The methods already described for isolating and identifying B. suis in samples taken from pigs are equally
applicable to B. suis biovar 4 in samples taken from reindeer. Biovar 4 grows well on all the usual media used for
the culture of Brucella. It reacts positively with both anti-A and -M monospecific sera. For serology, the tube
agglutination test has been reported to be satisfactory, with titres of 1/20 or greater being considered to be
diagnostic. The CFT has also been used but the clinical interpretation of these tests in reindeer has not been
established.
Vaccination of reindeer with B. abortus S19 vaccine, or alternatively with B. abortus 45/20 adjuvant vaccine, has
been tried experimentally without any clear-cut result. In the case of S19, the reaction to vaccination was rather
severe and immunity in the vaccinated animals could only be demonstrated against challenge with very small
doses of B. suis biovar 4. Gall et al. (2001) compared several serological tests and found that the specificity
values for the BPAT and CFT using reindeer/caribou sera was lower than the I-ELISA, C-ELISA and the FPA,
while sensitivity values were similar for all tests.
2.
Brucella suis infection in other nonporcine species
There are two different types of epidemiological situation with regard to B. suis infection in other nonporcine
species. In the first case, B. suis infection occurs in animals that are not the natural host of the particular infection
through the ingestion of contaminated materials or by co-habitation with infected natural hosts. For example,
Arctic foxes and wolves may contract B. suis biovar 4 from reindeer; dogs and rodents, such as rats and mice,
may acquire other B. suis biovars by cohabitation with infected hosts. Cattle and horses may become infected by
cohabitation or interaction with infected swine (Cvetnic et al., 2005). The infecting bacteria are invariably the well
defined biovars of the natural host species.
In the second case, wildlife species that are natural hosts for B. suis or B.-suis-like infections become infected.
One example is the so-called murine brucellosis of the former USSR, where small rodents are infected with
B. suis biovar 5. Other similar situations have been reported from Queensland, Australia and from Kenya. In all
three cases, B. suis strains with different characteristics were involved, and at least one of them was difficult to
classify.
Brucellosis caused by B. suis biovar 2 in the European hare (Lepus capensis) (Szulowski et al., 1999), is
characterised by the formation of nodules, varying in size from that of a millet seed to a cherry or even larger;
these often become purulent. Such nodules may occur in almost any location, sometimes subcutaneously or
intramuscularly, in the spleen, liver or lung and in the reproductive organs of either sex. The bodily condition of
the hare may be surprisingly unaffected. Other species may also become infected by cohabitation with B. suis
biovar 2 infected swine, wild boars or hares. Gutting or skinning wild boars in cattle sheds could be a route of
transmission to cattle (Garin-Bastuji & Delcueillerie, 2001).
Serological investigations in nonporcine species are usually carried out for screening purposes. In these particular
circumstances, specificity is more important than sensitivity. Here the CFT can be recommended, although the
RBT, may be also useful because of its simplicity. The indirect and competitive ELISAs appear to be very useful
2
Obtainable from the National Institute for the Control of Veterinary Products and Pharmaceuticals, Ministry of Agriculture,
30 Baishiqiao Road, Beijing 100081, China (Peoples Rep. of), or from AHVLA Weybridge, New Haw, Addlestone, Surrey
KT15 3NB, United Kingdom. Supply from the Weybridge laboratory needs the prior permission of the World Health
Organization.
1110
for epidemiological sero-surveys in wild boars as both are more sensitive and specific than the RBT and CFT. The
test has also been used successfully on blood samples that are in poor condition. When poor quality samples are
tested on other tests, the results may be uninterpretable. Another advantage of the ELISAs is that if serum is not
available, it is possible to test meat juice samples (Garin-Bastuji et al., 2004). However, in nonporcine species the
interpretation of serological results may be problematic. Where positive or equivocal serological results are
encountered, a bacteriological investigation should be conducted.
For bacteriological investigations in situations such as these, where the infecting organisms may have unusual
characteristics, it is advisable to duplicate the culture on selective media by culture on plain medium
supplemented with 5% serum, and to broaden the investigation by incubating the cultures in an atmosphere
containing 10% CO2. Colonies resembling Brucella can be tentatively identified by Gram staining, by slide
agglutination tests with monospecific anti-A and -M sera, and by anti-rough Brucella serum (chapter 2.4.3)
Brucella suis biovar 5 is unusual in that it reacts with anti-M monospecific serum, and not with anti-A monospecific
serum. Further identification is best carried out in a specialised laboratory.
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FERRIS R.A., SCHOENBAUM M.A. & CRAWFORD R.P. (1995). Comparison of serologic tests and bacteriologic culture
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FORBES L.B. (1991). Isolates of B. suis biotype 4 from animals and humans in Canada, 19821990. Can. Vet. J.,
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GARIN-BASTUJI B., CAU C., HARS J., BOU F. & TERRIER M.E. (2004). Utilisation compare du srum, du poumon et
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STOFFREGEN W.C., OLSEN S.C., BRICKER, B.J. (2006). Parenteral vaccination of domestic pigs with Brucella
abortus strain RB51. Am. J. Vet. Res., 67, 18021808.
SZULOWSKI K., IWANIAK W., PILASZEK J., TRUSZCZYNSKI M. & CHROBOCINSKA M. (1999). The ELISA for the
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VERGER J.M., GRAYON M., ZUNDEL E., LECHOPIER P. & OLIVER-BERNARDIN V. (1995). Comparison of the efficacy of
Brucella suis strain 2 and Brucella melitensis Rev.1 live vaccines against a Brucella melitensis experimental
infection in pregnant ewes. Vaccine, 13, 191196.
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XIE X. (1986). Orally administrable brucellosis vaccine: Brucella suis strain 2 vaccine. Vaccine, 4, 212216.
*
* *
NB: There are OIE Reference Laboratories for Porcine brucellosis
diagnostic tests, reagents and vaccines for porcine brucellosis
1112
CHAPTER 2.8.6.
PORCINE CYSTICERCOSIS
See Chapter 2.9.5. Cysticercosis.
*
* *
1113
CHAPTER 2.8.7.
PORCINE REPRODUCTIVE AND

RESPIRATORY SYNDROME
SUMMARY
Porcine reproductive and respiratory syndrome (PRRS) is characterised by reproductive failure of
sows and respiratory problems of piglets and growing pigs. The disease is caused by the PRRS
virus, a virus currently classified as a member of the order Nidovirales, family Arteriviridae, genus
Arterivirus. The primary target cell of the virus is the alveolar macrophage of the pig. Two major
antigenic types of the virus exist, the type 1 (European) and the type 2 (American). The virus is
primarily transmitted via infected pigs but also by faeces, urine, semen and fomites.
PRRS occurs in most major pig-producing areas throughout the world. The reproductive failure is
characterised by infertility, late fetal mummification, abortions, stillbirths, and the birth of weak
piglets that often die soon after birth from respiratory disease and secondary infections. Older pigs
may demonstrate mild signs of respiratory disease, usually complicated by secondary infections.
No other species are known to be naturally infected with PRRSV.
Identification of the agent: Virological diagnosis of PRRS virus infection is difficult; the virus can
be isolated from tissues such as serum, ascitic fluids, or organ samples, such as lungs, tonsil,
lymph nodes and spleen of affected pigs. As porcine alveolar macrophages are the most
susceptible culture system for virus of both antigenic types, these cells are recommended for virus
isolation. MARC-145 (MA-104 clone) cells are also suitable. There is variability between batches of
macrophages in their susceptibility to PRRS virus. Thus, it is necessary to identify a batch with high
susceptibility, and maintain this stock under liquid nitrogen until required. The virus is identified and
characterised by immunostaining with specific antisera. Additional techniques, such as
immunohistochemistry and in situ hybridisation on fixed tissues and reverse-transcription
polymerase chain reaction, have been developed for laboratory confirmation of PRRS virus
infection.
Serological tests: A wide range of serological tests is currently available for the detection of serum
antibodies to PRRS virus. The immunoperoxidase monolayer assay uses alveolar macrophages
and the indirect immunofluorescence assay uses MARC-145 cells that are usually infected using
either the European or the American antigenic type of the virus, respectively. Both assays can
however be designed with both cell and PRRS virus types. Commercial or in-house enzyme linked
immunosorbent assays (ELISA) are now often used. Several commercial ELISAs are specific for
both the European and American types of the virus. An indirect ELISA, a blocking ELISA and a
double ELISA that can distinguish between serological reactions to the European and the American
types have been described.
Requirements for vaccines: Vaccines can be of value as an aid in the prevention of reproductive
and respiratory forms of PRRS. Modified live vaccines are not suitable for use in pregnant sows
and gilts and in boars. Vaccination may result in shedding of vaccinal virus in semen. Modified live
virus vaccines can persist in vaccinated animals, and transmission to nonvaccinated animals and
subsequent vaccine-virus-induced disease have been reported.
A. INTRODUCTION
Porcine reproductive and respiratory syndrome (PRRS) is characterised by reproductive failure of sows and
respiratory disease in pigs. (Benfield et al., 1999). The disease was first recognised in 1987 in the United States
1114
Chapter 2.8.7. Porcine reproductive and respiratory syndrome
of America, and within a few years it became a pandemic. PRRS is caused by the PRRS virus (PRRSV). It was
discovered in 1991 in The Netherlands (Wensvoort et al., 1993) and is classified as a member of the order
Nidovirales, family Arteriviridae, genus Arterivirus (Brinton et al., 2000). PRRSV is a single-stranded positivesense RNA virus and the biology of the virus has been well characterised. Soon after the discovery of the virus it
became apparent the North American (NA, type 2) and European (EU, type 1) PRRSV isolates represented two
genotypes with antigenic differences (Larochelle & Magar, 1997c; Magar et al., 1997). Additional investigations
have demonstrated regional differences within each continent. These differences are now becoming blurred as
type 2 PRRSV has been introduced into Europe (in part through the use of a modified-live vaccine made from a
North American isolate) and type 1 virus has been discovered in North America. Most PRRSV isolates from South
America and much of Asia are of type 2 and it is assumed these viruses were introduced through the movement
of swine and/or semen. A reportedly highly virulent strain of type 2 virus in Southeast Asia is characterised by a
discontinuous 30 amino acid deletion in the NSP2 region of the genome.
There is an increasing diversity among strains of the two genotypes, which has been attributed to the high errorrate inherent in PRRSV replication (Chang et al., 2002) and recombination between strains (Van Vugt et al.,
2001) There have also been recent descriptions of East European strains of type 1 PRRSV with a high degree of
polymorphism, providing further insights into the emergence of the relatively new pathogen of pigs (Stadejek et
al., 2006; 2008). The effects of such diversity on diagnostics and vaccines are largely unknown, but do raise
concerns and should be considered.
The reproductive syndrome is recognised by late-gestation abortions and early or delayed farrowings that contain
dead and mummified fetuses, stillborn pigs, and weak-born pigs. An increase in repeat breeders during the acute
phase of the epizootic is commonly reported. Infrequently, there are reports of early- to mid-gestation reproductive
failure. In boars and unbred replacement gilts and sows, transient fever and anorexia may be observed. The
respiratory syndrome is recognised by dyspnoea (thumping), fever, anorexia, and listlessness. Younger pigs are
more affected than older animals with boars and sows (unbred) frequently having subclinical infection. An
increase in secondary infections is common and mortality can be high. In PRRSV-infected boars and boars that
have been vaccinated with live attenuated vaccine, PRRSV can be shed in semen, and changes in sperm
morphology and function have been described (Christopher-Hennings et al., 1997). The virus is primarily
transmitted directly via infected pigs and also by faeces, urine and semen. It can also be spread indirectly,
presumably via aerosol routes, leading to chronic re-infection of herds in swine dense areas, and possibly by
mechanical vectors. Gross and microscopic lesions consistent with PRRSV infection have been well described
(Halbur et al., 1995). In general, the lesions are more severe in younger animals than older ones. Differences in
virulence between PRRSV isolates within a genotype and between genotypes are believed to exist based on field
observations and some experimental studies (Halbur et al., 1995). This variability has been reinforced with the
emergence in 2006 of a PRRSV lineage in South-East Asia associated with porcine high fever disease, a
syndrome causing high mortality in all ages of swine (Tian et al., 2007). Although there is now an extensive body
of research completed since the discovery of PRRSV, there are still many gaps in the knowledge base about the
apparent link between PRRSV and other diseases as well as understanding the PRRSV immune response.
1.
Identification of PRRSV can be accomplished by virus isolation, the detection of nucleic acids, and the detection
of viral proteins. Following infection, swine develop a viraemia and lung infection that can persist for weeks in
young pigs and days in adult animals making serum and bronchoalveolar lung lavage ideal samples to collect for
detection of PRRSV. Isolation of PRRSV can be difficult as not all virus isolates (especially type 1 viruses) can
easily infect a cell line derived from the MA-104 monkey kidney cell line (Kim et al., 1993). Interestingly, this
continuous cell culture system has been the only one reported to sustain a PRRSV infection. Porcine alveolar
macrophages (PAM) will support replication of most, if not all PRRSV isolates. However, the collection of PAM is
not an easy task as only pigs of high health status and less than 8 weeks of age should be used as the source of
PAM (Wensvoort et al., 1993). Different batches of PAM are not always equally susceptible to PRRSV; thus it is
necessary to test each batch before use. PAM can be stored in liquid nitrogen until needed as described below.
Isolation of PRRSV using PAM is a technique that can be performed in most diagnostic laboratories. This
technique should be sensitive for isolation of all PRRSV strains and will be explained in detail. Detection of
PRRSV nucleic acid can be accomplished with reverse-transcription polymerase chain reaction (RT-PCR), nested
set RT-PCR, and real-time RT-PCR (Drew, 1995b; Kleiboeker et al., 2005; Larochelle & Magar, 1997a; Mardassi
et al., 1994; Wasilk et al., 2004). These tests are commonly used to detect nucleic acid in tissues and serum.
They are also useful when virus isolation is problematic, such as when testing semen (Christopher-Hennings et
al., 1997) and when testing tissues partially degraded by autolysis or by heat during transport of specimens for
virus isolation. A multiplex PCR assay has been designed to differentiate isolates of types 1 and 2 (Gilbert et al.,
1997). Restriction fragment length polymorphism analysis of PCR-amplified products has been developed for the
differentiation of field and vaccine PRRSV isolates (Wesley et al., 1998) and recently molecular epidemiological
1115
studies of PRRSV strains have been performed using phylogenetic analyses of specific structural gene
sequences. All of these nucleic acid tests are more rapid than virus isolation and do not require cell culture
infrastructure. Although seldom used for diagnostic purposes, in-situ hybridisation is capable of detecting and
differentiating type 1 and 2 PRRSV genotypes in formalin-fixed tissues (Larochelle & Magar, 1997c). The
sensitivity and specificity of the methods for detection of PRRSV genome can be compromised by the very high
genetic diversity of PRRSV, especially in type 1. Immunohistochemistry can be used to identify viral proteins
(Halbur et al., 1994; Larochelle & Magar, 1995) and when performed on formalin-fixed tissues enables the
visualisation of antigen together with histological lesions.
a)
Harvesting of alveolar macrophages from lungs

Lungs should preferably be obtained from SPF pigs or from a herd of pigs that is proven to be free from
PRRSV infection. Best results are obtained with pigs that are under 8 weeks of age. The macrophages
should be harvested from the lung on the same day that the pig is slaughtered. The lungs should be washed
three or four times with a total volume of approximately 200 ml sterile phosphate buffered saline (PBS). The
harvested wash fluid is then centrifuged for 10 minutes at 1000 g. The resulting pellet of macrophages is
resuspended in PBS and centrifuged (washed) twice more. The final pellet is resuspended in 50 ml PBS,
and the number of macrophages is counted to determine the cell concentration. The macrophages can then
be used fresh, or can be stored in liquid nitrogen according to standard procedures at a final concentration of
approximately 6 107 macrophages/1.5 ml. Macrophage batches should not be mixed.
b)
Batch testing of alveolar macrophages

Before a batch of macrophages can be used it should be validated. This should be done by titrating a
standard PRRSV with known titre in the new macrophages, and by performing an immunoperoxidase
monolayer assay (IPMA) with known positive and negative sera on plates seeded with the new
macrophages. The cells are suitable for use only if the standard PRRSV grows to its specified titre,
(TCID50 or 50% tissue culture infective dose). It is recommended that alveolar macrophages and fetal
bovine serum (FBS) to supplement culture medium be pestivirus free.
c)
Virus isolation on alveolar macrophages

Alveolar macrophages are seeded in the wells of flat-bottomed tissue-culture grade microtitre plates. After
attachment, the macrophages are infected with the sample. Samples can be sera or ascitic fluids, or 10%
suspensions of tissues, such as tonsils, lung, lymph nodes, and spleen. In general, the PRRSV gives a
cytopathic effect (CPE) in macrophages after 12 days of culture, but sometimes viruses are found that give
little CPE or give a CPE only after repeat passage. After a period of 12 days or once CPE has been
observed, the presence of PRRSV needs to be confirmed by immunostaining with a specific antiserum or
monoclonal antibody (MAb).
i)
Seeding macrophages in the microtitre plates

Defrost one vial containing 6 107 macrophages/1.5 ml. Wash the cells once with 50 ml PBS and
centrifuge the cell suspension for 10 minutes at 300 g (room temperature). Collect the cells in 40 ml
RPMI (Rose-Peake Memorial Institute) 1640 medium supplemented with 5% FBS and 10% antibiotic
mixture (growth medium). Dispense 100 l of the cell suspension into each well of a microtitre plate
(with one vial of cells, four plates can be seeded at a concentration of 105 cells in each well of the
plates).
ii)
Preparation of sample (serum, ascitic fluid, 10% tissue suspension) dilutions in a dummy plate
Dispense 90 l of growth medium into each well of a microtitre plate. Add 10 l samples to the wells
of rows A and E (duplicate 1/10 dilution). Shake the plates and transfer 10 l from rows A and E
to rows B and F (1/100 dilution). Shake the plates and transfer 10 l from rows B and F to rows C and
G (1/1000 dilution). Shake the plates and transfer 10 l from rows C and G to rows D and H
(1/10,000 dilution). Shake the plates.
iii)
Incubation of samples
Transfer 50 l of the sample dilutions from the dilution plates to the corresponding wells of the plate
with macrophages (first passage). Incubate for 25 days and observe daily for a CPE. At day 2, seed
macrophages in new microtitre plates (see above). Transfer 25 l of the supernatants from the plates
of the first passage to the corresponding wells of the freshly seeded plates (second passage). Incubate
for 25 days and observe daily for a CPE.
1116
iv)
Reading and interpreting the results

Wells in which macrophages show CPE in the first passage only are considered to be false positive
because of the toxicity of the sample. Wells in which macrophages show CPE in both passages or in
the second passage only are considered to be suspect positive. All wells with macrophage monolayers
that do not show CPE need to be identified as PRRSV negative by immunostaining with a PRRSVpositive antiserum or MAb. CPE-positive samples need to be identified as PRRSV positive by culturing
CPE-positive supernatant samples, or the original sample dilutions, for both 24 and 48 hours in
macrophages, followed by immunostaining with a PRRSV-positive antiserum or MAb.
v)
Immunostaining with a PRRSV-positive antiserum or MAb

Infect macrophages with 50 l of supernatant or tissue sample as described in Section B.2.a, and grow
the infected cells for 24 and 48 hours. Prepare an appropriate dilution of a PRRSV-positive serum in
dilution buffer, and immunostain the macrophages as described in Section B.2.a or B.2.b.
2.
Serological tests
A variety of assays for the detection of serum antibodies to PRRSV have been described. Serological diagnosis
is, in general, easy to perform, with good specificity and sensitivity, especially on a herd basis. Sera of individual
pigs sometimes cause difficulties because of nonspecific reactions, but this problem may be solved by resampling
the pig after 23 weeks. Serology is generally performed with a binding assay, such as the IPMA,
immunofluorescence assay, or the enzyme-linked immunosorbent assay (ELISA) of which many varieties are
described (Albina et al. 1992; Cho et al., 1997; Denac et al., 1997; Houben et al., 1995; Jusa et al., 1996; Nodelijk
et al., 1996; Wensvoort et al., 1993; Yoon et al., 1992). These tests are often performed with viral antigen of one
antigenic type, which means that antibodies directed against the other, heterologous, antigenic type may be
detected with less sensitivity. A blocking ELISA has been used extensively in Denmark and has been described
as a double ELISA set-up using both types 1 and 2 virus as antigen and thus it can distinguish between
serological reaction to both types (Sorensen et al., 1998). The first live attenuated vaccine for PRRS based on
type 2 virus has been observed to spread to nonvaccinated animals (Botner et al., 1997; Torrison et al., 1996),
and subsequent development in herds of vaccine-virus-induced PRRS reproductive failures has been reported in
Denmark (Botner et al., 1997; Madsen et al., 1998). Reaction to type 2 vaccine-like PRRSV can be anticipated in
countries using or having used this vaccine; European countries may therefore observe reactions and isolation of
both antigenic types (Botner et al., 1997; Madsen et al., 1998). The identification of type 1 strains of PRRSV in the
USA and Canada has also been reported (Fang et al., 2004), but the prevalence of infection by such strains is not
well documented. As both types of PRRSV are globally spread, serological tests should contain antigens of both
types.
Antibodies to the virus can be detected by antibody-binding assays as early as 714 days after infection, and
antibody levels reach maximal titres by 3050 days. Some pigs may become seronegative within 36 months, but
others remain seropositive for much longer. Antibodies to PRRSV have also been detected in meat juice and oral
fluid. Neutralising antibodies develop slowly and do not reach high titres. They can be detected from 3 to 4 weeks
after infection and they can persist for 1 year or more. The use of complement to make the serum virus
neutralisation test more sensitive has been reported (Dea et al., 1996). Extensive research into the duration of
antibody titres after infection has not yet been done, and the results probably depend on the test used. Maternal
antibodies have a half-life of 1214 days, and maternal antibody titre can, in general, be detected until 48 weeks
after birth, depending on the antibody titre of the sow at birth and the test used. In an infected environment, pigs
born from seropositive females can seroconvert actively from the age of 36 weeks.
This chapter describes the IPMA in detail as this test can easily be performed in laboratories where virus isolation
procedures using macrophages have been established, and can be used with virus of both antigenic types. This
assay can also be adapted to the MARC-145 cell line for both the European and American types (Jusa et al.,
1996). An indirect immunofluorescence assay (IFA) using MARC-145 cells can also be performed for PRRSV
serology and is included in the present chapter. Commercial ELISAs with good sensitivity and specificity are
available and have been compared (Drew, 1995a).
a)
Detection of antibodies with the immunoperoxidase monolayer assay

Alveolar macrophages are seeded in the wells of microtitre plates. After attachment, the macrophages are
infected with PRRSV. The object is to infect approximately 3050% of the macrophages in a well so as to be
able to distinguish nonspecific sera. After an incubation period, the macrophages are fixed and used as a
cell substrate for serology. An alternative method is to use MARC 145 cells instead of macrophage cells. On
each plate, 11 sera can be tested in duplicate. Test sera are diluted and incubated on the cell substrate. If
antibodies are present in the test serum, they will bind to the antigen in the cytoplasm of the macrophages.
In the next incubation step, the bound antibodies will be detected by an anti-species horseradish-peroxidase
1117
(HRPO) conjugate. Finally, the cell substrate is incubated with a chromogen/ substrate1 solution. Reading of
the test is done with an inverted microscope.
Seeding macrophages in the microtitre plates
i)
Defrost one vial containing 6 107macrophages/1.5 ml.
ii)
Wash the cells once with 50 ml of PBS and centrifuge the cell suspension for 10 minutes at 300 g
(room temperature).
iii)
Collect the cells in 40 ml RPMI 1640 medium supplemented with 5% FBS, 100 IU (International Units)
penicillin and 100 g streptomycin (growth medium).
iv)
Dispense 100 l of the cell suspension into each well of a microtitre plate (with one vial of cells, four
plates can be seeded at a concentration of 105 cells in each well of the plates).
v)
Incubate the plates for 1824 hours at 37C in a 5% CO2 incubator, under humid conditions.
Alternatively, use HEPES buffer (N-2-hydroxyethylpiperazine, N-2-ethanesulphonic acid) in the
medium.
Infection of cells with PRRSV
i)
Add to each well 50 l of a virus suspension containing 105 TCID50/ml, but leave two wells uninfected
to act as controls.
ii)
Incubate the plates for 1824 hours at 37C in a 5% CO2 incubator.
Fixation of the cells
i)
Discard the growth medium and rinse the plates once in saline.
ii)
Knock the plates gently on a towel to remove excess liquid and then dry them (without lid) for
45 minutes at 37C.
iii)
Freeze the plates (without a lid) for 45 minutes at 20C. (Plates that are not used immediately for
testing must be sealed and stored at 20C.)
iv)
Incubate the cells for 10 minutes at room temperature with cold 4% paraformaldehyde (in PBS).
Alternatively the cells could be fixed in ice-cold absolute ethanol for 45 minutes at 5C or in ice-cold
80% acetone for 45 minutes.
v)
Discard the paraformaldehyde and rinse the plates once in saline.
Preparation of serum dilutions in a dilution plate
i)
Dispense 180 l of 0.5 M NaCl with 4% horse serum and 0.5% Tween 80, pH 7.2 (dilution buffer), to
the wells of rows A and E of the dummy plate(s).
ii)
Dispense 120 l of dilution buffer to all other wells.
iii)
Add 20 l of the test serum or control sera to the wells of rows A and E (= 1/10 dilution), and shake.
iv)
Dilute the sera four-fold by transferring 40 l from rows A and E to rows B and F, and so on to provide
further dilutions of 1/40, 1/160 and 1/640.
Incubation of sera in the plate with fixed macrophages
i)
Transfer 50 l from each of the wells of the dummy plate(s) to the corresponding wells of the plate with
the fixed macrophages. Seal the plate(s) and incubate for 1 hour at 37C.
ii)
Discard the serum dilutions and rinse the plate(s) three times in 0.15 M NaCl + 0.5% Tween 80.
Incubation with conjugate
i)
Dilute the rabbit-anti-swine (or anti-mouse, if staining isolation plate with MAb) HRPO conjugate to a
predetermined dilution in 0.15 M NaCl + 0.5% Tween 80. Add 50 l of the conjugate dilution to all wells
of the plate(s). Seal the plate(s) and incubate for 1 hour at 37C. Rinse the plates three times.
Preparation of chromogen solution

Stock solution of chromogen (3-amino-9-ethyl-carbazole [AEC]): (a) 4 mg AEC; (b) 1 ml N,N-dimethyl-formamide.
Dissolve (a) in (b) and store the AEC stock solution at 4C in the dark.
Preparation of chromogen/substrate solution (prepare shortly before use)
Prepare 0.05 M sodium acetate buffer, pH 5.0, as follows: Dissolve 4.1 g sodium acetate in 1 litre distilled water. Adjust
the pH to 5.00 with 100% acetic acid. Add 1 ml AEC stock solution to 19 ml of 0.05 M sodium acetate buffer. Add 10 l
30% H2O2 for each 20 ml of chromogen/substrate solution. Filter the solution through a 5 m filter.
1118
Staining procedure
i)
Dispense 50 l of the filtered chromogen/substrate (AEC) solution to all wells of the plate(s) (see
footnote 1).
ii)
Incubate the AEC for at least 30 minutes at room temperature.
iii)
Replace the AEC with 50 l of 0.05 M sodium acetate, pH 5.0 (see footnote 1).
If antibodies are present in the test serum, the cytoplasm of approximately 3050% of the cells in a well are
stained deeply red by the chromogen. A negative test serum is recognised by cytoplasm that remains
unstained. A serum that reacts nonspecifically might stain all cells in a well (compared with a positive control
serum). The titre of a serum is expressed as the reciprocal of the highest dilution that stains 50% or more of
the wells. A serum with a titre of <10 is considered to be negative. A serum with a titre of 10 or 40 is
considered to be a weak positive. Often nonspecific staining is detected in these dilutions. A serum with a
titre of 160 is considered to be positive.
b)
Detection of antibodies with the indirect immunofluorescence assay

Although there is no single standard accepted immuofluorescence assay in use at this time, several
protocols have been developed and are used by different laboratories in North America. The IFA can be
performed in microtitre plates or eight-chamber slides using the MARC-145 cell line and a MARC-145 cellline-adapted PRRSV isolate. To prevent cross-reactivity with pestivirus, it is recommended that cells and
FBS, to supplement culture medium, be pestivirus free. After an incubation period, PRRSV-infected cells are
fixed and used as a cell substrate for serology. Serum samples are tested at a single screening dilution of
1/20 and samples are reported as being negative or positive at this dilution. Each porcine serum to be tested
is added to wells or chambers containing PRRSV-infected cells. Antibodies to PRRSV, if present in the
serum, will bind to antigens in the cytoplasm of infected cells. Following this step, an anti-porcine-IgG
conjugated to fluorescein is added, which will bind to the porcine antibodies that have bound to PRRSV
antigens in the infected cells. The results are read using a fluorescence microscope. Microtitre plates may
also be prepared for serum titration purposes (see Section b1 below).
Seeding and infection of MARC-145 cells in microtitre plates
i)
Add 50 l of cell culture medium (e.g. Minimal Essential Medium [MEM] containing 2 mM L-glutamine,
1 mM sodium pyruvate, 100 IU penicillin and 100 g streptomycin) without FBS to each well of columns
2, 4, 6, 8, 10 and 12 of a 96-well plate using a multichannel pipettor.
ii)
Trypsinise confluent MARC-145 cells (grown in culture flasks) to be used for seeding 96-well microtitre
plates and resuspend cells in cell culture medium containing 8% FBS at a concentration of 100,000
125,000 cells/ml. The MARC-145 cells are trypsinised from culture flasks for IFA once a week using
trypsin/EDTA (ethylene diamine tetra-acetic acid) and are seeded in culture flasks at a concentration of
250,000 cells/ml. After 4 days in culture flasks, new cell culture medium containing 2% FBS is added
for 3 additional days.
iii)
Using a multichannel pipettor, add 150 l of the cell suspension to each well of the 96-well plate.
iv)
Dilute PRRSV preparation in MEM without FBS to 102.2 TCID50/50 l and distribute 50 l in each well
of columns 1, 3, 5, 7, 9 and 11.
v)
Incubate the plates for approximately 4872 hours at 37C in a humidified 5% CO2 incubator to obtain
a monolayer with approximately 4050% of the cells infected as determined by indirect immunofluorescence. Alternatively, microtitre plates may first be seeded with MARC-145 cell suspensions (e.g.
concentration of 100,000 cells/ml in medium supplemented with 510% FBS) and incubated for up to
72 hours until they are confluent. Then volumes of 50 l of PRRSV preparations (e.g. 105 TCID50/ml)
are added per well and the plates are incubated for an additional 4872 hours prior to fixation. The use
of organic buffers such as HEPES in medium has been suggested to stabilise the pH when CO2
incubators are not available.
Seeding and infection of MARC-145 cells in eight-chamber glass slides
i)
Add 500 l of a MARC-145 cell suspension (e.g. in MEM supplemented with 10% FBS) at a
concentration of 100,000 cells/ml to each chamber of eight-chamber glass slides.
ii)
Incubate the cells for approximately 4872 hours at 37C in a humidified 5% CO2 incubator until they
are confluent.
1119
iii)
Add to each chamber 50 l of PRRSV suspension containing 105 TCID50/ml and further incubate cells
for approximately 18 hours at 37C in a humidified 5% CO2 incubator . At this time 1520 infected cells
per field of view may be observed by indirect immunofluorescence.
Fixation of the cells
i)
Discard the medium, rinse once with PBS and discard the PBS. For chamber slides, remove the plastic
chamber walls, leaving the gasket intact.
ii)
Add volumes of 150 l cold (4C) acetone (80% in water) to each well of the 96-well plate. Incubate the
plates at 4C for 30 minutes. For chamber slides, acetone (80100%) at room temperature is used to
fix the cells for 1015 minutes at room temperature. Some manufactured brands of acetone will
degrade the chamber slide gasket leaving a film on the slide. It is recommended to check the acetone
before using for routine fixation.
iii)
Discard the acetone and dry the plates and slides at room temperature.
iv)
The plates can then be placed in a plastic bag, sealed and stored at 70C until use. Chamber slides
can be kept similarly in slide cases.
Preparation of serum dilutions
i)
Dilute serum samples to a 1/20 dilution in PBS (0.01 M; pH 7.2) in separate 96-well plates (e.g. add
190 l of PBS using a multichannel pipettor followed by 10 l of the sera to be tested).
ii)
Include as controls reference PRRSV antibody positive and negative sera of known titre.
Incubation of sera with fixed MARC-145 cells
i)
Stored plates are removed from the 70C freezer and when the plates reach room temperature
rehydrate the cells with 150 l PBS for a few minutes. Discard the PBS by inverting the plates and
blotting dry on paper towels. Cells of eight-chamber slides are not rehydrated.
ii)
Add volumes of 50 l of each diluted serum to one well containing the fixed noninfected cells and to
one well containing the fixed infected cells. Add similar volumes for each serum to a single chamber.
iii)
Add volumes of 50 l of the negative control serum and positive control serum dilutions in the same
manner.
iv)
Incubate the plates with their lids on at 37C for 30 minutes in a humid atmosphere. Slides should be
incubated similarly in boxes or slide trays with a cover.
v)
Remove the serum samples and blot the plates dry on paper towels. A total of six washes using 200 l
of PBS are performed. The PBS is added to each well, followed by inversion of the plates to remove
the PBS. After removing serum samples, slides are rinsed in PBS followed by a 10-minute wash.
Incubation with conjugate
i)
Add volumes of 50 l of appropriately diluted (in freshly prepared PBS) rabbit or goat anti-swine IgG
(heavy and light chains) conjugated with FITC (fluorescein isothiocyanate) to each well using a
multichannel pipettor. Similar volumes are added to individual chambers.
ii)
Incubate plates or slides with their lids on at 37C for 30 minutes in a humid atmosphere.
iii)
Remove the conjugate from the plates and blot the plates dry on paper towels. A total of four washes
using PBS are performed as described above. Discard the conjugate from the slides, rinse in PBS,
wash for 10 minutes in PBS and rinse in distilled water. Tap the slides on an absorbent pad to remove
excessive water.
iv)
The plates and the slides are read using a fluorescence microscope.
The presence of a green cytoplasmic fluorescence in infected cells combined with the absence of such a
signal in noninfected cells is indicative of the presence of antibodies to PRRSV in the serum at the dilution
tested. The degree of intensity of fluorescence may vary according to the amount of PRRSV-specific
antibody present in the serum tested.
Absence of specific green fluorescence in both infected and noninfected cells is interpreted as absence of
antibody to PRRSV in that serum at the dilution tested. The test should be repeated if the fluorescence is not
seen with the use of the positive control sera on infected cells or if fluorescence is seen using the negative
1120
control serum on infected cells. No fluorescence should be seen on noninfected cells with any of the control
sera. Any test serum giving suspicious results should be retested at a 1/20 dilution and if results are still
unclear, a new serum sample from the same animal is requested for further testing.
b1) Evaluation by IFA of sera for antibody titres

Microtitre plates and IFA may also be used for serum titration purposes. Up to 16 sera may be titred per 96well microtitre plate.
i)
Seed 96-well microtitre plates with MARC-145 cells and incubate at 37C in a humidified 5% CO2
incubator until they are confluent.
ii)
Inoculate all wells with the PRRSV preparation except the wells of columns 1, 6 and 11, and incubate
the plates at 37C in a humidified 5% CO2 incubator for 4872 hours.
iii)
Discard culture medium and rinse the monolayers once with PBS (0.01 M, pH 7.2). Fix the monolayers
with cold acetone (80% aqueous solution) for 10 minutes at ambient temperature. Discard the acetone,
air-dry the plates and keep the plates with lids at 20C for short-term storage or 70C for long-term
storage, until use.
iv)
Serially dilute sera including a PRRSV-positive control serum using a four-fold dilution in PBS,
beginning at 1/16 or 1/20. Dilute a negative control serum at 1/16 or 1/20 dilution. Dispense 50 l of
each dilution (1/16, 1/64, 1/256, 1/1024 or 1/20, 1/80, 1/320, 1/1280) in wells containing viral antigen of
columns 2, 3, 4, 5 or 7, 8, 9, 10. For each serum, also dispense 50 l of dilution 1/16 or 1/20 in control
wells of columns 1 and 6. Similarly dispense dilutions of positive and negative control sera in wells of
columns 11 and 12.
v)
Incubate the plates at 37C for 30 minutes in a humid chamber. Discard the sera and rinse the plates
three times using PBS.
vi)
Add 50 l of appropriately diluted anti-swine IgG conjugated with FITC and incubate plates at 37C for
30 minutes in a humid chamber. Discard conjugate, rinse plates several times and tap the plates on
absorbent material to remove excessive liquid.
Following examination with a fluorescence microscope, the titre of a serum is recorded as the reciprocal of
the highest serum dilution in which typical cytoplasmic fluorescence is observed. For paired serum samples,
a four-fold increase in titre with a 2-week interval is indicative of active infection in an individual animal. No
specific fluorescence should be observed with test sera or positive and negative control sera on noninfected
control cells. No fluorescence should be seen on infected cells with negative control serum. Specific
fluorescence should be observed on infected cells with positive control serum at appropriate dilutions. The
IFA end-point may vary among laboratories. Test results may also vary depending on the PRRSV isolate
used in the test because of antigenic diversity.
c)
Detection of antibodies with the enzyme-linked immunosorbent assay

Several laboratories have developed ELISAs (indirect or blocking) for serological testing (Albina et al. 1992;
Cho et al., 1997; Denac et al., 1997; Houben et al., 1995; Sorensen et al., 1998). A double-blocking ELISA
format that can distinguish between serological reactions to the European and the American antigenic type
has been described (Sorensen et al., 1998). ELISA kits are available commercially to determine the
serological status of swine towards PRRSV. These kits use as antigens either the European or the American
PRRSV types separately or as combined antigens. Their main advantage is the rapid handling of a large
number of samples. Commercial ELISAs are available that use recombinant proteins of both PRRSV types
as antigens.

1.
Background
a)

Modified-live (MLV) PRRSV vaccines are commercially available in many countries for the control of the
reproductive and/or respiratory forms of PRRS. In the USA and Europe, a killed virus vaccine is licensed as
an aid in the reduction of abortions and weak piglets caused by the reproductive form of PRRS. All PRRS
vaccines currently licensed in the USA contain the antigenic type 2. In Europe, three MLV vaccines are
licensed and available commercially: one is based on antigenic type 2 and two others are type 1. It is
1121
assumed the most benefit from vaccination occurs when the vaccine virus is more closely related
antigenically to the field virus (Larochelle & Magar, 1997b; Scortti et al., 2006). Although vaccination of pigs
does not prevent PRRSV infection, it may be helpful in herds experiencing problems with PRRS or herds at
high risk of PRRSV infection. MLV vaccines are not intended to be used in naive herds, pregnant sows or
gilts or boars of breeding age. MLV vaccines are intended to be used in sows and gilts 36 weeks prior to
breeding and in piglets from 3 weeks of age or older as an aid in the reduction of diseases caused by PRRS.
Vaccine virus can persist in boars and be disseminated through semen (Christopher-Hennings et al., 1997).
MLV vaccine virus may be shed and transmitted to nonvaccinated contact pigs (Torrison et al., 1996).
vaccine production. The guidelines given here and in chapter 1.1.6 are intended to be general in nature and
may be supplemented by national and regional requirements.
2.
a)

i)
The isolate of PRRSV used for vaccine production must be accompanied by a history describing its
origin and passage history. The master seed virus (MSV) must be safe in swine at the intended age of
vaccination and provide protection against challenge. Isolates for a MLV vaccine must be shown not to
revert to virulence after passage in host animals.
ii)

The MSV should be free from bacteria, fungi and mycoplasma. The MSV must be tested for and free
from extraneous viruses, including transmissible gastroenteritis virus, porcine adenovirus, porcine
circovirus type 1 and 2, porcine haemagglutinating encephalitis virus, porcine parvovirus, bovine viral
diarrhoea virus, reovirus, and rabies virus by the fluorescent antibody technique. The MSV must be
free from extraneous virus by CPE and haemadsorption on the Vero cell line and an embryonic swine
cell type.
b)
i)
Procedure
The PRRSV is propagated in a continuous African green monkey kidney cell line, such as MA-104 or
Vero. Viral propagation should not exceed five passages from the master seed virus (MSV) unless
further passages prove to provide protection in swine
The African green monkey kidney cell line is seeded into suitable vessels. MEM supplemented with
FBS is used as the medium for production. Cell cultures are inoculated directly with PRRS working
virus stock, which is generally from 1 to 4 passages from the MSV. Inoculated cultures are incubated
for 18 days before harvesting the culture medium. During incubation, the cultures are observed daily
for CPE and bacterial contamination.
Killed virus vaccines are chemically inactivated with either formalin or binary ethylenimine and mixed
with a suitable adjuvant. MLV vaccines are generally mixed with a stabiliser before bottling and
lyophilisation. If formalin is used as an inactivant, the final product should be tested for residual
formaldehyde concentration and should not exceed 0.74 g/litre.
ii)

The FBS must be free from pestivirus or antibodies to pestivirus and free from bovine spongiform
encephalopathy risk.
iii)
In-process control
Production lots of PRRSV must be titrated in tissue culture for standardisation of the product. Lowtitred lots may be concentrated or blended with higher-titred lots to achieve the correct titre.
iv)

Final container samples are tested for purity, safety and potency. MLV vials are also tested for the
maximum allowable moisture content.
Samples are examined for bacterial, fungal and pestivirus contamination. To test for bacteria in a MLV
vaccine, ten vessels, each containing 120 ml of soybean casein digest medium, are inoculated with
0.2 ml from ten final-container samples. The ten vessels are incubated at 3035C for 14 days and
observed for bacterial growth. To test for fungi, ten vessels, each containing 40 ml of soybean casein
digest medium, are inoculated with 0.2 ml from ten final-container samples. The vessels are incubated
1122
at 2025C for 14 days and observed for fungal growth. Killed vaccines require 1.0 ml from ten final
container samples be inoculated into the appropriate ten vessels of media.
Safety
Safety tests can be conducted in a combination of guinea-pigs, mice or pigs.
Batch potency
Final container samples of an MLV vaccine are titrated (log10) in microtitre plates for determination of
the titre.
Test procedure
i)
Prepare tenfold dilutions from 101 through 105 by using 0.2 ml of rehydrated test vaccine and
1.8 ml of MEM. An internal positive control PRRSV should be titrated in the appropriate range.
ii)
Inoculate 0.1 ml/well from each dilution into five wells of a 96-well plate containing African green
monkey kidney monolayers.
iii)
Incubate the plate at 37C in a CO2 atmosphere for 57 days.
iv)
Read the plates microscopically for CPE. The internal positive control PRRSV should give a titre
within 0.3 log10 TCID50 from its predetermined mean.
v)
Determine the TCID50/dose by the SpearmanKrber method. The release titre must be at least
1.2 logs higher than the titre used in the immunogenicity trial. The 1.2 logs include 0.5 logs for
stability throughout the shelf life of the product and 0.7 logs for potency test variability.
Killed virus vaccines may use host animal or laboratory animal vaccination/serology tests or
vaccination/challenge tests to determine potency of the final product. Parallel-line assays using ELISA
antigen-quantifying techniques to compare a standard with the final product are acceptable in
determining the relative potency of a product. The standard should be shown to be protective in the
host animal.
c)

i)
Safety requirements
Field trial studies should be conducted to determine the safety of the vaccine. Nonvaccinated sentinel
pigs should be included at each site for monitoring the shed of the attenuated virus.
MSV must be shown not to revert to virulence after several passages in host animals, although the
definition of virulence with such a virus is difficult. Attenuated PRRSV isolates are known to cause
viraemia and will transmit to susceptible animals. The MSV should be shown to be avirulent in weaned
piglets and pregnant animals by five serial passages (up to ten passages depending on country) of the
MSV through susceptible swine using the most natural route of infection.
Not applicable
ii)
In an immunogenicity trial, the MSV at the highest passage level intended for production must protect
susceptible swine against a virulent, unrelated challenge strain. For the respiratory form, 3-week-old
piglets are vaccinated with the highest passage level of MSV. The piglets are challenged with a virulent
isolate of PRRSV 216 weeks later to determine protection from respiratory clinical signs of PRRS. To
determine protection from the losses caused by the reproductive form of PRRS, vaccinated animals
are challenged at approximately 85 days gestation. A prevented fraction, the proportion of potential
PRRS disease occurrence reduced due to vaccination,is calculated to determine if there is acceptable
protection, based on the proposed label claims, in the vaccinates from the clinical signs of reproductive
disease, including fetal mummification, stillborn piglets and/or weak piglets, when compared with the
controls.
Duration of immunity studies are conducted before the vaccine receives final approval. For the
respiratory form of PRRS, duration should be shown up to the market age in pigs. Duration of immunity
for the reproductive form should be shown through weaning of the piglets.
1123

Not applicable
iii)
Stability
All vaccines are initially given a shelf life of 24 months before expiry. Real-time stability studies are
then conducted to confirm the appropriateness of the expiry date.
Multiple batches of MLV vaccines should be re-titrated periodically throughout the shelf-life to
determine vaccine variability. The release value should be adjusted if the titres are insufficient or highly
variable.
Killed vaccines using in-vivo potency tests should be retested at expiry to demonstrate stability.
Parallel-line assays using ELISA antigen-quantifying techniques should demonstrate the stability of the
standard.
3.
a)

None.
b)

None.
REFERENCES
ALBINA E., LEFORBAN Y., BARON T., PLANA DURAN J. & VANNIER P. (1992). An enzyme linked immunosorbent assay
(ELISA) for the detection of antibodies to the porcine reproductive and respiratory syndrome (PRRS) virus. Ann.
Rech. Vet., 23, 167176.
BENFIELD D.A., COLLINS J.E., DEE S.A., HALLBUR P.G., JOO H.S., LAGER K.M., MENGELING W.L., MURTAUGH M.P.,
ROSSOW K.D., STEVENSON G.W. & ZIMMERMAN J.J. (1999). Porcine reproductive and respiratory syndrome. In:
Diseases of Swine, Eight Edition, Straw B.E., DAllaire S., Mengeling W.L. & Taylor D.J., eds. Iowa State
University Press. Ames, Iowa, USA, 201232.
BOTNER A., STRANDBYGAARD B., SORENSEN K.J., HAVE P., MADSEN K.G., MADSEN E.S. & ALEXANDERSEN S. (1997).
Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine. Vet.
Rec., 141, 497499.
BRINTON M.A., GODENY E.K., HORZINEK M.C., MEULENBERG J.J.M., MURTAUGH M.P., PLAGEMANN P.G.W. & SNIJDER
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*
* *
NB: There are OIE Reference Laboratories for Porcine reproductive and respiratory syndrome
diagnostic tests, reagents and vaccines for porcine reproductive and respiratory syndrome
1126
CHAPTER 2.8.8.
SWINE INFLUENZA
SUMMARY
Swine influenza is a highly contagious viral infection of pigs. Swine influenza virus (SIV) infections
cause respiratory disease characterised by coughing, sneezing, nasal discharge, elevated rectal
temperatures, lethargy, breathing difficulty, and depressed appetite. In some instances, SIV
infections are associated with reproductive disorders such as abortion. Clinical signs and nasal
shedding of virus can occur within 24 hours of infection. Morbidity rates can reach 100% with SIV
infections, while mortality rates are generally low. Secondary bacterial infections can exacerbate
the clinical signs following infection with SIV. Transmission is through contact with SIV-containing
secretions such as nasal discharges and aerosols created by coughing or sneezing.
Identification of the agent: Samples for virus identification should be collected within 2472 hours
after development of clinical signs. The animal of choice is an untreated, acutely ill pig with an
elevated rectal temperature. Virus can readily be detected in lung tissue and nasal swabs. Virus
isolation can be conducted in embryonated chicken eggs and on continuous cell lines. Isolated
viruses can be subtyped using the haemagglutination inhibition (HI) and the neuraminidase
inhibition
tests,
or
by
reverse
transcription-polymerase
chain
reaction
assays.
Immunohistochemistry can be conducted on formalin-fixed tissue and a fluorescent antibody test
can be conducted on fresh tissue. Enzyme-linked immunosorbent assays (ELISA) are commercially
available for detection of type A influenza viruses.
Serological tests: The primary serological test for detection of SIV antibodies is the HI test
conducted on paired sera. The HI test is subtype specific. The sera are generally collected 10
21 days apart. A four-fold or greater increase in titre between the first and second sample is
suggestive of a recent SIV infection. Additional serological tests that have been described are the
agar gel immunodiffusion test, indirect fluorescent antibody test, virus neutralisation, and ELISA.
Requirements for vaccines: Inactivated, adjuvanted SIV vaccines are commercially available.
Vaccines may be in the form of a single SIV subtype or may contain multiple SIV subtypes.
Vaccines should reflect the current antigenic profile of field viruses, containing subtypes and strains
that are changed as needed to assure protection. The finished vaccine must be shown to be pure,
safe, potent, and efficacious.
A. INTRODUCTION
Swine influenza is a highly contagious viral infection of pigs that can have significant economic impact on an
affected herd (Olsen et al., 2005). The swine influenza virus (SIV) is a type A orthomyxovirus with a segmented
RNA genome. The type A swine influenza viruses are further subdivided based on their haemagglutinin and
neuraminidase proteins. Subtypes of SIV that are most frequently identified in pigs include classical and avian
H1N1, human (hu) H1N1 and H1N2, reassortant (r) H3N2, and rH1N2 (Choi et al., 2004; Gramer, 2007; Gregory
et al., 2001; 2003; Marozin et al., 2002; Olsen et al., 2002; Schrader & Sss, 2004). Other subtypes that have
been identified in pigs include rH1N7, rH3N1, H2N3, avian (av) H4N6, avH3N3, and avH9N2 (Brown et al., 1997;
Karasin et al., 2000a; Karasin et al., 2004; Ma et al., 2007; Olsen et al., 2005; Peiris et al., 2001). The H1N1,
H1N2 and H3N2 viruses found in Europe are antigenically and genetically different from those found in America
(Brown et al., 1998; Castrucci et al., 1993; Done & Brown, 1997; Karasin et al., 2000a; 2000b; 2002; 2004; Noble
et al., 1993; Olsen, 2002; Sheerar et al., 1989; Webby et al., 2000; 2004; Zhou et al., 1999). Pigs have receptors
in their respiratory tract that will bind swine, human, and avian influenza viruses. Consequently, pigs have been
called mixing vessels for the development of new influenza viruses when swine, avian, and/or human influenza
viruses undergo genetic reassortment in pigs. SIV infections are described as causing respiratory disease
characterised by coughing, sneezing, nasal discharge, elevated rectal temperatures, lethargy, breathing difficulty
1127
Chapter 2.8.8. Swine influenza
and depressed appetite. Other agents that may cause respiratory disease in pigs include porcine reproductive
and respiratory syndrome virus, Aujeszkys disease (pseudorabies) virus, porcine respiratory coronavirus,
Actinobacillus pleuro-pneumoniae, Mycoplasma hyopneumoniae and other bacterial agents. However, most of
these have other signs that do not mimic swine influenza. Actinobacillus pleuropneumoniae, in the acute form of
the infection, has clinical signs most similar to swine influenza, such as dyspnoea, tachypnoea, abdominal
breathing, coughing, fever, depression and anorexia. Clinical signs and nasal shedding of SIV can occur within
24 hours of infection, and shedding typically ceases by day 710 after infection. Two forms of the disease occur in
swine, epidemic or endemic. In the epidemic form, the virus quickly moves through all phases of a swine unit with
rapid recovery, provided there are not complicating factors such as secondary bacterial infections. In the endemic
form, clinical signs may be less obvious and not all pigs may demonstrate traditional clinical signs of infection.
Morbidity rates can reach 100% with SIV infections, while mortality rates are generally low. The primary economic
impact is related to retarded weight gain resulting in an increase in the number of days to reach market weight.
Transmission is through contact with SIV containing secretions such as nasal discharges and aerosols created by
coughing or sneezing. Human infections with SIV can occur and a limited number of deaths have been reported
(Myers et al., 2007; Olsen et al., 2002). Precautions should be taken to prevent human infection as described in
Chapter 1.1.3 Biosafety and biosecurity in the veterinary microbiological laboratory and animal facilities.
Conversely, human influenza viruses can occasionally be transmitted from animal caretakers to pigs. Similarly,
influenza virus can also be transmitted occasionally from poultry to pigs as well as from pigs to poultry. In the
spring of 2009 a newly identified H1N1 virus was detected in people in the Western Hemisphere. This novel virus
was composed entirely of swine genes the origins of which were of swine, avian, and human lineages. The matrix
and neuraminidase were from European H1N1 swine influenza viruses of avian lineage and the remaining genes
were from North American swine influenza viruses of swine, avian, and human lineage. The virus spread rapidly
throughout the world through human-to-human transmission. Swine cases in both Northern and Southern
Hemispheres have occurred since the virus was first recognised in humans (http://www.oie.int/eng/en_index.htm).
Current information on recent research can be found at a number of websites, including, but not limited to
http://www.defra.gov.uk/vla/science/sci_si.htm and http://www.ars.usda.gov/2009H1N1/.
1.
Because SIV is a potential human pathogen, all work with infectious tissues, swabs, embryonated eggs, and cell
cultures should be done in a class II biological safety cabinet. Additional safety precautions (personal protective
equipment) may be considered such as the use of respirators during laboratory work.
a)
Culture
Sample processing
Lung tissue can be processed for virus isolation in a variety of ways, for example with a mortar and pestle,
stomacher, homogeniser, or mincing with a scalpel blade or scissors. Processing of the tissue is done in cell
culture medium with antibiotic supplement (e.g. 10 working strength), at a final concentration of 1020%
weight to volume. Nasal swabs should be collected in cell culture medium or phosphate buffered saline
(PBS), supplemented with antibiotics and bovine serum albumin (5 mg/ml). Fetal bovine serum should not
be included. Samples should ideally be shipped to a diagnostic laboratory overnight on wet ice, not frozen
(see http://offlu.net for guidance on sample collection and sample shipment). Upon receipt at the laboratory,
the nasal swabs are vigorously agitated by hand or on a vortex mixer. The nasal swab and lung materials
are centrifuged at 15001900 g for 1530 minutes at 4C. The supernatant is collected and maintained at
4C until inoculated. If supernatant is to be held for longer than 24 hours before inoculation, it should be
stored at 70C. Lung supernatant is inoculated without further dilution. Nasal swab supernatant can also be
inoculated without dilution or diluted 1/3 in cell culture medium. Antibiotics are added to the cell culture
medium used for processing and/or the supernatant can be filtered to reduce bacterial contamination, but
this may decrease virus titre. As an alternative, the virus preparation may be treated with antibiotics such as
gentamicin (100 g/ml) or penicillin (10,000 units/ml: streptomycin (10,000 units/ml) and 2% fungizone
(250 mg/ml) for 3060 minutes at 4C prior to inoculating the embryos or cell culture.
Cell culture virus isolation
i)
Virus isolation can be conducted in cell lines and primary cells susceptible to SIV infection. Madin
Darby canine kidney (MDCK) is the preferred cell line, but primary swine kidney, swine testicle, swine
lung, or swine tracheal cells can be used.
ii)
Wash confluent cell monolayers (4872 hours post-seeding) three times with cell culture medium
containing a final concentration of 1 g/ml of TPCK1-treated trypsin; however, the concentration will
TPCK: tosylphenylalanylchloromethane
1128
depend on the type of trypsin and the cells used (0.310 g/ml may be used). The cell culture medium
can be supplemented with antibiotics, but is not supplemented with fetal bovine serum.
iii)
Inoculate cell cultures with an appropriate amount of tissue suspension or swab supernatant. Note: The
volume of inoculum will vary with the size of the cell culture container. In general, 100200 l are
inoculated in each well of a 24-well culture plate, 1 ml in each Leighton tube, and 12 ml into a 25 cm2
flask.
iv)
Incubate inoculated cell cultures for 12 hours at 37C with occasional rocking. When using cell culture
containers that are open to the environment, such as culture plates, incubation should be done in a
humidified incubator with 5% CO2.
v)
Remove the inoculum and wash the cell monolayer three times with the cell culture medium containing
trypsin.
vi)
Add an appropriate volume of the cell culture maintenance medium (as noted in ii above) to all
containers and incubate at 37C for 57 days with periodic examination for cytopathic effect (CPE). If
CPE is not observed at the end of the incubation period, the cell culture container can be frozen at
70C, thawed, and blind passaged as described above (step iii). If CPE is observed, an aliquot of the
cell culture medium can be tested for haemagglutinating viruses or by reverse transcription-polymerase
chain reaction (RT-PCR) for conserved influenza virus genes such as nucleoprotein or matrix, and can
be collected and used as inoculum for confirmation by the fluorescent antibody technique (see Section
B.1.e below). Cover-slips (Leighton tube, 24-well cell culture plate) or chamber slides with MDCK (or
other appropriate cell) monolayer can be inoculated for this purpose. The isolation procedure is as
described above (step iii). In some instances, it may be necessary to make tenfold dilutions of the cell
culture virus in order to have appropriate CPE on the cover-slip. Influenza subtypes can be determined
by the haemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests, or by RT-PCR with
primers validated for sensitive and specific amplification of individual HA and NA genes (Hoffman et al.,
2001; Phipps et al., 2004).
Egg inoculation (Senne, 1998)
i)
Use 1011-day-old embryonated chicken eggs (Senne, 1998).
ii)
Inoculate 0.10.3 ml of inoculum into the allantoic cavity and amniotic sac; many laboratories only
inoculate via the allantoic route with similar sensitivity. Generally, 34 eggs are inoculated per sample.
iii)
Incubate eggs at 3537C for 34 days and candle daily. Eggs with embryos that have died within
24 hours of inoculation are discarded.
iv)
Refrigerate eggs with embryos that have died later than 24 hours after inoculation. Harvest amniotic
and allantoic fluids from eggs with dead embryos and from eggs with viable embryos at the end of the
incubation period. All egg materials should be considered to be potentially infectious and should be
treated accordingly to prevent SIV exposure to the laboratory worker.
v)
Centrifuge fluids at 15001900 g for 1020 minutes at 4C. Transfer the supernatant to another tube
for testing.
vi)
Fluids are evaluated for the presence of SIV with the haemagglutination (HA) test (see below).
vii)
Repass fluids negative for haemagglutinating activity (negative for SIV) in eggs or on cell lines as
described above. Isolation may be improved by making tenfold dilutions of the fluid in cell culture
medium. Antibiotics may be added to the cell culture fluid.
Haemagglutination test
i)
Prepare a 0.5% erythrocyte suspension from male turkey or chicken blood. Dispense whole blood into
a tube and add PBS. For example, 1020 ml whole blood in a 50 ml centrifuge tube to which PBS is
added to fill the tube. Gently invert the tube several times to wash the erythrocytes. Centrifuge at 800 g
for 10 minutes in a refrigerated centrifuge. Aspirate PBS and buffy coat (white blood cell layer) from the
tube. Refill the tube with fresh PBS and resuspend erythrocytes thoroughly. Repeat the washing and
centrifugation cycle two additional times. Once washing is complete, add sufficient erythrocytes to PBS
to make a 0.5% solution. Certain virus strains agglutinate turkey rather than chicken erythrocytes to
greater or lesser degrees. Therefore, it may be necessary to choose the species of erythrocytes based
on the strains circulating in a given area. Washed erythrocytes and 0.5% suspensions of erythrocytes
can be stored at 4C for up to 1 week. Discard if haemolysis is observed.
ii)
Dispense 50 l PBS in a row of 812 wells on a 96-well V- or U-bottom microtitre plate for each
unknown virus. U-bottom plates are generally preferred over V-bottom plates. One additional row of
wells should be included for a positive control.
iii)
Add 50 l of undiluted isolate to the first well of each corresponding row.
1129
b)
iv)
Serially dilute the isolate with a micropipette set to deliver 50 l. The resulting dilutions will range from
1/2 (well 1) to 1/2048 (well 11). Well 12 contains PBS only and serves as a cell control.
v)
Add 50 l of 0.5% erythrocyte suspension to each well and agitate the plate to mix thoroughly. Note:
keep erythrocytes thoroughly suspended during the dispensing process.
vi)
Cover the plate with sealing tape and incubate at room temperature until a distinct button has formed
(3060 minutes) in the control well.
vii)
Wells with complete haemagglutination (positive HA, SIV present) will have erythrocytes spread
throughout the well in a mat type appearance. Wells with a distinct button of erythrocytes at the
bottom of the well are negative for haemagglutinating activity (negative for SIV). Incomplete HA activity
is demonstrated by partial buttons characterised by fuzzy margins or donut-like appearance. When
interpretation between negative and incomplete inhibition is doubtful, tilt the microtitre plate to about a
45-degree angle for 2030 seconds and look for streaming, which produces a tear-drop appearance
and translucency around the cells in wells with negative hemagglutination. Wells with partial inhibition
will not produce a tear drop.
Typing SIV isolates
i)
Dilute reference HA antigens (H1, H3, etc.) to a concentration of 8 HA units (HAU) per 50 l
(4 HAU/25 l) in 0.01 M PBS, pH 7.27.4.
ii)
Standardise unknown influenza A viruses to contain 8 HAU in 50 l.
iii)
Conduct a back titration (HA test) for all unknown isolates and the H subtype antigens to assure that
the correct HAUs are present. The back titration is performed as described in the HA procedures
except that six well dilutions are used instead of eleven.
iv)
Treat each reference serum (specific for an individual HA subtype) with RDE (receptor-destroying
enzyme); add 50 l serum to 200 l RDE (1/10 dilution in calcium saline solution equalling 100 units
per ml). Incubate overnight (1218 hours) in a 37C water bath. Add 150 l 2.5% sodium citrate
solution and heat inactivate at 56C for 30 minutes. Combine 200 l treated sample and 25 l PBS.
Note: RDE treatment is recommended as it will reduce nonspecific reactions and will enhance the
identification of H1N2 and H3N2 isolates.
v)
Remove natural serum agglutinins from the sera by treating diluted serum with 0.1 ml packed, washed
erythrocytes per 1 ml diluted serum. Incubate for 30 minutes at room temperature with occasional
mixing to keep the erythrocytes suspended. Centrifuge the treated serum at 800 g for 10 minutes and
then retain the serum.
vi)
Dispense 25 l of standardised antigen (unknown isolate or positive control antigen) into three wells of
a 96-well V- or U-bottom microtitre plate. Add 50 l of PBS to several wells to serve as an erythrocyte
cell control. Note: 25 l of PBS can be used in place of the 25 l of standardised antigen.
vii)
Add 25 l of the appropriate reference serum to the first well of the H subtype being evaluated. Serially
dilute the antiserum in 25 l volumes in the antigen wells with a pipette set to deliver 25 l. Repeat this
procedure for each H subtype being evaluated. Note: If 25 l of PBS was used in place of the 25 l of
standardised antigen in step vi, add 25 l of standardised antigen to each well containing the reference
serum.
viii) Cover plate(s) and incubate at room temperature for 1030 minutes.
ix)
Add 50 l 0.5% erythrocyte suspension to each well and shake/agitate the plate(s) to mix thoroughly.
Keep the erythrocytes thoroughly suspended during the dispensing process.
x)
Cover the plate(s) with sealing tape and incubate at room temperature until a distinct button has
formed in the positive control wells (usually 3060 minutes). Observe the plates after about 20 minutes
incubation for evidence of haemagglutination as some isolates may begin to elute (detach from
erythrocytes) in 30 minutes.
xi)
Read test results as described above for the HA test. A sample is considered positive for a specific H
subtype if haemagglutination is inhibited. The test is considered valid if the positive reference antigen
and its homologous antiserum demonstrate the expected HI titre and the back titration of each antigen
(unknown and positive control) is 8 HAUs. If these conditions are not met, the test should be repeated.
xii)
If erythrocytes in the cell control wells do not settle into a well-defined button, check the following as
possible causes: incorrect formulation of PBS, excessive evaporation from the plates, erythrocytes too
old, or incorrect concentration of erythrocytes.
Neuraminidase inhibition test
Subtype identification based on the NI test is beyond the scope of many laboratories. Reference laboratories
should be consulted for N typing of isolates.
1130
c)
d)

i)
This technique can be used for tissue sections, cover-slips/slides, or 96-well plates of infected cell
monolayers (Vincent et al., 1997). Positive and negative controls should be included with all staining
procedures.
ii)
Inoculated cells are incubated for an appropriate length of time to allow 1025% of the cells to become
productively infected with virus. Rinse the cover-slip or slide once in PBS, place in 100% acetone for
510 minutes and air-dry. Acetone should be used in a vented hood.
iii)
Prepare frozen tissue sections on glass slides. Fix the glass slides in acetone for 510 minutes and airdry.
iv)
Apply conjugate (fluorescein-labelled swine influenza antibody) and incubate in a humid chamber at
37C for 30 minutes. Preferably the conjugate contains Evans blue for counter staining.
v)
Rinse in PBS, pH 7.2, soak for 510 minutes in fresh PBS, rinse in distilled water, and air-dry.
vi)
Place cover-slips on glass slides, cell side down, with mounting fluid. Remove the rubber gasket from
chamber slides and add mounting fluid followed by a glass cover-slip. Mounting fluid followed by a
glass cover-slip is also placed over tissue sections on the slide. If 96-well plates are used, mounting
medium and cover-slips are not required.
vii)
Observe stained slides in a darkened room with the use of an ultraviolet microscope. Cells infected
with SIV are identified by the presence of bright apple-green fluorescence. It is recommended that the
person examining the slides receive training in reading fluorescein-labelled slides as they can be
difficult to interpret. It is also important to use an antibody that recognises all possible viruses
circulating in the area (e.g. a pan-anti-influenza A nucleoprotein antibody).
Immunohistochemistry (Vincent et al., 1997)

i)
Slice formalin-fixed, paraffin-embedded lung in 4-m thick sections and place on poly-L-lysine-coated
slides. Positive and negative control tissues should be included with all tests.
ii)
Heat slides at 60C for 15 minutes, deparaffinise, and rehydrate through immersions in decreasing
concentrations of ethanol and then in distilled water.
iii)
Treat samples with 3% hydrogen peroxide for 10 minutes and rinse twice in distilled water.
iv)
Digest samples with 0.05% protease for 2 minutes and rinse twice for 2 minutes in 0.1 M Tris/PBS
buffer, pH 7.2, at room temperature.
v)
Apply primary mouse anti-SIV monoclonal antibody (directed against the viral nucleoprotein) to each
slide and incubate at room temperature for 1 hour or overnight at 4C. Rinse slides with Tris/PBS
buffer.
vi)
Apply secondary antibody (biotinylated goat anti-mouse antibody) for 10 minutes at room temperature.
Rinse with Tris/PBS buffer.
vii)
Apply tertiary antibody (peroxidase-conjugated streptavidin) for 10 minutes at room temperature. Rinse
with Tris/PBS buffer.
viii) Apply diaminobenzidine tetrahydrochloride solution for 5 minutes at room temperature. Rinse twice in
distilled water.
e)
ix)
Counterstain slides in Gills haematoxylin for 1030 seconds, wash in water for 2 minutes, dehydrate,
clear, and add cover-slips.
x)
SIV-infected tissues are identified by the presence of brown staining in bronchiolar epithelium and
pneumocytes.
Antigen-capture enzyme-linked immunosorbent assays

Type A antigen-capture enzyme-linked immunosorbent assays (ELISAs) are commercially available for
detection of human and animal influenza viruses. These types of assays have been used for detection of
SIV in lung tissue and nasal swabs (Swenson et al., 2001). The assays are generally available through
human health and animal health care companies.
f)

RT-PCR tests have been developed for the diagnosis of swine influenza and for hemagglutinin and
neuraminidase typing (Landolt et al., 2004). With the identification of the novel (pandemic) H1N1 in 2009,
molecular assays based on an avian influenza matrix real-time PCR (Spackman et al., 2002) were adapted
for use in swine. Modifications to the assay vary by country and a swine influenza reference laboratory
should be consulted (http://offlu.net) for the most suitable matrix PCR assay. Additional real-time PCR
1131
assays that can differentiate the novel H1N1 from conventional H1N1 based on differentiable matrix realtime or N1 real-time assays have also been developed for use in North America. In many instances it is
necessary to conduct partial or complete gene sequencing of one or more of the SIV genes (i.e. matrix,
neuraminidase, haemagglutinin) to ascertain the subtype of detected virus. Population-wide validation data
for these tests are not currently available.
2.
Serological tests
The primary serological test for detection of SIV antibodies is the HI test and it is subtype specific. It should be
conducted on paired sera collected 1021 days apart. A four-fold or greater increase in titre between the first and
second sample is suggestive of a recent SIV infection. Additional serological tests that have been described but
not commonly used are the virus neutralisation, agar gel immunodiffusion test, and indirect fluorescent antibody
test. ELISA technology for detection of SIV antibodies has been described in the literature and commercial kits
have been marketed (Lee et al., 1993).

i)
Dilute reference HA antigens (H1, H3, etc.) to a concentration of 48 HAU/25 l in 0.01 M PBS, pH 7.2.
ii)
H1N1 test: Heat inactivated sera for 30 minutes at 56C. Dilute 1/10 in PBS. Add 0.1 ml packed,
washed erythrocytes to 1 ml of heat-inactivated, diluted serum and mix. Incubate at room temperature
for 30 minutes with periodic shaking every 1015 minutes. Centrifuge at 800 g for 10 minutes at 4C.
Note: Sera can be treated with RDE and erythrocytes as described below in step iii as an alternative to
heat inactivation and treating with packed erythrocytes.
iii)
H1N2 and H3N2 test: Add 50 l serum to 200 l RDE (1/10 dilution in calcium saline solution equalling
100 units per ml). Incubate overnight (1218 hours) in a 37C water bath. Add 150 l 2.5% sodium
citrate solution and heat inactivate at 56C for 30 minutes. Combine 200 l treated sample and 25 l
PBS. Add 50 l of 50% erythrocytes. Shake and incubate for 30 minutes at room temperature or
overnight at 4C. Centrifuge at 800 g for 10 minutes at 4C.
iv)
Dispense 50 l treated serum into two wells of a V- or U-bottom 96-well plate. Dispense 25 l of
treated serum into two wells to be used as a serum control. Positive and negative control sera are
treated in the same way as the unknown sera.
v)
Dispense 25 l PBS in the serum control wells and all empty wells except two wells identified as the
cell control wells. Add 50 l PBS in the cell control wells.
vi)
Make serial twofold dilutions of the serum in 25 l volumes in the plate and then add 25 l of
appropriate antigen to all test wells except the serum control wells and the cell control wells.
vii)
Incubate covered plates at room temperature for 3060 minutes.
viii) Add 50 l of 0.5% erythrocyte suspension to each well, shake, and incubate at room temperature for
2030 minutes until a distinct button forms at the bottom of the cell control wells. Keep erythrocytes
thoroughly suspended during the dispensing process.
b)
ix)
Conduct a HA test using the HI test antigens prior to and simultaneously to conducting the HI test to
verify that antigen concentrations are appropriate.
x)
For the test to be valid, there should be no haemagglutination in the serum control well, no inhibition of
haemagglutination with the negative serum, the positive serum should have its anticipated HI titre and
the HA back titration should indicate 48 HAU per 25 l.
Enzyme-linked immunosorbent assay (Lee et al., 1993)

ELISA technology for detection of SIV antibodies has been described in the literature and ELISAs are
available as commercially produced kits.
C. REQUIREMENT FOR VACCINES

1.
Background
1132
a)

Swine influenza can cause significant economic impact for producers because of reduced feed intake during
illness resulting in decreased weight gain, increased days to market, and decreased feed efficiency. Where
vaccination is practiced, vaccine is used to reduce the economic impact of disease by reducing the severity
and duration of clinical signs. In addition, vaccines can reduce the level of viral shedding and the duration of
viral shedding. Decreasing the amount of virus shed and duration of shedding can be important in
minimising the risk of exposure for swine caretakers.
2.
a)

i)
Strains used in vaccine production should be antigenically relevant to SIV strains circulating in the field.
Haemagglutination inhibition and neutralisation tests demonstrating cross-reactivity between antisera
from animals vaccinated with the candidate vaccine strain and current field isolates can be used for the
selection.
Identity of the seed should be well documented, including the source and passage history of the
organism. All defining characteristics such as haemagglutinin and neuramindase subtype should be
established. Haemagglutination inhibition and neuraminidase inhibition by subtype-specific antisera or
RT-PCR and sequencing can be used to establish the H and N subtypes. Also, aliquots of the master
seed virus (MSV) can be neutralised with specific antiserum, e.g. antiserum produced against H1N1 or
H3N2 SIV, then inoculated into the allantoic sac of 10-day old embryonated chicken eggs or on to
susceptible cell lines such as the MDCK cell line. Allantoic fluid or cell culture supernatant is harvested
7296 hours post-inoculation and tested for HA activity. Identity is demonstrated by the lack of HA
activity in the neutralised seed, and the presence of HA activity in the non-neutralised seed. Significant
antigenic differences present in a given strain that set it apart from other members of its subtype, and
that purportedly have a beneficial impact on its use as a vaccine, should be confirmed.
Factors that may contribute to instability during production, such as replication on an unusual cell line,
should be investigated. If production is approved for five passages from the master seed, then
sequencing of the genes for H and N at the maximum passage may be warranted to confirm the stability
of the viral seed.
ii)

The purity of the seed and cells to be used for vaccine production must be demonstrated. The MSV
should be free from adventitious agents, bacteria, or Mycoplasma, using tests known to be sensitive for
detection of these microorganisms. The test aliquot should be representative of a titre adequate for
vaccine production, but not such a high titre that hyperimmune antisera are unable to neutralise seed
virus during purity testing. Seed virus is neutralised with monospecific antiserum or monoclonal
antibody against SIV and the virus/antibody mixture is cultured on several types of cell line monolayers.
Cultures are subpassaged at 7-day intervals for a total of at least 14 days, then tested for
cytopathogenic and haemadsorbing agents.
b)
i)
Procedure
Once the vaccine is shown to be efficacious, and the proposed conditions for production are
acceptable to regulatory authorities, approval may be granted to manufacture vaccine. SIV can be
grown in eggs or in cell culture. Selection of a culture method is dependent on the degree of virus
adaptation, growth in medium, rate of mutation, and viral yield in the specific culture system. SIV
vaccine products should be limited to five passages from the MSV to avoid genetic/antigenic variation.
Generally, large-scale monolayer or suspension cell systems are operated under strict temperaturecontrolled, aseptic conditions and defined production methods, to assure lot-to-lot consistency. When
the virus has reached its maximum titre, as determined by HA, CPE, fluorescent antibody assay, or
other approved technique, the virus is clarified, filtered, and inactivated. Several inactivating agents
have been used successfully, including formalin or binary ethylenimine. Typically, adjuvant is added to
enhance the immune response.
ii)

Cells are examined for adventitious viruses that may have infected the cells or seed during previous
passages. Potential contaminants include bovine viral diarrhoea virus, reovirus, rabies virus,
Aujeszkys disease (pseudorabies) virus, transmissible gastroenteritis virus, porcine respiratory
coronavirus, porcine parvovirus, porcine adenovirus, haemagglutinating encephalomyelitis virus,
porcine rotavirus, porcine circovirus, and porcine reproductive and respiratory syndrome virus. Cell
1133
lines on which the seed is tested include: an African green monkey kidney (Vero) cell line (rabies and
reoviruses), a porcine cell line, a cell line of the species of cells used to propagate the seed, if not of
porcine origin, and cell lines for any other species through which the seed has been passaged.
Additionally, a cell line highly permissive for bovine viral diarrhoea virus, types 1 and 2, is
recommended. Bovine viral diarrhoea virus is a potential contaminant introduced through the use of
fetal bovine serum in cell culture systems.
iii)
In-process controls
Cell cultures should be checked macroscopically for abnormalities or signs of contamination and
discarded if unsatisfactory. A lot is ready to harvest when viral CPE has reached 8090%. Virus
concentration can be assessed using antigenic mass or infectivity assays.
iv)

Vaccine candidates should be shown to be pure, safe, potent, and efficacious.
During production, tests for bacteria, Mycoplasma, and fungal contamination should be conducted on
both inactivated and live vaccine harvest lots and confirmed on the completed product (see Chapter
1.1.7 Tests for sterility and freedom from contamination of biological materials).
Safety
An inactivation kinetics study should be conducted using the approved inactivating agent on a viral lot
with a titre greater than the maximum production titre and grown using the approved production
method. This study should demonstrate that the inactivation method is adequate to assure complete
inactivation of virus. Samples taken at regular timed intervals during inactivation, and then inoculated
on to a susceptible cell line or into the allantoic sac of embyonated eggs, should indicate a linear and
complete loss of titre by the end of the inactivation process. This is represented as less than one
infectious particle per 104 litres of fluids following inactivation.
Batch potency
During production, antigen content is measured to establish that minimum bulk titres have been
achieved. Antigen content is generally measured before inactivation and prior to further processing.
Relative potency ELISA, HA, and HI are among the assays that can be used to determine antigen
content in final product. It is necessary to confirm the sensitivity, specificity, reproducibility, and
ruggedness of such assays.
The potency assay established at the time of the minimum antigen protection study should be used to
evaluate new lots for release. The assay needs to be specific and reproducible. It must reliably detect
vaccines that are not sufficiently potent. If laboratory animal serology is used instead of swine serology, it
should first be demonstrated that vaccination of the laboratory animal induces a specific, sensitive, dosedependent response as measured in the potency assay and is correlated to protection in swine
c)

i)
Safety requirements
Final container samples of completed product from inactivated vaccines should be tested in young
mice for safety. Generally, healthy pigs of weaning age or older and pregnant sows at any stage of
gestation may be safely vaccinated with inactivated SIV vaccines. Final product may be evaluated in
the host animal using two animals of the minimum age recommended for use, according to the
instructions given on the label; the animals are observed for 21 days. Field safety studies conducted on
vaccinates, in at least three divergent geographical areas, with at least 300 animals per area, are also
recommended.
Reversion-to-virulence for live viral vaccines is often demonstrated by back passage through
susceptible species. Virus is isolated from the vaccinated animal and the isolated virus is then used to
inoculate additional animals. Sequential passage through animals should show that animals remain
clinically healthy with no demonstration of typical vesicular stomatitis lesions.
Inactivated SIV vaccines present no special danger to the user, although accidental inoculation may
result in an adverse reaction caused by the adjuvant and secondary components of the vaccine.
1134
Modified live virus vaccines may pose a hazard to the user depending on the level of inactivation of the
virus.
Preservatives should be avoided if possible, and where not possible, should be limited to the lowest
concentration possible. The most common preservative is thimerosol, at a final concentration not to
exceed 0.01% (1/10,000). Antibiotics may be used as preservatives in SIV vaccines but are limited as
to kinds and amounts. Also restricted are residual antibiotics from cell culture media that may be
present in the final product. For example, the total amount of preservative and residual gentamicin is
not to exceed 30 mcg per ml of vaccine.
Vaccine bottles, syringes, and needles may pose an environmental hazard for vaccines using
adjuvants or preservatives and for modified live virus vaccines. Instructions for disposal should be
included within the vaccine packaging information and based on current environmental regulations in
the country of use.
ii)
A vaccination/challenge study in swine, using homologous and heterologous challenge strains, will
indicate the degree of protection afforded by the vaccine. Swine used in vaccination/challenge studies
should be free of antibodies against SIV at the start of the experiments. Vaccination/challenge studies
should be conducted using virus produced by the intended production method, at the maximum viral
passage permitted, and using swine of the minimum recommended age listed on the label. Initially, lots
are formulated to contain varying amounts of viral antigen. The test lot containing the least amount of
antigen that demonstrates protection becomes the standard against which future production lots are
measured. The most valuable criterion for blind trial evaluations of treatment groups is a statistically
significant reduction of virus (titres and duration of shedding) in the respiratory tract of vaccinated pigs.
Differences in clinical observations and lung lesions are also among the criteria used in evaluation of a
successful trial. If in-vivo or in-vitro test methods are to be used to determine the potency of each
production lot of vaccine, those assays should be conducted concurrent with the minimum antigen
studies in order to establish the release criteria. Combination vaccines containing more than one strain
of SIV are available in some countries. The efficacy of the different components of these vaccines must
each be established independently and then as a combination in case interference between different
antigens exists.
The duration of immunity and recommended frequency of vaccination of a vaccine should be
determined before a product is approved. Initially, such information is acquired directly using host
animal vaccination/challenge studies. The period of demonstrated protection, as measured by the
ability of vaccinates to withstand challenge in a valid test, can be incorporated into claims found on the
vaccine label. Once a suitable potency assay has been identified, should antigenic drift require
replacement of strains within the vaccine, strains of the same subtype can be evaluated in either the
host animal or a correlated laboratory animal model. However, circulating strains may show significant
antigenic differences from the vaccine strain, but the vaccine strain may still provide protection. Also,
the vaccine may not protect against a new strain that appears to be antigenically similar to the vaccine.
Other factors that play a role include the adjuvant and the antigenic dose. Consequently, it would
appear that the efficacy of a vaccine will always have to be evaluated in swine.
If the vaccine is to be used in swine destined for market and intended for human consumption, a
withdrawal time consistent with the adjuvant used (generally 21 days) should be established by such
means as histopathological examination submitted to the appropriate food safety regulatory authorities.
The same principles apply as for animal production usage. In addition, it should be noted that antibody
responses in vaccinated animals may not be differentiated from animals exposed to field virus.
Therefore, vaccinated animals will need to be clearly identified if serological methods will be used in
conjunction with compatible clinical signs to assess field virus exposure.
iii)
Stability
Vaccines should be stored with minimal exposure to light at 4C2C, or as approved by the
designated regulatory authorities. The shelf life should be determined by use of the approved potency
test over the proposed period of viability.
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*
* *
NB: There are OIE Reference Laboratories for Swine influenza
diagnostic tests, reagents and vaccines for swine influenza
1137
CHAPTER 2.8.9.
SWINE VESICULAR DISEASE
SUMMARY
Swine vesicular disease (SVD) is a contagious disease of pigs, caused by an enterovirus and
characterised by vesicles on the coronary bands, heels of the feet and occasionally on the lips,
tongue, snout and teats. Strains of SVD virus may vary in virulence, and the disease may be
subclinical, mild or severe, the latter usually only being seen when pigs are housed on abrasive
floors in damp conditions. The main importance of SVD is that it is clinically indistinguishable from
foot and mouth disease (FMD), and any outbreaks of vesicular disease in pigs must be assumed to
be FMD until investigated by laboratory tests and proven otherwise. However, subclinical infection
has been the most frequent condition observed during recent years.
Identification of the agent: Where a vesicular condition is seen in pigs, the demonstration by
enzyme-linked immunosorbent assay (ELISA) of SVD viral antigen in a sample of lesion material or
vesicular fluid is sufficient for a positive diagnosis. If the quantity of lesion material submitted is not
sufficient (less than 0.5 g), or if the test results are negative or inconclusive, a more sensitive test,
such as the reverse transcription polymerase chain reaction (RT-PCR) or isolation of virus (VI) in
porcine cell cultures, may be used. If any inoculated cultures subsequently develop a cytopathic
effect, the demonstration of SVD viral antigen by ELISA or by RT-PCR will suffice to make a
positive diagnosis. Subclinical infection may be detected by random sampling of pen-floor faeces
followed by identification of SVD viral genome using RT-PCR or VI tests.
Serological tests: Serological tests can be used to help confirm clinical cases as well as to identify
subclinical infections. Specific antibody to SVD virus can be identified using the microneutralisation
test or ELISA. Although the microneutralisation test requires 23 days to complete, it remains the
definitive test for antibody to SVD virus. A small proportion (up to 0.1%) of normal, uninfected pigs
will react positively in serological tests for SVD. The reactivity of these singleton reactors is
transient, so that they can be differentiated from infected pigs by resampling of the positive animal
and its cohorts.
Requirements for vaccines and diagnostic biologicals: There are currently no commercial
vaccines available against SVD. Diagnostic and standard reagents are available from regional
reference laboratories.
A. INTRODUCTION
Swine vesicular disease (SVD) can be a subclinical, mild or severe vesicular condition depending on the strain of
virus involved, the route and dose of infection, and the husbandry conditions under which the pigs are kept.
Clinically, SVD is indistinguishable from foot and mouth disease (FMD) and this is its main importance. It is
therefore urgent that cases of SVD be distinguished from FMD by laboratory investigation. Recent outbreaks of
SVD have been characterised by less severe or no clinical signs; infection has been detected when samples are
tested for a serosurveillance programme or for export certification.
The incubation period for SVD is between 2 and 7 days, after which a transient fever of up to 41C may occur.
Vesicles then develop on the coronary band, typically at the junction with the heel. These may affect the whole
coronary band resulting in loss of the hoof. More rarely, vesicles may also appear on the snout, particularly on the
dorsal surface, on the lips, tongue and teats, and shallow erosions may be seen on the knees. Affected pigs may
be lame and off their feed for a few days. Abortion is not a typical feature of SVD. Recovery is usually complete in
23 weeks, with the only evidence of infection being a dark, horizontal line on the hoof where growth has been
temporarily interrupted. The clinical signs vary according to the age of pigs affected, the conditions under which
they are kept and the strain of SVD virus involved (Loxam & Hedger, 1983). Disease caused by mild strains may
1138
Chapter 2.8.9. Swine vesicular disease
remain unobserved, particularly in pigs kept on grass or housed on deep straw. Younger animals are more
severely affected, although mortality due to SVD is very rare, in contrast with FMD in young stock. Nervous signs
have been reported, but are unusual. Affected pigs may excrete virus from the nose and mouth and in the faeces
up to 48 hours before the onset of clinical signs. Most virus is produced in the first 7 days after infection, and virus
excretion from the nose and mouth normally stops within 2 weeks. Virus may continue to be shed for up to
3 months in the faeces. The SVD virus is extremely resistant to inactivation in the environment, and is stable in
the pH range 2.512.0 (Mann, 1981). This is in contrast to the FMD virus, which is very labile outside the pH
range 6.08.0.
Because SVD may be mild or subclinical, it is essential when submitting samples from suspect clinical cases that
serum samples from both the suspect pigs and other apparently unaffected animals in the group be included. It is
possible for SVD to circulate unnoticed until it affects a particularly susceptible group, and therefore, in order to
ascertain how long infection has been present, it is necessary to look for seroconversion to SVD virus in
apparently healthy animals. Also the identification of the isotype of the immunoglobulins (M or G) to SVD virus
may help to ascertain the time of exposure to infection.
SVD is clinically very similar to FMD. Samples for virus isolation or antigen detection must be handled and
submitted as though they contained FMD virus and must be transported in phosphate buffered saline (PBS)
mixed with glycerol (1/1), pH 7.27.6, with antibiotics such as (final concentration per ml) penicillin
(1000 International Units [IU]), neomycin sulphate (100 IU), polymyxin B sulphate (50 IU), and mycostatin (100 IU)
(Kitching & Donaldson, 1987).
SVD virus has been classified as a pig enterovirus, in the family Picornaviridae. All isolates are classified in a
single serotype, with four distinguishable antigenic/genomic variants (Brocchi et al., 1997). Antigenically, SVD
virus is related to the human virus coxsackievirus B5. There are reports of seroconversion to SVD virus in
laboratory workers handling the agent. Clinical disease was reported to be mild with the exception of a single
case of meningitis associated with SVD virus infection. However, there have been no reported cases of
serconversion or disease in farmers or veterinarians working with infected pigs. Under experimental conditions, it
has not been possible to show transmission of coxsackievirus B5 between pigs.
1.
Any vesicular condition in pigs may be FMD. Once suspicion of FMD has been eliminated, the diagnosis of SVD
requires the facilities of a specialised laboratory. Countries that lack such a facility should send samples for
investigation to an OIE Reference Laboratory for SVD1. In the Americas, parallel testing for vesicular stomatitis
viral antigen should also be conducted.
The detection of antigens or genome of SVD virus by means of enzyme-linked immunosorbent assay (ELISA) and
reverse transcription polymerase chain reaction (RT-PCR) has the same diagnostic value as virus isolation. Due
to their speed, ELISA and RT-PCR make suitable screening tests. However, virus isolation is the reference
method and should be used if a positive ELISA or RT-PCR result is not associated with the detection of clinical
signs of disease, the detection of seropositive pigs, or a direct epidemiological connection with a confirmed
outbreak.
If there are clinical signs, investigation should start with the examination of a 10% suspension of lesion material in
PBS. Faecal samples are the specimen of choice for the detection of virus where subclinical SVD is suspected.
Faecal samples can be collected from individual pigs or from the floor of premises suspected to contain, or to
have contained, pigs infected with SVD. The level of virus in faeces is usually insufficient for detection by ELISA
and the use of RT-PCR and/or virus isolation is required. A significant proportion of faecal samples inoculated into
cell cultures will give rise to the growth of other enteroviruses. These can be differentiated from SVD virus by
ELISA or RT-PCR, but they may also outgrow SVD virus that is present, and give rise to false negative results.
Preparation of samples
Lesion material: a suspension is prepared by grinding the sample in sterile sand in a sterile pestle and
mortar with a small volume of tissue culture medium and antibiotics. Further medium should be added to
obtain approximately a 10% suspension. This is clarified by centrifugation at 10000 rpm for 2030 minutes in
a high speed centrifuge and the supernatant is harvested.
FAO (Food and Agriculture Organization of the United Nations) World Reference Laboratory for FMD, Institute for Animal
Health, Pirbright, Woking, Surrey GU24 0NF, United Kingdom (also an OIE Reference Laboratory for FMD and SVD).
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna (IZSLER), Brescia, Via Bianchi 9, Italy.
1139
Faecal samples: faecal material (approximately 20 g) is resuspended in a minimal amount of tissue culture
medium or phosphate buffer (0.04 M phosphate buffer or PBS). The suspension is homogenised by
vortexing and clarified by centrifugation at 10000 rpm for 2030 minutes in a high speed centrifuge; the
supernatant is harvested and filtered through 0.45 m filter.
a)
Virus isolation
A portion of the clarified epithelial or faecal suspension is inoculated on to monolayers of IB-RS-2 cells or
other susceptible porcine cells, grown in appropriate containers (25 cm2 flasks, rolling tubes, 24-, 12-, 6-well
plates). For differential diagnosis (e.g. FMD) in case of clinical lesion bovine cell culture systems should also
be employed. Generally SVD virus will grow in cells of porcine origin only, however there is a report that the
virus can be isolated in secondary lamb kidney cells. Tissue culture medium is supplemented with, 10%
bovine serum for cell growth, with 3% bovine serum for maintenance, and with antibiotics.
Cultures are examined daily. If a cytopathic effect (CPE) is observed, the supernatant fluid is harvested and
virus identification is performed by ELISA (or other appropriate test, e.g. RT-PCR). Negative cultures are
blind-passaged after 48 or 72 hours, and observed for a further 23 days. If no CPE is evident after the
second passage, the sample is recorded NVD (no virus detected). When isolating virus from faeces in
which the amount of virus present may be low, a third tissue culture passage may be required.
b)
The detection of SVD viral antigen by an indirect sandwich ELISA has replaced the complement fixation test
as the method of choice. The test is the same as that used for FMD diagnosis. Wells of ELISA plates are
coated with rabbit antiserum to SVD virus. This is the capture serum. Test sample suspensions are added
and incubated. Appropriate controls are also included. Guinea-pig detection serum is added at the next
stage followed by rabbit anti-guinea-pig serum conjugated to horseradish peroxidase. Extensive washing is
carried out between each stage to remove unbound reagents. A positive reaction is indicated if there is a
colour reaction on the addition of chromogen (for example orthophenylenediamine) and substrate (H2O2).
With strong positive reactions this will be evident to the naked eye, but results can also be read
spectrophotometrically at the appropriate wavelength, in which case an absorbance reading 0.1 above
background indicates a positive reaction. As an alternative to guinea-pig and rabbit antisera, suitable
monoclonal antibodies (MAbs) can be used, coated to the ELISA plate as the capture antibody, or
peroxidase conjugated as tracing antibody.
An MAb-based ELISA can also be used to study antigenic variation among strains of SVD virus. Tissueculture grown viral strains are trapped by a rabbit hyperimmune antiserum to SVD virus adsorbed to the
solid phase. Appropriate panels of MAbs are then reacted and the binding of MAbs to field strains is
compared with the binding of MAbs to the parental strains. Strong binding indicates the presence of epitopes
shared between the parental and the field strains (Brocchi et al., 1997).
c)

Reverse transcription followed by the PCR (RT-PCR) is a useful method to detect SVD viral genome in a
variety of samples from clinical and subclinical cases. Several methods have been described (Callens & De
Clercq, 1999; Fallacara et al., 2000; Lin et al., 1997; Nunez et al., 1998; Reid et al., 2004a; 2004b;
Vangrysperre & De Clercq, 1996), employing different techniques for RNA extraction, targeting different
parts of the SVD virus genome and using different approaches to detect the DNA products of amplification.
In the case of faecal samples, an immunocapture technique using a SVD virus-specific MAb has been
shown to be effective (Fallacara et al., 2000). This method (described below) is suitable for laboratories
without sophisticated equipment for real-time detection of DNA amplification products, but where such
facilities are available an approach such as that described by Reid et al. (2004a; 2004b) offers advantages
in terms of ease of use and reduced risk of laboratory contamination by PCR products.
i)
RNA Immune-extraction: Coat wells of an ELISA plate with a saturating solution of MAb 5B7
(200 l/well, diluted in carbonate-bicarbonate buffer) by overnight incubation at 4C. Wash plates three
times with PBS. Use plates immediately or store at 20C for up to 23 weeks, or more if stabilised.
NOTE: As an alternative to immunocapture, RNA extraction can be performed using a suitable
commercially available kit (such as RNeasy kit from Qiagen).
ii)
Distribute each sample (faeces suspension) into three wells of the 5B7-coated plate (200 l/well, 600 l
of sample in total).
iii)
After incubation for 1 hour at 37C with very slow shaking, wash wells three times with PBS. Washing
is performed manually, in order to avoid cross-contamination between wells.
1140
iv)
RNA is extracted from each sample by adding approximately 100 l/well of lysis buffer (4 M guanidine
thiocyanate, 25 mM sodium citrate, pH 7, 0.5% Sarkosyl). Incubate wells for 35 minutes and recover
the sample from the three wells (300350 l total), and transfer into a single tube.
v)
RNA is then precipitated by adding a mixture of 750 l of absolute ethanol and 35 l of 3 M sodium
acetate (pH 5.2); vials are vortexed and incubated at 20C for a minimum of 1 hour (prolonged
overnight precipitation at 20C may also be suitable).
vi)
Centrifuge the sample at 13,000 rpm for 30 minutes at 4C, after which a pellet should be visible which
should be washed with 500 l of 70% cold ethanol (centrifuged at 13000 rpm for 10 minutes at 4C)
and dried.
vii)
Assemble the reaction mixes for the reverse transcription of SVDV RNA. Mixes (20 l total volume)
contain 4 l of 5 AMV reaction buffer (final reaction concentration is 8 mM MgCl2, 30 mM KCl, 1 mM
dithiothreithol, 50 mM Tris/HCl, pH 8.5), 2 l 10 mM dNTP mix, 1 l Random Primer pd(N)6 (100 pmol),
0.3 l RNase Inhibitor (12 units), 0.2 l AMV reverse transcriptase (5 units) 0.5 l BSA (0.5 g) and
12 l of nuclease-free water.
viii) Resuspend the RNA pellet in 20 l of reverse transcription mix (see above).
ix)
Incubate reactions at 42C for 60 minutes followed by 95C for 3 minutes.
x)
PCR for SVDV Genome: Add 5 l of prepared cDNA to 20 l PCR amplification mix containing 0.55 l
2M KCl (final reaction concentration is 44 mM), 2 l 10 mM dNTP mix, 1 l pSVDV-SA2 rev primer
(10 pmol: 5-TCA-CGT-TTG-TCC-AGG-TTA-CC-3), 1 l pSVDV-SS4 dir primer (10 pmol: 5-TTCAGA-ATG-ATT-GCA-TAT-GGG-G-3), 0.25 l Taq polymerase (1.24 units) and 15.2 l of nuclease-free
water.
xi)
Place the plate in a PCR thermocycler and run the following programme:
40 Cycles of 94C for 20 seconds, 60C for 20 seconds, 72C for 30 seconds,
xii)
Mix a 20 l aliquot of each sample with 4 l of staining solution and load onto a 2% agarose gel. After
electrophoresis, a positive result is indicated by the presence of a 154 bp fragment of SVDV RNA
polymerase (3D) gene in the gel.
Comparative analysis of sequences of the viral genome is useful to establish relationships between isolates
of SVD virus. By sequencing the 1D gene, which codes for the major structural protein VP1, it has been
possible to group strains of SVD virus according to their sequence homology, and to relate epidemiologically
strains causing disease in different regions or at different times (Brocchi et al., 1997). A database of ID gene
sequences of SVD viruses is held at the OIE Reference Laboratory, Pirbright, UK.
2.
Serological tests
These are used in the laboratory confirmation of outbreaks, for serological surveillance and for export certification
of pigs. SVD is often diagnosed solely on the evidence of serological tests. Because of the subclinical or mild
nature of the disease, it is often first suspected following routine serology for disease surveillance or export
certification. The virus neutralisation (VN) test, the double immunodiffusion test, the radial immunodiffusion test,
the counter immunoelectrophoresis test and the ELISA have all been described for the detection of antibodies to
SVD virus (Brocchi et al., 1995; Donaldson et al., 1983; Golding et al., 1976). However, the VN test and the
ELISA are the only techniques commonly used. The VN test is the accepted standard test, but has the
disadvantage that it takes 23 days to complete and requires tissue culture facilities. The ELISA is more rapid and
can be more easily standardised. A small proportion of sera from animals with no previous exposure to SVD virus
will react positively in serological tests for antibody to SVD virus. The 5B7 MAb competitive ELISA (MAC-ELISA)
is a reliable technique for detecting SVD antibody (Brocchi et al., 1995; Heckert et al., 1998) and similar results
have been obtained with other ELISAs (Chenard et al., 1998; Ko et al., 2005). Results from a small proportion,
0.25%-0.45%, of sera from normal pigs are borderline or positive by the MAC-ELISA and should be retested by
the VN test. Up to approximately 50% of these sera will also be positive by the VN test (i.e. 0.10.2% of the
original population). Animals that test positive by ELISA but negative by VN test can be regarded as uninfected.
Repeat samples should be collected from animals positive in both tests and from cohorts. A constant or declining
titre in the positive animal and the absence of antibody to SVD virus in cohorts confirms the status of the positive
animal as a singleton reactor. The factors responsible for singleton reactors are unknown. Serological crossreactivity with SVD virus might arise due to infection with another, as yet unidentified, picornavirus or may be due
to other non-specific factors present in the serum. Identification of the isotype of antibody present in positive sera
(Brocchi et al., 1995) can be helpful as sera from infected pigs usually contain specific IgG alone or both IgG and
IgM, whereas sera from singleton reactors usually contain exclusively IgM and do not convert to IgG (De Clercq,
1998). IgM/IgG isotype-specific ELISAs are also helpful in assessing the time of infection in the pig or on the
1141
infected premises. The presence of IgM, alone or together with IgG, is evidence of recent infection and indicative
of virus shedding, while detection of IgG alone suggests an older exposure to infection (Brocchi et al., 1995).
a)
Virus neutralisation (the prescribed test for international trade)

The quantitative VN microtest for antibody to SVD virus is performed using IB-RS-2 cells (or suitable
susceptible porcine cells) in flat-bottomed tissue-culture grade microtitre plates.
Virus is grown on IB-RS-2 cell monolayers and stored at 20C after the addition of an equal volume of
glycerol. SVD virus has been found to be stable under these conditions for at least 1 year. The sera are
inactivated at 56C for 30 minutes before testing. A suitable medium is Eagles complete medium/LYH with
antibiotics.
The test is an equal volume test in 50 l volumes:
i)
Starting from a 1/4 dilution, sera are diluted in a twofold dilution series across the plate, two rows of
wells per serum.
ii)
Previously titrated virus is added; each 50 l unit volume of virus suspension contains about
100 TCID50 (50% tissue culture infective dose).
iii)
Controls include at least a weak positive serum and a negative serum, a cell control, a medium control
and a virus titration used to calculate the actual virus titre used in the test.
iv)
Incubate at 37C for 1 hour with the plates covered.
v)
A cell suspension at 106 cells/ml is prepared in medium containing 10% bovine serum for cell growth.
50 l of cell suspension is added to each well.
vi)
Plates are sealed with pressure-sensitive tape and incubated at 37C for 23 days. Alternatively, the
plates may be covered with loosely fitting lids and incubated in an atmosphere of 5% carbon dioxide at
37C for 23 days.
vii)
Microscopic readings are feasible after 4872 hours; the plates may be finally fixed and stained
routinely on the third day. Fixation is effected with 10% formalin/saline for 30 minutes; staining is done
by immersion in 0.05% methylene blue in 10% formalin for 30 minutes. The plates are rinsed in
tapwater.
viii) Positive results are blue-stained cell sheets; the negative wells are empty. Titres are expressed as the
final dilution of serum present in the serum/virus mixture at the 50% end-point. The test is considered
to be valid when the amount of virus actually used per well is between 101.5 and 102.5 TCID50, and the
positive standard sera are within twofold of their expected titre.
ix)
b)
Interpretation of the results: At the Pirbright, OIE Reference Laboratory for SVD (see footnote 1), VN
titres less than or equal to 1/11 are considered to be negative. Titres of 1/16 to 1/32 are doubtful and
VN titres of 1/45 or more are regarded as positive. However, as titres depend on the cell system used,
laboratories should establish their own criteria by reference to standard reagents available from the
OIE Reference Laboratory.

In the ELISA developed by Brocchi et al. (1995), the SVD viral antigen is trapped to the solid phase using
the MAb 5B7. The ability of test sera to inhibit the binding of peroxidase-conjugated MAb 5B7 to the trapped
antigen is then evaluated. Finally, the amount of conjugated MAb bound is detected by the addition of
substrate and chromogen.
i)
ELISA plates are coated with 50 l/well of MAb 5B7 at a saturating dilution in carbonate/bicarbonate
buffer, pH 9.6, by overnight incubation at 4C.
ii)
The plates are washed three times with PBS containing 0.05% Tween 20, and 50 l of SVD antigen
(SVD virus grown in IB-RS-2 cells, clarified, filtered and inactivated) at a predetermined optimal
dilution, is added to each well. The optimal dilution of antigen is determined by checkerboard titrations
of antigen and conjugated MAb that define the working dilution giving an absorbance on the upper part
of the linear region of the antigen titration curve (between 1.5 and 2.0 optical density units). Plates are
then incubated for 1 hour at 37C.
iii)
After three additional washes, 50 l of diluted test sera (not inactivated) and control sera are incubated
with the trapped antigen for 1 hour at 37C. Three-fold dilution series of sera are obtained directly in
ELISA wells by adding 10 l of serum to 65 l of buffer (1/7.5 dilution) then transferring 25 l to
sequential wells containing 50 l of buffer, mixing, and finally discarding 25 l.
1142
iv)
After incubation for 1 hour, 25 l of an optimal dilution of peroxidase-conjugated MAb 5B7 (see step ii
above) is added to each well and the plates are incubated at 37C for a further 1 hour.
v)
After a final series of washes, the colorimetric reaction is developed by distributing 50 l per well of the
substrate solution (for example 0.5 mg/ml orthophenylene-diamine in phosphate/citrate buffer, pH 5,
containing 0.02% H2O2).
vi)
The reaction is stopped after 10 minutes by adding 50 l of 2 N H2SO4. The absorbance is read at the
appropriate wavelength using a microplate reader.
Antigen, sera and conjugate are diluted in PBS, pH 7.4, containing 0.05% Tween 20 and 1% yeast extract;
the dilution buffer for sera contains, in addition, 1.0% mouse serum to prevent nonspecific binding of pig
serum to MAb 5B7 either coated to the plate or conjugated to peroxidase.
vii)
Controls: Four wells on each plate containing all reactants except test serum confirm the maximum
absorbance reading for the antigen; convalescent pig serum at four selected dilutions; negative pig
serum; a low positive standard pig serum.
viii) Interpretation of the results: Reactions are expressed as the percentage inhibition by each test serum
of the MAb reaction with the SVD antigen. Sera are considered to be positive when producing an
inhibition 80% at the 1/7.5 dilution; negative when producing an inhibition <70% at the 1/7.5 dilution;
doubtful when producing an inhibition 70% and <80% at the 1/7.5 dilution. The second dilution
(1/22.5) provides an indication of the level of antibodies: strongly positive sera show >80% inhibition at
both 1/7.5 and 1/22.5 dilutions, while Sera registering >80% inhibition at the 1/7.5 dilution but <50%
inhibition at the 1/22.5 dilution are considered to be low positive or borderline. All positive, borderline
and doubtful sera should be confirmed using the VN test.
STANDARD REFERENCE SERA FOR SVD SEROLOGY

The OIE Reference Laboratory, Pirbright, UK maintains a panel of reference sera that have been extensively
validated by the National SVD Reference Laboratories of the Member States of the European Union.

No commercial SVD vaccines are currently available. Standard sera can be obtained from the OIE/FAO WRL for
FMD (see footnote 1). MAb 5B7 is available from the OIE Reference Laboratory for swine vesicular disease in
Italy (see Table given in Part 4 of this Terrestrial Manual).
REFERENCES
BROCCHI E., BERLINZANI A., GAMBA D. & DE SIMONE F. (1995). Development of two novel monoclonal antibodybased ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular
disease virus. J. Virol. Methods, 52, 155167.
BROCCHI E., ZHANG G., KNOWLES N.J., WILSDEN G., MCCAWLEY J.W., MARQUARDT O., OHLINGER V.F. & DE SIMONE F.
(1997). Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically
distinct variants in Europe, 19871994. Epidemiol. Infect., 118, 5161.
CALLENS M. & DE CLERCQ K. (1999). Highly sensitive detection of swine vesicular disease virus based on a single
tube RT-PCR system and DIG-ELISA detection. J. Virol. Methods, 77, 8799.
CHENARD G., BLOEMRAAD M., KRAMPS J.A., TERPSTRA C. & DEKKER A. (1998). Validation of a monoclonal antibodybased ELISA to detect antibodies directed against swine vesicular disease virus. J. Virol. Methods, 75, 105112.
DONALDSON A.I., FERRIS N.P., KNOWLES N.J. & BARNETT I.T.R. (1983). Comparative studies of United Kingdom
isolates of swine vesicular disease virus. Res. Vet. Sci., 35, 295300.
DE CLERCQ K. (1998). Reduction of singleton reactors against swine vesicular disease virus by a combination of
virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA. J. Virol.
Methods, 70, 718.
1143
FALLACARA F., PACCIARINI M., BUGNETTI M., BERLINZANI A. & BROCCHI E (2000). Detection of swine vesicular disease
virus in faeces samples by immune-PCR assay. In: Veterinary Virology in the New Millennium, Proceedings of the
5th International Congress of the European Society for Veterinary Virology, Brescia, Italy, 2730 August 2000, pp
173174.
GOLDING S.M., HEDGER R.S., TALBOT P. & WATSON J. (1976). Radial immunodiffusion and serum neutralisation
techniques for the assay of antibodies to swine vesicular disease. Res. Vet. Sci., 20, 142147.
HECKERT A., BROCCHI E., BERLINZANI A. & MACKAY D. (1998). An international comparative analysis of a competitive
ELISA for the detection of antibodies to swine vesicular disease virus. J. Vet. Diagn. Invest., 10, 295297.
KITCHING R.P. & DONALDSON A.I. (1987). Collection and transportation of specimens for vesicular virus
investigation. Rev. sci. tech. Off. int. Epiz., 6, 263272.
KO Y.-J., CHOI K.-S., NAH J.-J., PATON D.J., OEM J.-K., WILSDEN G., KANG S.-Y., JO N.-I., KIM J.-H., LEE H.-W. & PARK
J.-M. (2005). Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in
pigs by enzyme linked immunosorbent assay. Clin. Diagn. Lab. Immunol., 12, 922929.
LIN F., MACKAY D.K.J. & KNOWLES N.J. (1997). Detection of swine vesicular disease virus RNA by reverse
transcription-polymerase chain reaction. J. Virol. Methods, 65, 111121.
LOXAM J.R. & HEDGER R.S. (1983). Swine vesicular disease: clinical signs, diagnosis, epidemiology and control.
Rec. sci. tech. Off. int. Epiz., 2, 1124.
MANN J.A. (1981). Swine vesicular disease. In: Virus Diseases of Farm Animals, Vol. 2, Gibbs E.P.J., ed.
Academic Press, London, UK, 365381.
NUNEZ J.I., BLANCO E., HERNANDEZ T., GOMEX-TEJEDOR C., MARTIN M.I., DOPAZO J. & SOBRINO F. (1998). A RT-PCR
assay for the differential diagnosis of vesicular viral diseases of swine. J. Virol. Methods, 72, 227235.
REID S.M., FERRIS N.P., HUTCHINGS G.H., KING D.P. & ALEXANDERSEN S. (2004a). Evaluation of real-time reverse
transcription polymerase chain reaction assays for the detection of swine vesicular disease virus. J. Virol.
Methods, 116, 169176.
REID S.M., PATON D.J., WILSDEN G., HUTCHINGS G.H., KING D.P., FERRIS N.P. & ALEXANDERSEN S. (2004b). Use of
automated real-time RT-PCR to monitor experimental swine vesicular disease virus infection in pigs. J Comp.
Pathol., 131, 308317.
VANGRYSPERRE W. & DE CLERCQ K. (1996). Rapid and sensitive polymerase chain reaction based on detection and
typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous
differentiation with other genomically and/or symptomatically related viruses. Arch. Virol., 141, 331344.
*
* *
NB: There are OIE Reference Laboratories for Swine vesicular disease
diagnostic tests and reagents for swine vesicular disease
1144
CHAPTER 2.8.10.
TESCHOVIRUS ENCEPHALOMYELITIS
(previously enterovirus encephalomyelitis or
Teschen/Talfan disease)
SUMMARY
Teschovirus encephalomyelitis was first described as a particularly virulent, highly fatal
encephalomyelitis of pigs and was previously known as Teschen disease (or enterovirus
encephalomyelitis). It is caused by strains of porcine teschovirus serotype 1 (PTV-1) of the genus
Teschovirus, family Picornaviridae. Less severe forms of the disease were first recognised in the
United Kingdom, where it was called Talfan disease, and in mainland Europe, where it was called
poliomyelitis suum or benign enzootic paresis. In addition to PTV-1 strains, the milder form of the
disease can be caused by other PTV serotypes, including PTV-2, -3, -4, -5, -6, -9 and -10.
The disease was first described in Teschen, Czechoslovakia in 1929. During the 1940s and 1950s
it caused serious losses in European countries and was spread to other continents. The clinical
disease is now rare and has not been reported in Western Europe since 1980. However, there has
been serological evidence that virus variants, that are not pathogenic or of low pathogenicity,
circulate in pig populations.
Identification of the agent: The virus has affinity for the central nervous system and therefore
suspensions of brain and spinal cord from affected pigs are used as inocula for virus isolation. The
virus propagates successfully on monolayers derived from swine tissue, in particular from kidney. If
PTV is present, it gives rise to specific cytopathic effects characterised by rounded refractile cells.
For PTV identification and serotyping, suitable tests are employed using specific antisera or
monoclonal antibodies against standard strains of PTV. Virus neutralisation tests and indirect
fluorescent antibody tests are preferred. Reverse-transcription polymerase chain reaction
amplification of parts of the viral genome is possible, but as yet no specific tests have formally been
accepted for diagnosis.
Serological tests: Because the seroprevalence of PTV-1 may exceed 60% in healthy pig
populations in Central Europe, and identical clinical signs may be caused by other viruses,
including other serotypes of PTV, a single serological test for PTV-1 giving positive results does not
indicate that the neurological signs observed are actually caused by a PTV-1 infection. A four-fold
rise in titre together with typical signs should be considered to be an indication that PTV-1 infection
caused clinical disease. For screening for specific antibodies in pig populations, it is recommended
to use the virus neutralisation test in microtitre plates or the enzyme-linked immunosorbent assay.
Requirements for vaccines and diagnostic biologicals: When clinical disease was common,
vaccines were available and used; however, as the disease is now rare, vaccines are no longer
available.
A. INTRODUCTION
Teschovirus encephalomyelitis (previously Teschen/Talfan diseases, and later enterovirus encephalomyelitis) is
an acute condition of pigs characterised by central nervous system (CNS) disorders. Teschen is the name of the
town in the Czech Republic where the disease was first recognised in 1929 (Klobouk, 1931; 1933). In the 1950s,
the disease spread throughout Europe and caused huge losses to the pig breeding industry. Less severe forms of
the disease were first recognised in the United Kingdom, where it was called Talfan disease, and in Denmark,
where it was called poliomyelitis suum; these were benign enzootics of swine. Teschovirus encephalomyelitis has
not been reported in Western Europe since 1980 (Austria) and the disease is now considered rare. In the last
1145
Chapter 2.8.10. Teschovirus encephalomyelitis (previously enterovirus encephalomyelitis or Teschen/Talfan disease)
12 years (since 1996) disease was reported to the OIE by the following countries: Belarus (1996, 1999 and 2005),
Japan (2002), Latvia (1997 and 20002002), Madagascar (19962000, 2002 and 20042005), Moldavia (2002
2004), Romania (2002), Russia (2004), Uganda (2001) and Ukraine (19962005). In most of these cases it is not
known if diagnosis was made purely on clinical grounds or in conjunction with laboratory tests; the exception
being in Japan in 2002 (Yamada et al., 2004).
The causal agent of teschovirus encephalomyelitis is porcine teschovirus serotype 1 (PTV-1), which belongs to
the species Porcine teschovirus, genus Teschovirus, family Picornaviridae (Betts, 1960; Klobouk, 1933).
Originally the PTVs were classified within the genus Enterovirus and the original 11 porcine enterovirus (PEV)
serotypes, PEV-1 to PEV-11, were placed in three groups I, II and III on the basis of cytopathic effect (CPE)
produced, serological assays and replication in different cell cultures (Knowles et al., 1979). PEV-1 to PEV-7 and
PEV-11 to PEV-13 were identified as group I. Based on nucleotide sequencing and phylogenetic analysis, the
PEV group I viruses have now been placed in the genus Teschovirus. PEV-1 to -7 have been renamed PTV-1 to
-7 and PEV-11 to -13 were renamed PTV-8 to 10; an additional serotype, PTV-11, has also recently been
described (Krumbholz et al., 2002; Stanway et al., 2005). PEV group II contains PEV-8 (species Porcine
enterovirus A) and group III consists of PEV-9 and PEV-10 (species Porcine enterovirus B). These two groups
currently belong to the genus Enterovirus (Stanway et al., 2005; Zell et al., 2000), although it has been suggested
that PEV-8 may be reclassified in another new picornavirus genus (Krumbholz et al., 2002).
PTV-2, -3, -4, -5, -6, -9 and -10 have been isolated from pigs with milder forms of the disease (Witte von et al.,
1994). PTV infections often do not produce clinical signs. Serotypes may be differentiated using a virus
neutralisation (VN) test (Betts, 1960; Knowles et al., 1979), complement fixation test (Knowles & Buckley, 1980)
or indirect fluorescent antibody (IFA) test (Auerbach et al., 1994; Romanenko et al., 1982).
PTV infections only occur in swine; other animal species are not known to be susceptible.
Differential diagnoses include pseudorabies (Aujeszkys disease) and classical swine fever (acute form). In
addition, Japanese encephalitis, Streptococcus suis and haemagglutinating encephalomyelitis may occasionally
produce similar clinical signs. Non-infectious aetiologies, in particular toxicities, must also be considered.
PTV may be identified serologically using standard antisera that have been prepared by hyperimmunisation of
guinea-pigs, rabbits, or colostrum-deprived piglets with standard strains of PTV serotypes 111.
The virus enters the animal via the oral or nasal cavity. The incubation period is about 14 days. The main signs of
the prodromal stage are fever up to 41.5C, lassitude, anorexia and locomotor disturbances. This stage is
followed by hypersensitivity, tremors, clonic spasms of the legs, flaccid paralysis, opisthotonos and nystagmus;
convulsions may be observed in young pigs. In the final clinical stage, paralysis proceeding from the hind part
through the loins to the fore part of the body is observed. Paralysis of the thermoregulatory centre results in
hypothermy. When respiratory muscles are paralysed, the animal dies of suffocation.
Laboratory diagnosis of the disease is based on typical clinical signs plus histological lesions of the brain and
spinal cord, identification of the virus in the CNS of affected pigs, and on the detection of specific antibodies in the
blood of convalescent animals.
1.
Histological examination and immunohistochemistry
For histological diagnosis, samples of cerebrum, cerebellum, diencephalon, medulla oblongata and cervical and
lumbar spinal cord are collected. The samples are fixed in formaldehyde and sections are stained using
conventional histological methods. The virus multiplies in the CNS causing a nonsuppurative polioencephalomyelitis with lymphocytic perivasculr cuffs, especially in the spinal cord (Klobouk, 1931). Pathological
changes are observed in the grey matter of the diencephalon, cerebellum, medulla oblongata and in the ventral
horns of the spinal cord, consistently including dorsal root ganglia and trigeminal ganglia (ganglioneurites) and to
a lesser extent in the cerebral hemispheres. Lesions may involve the dorsal horns of the spinal cord in very young
animals. Degeneration of neurons (swelling, chromatolysis, necrosis, neuronophagia, axonal degeneration) and
their replacement by microgliosis (astrobytosis, astrogliosis) develops in the late stage of the disease.
Detection of teschovirus antigens by immunohistochemistry on fixed, paraffin-embedded CNS sections is very
difficult and not consistently possible. If suitable specific antisera or monoclonal antibodies are available, as well
as specific detection techniques, correlation of pathological changes with the location of the agent may be
possible on fixed, paraffin-embedded sections of the CNS.
1146
2.
a)
Isolation of the virus

Progress in diagnosis of teschovirus encephalomyelitis and vaccine production has been made possible by
the propagation of virus in cell culture (Madr, 1959; Mayr & Schwoebel, 1957).
Samples of brain and spinal cord are collected from pigs slaughtered at an early clinical stage of the
disease. When not processed immediately, the samples should be placed in a solution prepared from equal
parts of phosphate buffered isotonic saline solution (PBS), pH 7.4, and glycerol. Pieces of tissue are minced
to prepare a 10% (w/v) suspension in PBS. The suspension is centrifuged at 800 g for 10 minutes and the
supernatant fluid is used for inoculation of cell cultures. Monolayer cultures of primary porcine kidney or
established cell lines derived from porcine tissue are suitable for isolation of PTV.
b)
Test procedure
i)
Test tubes or tissue culture vessels with monolayer cell cultures are used. Growth medium is discarded
and tubes or vessels are inoculated with 0.1 ml of suspect tissue homogenate.
ii)
Inoculated test tubes are placed on a roller drum or tissue culture vessels are placed on a tray and
incubated for 1 hour at 37C.
iii)
The inoculum is discarded; the tubes or tissue culture vessels are washed with PBS and replenished
with 120 ml (depending on the type of tissue culture vessel used) of maintenance medium without calf
serum.
iv)
The tubes are examined microscopically each day. If the sample contains PTV, characteristic CPE will
be seen after 34 days. The CPE is characterised by small foci of rounded refractile cells. After several
passages the virus grows better and produces complete CPE. The identity of PTV can be confirmed by
the use of specific antiserum or monoclonal antibodies. The VN or the IFA test is best suited to this
purpose. Once an isolate has been identified serologically as PTV, piglet inoculation is the only certain
means of determining that the given isolate is pathogenic.
Virus neutralisation test for porcine teschovirus identification

The virus harvested from cell cultures is diluted in cell culture maintenance medium over the range 101 to
106 in tenfold steps. For teschovirus serotyping, 12 rows of each dilution are prepared; 50 l of standard
antisera to PTV-111 diluted 1/10 is added to rows 111 and 50 l of negative serum is added to the last
row. Mixtures are incubated overnight at 4C or for 1 hour at 37C and thereafter inoculated into roller tube
cultures or into wells of microtitration plates with confluent monolayer cell cultures. The inoculated cell
cultures are incubated at 37C. Assessment is carried out 72 hours later and every following day up to day
10, depending on when the CPE is seen. The identification of a PTV serotype is confirmed if the titre of the
isolated virus in the presence of that antiserum is at least 103 lower than that virus incubated with negative
serum.
c)
Indirect fluorescent antibody test for the confirmation of porcine teschovirus antigen in cells
The IFA test is based on the reaction of the antigens in infected cells with specific antibodies in positive
serum (Romanenko et al., 1982). The reaction is visualised by a fluorescein isothiocyanate (FITC)conjugated antiglobulin, using a microscope with a UV or a blue light source. The antigen is detectable in
cells 12 hours after the infection with PTV, i.e. before the development of CPE. Polyclonal antisera often
show cross-reactivity with different PTV types, which can confuse the interpretation of results.
Test procedure
i)
Monolayers of porcine kidney cells on cover-slips are inoculated with the suspected material. Positive
and negative controls should be processed in parallel with the test specimens.
ii)
After incubation for 1216 hours, the cover-slips are removed, washed twice in PBS, air-dried and fixed
in cold acetone for 515 minutes.
iii)
The cover-slips are placed into a wet box and flooded with rabbit or pig hyperimmune anti-PTV serum
optimally diluted 1/10 with PBS or with PTV-specific monoclonal antibody at working dilution.
iv)
The wet box is closed and incubated at 37C for 60 minutes.
v)
The cover-slips are removed and washed three times in PBS, then flooded with FITC-conjugated antirabbit or anti-pig goat serum, at a previously assessed working dilution, and incubated at 37C for
30 minutes.
1147
vi)
The cover-slips are then washed three times with PBS, air-dried and mounted in 0.1 M Tris-buffered
glycerol, pH 8.6.
After processing, the cover-slips are examined microscopically. The control slides are examined first to
confirm that the fluorescence observed is specific. The fluorescence is apple green in colour and occurs in
the cell cytoplasm and at the periphery of the nucleus. Instead of cover-slips, multispot slides or multiwell
plates can also be used.
d)
Reverse-transcription polymerase chain reaction

The reverse-transcription polymerase chain reaction (RT-PCR) provides a method for detection and
differentiation of specific gene regions of porcine teschoviruses (Palmquist et al., 2002; Zell et al., 2000).
The nested RT-PCR with specific primer sets has been used to differentiate between PTVs and PEVs (Zell
et al., 2000). PCR is more rapid and less laborious than virus isolation by tissue culture technique and
serotyping. However, the PCR technique is currently restricted to specialised laboratories.
3.
Serological tests
Because the seroprevalence of PTV-1 may exceed 60% in healthy pig populations in some countries of Central
Europe, and identical clinical signs may be caused by other viruses including other serotypes of PTV a single
serological test for PTV-1 giving positive results does not indicate that neurological signs observed are actually
caused by PTV-1. A four-fold rise in titre together with typical signs should be considered to be an indication that
PTV-1 infection caused clinical disease. Another reason that paired serum samples are needed for confirmation
of the significance of titres is that cross-reactions have been reported with orphan teschoviruses.
Pigs that have recovered from disease, or those with inapparent disease, produce specific antibodies. Several
serological methods are available for their detection, of which the microtitre VN test using pig kidney cell cultures
is the most useful (Mayr & Bibrack, 1971). An ELISA has been developed that is more sensitive and rapid
(Hubschle et al., 1983).
For serological diagnosis it is necessary to have standard strains of PTV serotypes propagated in cell cultures
and hyperimmune serum monospecific for PTV types.
Standard strains of porcine teschoviruses
Characteristics: Following long experience, the strain Zabreh, isolated in Czechoslovakia during the period of
peak incidence of the disease, was selected as the standard strain to generate the severe form of teschovirus
encephalomyelitis. The pathogenicity of the strain is maintained by intracerebral passages in healthy, colostrumdeprived piglets. The virus produces typical signs of teschovirus encephalomyelitis after an incubation period of
57 days. For serological diagnosis, the following strains of PTV serotypes should be used as standard strains:
type 1: Talfan, type 2: T80, type 3: O2b, type 4: PS36, type 5: F26, type 6: PS37, type 7: F43, type 8:
UKG/173/74, type 9: Ger-2899/84, type 10: Ger-460/88, type 11: Dresden.
Stock virus: Standard strains are propagated on monolayers of cell culture either from primary porcine kidney or
testes or on an established cell line, for example PK-15. A 10% suspension in PBS, pH 7.4, is prepared from the
brain and spinal cord of piglets infected experimentally with PTV. Some types are isolated from faeces. The
suspension is centrifuged and the supernatant is used for the inoculation of cell cultures. The procedure for the
cultivation of PTV in cell cultures is as follows:
The growth medium is removed from the cell culture and after rinsing with buffered saline, cells are inoculated
with the virus suspension at 37C. The size of the inoculum should be equal to 10% of the growth medium. After
1 hour of incubation at 37C, the inoculum is decanted, the culture vessel is rinsed with buffered saline, and the
cells are overlayed with the appropriate volume of serum-free medium supplemented with antibiotics. CPE is
apparent within 48 hours, and the monolayer disintegrates more or less completely during the next 4872 hours.
In the subsequent three to five passages in cell culture, the development of the CPE accelerates and the
concentration of virions increases. Titration of the virus is performed in tube cultures or on microtitre plates. A celladapted strain usually reaches TCID50 (50% tissue culture infective dose) titres of 106107 per ml.
The fluid harvest is checked for specificity using known specific hyperimmune antiserum. Treatment with 5%
chloroform and cultivation in human and bovine cell cultures and chicken embryos is used to exclude
contamination with other viruses. PTV is chloroform resistant and multiplies only in cultures of swine origin.
Immunofluorescent antibody staining is useful to detect possible contaminants that are also chloroform resistant
and propagate on cells of swine origin (e.g. parvovirus), or that are non-cytopathic. The stock virus should be
dispensed into small aliquots and preserved at 60C. Frozen virus retains its properties for several years. For
stock virus that is to be used in the neutralisation test, a constant dose of 100 TCID50 is recommended.
1148
Specific hyperimmune serum
Specific hyperimmune serum is obtained by repeated immunisation of guinea-pigs, rabbits or colostrum-deprived

piglets with PTV. Although the animals are selected from specific pathogen free breeds, they are nonetheless
tested before immunisation for absence of antibodies against PTV. The standard strains should be used. Rabbits
are immunised either intravenously, using virus suspension alone, or subcutaneously or intraperitoneally, using
the virus suspension with 10% oil adjuvant. Good results may be obtained by administering three doses of 2 ml of
virus suspension plus 0.2 ml oil adjuvant, at intervals of 2 weeks. The rabbits are bled 10 days after the last
immunisation. Piglets are immunised in the same way. The harvested sera are clarified by centrifugation and
stored in small aliquots at 20C. The sera are titrated using a neutralisation test and constant antigen. Only sera
with an antibody titre of at least 1/256 can be used for the identification of the virus.
a)
Virus neutralisation test in microtitre plates

The test is performed in flat-bottomed cell culture microtitre plates, using low passage porcine kidney or
testes cells or cell lines derived from porcine cells. Stock virus is grown in cell monolayers. The virus
harvested from cell cultures is clarified by centrifugation and stored in aliquots at 20C. Culture medium,
such as Eagles complete medium or LYH (Hanks balanced salt solution with yeast extract, lactalbumin and
antibiotics), is used as diluent. The virus harvested from cell cultures is clarified by centrifugation and stored
in aliquots at 70C or as 50/50 mixture with glycerol and can be stored at 20C.
Test procedure
i)
Inactivate swine sera for 30 minutes at 56C.
ii)
The sera to be tested are diluted in cell culture medium in twofold steps from 1/2 to 1/64, four wells per
dilution and 50 l volumes per well.
iii)
Controls include positive and negative sera, cells and medium control.
iv)
Add to each well 50 l of virus stock previously diluted in culture medium to provide 100 TCID50.
v)
Incubate for 1 hour at 37C with the plates covered. The residual virus stock is also incubated.
vi)
Make back titrations of the residual virus stock in four tenfold dilution steps using 50 l per well and
four wells per dilution.
vii)
Add 50 l of porcine kidney cell suspension at 5 105 cells per ml.
viii) After further shaking, lids are put on and the plates are incubated at 37C in a 5% CO2 atmosphere for
23 days or longer, to a maximum of 8 days.
b)
ix)
Examine the plates microscopically for CPE. The test should be validated by checking the back titration
of virus and titration of positive control serum. Virus should give a value of 100 TCID50 with a
permissible range of 30300. The standard positive serum should give a titre within 0.3 log10 units from
its predetermined mean. A negative serum should give no neutralisation at the lowest dilution tested,
i.e. 1/2.
x)
The VN results are determined by the SpearmanKrber method as the dilution of serum that
neutralised the virus in 50% of the wells.
xi)
Virus neutralisation titres are regarded as positive if the corresponding serum neutralises the virus at
an initial serum dilution of 1/8 or higher.

An alternative method for the detection and titration of specific antibodies against PTV is the ELISA
technique (Hubschle et al., 1983). The test is performed in microtitre plates using PTV grown on cell cultures
as antigen. The technique can be carried out using the following steps.
Antigen preparation
i)
Virus is propagated on monolayers of cell culture either from primary porcine kidney or testes or on an
established cell line, for example PK-15. The growth medium is removed from the cell culture and after
rinsing with buffered saline, cells are inoculated with virus suspension at a low multiplicity of infection.
After 30 min of incubation at 37C, cells are overlayed with the appropriate volume of serum-free
medium supplemented with antibiotics. Incubation at 37C is continued with daily microscopic
observations. CPE should be apparent within 48 hours, and the monolayer disintegrates more or less
completely during the next 4872 hours. A cell-adapted strain usually reaches TCID50 (50% tissue
culture infective dose) titres of 106107 per ml.
1149
ii)
The harvested virus is clarified by centrifugation at 200 g for 15 minutes, and then precipitated with a
final 50% saturated (NH4)2SO4 for 120 minutes at 4C.
iii)
After centrifugation at 2000 g, the resulting precipitate is suspended in TEN buffer (Tris-hydroxymethylmethylamine [0.01 M], ethylene diamine tetra-acetic acid [1 mM] and NaCl [0.15 M]), pH 7.4, to 1/100
of the initial volume.
iv)
The concentrated viral suspension is extracted by shaking with freon 3/1 for 10 minutes at 4C.
v)
Following further centrifugation, the supernatant is divided into two separate phases. The upper
aqueous phase, containing the viral antigen, is desalinated by passage through a 2.5 40 cm cylinder
packed with sephadex G 25.
vi)
The viral solution is finally concentrated by ultracentrifugation at 160,000 g for 3 hours.
vii)
The pellet is suspended in TEN buffer, pH 7.4, in approximately 1000th the initial volume of virus.
viii) Insoluble proteins are separated by light centrifugation, and the supernatant is used as the positive
antigen in the ELISA.
Test procedure
i)
Plates are sensitised with pre-diluted antigen in phosphate buffered saline (PBS), pH 7.2, by adding
100 l to each well. The adsorption of antigen to the surface of the plate takes place overnight at 4C.
Parallel rows of the plate should be treated with negative antigen.
ii)
The plate is washed five times in PBS to remove excess antigen.
iii)
Test sera are diluted 1/20 with PBST (PBS solution containing 0.05% Tween 20). 50 l of the diluted
sera is placed into each of two wells with positive antigen and into two wells with negative antigen.
(Negative antigen is prepared as described above except that the tissue culture is not inoculated with
virus and cells are disrupted by freezing.) The plate is incubated for 1 hour at 37C.
iv)
The plates are washed five times with PBST.
v)
A predetermined dilution of horseradish peroxidase conjugated with anti-swine immunoglobulin

prepared in rabbits is added in 50 l quantities to each well. The plates are further incubated for a
further 1 hour at room temperature.
vi)
The plates are washed five times in PBS.
vii)
Substrate solution (0.1% ortho-phenylendiamine with 0.03% hydrogen peroxide in PBS, pH 6.0) is
added in 100 l quantities to each well.
viii) After the addition of substrate, positive samples change colour to dark brown. When a sufficient degree
of colour reaction is seen in the wells of known positive sera, the reaction is stopped by addition of
50 l of 2 M sulphuric acid to each well. The absorbance of the wells is measured at a wavelength of
492 nm, preferably using an automatic multi-channel spectrophotometer with print-out mechanism.
Positive and negative sera and non-infected cells should be processed as controls in parallel with the
test specimens.
ix)
The absorbance of a serum is the mean reading of two wells with positive antigen minus the mean
reading of two wells with negative antigen. Absorbance readings of test sera that exceed by more than
twofold the mean reading of standard negative sera are regarded as positive.
Vaccines against teschovirus encephalomyelitis
During the period of highest incidence of the disease in central Europe and Madagascar, active
immunoprophylaxis was an important means for the control of this infection (Traub, 1942). As severe clinical
disease has disappeared, vaccination has been discontinued and the vaccine is no longer being produced or
used anywhere in the world.
REFERENCES
AUERBACH J., PRAGER D., NEUHAUS S., LOSS U. & WITTE K.H. (1994). Grouping of porcine enteroviruses by indirect
immunofluorescence and description of new serotypes. J. Vet. Med. [B], 41, 277282.
1150
BETTS A.O. (1960). Studies on enteroviruses of the pig. VI. The relationship of the T 80 strain of a swine
polioencephalomyelitis virus to some other viruses as shown by neutralization tests in tissue cultures. Res. Vet.
Sci., 1, 296300.
HUBSCHLE O.J.B., RAJOANARISON J., KOKO M., RAKOTONDRAMARY E. & RASOLFOMANANA P. (1983). ELISA for
detection of Teschen virus antibodies in swine serum samples. Dtsch Tierarztl. Wochenschr., 90, 8688.
KLOBOUK A. (1931). Encephalomyelitis enzootica suum. Zverolekarsky obzor, 24, 436480.
KLOBOUK A. (1933). Aetiology of the so-called Teschen disease Encephalomyelitis enzootica suum.
Zverolekarske rozpravy, 8, 8596.
KNOWLES N.J. & BUCKLEY L.S. (1980). Differentiation of porcine enterovirus sertoypes by complement fixation.
Res. Vet. Sci., 29, 113115.
KNOWLES N.J., BUCKLEY L.S. & PEREIRA H.G. (1979). Classification of porcine enteroviruses by antigenic analysis
and cytopathic effects in tissue culture: description of 3 new serotypes. Arch. Virol., 62, 201208.
KRUMBHOLZ A., DAUBER M., HENKE A., BIRCH-HIRSCHFELD E., KNOWLES N.J., STELZNER A. & ZELL R. (2002).
Sequencing of porcine enterovirus groups II and III reveals unique features of both virus groups. J. Virol., 76,
58135821.
MADR V. (1959). Propagation of the Teschen disease virus in cell cultures. Veterinarstvi, IX, 298301.
MAYR A. & BIBRACK B. (1971). Demonstration of Teschen Talfan infection using a micromodification of
neutralization test. Zentralbl. Veterinarmed. [B], 18, 657664.
MAYR A. & SCHWOEBEL W. (1957). Propagation of the Teschen disease virus in porcine kidney cell cultures and
properties of the cultured virus. 1.2.3. part. Zentralbl. Bakteriol. [I. Orig.], 168, 329359.
PALMQUIST J., MUNIR S., TAKU A., KAPUR V. & GOYAL S.M. (2002). Detection of porcine teschovirus and enterovirus
type II by reverse transcription-polymerase chain reaction. J. Vet. Diagn. Invest., 14, 476480.
ROMANENKO V.F., PRUSS O.G., BELYI YU.A. & KUPNOVSKAYA L.V. (1982). Immunofluorescent diagnosis of porcine
encephalomyelitis. Veterinariia, 4, 6972.
STANWAY G., BROWN F., CHRISTIAN P., HOVI T., HYYPI T., KING A.M.Q., KNOWLES N.J., LEMON S.M., MINOR P.D.,
PALLANSCH M.A., PALMENBERG A.C. & SKERN T. (2005). Family Picornaviridae. In: Virus Taxonomy. Eighth Report
of the International Committee on Taxonomy of Viruses, Fauquet C.M., Mayo M.A., Maniloff J., Desselberger U. &
Ball L.A., eds. Elsevier/Academic Press, London, UK, 757778.
TRAUB E. (1942). Active immunization against Teschen disease using vaccines adsorbed on aluminium hydroxide.
Arch. Tierheilkd, 77, 5266.
WITTE VON K.H., AUERBACH J., LOSS K.U., NEUHAUS S. & PRAGER D. (1994). Typisierung von 17 porzinen
Enterovirusisolaten aus Polioenzephalomyelitisfllen der Jahre 19831991. DTW Dtsch. Tierarztl. Wochenschr.,
101, 453492.
YAMADA M., KOZAKURA R., IKEGAMI R., NAKAMURA K., KAKU Y., YOSHII M. & HARITANI M. (2004). Enterovirus
encephalomyelitis in pigs in Japan caused by porcine teschovirus. Vet. Rec., 155, 304306.
ZELL R., DAUBER M., KRUMBHOLZ A., HENKE A., BIRCH-HIRSCHFELD E., STELZNER A., PRAGER D. & WURM R. (2001).
Porcine teschoviruses comprise at least eleven distinct serotypes: molecular and evolutionary aspects. J. Virol.,
75, 16201631.
ZELL R., KRUMBHOLZ A., HENKE A., BIRCH-HIRSCHFELD E., STELZNER A., DOHERTY M., HOEY E., DAUBER M., PRAGER D.
& WURM R. (2000). Detection of porcine enteroviruses by nRT-PCR: differentiation of CPE groups IIII with
specific primer sets. J. Virol. Methods, 88, 205218.
*
* *
1151
CHAPTER 2.8.11.
TRANSMISSIBLE GASTROENTERITIS
SUMMARY
Transmissible gastroenteritis (TGE) is an enteric disease of pigs caused by TGE virus (TGEV), a
member of the Coronaviridae. Since 1984, a distinct respiratory variant (porcine respiratory
coronavirus or PRCV) has spread throughout many parts of the world. This virus is probably a
deletion mutant of TGEV. PRCV does not appear to be an important primary pathogen, but it
contributes to the porcine respiratory disease complex and it has greatly complicated the diagnosis
of TGE, particularly by serological means.
Laboratory diagnosis is made by demonstrating the presence of virus, viral antigens or viral nucleic
acid in material from suspected cases, or by demonstrating virus-specific humoral antibodies.
Identification of the agent: Virus may be identified by virus isolation in tissue culture, electron
microscopy, various immunodiagnostic assays, and more recently by specific detection of viral
RNA. The most commonly employed rapid assays are probably the immunodiagnostic ones,
particularly enzyme-linked immunosorbent assays (ELISAs) on faeces and fluorescent antibody
tests on cryostat sections of intestine. Another enteric disease, porcine epidemic diarrhoea, is
caused by a serologically distinct coronavirus that nevertheless has an identical appearance under
the electron microscope. Diagnostically, immune electron microscopy circumvents this problem.
Serological tests: The most widely used methods are virus neutralisation tests and ELISAs. Only
in the latter case is differentiation from PRCV possible, as TGEV and PRCV antibodies show
complete cross-neutralisation.
Requirements for vaccines and diagnostic biologicals: There are no commercial biological
products available internationally. However, several countries practise vaccination, and in the
United States of America, licences have been issued authorising the production and distribution of
monovalent and combined vaccines.
A. INTRODUCTION
Transmissible gastroenteritis (TGE) is an enteric disease of pigs caused by TGE virus (TGEV), a member of the
Coronaviridae. Since 1984, a distinct respiratory variant (porcine respiratory coronavirus or PRCV) has spread
throughout many parts of the world and is now found in most countries where surveys for it have been conducted,
one exception being Oceania. Occurrences of TGE have become more sporadic. The disease is still reported on
an occasional basis from parts of Europe, North America and Asia. TGEV multiplies in and damages the
enterocytes lining the small intestine, producing villous atrophy and enteritis. Diarrhoea and vomiting occur in pigs
of all ages; mortality is highest in neonates. Extra-intestinal sites of virus multiplication include the respiratory tract
and mammary tissues (Kemeny et al., 1975), but the virus is most readily isolated from the intestinal tract and
from faeces. By contrast, PRCV is most readily isolated from the upper respiratory tract, the trachea, tonsils or the
lungs, and little enteric multiplication of virus occurs (Cox et al., 1990; OToole et al., 1989; Pensaert et al., 1986)
although PRCV can be detected by nested reverse-transcription polymerase chain reaction (RT-PCR) in nasal
swabs and faeces of PRCV-infected swine (Costantini et al., 2004). PRCV is probably a deletion mutant of TGEV
(Rasschaert et al., 1990) as confirmed by recent data comparing the complete 30 Kb genome sequences of
TGEV and PRCV strains (Zhang et al., 2007).
As TGE is a contagious disease that can occur as explosive epizootics, rapid diagnostic methods for its
confirmation are particularly important. The disease can also take the form of a low-level endemic problem of
post-weaning diarrhoea, which is more difficult to diagnose. The occurrence of TGEV in PRCV-immune herds
also leads to milder and sporadic clinical cases of TGEV, further complicating TGEV diagnosis in such scenarios
(Kim et al., 2000b).
1152
Chapter 2.8.11. Transmissible gastroenteritis
Possible wild and domestic animal reservoirs for TGEV have been suggested. Wild and domestic carnivores
(foxes, dogs, possibly mink) and cats seroconvert to TGEV and are suggested as potential subclinical carriers of
TGEV, serving as reservoirs between seasonal (winter) epidemics. However only virus excreted by serially
TGEV-infected dogs has been confirmed as infectious for pigs (Saif & Sestak, 2006). Based on genetic and
antigenic similarities, it has been proposed that TGEV, PRCV, feline and canine coronaviruses represent hostrange mutants of an ancestral coronavirus. Wild birds (Sturnus vulgaris) and flies (Musca domestica) have been
proposed as mechanical vectors for TGEV, excreting virus for 3272 hours, respectively (Saif & Sestak, 2006).
1.
Virus may be identified by virus isolation in tissue culture (Dulac et al., 1977), immunofluorescence, reversed
passive hemagglutination, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassay (RIA),
hybridisation with DNA probes, electron microscopy, and, more recently, by specific detection of viral RNA
(Enjuanes & Van der Zeijst, 1995; Kim et al., 2000a; Paton et al., 1997; Saif & Sestak, 2006; Sirinarumitr et al.,
1996; Woods, 1997). Molecular techniques such as RT-PCR and nested RT-PCR developed in the past few
years have increased the sensitivity and specificity of detection and differentiation of TGEV and PRCV directly
from field samples (Costantini et al., 2004; Kim et al., 2000a; 2000b; Paton et al., 1997). An alternative diagnostic
method that has been recommended for laboratories lacking facilities for specialised tests is the oral dosing of
susceptible TGEV/PRCV seronegative piglets with suspect intestinal contents. However, laboratory tests are still
required to confirm susceptibility of the pigs prior to inoculation and to show that any illness induced in these
animals is due to TGE. The most commonly employed rapid assays are probably the immunodiagnostic ones,
particularly enzyme-linked immunosorbent assays (ELISAs) on faeces (Bernard et al., 1986; Lanza et al., 1995;
Van Nieuwstadt et al., 1988b), fluorescent antibody tests (FAT) on cryostat sections of intestine (Pensaert et al.,
1968) and immunohistochemistry (IHC) on formalin-fixed, paraffin sections (Shoup et al., 1996). Detection of virus
by reversed passive hemagglutination has also been described (Asagi et al., 1986). Another enteric disease,
porcine epidemic diarrhoea (PED), is caused by a serologically distinct coronavirus that nevertheless has an
identical appearance under the electron microscope. Diagnostically, immune electron microscopy circumvents
this problem (Saif et al., 1977; Van Nieuwstadt et al., 1988a) as does the application of PED virus-specific
detection assays (Kim et al., 2001).
a)
Virus isolation in tissue culture

Apart from the inoculation of live piglets (Dulac et al., 1977), this is the most definitive method of diagnosis.
However, for routine use it is slow and laborious. TGEV does not grow well in cell culture, making this
technique impractical as a routine diagnostic procedure. Moreover isolation of TGEV from pigs in PRCV
seropositive herds is also problematic and often requires placement of TGEV/PRCV seronegative pigs in the
suspect herd to serve as sentinels, followed by collection of samples from the sentinel pigs for TGEV
isolation or detection (Costantini et al., 2004; Kim et al., 2000b). PRCV can be isolated in tissue culture
using similar cell types and techniques as for TGEV, but using nasal cells or fluids and tracheal, tonsil or
lung tissues or homogenates as the optimal specimens (Costantini et al., 2004; Pensaert et al., 1986).
For TGEV, isolation is usually attempted ante-mortem from faeces or post-mortem from the small intestine.
Loops of affected small intestine, ligated at each end to retain the contents, or mucosal impression smears
of the the small intestinal luminal surface are the preferred specimens. As the virus is heat labile, all samples
should be fresh or chilled.
Sample material is homogenised in cell culture medium or phosphate buffered saline (PBS), pH 7.2,
containing antibiotics, e.g. penicillin (1000 U/ml), dihydrostreptomycin (1000 g/ml), and mycostatin
(20 U/ml), to produce a 10% suspension. This is allowed to stand out of direct sunlight for 30 minutes at
room temperature. The suspension is then sonicated and clarified by low-speed centrifugation. The
supernatant fluid may be mixed with an equal volume of heat-inactivated bovine serum in order to reduce
the cytotoxic effect of the material and it is then used to inoculate susceptible tissue cultures, such as 3
4-day-old primary or secondary pig kidney monolayers. Other low passage porcine cultures (such as thyroid
or testis) and some cell lines (Honda et al., 1990; McClurkin & Norman, 1966) may also be used for primary
virus isolation. After incubation at 37C for 1 hour, the cell sheets are overlaid with a medium, such as
Earles yeast lactalbumin (EYL) balanced salt solution, containing sodium bicarbonate and antibiotics, e.g.
penicillin (100 U/ml), dihydrostreptomycin (100 g/ml), mycostatin (20 U/ml), and 1% fetal calf serum.
Incorporation of trypsin into the culture medium may enhance primary viral recovery (Bohl, 1979; Honda et
al., 1990). Uninoculated control cultures are established concurrently and all cultures are incubated at 37C.
Viral cytopathic effect (CPE) may be observed after 37 days, characterised by cells rounding, enlarging,
forming syncytia and detaching into the medium. Plaque formation is sometimes more reliable and easier to
recognise. A suitable plaquing overlay is 1.6% noble agar in 2 minimal essential medium with 1% NaCO3,
1153
antibiotics (as above), 0.7% neutral red and 1% DEAE (diethylaminoethyl) (100 g/ml). Wild-type TGEV
does not grow readily in tissue culture, so several subpassages may be necessary before these distinctive
changes become apparent. Cytopathic isolates must be confirmed as TGEV by immunostaining or by in-vitro
neutralisation tests using appropriate TGEV-specific antisera (Bohl, 1979). If suitable monoclonal antibodies
(MAbs) are available they can be used to distinguish between TGEV and PRCV by immunostaining methods
(Garwes et al., 1988; Simkins et al., 1992). Differentiation of TGEV from PRCV can also be accomplished by
TGEV-specific cDNA probes (Bae et al., 1991) or by discriminatory RT-PCR or nested RT-PCR (Costantini
et al., 2004; Kim et al., 2000a; 2000b; Paton et al., 1997).
b)
Fluorescent antibody test for viral antigens

The fluorescent antibody test is a rapid, sensitive and specific means of identifying TGE viral antigens in
cryostat sections of intestine. A freshly dead pig is required, and the ideal animal should be under 4 weeks
of age (preferably less than 1 week) and just starting to show clinical signs of the disease (that is, within 24
28 hours of infection). Within 30 minutes of death, 2 cm lengths from four different regions of the posterior
part of the small intestine should be removed. Lengths of 510 mm are cut from these for snap freezing with
solid CO2. Correct orientation of the material is important to ensure that subsequent cutting by cryostat
yields true transverse sections. Sections are cut 6 m thick, mounted on cover-slips, air-dried and fixed in
acetone. An alternative and faster procedure is to excise and longitudinally cut open a piece of the distal
small intestine, gently wash the mucosal surface with PBS and prepare impression smears of the luminal
intestinal surface on ethanol-cleaned microscope slides followed by air drying and acetone fixation (Bohl,
1979). The slides are then processed and stained like the cryostat sections as follows. Fixed positive and
negative control sections or smears are stored at 20C for staining in parallel. After washing with Tris
buffer, pH 8.7, or PBS, the sections are stained with a diluted solution of fluorescein isothiocyanate (FITC)conjugated TGEV antibody, and placed in a humid incubator at 37C for 30 minutes. Any unbound stain is
removed by washing in Tris buffer. If desired, the sections are counterstained with a 105 dilution of Evans
blue in Tris buffer and mounted in glycerol.
Stained sections or smears should be examined by ultraviolet light microscopy as soon as possible. The
quality of the staining is assessed by reference to the controls. An accurate interpretation depends on the
preservation of the villous architecture, the epithelial cells of which are examined for intracytoplasmic
fluorescence.
A peroxidaseantiperoxidase IHC method for the demonstration of TGEV has been developed for detection
of TGEV and PRCV in both frozen and formalin-fixed, paraffin-embedded tissues (Jean et al., 1987; Shoup
et al., 1996). The IHC applied to formalin-fixed tissues is advantageous because it can be done
prospectively or retrospectively on the same formalin-fixed tissues used for histopathology and the fixed
tissues or slides can be more readily shipped as they are stable and they do not contain live virus (Shoup et
al., 1996).
c)
Enzyme-linked immunosorbent assay detection of faecal virus antigens

A double antibody-sandwich system may be used, for instance with a capture MAb and a polyclonal
enzyme-linked detector antibody (Lanza et al., 1995; Sestak et al., 1996). This test is based on capture of
the viral antigen from the faecal sample by three MAb, two specific for the S protein (site A and D) and one
for the nucleoprotein N (Lanza et al., 1995; Sestak et al., 1996). A negative coating is used as control for the
specificity of the test, consisting of antibodies purified from the ascitic fluid of mice inoculated with SP2/0
myeloma cells that do not recognise TGEV. MAbs are applied to 96-well microplates in a bicarbonate buffer,
pH 9.6, and incubated overnight at 37C. All samples are tested in duplicate wells, one containing positive
coating (TGEV MAbs) and one containing the negative coating. Faecal samples are diluted in cell culture
medium (1/10), vortexed and centrifuged at low speed (2000 g) for 15 minutes. Then the supernatant is
decanted into sterile tubes and tested or stored frozen. Plates are washed twice with washing buffer (PBS
containing 0.05% Tween 20) before adding the prepared faecal samples. The plates are incubated overnight
at 37C. After washing four times, a biotinylated polyclonal anti-TGEV serum is added in PBS buffer
containing 0.05% Tween 20. The plates are incubated at 37C for 1 hour. The plates are washed four times
before adding a horseradish peroxidase-labelled streptavidin conjugate and incubated at 37C for 1 hour.
The plates are washed six times before adding the enzyme substrate, which is ABTS (2,2-azino-di-[3-ethylbenzthiazoline]-6-sulphonic acid) with 0.03% H2O2 in 0.1 M citrate buffer, pH 4.2. The reaction is stopped
after 30 minutes at room temperature by the addition of 5% sodium dodecyl sulphate and the absorbance
determined in an ELISA reader at 405 nm. TGEV negative and positive faecal samples are included on each
plate.
d)

In-situ hybridisation (ISH) and RT-PCR methods have been described for the direct detection of TGEV in
clinical samples, with differentiation from PRCV (Kim et al., 2000a; Paton et al., 1997; Sirinarumitr et al.,
1996). A second round of nested PCR may significantly enhance the sensitivity (Costantini et al., 2004; Kim
et al., 2000a; 2000b; Paton et al., 1998). Differentiation between TGE viruses may be achieved by analysing
1154
PCR products with restriction endonuclease enzymes (Woods, 1997) or by sequencing (Costantini et al.,
2004; Kim et al., 2000b; McGoldrick et al., 1999; Paton & Lowings, 1997; Zhang et al., 2007). Duplex RTPCR for the combined detection of TGEV and porcine epidemic diarrhoea virus has been described (Kim et
al., 2001).
2.
Serological tests
Serology may be diagnostic if a rising titre of antibody can be demonstrated. In addition, a single seropositive
result has diagnostic value if collected from a population previously known to be seronegative. As the possibility of
acquiring carrier virus status among pigs can be reduced by accepting only seronegative animals, serological
testing is also a common precondition for importation.
Following infection with TGEV or PRCV, viral antibodies can be detected in serum from 6 or 7 days post-infection,
and such antibodies persist at least for many months. Although PRCV and TGEV antibodies show complete
neutralisation of either virus, there are differences in the specificities of some of the non-neutralising antibodies
(Callebaut et al., 1988; Enjuanes & Van der Zeijst, 1995; Garwes et al., 1988; Saif & Sestak, 2006; Simkins et al.,
1992), as PRCV lacks certain epitopes present on the TGEV. However, virus neutralisation (VN) is not a practical
method to differentiate PRCV from TGEV infection. MAbs to such regions can be incorporated into competitive
ELISAs to detect serum antibody that is entirely TGEV specific. While such tests are reliable in that they do not
produce false-positive results with PRCV antisera, false negatives may occur because of a reduced sensitivity
compared with neutralisation tests, and because of strain variation among TGE viruses, such that a single TGEVspecific MAb may not recognise all strains (Brown & Paton, 1991; Simkins et al., 1992). The problem of
insensitivity can be reduced by using the tests on a group or herd basis. These MAb-based ELISAs are the
method of choice for differentiating PRCV from TGEV to qualify animals for export.
In addition, using such tests for differential diagnosis less than 3 weeks after exposure to PRCV produced
inconsistent and unreliable results (Sestak et al., 1999b). More accurate results were also achieved by testing
paired serum samples (acute and convalescent) in the assays and by using the recombinant spike (S) protein of
TGEV as the coating antigen in place of TGEV-infected, fixed swine testicular cells (Sestak et al., 1999b).
a)
Transmissible gastroenteritis virus/porcine respiratory coronavirus tests

These tests detect antibody to both TGEV and PRCV, and include VN tests, indirect ELISAs (Hohdatsu et al.,
1987; Huang et al., 1988; Liu et al., 2001; McGoldrick et al., 1999; Rukhadze et al., 1989) and competitive
ELISAs based on TGEV/PRCV group-specific MAbs (Paton et al., 1991).
VN tests can be performed with a variety of cell types and viral strains. Commonly used cell lines include
swine testes (McClurkin & Norman, 1966) or primary or continuous porcine kidney cells. Such tests have
been very widely used for many years and are commonly regarded as standards against which to assess
new assays. A plaque reduction VN assay using swine testes cell monolayers in 6-well plastic plates and the
atttenuated Purdue strain of TGEV is commonly used (Bohl, 1979). A modification of the method of Witte
(Witte, 1971) described below, uses flat-bottomed tissue-culture grade microtitre plates, a cell line of A72
cells derived from a dog rectal tumour, and a field strain of virus adapted to grow in such cells: 100 TCID50
(50% tissue culture infective dose) of virus is incubated with heat-inactivated test sera, and neutralisation is
indicated by absence of CPE after further incubation with A72 cells in Leibovitz 15 medium (Sigma, United
Kingdom) with added antibiotics, 10% fetal calf serum and 1% L-glutamine. The total volume of reagents in
all wells should be 150 l.
Virus neutralisation: test procedure
i)
Sera are inactivated for 30 minutes in a water bath at 56C.
ii)
Doubling dilutions of test sera are made in cell culture medium beginning with undiluted serum (this
gives a neutralisation stage dilution of 1/2 when mixed with an equal volume of virus). The dilutions are
prepared in a 96-well flat-bottomed cell-culture grade microtitre plate using, optimally, three wells per
dilution and 25 l volumes per well. Positive and negative control sera are also included in the test. No
standard sera are available, but internal positive standards should be prepared and titrated in an
appropriate range.
iii)
25 l TGEV stock is added to each well at a dilution in culture medium calculated to provide
100 TCID50 per well. Virus should be added to two out of the three wells containing serum at each
dilution. The third well serves as a serum-only control and should receive 25 l per well of culture
medium instead of virus.
iv)
The residual virus is back titrated in four tenfold steps using 25 l per well and at least four wells per
dilution; 25 l of culture medium is added to each of the back-titration wells to compensate for the
absence of a test serum.
v)
The plates are agitated briefly and then incubated for 1 hour in a 5% CO2 atmosphere at 37C.
vi)
100 l of, for example, A72 cell suspension at 2 105 cells per ml is added to each well.
1155
vii)
The plates are incubated for 37 days in a 5% CO2 atmosphere at 37C; the test can be performed
successfully, if the plates are incubated without CO2.
viii) The plates are read microscopically for CPE. The test is validated by checking the back titration of virus
(which should give a value of 100 TCID50 with a permissible range of 50200 TCID50) and the control
sera. The standard positive serum should give a value within 0.3 log10 units either side of its
predetermined mean. Readings of each test serum dilution should be made with reference to the
appropriate serum-only control to distinguish viral CPE from serum-induced cytotoxicity or
contamination.
b)
ix)
The test serum results are determined by the SpearmanKrber method as the dilution of serum that
neutralised the virus in 50% of wells.
x)
A negative serum should give no neutralisation at the lowest dilution tested (i.e. undiluted serum,
equivalent to a dilution of 1/2 at the neutralisation stage).
Transmissible gastroenteritis virus-specific tests to differentiate TGEV- from PRCV-infected pigs

TGEV-specific tests are blocking or competition ELISAs that use an MAb that recognises TGEV but not
PRCV (Brown & Paton, 1991; Callebaut et al., 1989; Sestak et al., 1999b; Simkins et al., 1992; Van
Nieuwstadt & Boonstra, 1991) and are the tests of choice for qualifying animals for export. Test sera from
pigs previously infected with a strain of TGEV recognised by the MAb will contain antibodies of the same
specificity that can compete with it for binding to TGEV antigen-coated ELISA plates. Pigs infected with
PRCV that does not contain the TGEV unique epitope will not produce antibodies to this epitope; hence,
PRCV antibodies will not compete with or block binding of the TGEV-specific MAb (Brown & Paton, 1991;
Callebaut et al., 1989; Sestak et al., 1999b; Simkins et al., 1992; Van Nieuwstadt & Boonstra, 1991). ELISA
antigens may be prepared from cell lysates of kidney cell lines that were either inoculated with tissueculture-adapted strains of TGEV, or uninfected. Alternatively TGEV-infected or uninfected swine testes cells
fixed in 80% acetone have been used as an antigen source, or antigens may be prepared from recombinant
S (rec-S) protein harvested in soluble form from an insect (Sf9) cell line infected with a recombinant
baculovirus expressing a TGEV S protein containing the four major antigenic sites (Sestak et al., 1999b;
Simkins et al., 1992). Positive and negative antigens are coated to alternate rows of microtitre plates using
bicarbonate buffer, pH 9.6. Diluted test sera, including known TGEV positive and known TGEV/PRCV
negative controls, as well as known PRCV positive (negative in this test, positive in VN test) are added to
appropriate wells and incubated overnight before further addition of diluted MAb to all wells. Bound MAb is
detected by a peroxidase-conjugated anti-mouse antibody that induces a colour reaction in the presence of
an appropriate substrate. The colour changes are measured using spectrophotometer, and for each test
sample the net result is the difference in absorbance between the positive and negative antigen wells,
expressed as a percentage of the result obtained with the negative control serum. The negativepositive cutoff value for the test must be determined by previous testing of known negative and positive populations.
There are several commercial kits available that are TGEV specific.
Haemagglutination-based tests described to date (Labadie et al., 1977; Noda et al., 1987; Shimizu &
Shimizu, 1977) were validated before the appearance of PRCV. However, they may be TGEV specific as
TGEV, but not PRCV, is haemagglutinating (Schultze et al., 1996).

Vaccination against TGE is carried out in several countries.
Information on experimental work or field trials of TGEV vaccines licensed for use in the United States of America
(USA) has been reviewed, including possible limitations in their field efficacy and concepts related to the design of
optimal TGEV vaccines (Saif, 1993; Saif & Jackwood, 1990; Saif & Sestak, 2006). Several manufacturers are
licensed to produce TGEV vaccines in the USA: the vaccines include modified live and inactivated vaccines. The
modified live vaccines are used for oral administration to pregnant sows (to induce passive immunity) or have also
been licensed for oral administration to nursing or weaned pigs (to induce active immunity). Inactivated TGEV
vaccines are licensed for parenteral inoculation of pregnant sows by the intramuscular route or for intraperitoneal
administration to nursing or weaned pigs. In general, these vaccines induced marginal passive protection against
TGEV challenge of nursing piglets when evaluated under controlled experimental conditions or in the field in
TGEV/PRCV herds. Although they fail to adequately protect against epizootic TGE, data suggest that these
vaccines may provide some efficacy against enzootic TGEV by stimulating an anamnestic antibody response to
TGEV in serum and milk (Saif & Jackwood, 1990; Saif & Sestak, 2006).
1156
The main reason proposed for TGEV vaccine failures was their inability to stimulate high levels of secretory IgA
(SIgA) antibodies in milk analogous to the SIgA antibody responses found in the milk of sows naturally infected
with TGEV (Saif & Jackwood, 1990; Saif & Sestak, 2006). Furthermore, these vaccines did not adequately protect
the seronegative sow against TGE, such that illness in the sow often resulted in anorexia, agalatica and failure to
passively protect her piglets. Thus the modified live vaccines may fail to replicate to the extent required to induce
protective immunity in the intestine, or if given to seronegative neonatal animals, concerns exist regarding their
possible reversion to virulence. Killed vaccines given parenterally do not induce SIgA antibodies; cell-mediated
immune responses are often poor and the duration of immunity may be short-lived. Although use of PRCV strains
as vaccine candidates for TGE has been proposed, experimental studies regarding their efficacy against TGEV
have shown a lack of efficacy (Paton & Brown, 1990) or only partial cross-protection (Bernard et al., 1989; Cox et
al., 1993; Van Cott et al., 1994). However, the widespread prevalence of PRCV infections in the swine population
in Europe appears to have dramatically reduced the incidence of epizootic TGE in Europe (Pensaert et al., 1986).
Newer recombinant DNA strategies for the development of TGEV vaccines include the possible use of an S
protein subunit vaccine (contingent upon the development of mucosal delivery systems and adjuvants) (Park et
al., 1998; Sestak et al., 1999a; Shoup et al., 1997) or the use of live recombinant viral or bacterial vectors that
express TGEV genes important for the induction of immunity (Enjuanes et al., 2001; Saif, 1993; Saif & Sestak,
2006; Smerdou et al., 1996; Torres et al., 1996; Yount et al., 2000).
There are a number of general requirements (e.g. produced in a licensed facility, label rules, tracking capability,
etc.) that apply to all biological products including vaccines. A set of regulations exist (called standard
requirements, or SRs) that describe testing to be done on the vaccine and parent materials. Detailed information
on SRs for vaccines in the USA are contained in the Code of Federal Regulations (CFR) Title 9, Volume 1, Part
113 (abbreviated below as 9 CFR, 113) (United States Department of Agriculture [USDA], 1995). The general
European Pharmacopoeia monograph and EMEA (European Medicines Agency) guidelines are applicable to
TGEV vaccines, even though no vaccines are currently used in the European Union.
1.
Seed management
a)

The seed virus must be tested for purity and identity. The purity includes freedom from bacteria and fungi
(9 CFR 113:27), mycoplasmas (9 CFR 113:28), and extraneous viruses (9 CFR 113:55) (USDA, 1995). The
demonstration of identity is usually accomplished by VN or FAT. Genetically engineered vaccines or
naturally selected vaccines with claims of antigen-coding gene deletion/inactivation are required to provide
evidence (genotypic and/or phenotypic) of that identity.
b)
Method of culture
Culture must be carried out on proven uncontaminated (approved) cells, and the number of cell culture
passages is limited (usually to five). It is not required that the species of origin of the cell line be that of the
target species.
c)
Vaccine validation takes two forms. The master seed is considered to be immunogenic if a vaccine made at
the highest passage, and according to the outline of production, is shown to be protective. The lowest
antigenic level (modified live virus titre or inactivated antigen mass) shown to be protective becomes the
baseline for all future serials (lots) of the product. In the case of live products, factors for titration variation
and the death curve over time would be added. These trial vaccines should be tested for purity, safety, and
efficacy by the manufacturer. Protection must be shown against the natural disease with the virulent
challenge virus. Virulent challenge virus is defined as the dose that causes disease in 95% of the
susceptible controls. Three prelicense serials must subsequently be made and tested by the manufacturer
and by the licensing authority, for potency, sterility and safety.
2.
This is proprietary information for each manufacturer and hence not available.
3.
In-process control
This is largely proprietary. Some in-process controls refer directly to production (e.g. O2 concentration in the
fermenter). Another category, however, includes tests similar to the final container potency test. For all vaccines,
the simpler the final batch or container potency test, the more likely it is that it may be used as a
monitoring/blending test: for example, virus titration on sub-batches may be used to predict final blended batch
titre. Ingredients of animal origin must be sterilised or shown to be free from contamination.
1157
4.
Batch control
Batches must be blended to the final specifications and bottling specifications (e.g. fermentation runs may be
pooled, or one run may be split and pooled with each of three others, etc.). In some countries, bulk and process
control define the product and are the subject of intense regulation and scrutiny. The emphasis in the USA is on
the final product. Batch control techniques must be detailed in the outline of production and must be meaningful,
trackable, and the manufacturer must discard product that fails to meet specifications. If a batch is to be exported
to another country for bottling or blending, then it is subjected to all the testing as though it were final product.
a)
Sterility
All products must be tested for sterility. The manufacturer may also run sterility tests on batches for
monitoring. Tests are similar to those described in Section C.1.a.
b)
Safety
Safety tests are done before the licence is granted, and then on the final container (Sections C.5.a and
C.5.b).
c)
Potency
Potency would normally only be done if the potency test were a simple test (e.g. ELISA) to confirm the
blending calculations before bottling.
d)
Duration of immunity is tested in the prelicence (efficacy) serial test, not the batch control. New products are
required to support label claims for revaccination schedules with efficacy trials (challenge) at the specified
time after vaccination.
e)
Stability
Stability is established before the licence is granted. Usually accelerated ageing (37C) is used to estimate
the lifetime so that the products do not have to be kept at storage temperature (4C) for the real-time period.
This will be confirmed with real-time data later. The manufacturer is not required to do stability testing.
Manufacturers are required to state the amount of antigenic material that will be in their product throughout
the shelf life. Samples of product are selected (usually live) and tested within 30 days of expiration to see if,
for example, the titre is at the level stated in the manufacturers outline. Stability is also affected by moisture.
Moisture left in a desiccated product can shorten its life, so this has to be tested in the final product or inprocess.
f)
Preservatives
There are restrictions on the maximum amounts of antibiotics that can be in a product. Restrictions on some
vaccine components are related to their safety and to whether the stated withdrawal period is long enough
for the component to have cleared before the animal is slaughtered. Preservatives used are proprietary.
g)
Any risks to vaccinates need to be clearly stated on the label. This usually applies to pregnancy warnings for
abortogenic live viruses, and the general anaphylaxis warning, but may also attempt to warn the user about
soreness or swelling at the injection site, or transient fever or inappetence in some cases. No unusual label
precautions apply to the TGE vaccines currently licensed.
5.
a)
Safety
Usually this will be a mouse and/or a guinea-pig or swine safety test (9 CFR 113:33, and Witte, 1971).
Sterility tests are also carried out on the final product.
b)
Potency
There is no single test for release potency. Whatever test is used must be correlated to protection in the host
animal (the efficacy tests). The potency of live TGEV vaccines can be evaluated by in-vitro titration of the
viral infectious dose in cell culture (Saif, 1993). This titre must be correlated with the minimum viral titre
required to induce protective immunity against experimental challenge, and also against natural challenge
1158
under field conditions. The potency of killed vaccines is evaluated by vaccination and challenge tests using
different doses of the vaccine. Titres of neutralising antibodies induced by inoculation of laboratory animals
with the vaccine may be accepted if there is an established correlation with development of protective
immunity.
Particular viral antigens associated with the induction of neutralising antibodies and protection against
challenge can be quantified in killed vaccines using specific MAbs in ELISA, such as neutralising MAbs to
the S protein of TGEV (Saif, 1993).
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NODA M., YAMASHITA H., ICOIDE F., KODOI K., ORNON T., ASAGI M. & INABA Y. (1987). Haemagglutination with
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*
* *
NB: There is an OIE Reference Laboratory for Transmissible gastroenteritis
diagnostic tests, reagents and vaccines for transmissible gastroenteritis
1162
SECTION 2.9.
OTHER DISEASES1
CHAPTER 2.9.1.
BUNYAVIRAL DISEASES OF ANIMALS

(excluding Rift Valley fever)*
SUMMARY
A part of the large complex group of arboviruses is the Bunyaviridae family with over 300 members
distributed over five genera. Two of the genera of veterinary importance in the family are Nairovirus,
which contains the ruminant pathogen Nairobi sheep disease (NSD) virus, and the largest genus,
Orthobunyavirus, which is subdivided into 48 serogroups. This genus contains only a few viruses
that are significant pathogens of animals, among them Cache Valley virus (CVV) and Akabane
virus. Although they have been placed in different antigenic groups, these two viruses have a
tropism for fetal tissues and are responsible for embryonic and fetal losses and multiple congenital
deformities in domestic ruminants. Members of the Nairovirus and Orthobunyavirus genera are
single-stranded, enveloped spherical or pleomorphic RNA viruses, 80110 nm in diameter. A third
member of the Bunyaviridae family that is of veterinary importance is Rift Valley fever, a member of
the Phlebovirus genus, and is discussed in Chapter 2.1.14 Rift Valley fever.
Identification of the agent:
Cache Valley virus (CVV), a member of the Bunyamwera virus serogroup of the Orthobunyaviridae
genus, can be isolated from the blood of febrile or viraemic adult animals. Attempts at isolation from
the fetus at birth are generally unsuccessful due to virus clearance by the fetal immune response.
Cell lines derived from monkey or baby hamster kidney are employed for isolation of the virus or
alternatively, intracerebral inoculation of infant mice may be used. Virus or antigen is identified by
fluorescence (FAT), immunohistochemistry (IHC) or neutralisation tests. Group- and virus-specific
polymerase chain reaction (PCR) techniques have been developed for the Orthobunyaviruses.
Akabane virus can be isolated from the blood of viraemic animals and occasionally from fetal
material. Cell lines of monkey, baby hamster and mosquito are used. The virus produces
deformities in the developing chicken embryo. Yolk sac inoculation, as well as intracerebral
inoculation of suckling mice, is used. Virus or antigen is identified by FAT, IHC or neutralisation
tests. Nested and multiplex real-time reverse-transcriptase PCR techniques have been developed
for Akabane and related viruses.
Nairobi sheep disease (NSD) virus is best isolated from plasma from febrile animals, mesenteric
lymph nodes or spleen. Laboratory-reared sheep, 24-day-old unweaned mice inoculated
intracerebrally, or cell cultures may be used for primary isolation. Sheep are the most sensitive
animals for this, whereas a baby hamster kidney cell line and lamb or hamster kidney cell cultures
are the most sensitive cells. Subinoculation of plasma from an experimentally infected sheep into
cell cultures or mice is also recommended. Identification of the virus may be made by direct
immunofluorescence of inoculated tissue cultures or of mouse brain smears. The agar gel
immunodiffusion test can also be used to demonstrate the presence of NSD antigen in tissues but
1
The diseases in this section that are marked with an asterisk are included in some individual species sections of the OIE
List, but these Terrestrial Manual chapters cover several species and thus give a broader description.
1163
Chapter 2.9.1. Bunyaviral diseases of animals (excluding Rift Valley fever)
will cross react with other viruses of the Nairovirus genus. Infected tissue cultures or mouse brain
suspensions may be used as sources of complement-fixing or enzyme-linked immunosorbent
assay (ELISA) antigens.
Serological tests: For CVV and Akabane virus, haemagglutination inhibition, complement fixation
(CF) and serum neutralisation tests are used to detect antibodies. An ELISA, based on one used
for Rift Valley fever, has also been described. A competitive ELISA specific for Akabane has been
published. For NSD the most suitable test is the indirect fluorescent antibody test. CF and indirect
haemagglutination tests have also been used to confirm NSD outbreaks in the field. Virus
neutralisation tests give equivocal results, a feature that also occurs with other members of the
Nairovirus group. ELISAs are now being evaluated for NSD. Infected spleen may be used as a
source of antigen in immunodiffusion tests.
Requirements for vaccines and diagnostic biologicals: No vaccine is currently available for
CVV. Vaccines for domestic use against Akabane virus have been produced in both Japan and
Australia. For NSD, an experimental attenuated live virus vaccine has been investigated, and a
killed tissue culture vaccine has been shown to be immunogenic.
A. INTRODUCTION
Cache Valley virus
Cache Valley virus (CVV) is a teratogenic Orthobunyavirus of North America affecting mainly sheep. A recent
survey of cattle indicated that up to 28% were positive for specific antibodies to CVV (Sahu et al., 2002). It is a
member of the Bunyamwera serogroup of the Orthobunyavirus genus, family Bunyaviridae (Fauquet et al., 2005)
and is also the most common of the Orthobunyaviruses of North America (Calisher et al., 1986). CVV was first
isolated from a mosquito pool in Utah, United States of America (USA) in 1956 (Holden & Hess, 1959), but was
only linked to disease during an epizootic of neonatal loss and malformed lambs in a sheep flock in Texas in 1987
(Crandell et al., 1989). The virus has also been isolated from a horse and a clinically healthy cow.
Serological surveys have shown a widespread prevalence of antibodies in domestic and wild ruminants and
horses. Seroprevalence to CVV is high in deer, and the 13 day viraemia is sufficient to infect vectors allowing
deer to act as amplifying hosts (Blackmore & Grimstad, 1998). Vectors include both Culicoides midges and
mosquitoes of the Aedes, Anopheles, Coquillettidia and Culiseta groups.
CVV infection of adult animals is largely subclinical, and experimentally infected ewes show only a transient
febrile response, but with a detectable viraemia. Human disease has been reported on two occasions (Campbell
et al., 2006; Sexton et al., 1997).
CVV was the first North American Orthobunyavirus to be linked to fetal arthrogryposis and hydrancephaly,
however other related viruses have been shown experimentally to have the same potential. Fetal infection with
CVV is age dependent in its outcome. Malformations take place between 27 and 45 days gestation, with infection
at 2836 days giving rise to central nervous system (CNS) and musculoskeletal defects, and infection at 37
42 days giving rise to musculoskeletal deformities only. Infection after 50 days gestation does not result in lesions
and after 76 days the fetus is immunocompetent and antibodies are produced. Most CVV fetal deaths occur
between 27 and 35 days gestation. The fetus is, however, susceptible at any age demonstrating the tropism of
many Orthobunyaviruses for fetal tissues (Chung et al., 1990).
Gross pathology of the musculoskeletal system includes arthrogryposis of one or more limbs, torticollis, scoliosis
of the vertebral column and muscular hypoplasia. CNS lesions include hydrancephaly, hydrocephalus,
porencephaly, microencephaly, cerebral and cerebellar hypoplasia and micromelia (Chung et al., 1990; Edwards
et al., 1997). Dead embryos and stillborn or mummified lambs with no obvious defects are also found. Anasarca is
seen, as is oligohydramnion. This reduction in amniotic fluid is thought to contribute to restriction of fetal
movement and thus to the skeletal deformities seen. Limb defects are also due to neurodegenerative changes
seen histopathologically as areas of necrosis and loss of paraventricular neuropil in the brain together with a
reduction in the number of motor neurons. Skeletal muscle changes involve poorly developed myotubular
myocytes (Edwards et al., 1997).
Akabane virus
Akabane virus is a teratogenic Orthobunyavirus widely distributed across the world but not in New World
countries. It affects mainly cattle. It is a member of the Akabane serogroup, Orthobunyavirus genus, family
1164
Bunyaviridae (Karabatsos, 1985). Other Orthobunyaviruses that are potential pathogens are Aino (Shuni virus
seogroup), Peaton (Shamonda virus serogroup), Douglas (Sathuperi virus serogoup) and Tinaroo (Akabane virus
serogroup). Akabane virus is however, the best studied and most pathogenic of the Orthobunyaviruses and a
major cause of arthrogryposis and hydranencephaly.
Akabane virus was first isolated in Japan in 1959, initially from a mosquito pool and then a pool of Culicoides
midges. This was followed in 1972 by isolations from Culicoides in Australia and mosquito pool isolations in
Africa. Akabane virus antibodies have been demonstrated in sera from cattle, sheep, goats, horses, buffalo and
camels. Many indigenous game species in Africa south of the Sahara have Akabane virus neutralising antibodies.
The range of Akabane virus includes the Middle East, Asia, Cyprus and Africa, but it is in Australia and Japan
where regular epizootics of Akabane virus disease occur. Conditions favourable to such outbreaks are
susceptible animals in early pregnancy and a sudden increase in vector populations, particularly when the virus
has been absent from the area for a number of years.
Akabane virus infection in adult animals is usually subclinical, but encephalomyelitis has been recently associated
with Akabane virus infection in adult cattle (Lee et al., 2002). Cattle seroconvert after a 34-day viraemia.
In endemic areas, antibody in the female animal prevents fetal infection, but Akabane virus is capable of
establishing a long-term infection of the placenta in susceptible cattle and sheep. This takes place between
30 and 70 days gestation in the ewe and between 30 and 150 days gestation in the cow. Akabane virus has a
predilection for brain, spinal cord and muscle cells where non-inflammatory necrosis interferes with
morphogenesis.
Akabane virus infection has been studied experimentally in sheep and goats with the production of
arthrogryposis/hydroencephaly, kyphosis, scoliosis, micro- and porencephaly, stillbirths and abortions (Parsonson
et al., 1975). Natural infection of the ovine fetus has been described in Australia where perinatal lamb mortality
and congenital microencephaly were most often seen.
Experimental Akabane virus studies have been carried out in pregnant cattle and it was shown that the type of
abnormality is dependent on the gestational age of the fetus with hydroencephaly seen at 76104 days and
arthrogryposis at 103174 days gestation (Kirkland et al., 1988). This time differential in appearance of
abnormalities is clearly seen in bovine fetuses, whereas in sheep with a shorter gestation period, brain and
skeletal lesions appear concurrently in the same fetus. The sequence of events during an epizootic of Akabane
virus-induced fetal loss are the birth of uncoordinated calves, followed by those with arthrogryposis and dysplastic
muscle changes, and lastly those with hydrocephalus and other severe CNS lesions. These events may be
preceded by stillbirths and abortions (Shepherd et al., 1978). Akabane virus is responsible for severe neural and
muscular abnormalities and lesions are characterised by a nonpurulent encephalomyelitis, focal cerebral
degenerative encephalomyelopathy porencephaly, microencephaly, hydrocephalus, loss of ventral horn motor
neurons and axons, depletion of myelin in spinal cord motor tracts, necrosis and polymyositis in the myotubules
with parenchymal degeneration of skeletal muscles. Spinal cord abnormalities include scoliosis, and kyphosis and
arthrogryposis may affect almost any skeletal joint.
Nairobi sheep disease (NSD) is a disease of sheep and goats caused by a Nairovirus of the family Bunyaviridae
(Davies, 1988). It is characterised by a mortality rate, which may range between 40% and 90%, and should
always be suspected when animals have recently been moved from an area free from the disease into one where
it is endemic. Outbreaks also follow incursions of ticks into previously free areas, particularly following heavy rains
(Davies, 1997). The clinical signs are similar in both sheep and goats, although there are differences in
susceptibility among the various breeds and strains in their response to infection with NSD virus, some being
more susceptible than others. Cattle and game are refractory to infection with NSD virus (Zeller & Bouloy, 2000).
The incubation period for the disease varies from 2 to 5 days, when a temperature reaction of 4142C develops.
There is hyperventilation accompanied by severe depression, anorexia and a disinclination to move. Animals
stand with lowered head, and show a conjunctivitis and sero-sanguinous nasal discharge. Some of the superficial
lymph nodes, such as the prescapular and/or precrural, become palpable. Diarrhoea usually develops within 36
56 hours of the onset of the febrile reaction. This is at first profuse, watery and fetid, later haemorrhagic and
mucoid, and accompanied by colicky pains and tenesmus. Abortion is a common sequel to the infection.
Examination of the predilection sites for the attachment of ticks, such as the ears, head and body, is likely to
reveal the presence of the Ixodid tick Rhipicephalus appendiculatus.
Deaths can occur in peracute cases within 12 hours of the onset of the fever and at any time during the febrile
reaction, while the animal is acutely ill. Further deaths then follow the fall in temperature for a further 37 days,
associated with severe diarrhoea and dehydration.
The gross pathology of NSD can be misleading, for most deaths are likely to occur during the period of viraemia,
when the only signs are likely to be lymphadenitis with petechial and ecchymotic haemorrhages on the serous
1165
surfaces of the alimentary tract, spleen, heart and other organs. None of these signs allows a specific diagnosis of
NSD to be made or even suspected, for they are shared with many other febrile diseases of sheep in NSDendemic areas. Diseases with which NSD may be confused include Rift Valley fever, peste des petits ruminants,
rinderpest, salmonellosis and heartwater. Later in the course of the disease, a haemorrhagic gastroenteritis
becomes more obvious, with haemorrhages on the mucosa of the abomasum, especially along the folds, in the
region of the ileo-caecal valve, and most commonly in the colon and rectum. Zebra striping of the latter is often
seen. The gall bladder is usually enlarged and haemorrhagic. Inflammatory lesions with haemorrhage may be
seen in the female genital tract, if there has been abortion. However, in many animals dying from NSD, there may
be none of these gastroenteric lesions, and a tentative diagnosis based on post-mortem signs can rarely be
made. Common histopathological lesions are myocardial degeneration, nephritis and necrosis of the gall bladder.
The post-mortem signs following the early stage of NSD death are the nonspecific changes of congestion and
petechial and ecchymotic haemorrhages on serous surfaces, on lymph nodes, the spleen and other organs such
as the kidney, lungs and liver. Following death at later stages, haemorrhagic gastroenteritis becomes apparent,
with ulceration of the abomasum, duodenum, caecum and colon. The virus is principally transmitted by the tick
Rhipicephalus appendiculatus, and any infestation with such parasites should arouse suspicion of the presence of
the disease. NSD virus may also be transmitted by other species of the genus Rhipicephalus and by the bont tick
Ambylomma variegatum.
NSD is an apparently rare zoonotic agent in the field, causing mild influenza-like disease in humans. Laboratory
infection has been associated with fever and joint pains (Zeller & Bouloy, 2000).
1.
Cache Valley virus
At birth CVV cannot be isolated from the fetus but has been isolated from mosquito pools and the blood of
viraemic adult animals. This has been done on tissue culture using hamster and monkey kidney cell lines
including baby hamster kidney (BHK), African green monkey kidney (Vero) and LLC-MK2. Virus can be isolated
from a febrile animal using a 10% buffy coat suspension in minimal essential medium (MEM) and co-cultivation
with Vero cells in MEM supplemented with 2% fetal bovine serum.
Virus isolation is also commonly done in newborn or weaned mice by intracerebral or intraperitoneal inoculation.
Many Orthobunyaviruses have been sequenced as they are medically important pathogens associated with
encephalitis in humans in both North and South America. Polymerase chain reaction (PCR) technology has been
applied to mosquito-pool surveillance, instead of traditional isolation in infant mice, and sensitivity is reported to
be one positive mosquito in a pool of 100, which is undetectable by traditional plaque titration in cell culture
(Huang et al., 2001).
Group-specific and virus-specific primers have been designed, and using the reverse-transcription PCR (RTPCR) the Bunyamwera (BUN) and California (CAL) serogroup viruses can be distinguished. Using a nested RTPCR technique the CAL and most of the BUN serogroup viruses can be distinguished from other Orthobunyavirus
genus members (Kuno et al., 1996; Moreli et al., 2001).
Akabane virus
Diagnosis of infection is rarely made by virus isolation, but rather by histopathology and serology. Virus has
however been isolated from viraemic sentinel animals using plasma or buffy coat suspensions from vector pools
and occasionally from fetal material. RT-PCR has been described for the detection of Akabane virus and
differentiation from Aino. Use of this assay could contribute to diagnosis, but the diversity of the Orthobunyavirus
genus will require validation to confirm specificity of the test as there is evidence of reassortment.
Suckling mice, 12 days old, may be used and inoculated intracerebrally with 0.01 ml of a clarified 10%
suspension of the test material. A sensitive system for recovery of this serogroup of viruses is intravenous
inoculation of embryonating chick eggs (ECE), followed by passage of harvested material in insect cells (C6/36)
then mammalian cell lines (Gard et al., 1988; Weir, 2003). Virus isolation in tissue culture is frequently done using
Vero, BHK-21 and HmLu-1 cell lines. If C6/36 mosquito cells are used, cultures are left stationary for 7 days and
material is repassaged on to a hamster or Vero cell line where cytopathic changes in the cultures become visible.
1166
Methods employed for specific identification of Akabane virus using monospecific antibodies have included virus
neutralisation, and immunofluorescence (Blacksell et al., 1997; Gard et al., 1988). Antigen detection in formalinfixed material by peroxidase staining of bovine and ovine fetal material also in naturally infected newborn calves
(Noda et al., 2001). Nucleic acid detection methods have also been developed for the differentiation of Aino and
Akabane viruses using a nested RT-PCR technique followed by restriction enzyme digests to differentiate
Akabane and Tinaroo viruses in the same serogroup and Aino and Peaton viruses in the Shuni and Shamonda
serogroups (Akashi et al., 1999). A multiplex real time RT-PCR has also been described using Taq Man probes
which is said to reliably identify akabane and Aino viruses accurately (Stram et al., 1978).
Nairobi sheep disease virus
NSD virus may be isolated from material collected from field cases by the use of laboratory animals or cell
cultures (Davies et al., 1977a). Safety precautions against aerosol infections should be taken when working with
this agent. Uncoagulated blood, mesenteric lymph nodes and spleen tissue submitted with frozen gel packs are
the optimal samples to be collected from febrile or dead animals. The plasma can be used directly as inoculum,
and the lymph nodes or spleen should be homogenised to make an approximate 10% (w/v) suspension in a
transport medium. This medium can be Hanks medium with 0.5% lactalbumin hydrolysate or 0.75% bovine
serum albumin, and containing penicillin (500 International Units/ml), streptomycin sulphate (500 g/ml), and
mycostatin (50 units/ml) or fungizone (2.5 g/ml).
A recommended initial procedure is to inoculate an NSD-susceptible sheep held in isolation with 12 ml of the
tissue suspensions or plasma. Any pyrexia and clinical disease that develops permits a tentative diagnosis of
NSD and, at the same time, furnishes excellent samples for virus isolation. This is especially valuable where the
original field samples have been transported in hot climates where some loss of virus has inevitably occurred.
Sheep are at least 100 times more sensitive than mice to NSD virus infection.
Infant mice, 24 days old, can be inoculated intracerebrally with 0.01 ml of a 1/10 dilution of plasma or of the
tissue suspension. Two litters, each of 810 suckling mice, should be used for each sample and one blind
passage is made routinely. The mice become debilitated and die within 59 days post-inoculation. Their brains
should be harvested aseptically, pooled and diluted 1/100 for passage into additional mice.
Cell cultures may be used in conjunction with mouse inoculation for the primary isolation of NSD virus, as they
have shown levels of sensitivity similar to that of the intracerebral inoculation of unweaned mice. The BHK-21C13 cell line is especially valuable; the Vero cell line (Shepherd et al., 1978) and primary and secondary lamb or
hamster kidney cells have also been used. Most strains of NSD virus produce a cytopathic effect (CPE) on first
passage in BHK cells; others produce a more obvious CPE only after subinoculation. The appearance of a CPE is
not such a regular finding with lamb testis and kidney cells, although it is usually seen on the second passage in
lamb kidney cells. Tube cultures should be used both with and without flying cover-slips, or if plastic bottles are
used for isolation, microwell slide cultures should also be prepared. Approximately 0.2 ml should be inoculated
and a period of 12 hours allowed for adsorption. The CPE becomes evident in roller cultures as foci of granular
rounded cells after 2448 hours in BHK cells, and in a further 2448 hours in other cell types. The CPE is not
specific for NSD virus, which is identified in cover-slip cultures by immunofluorescence or by staining with
haematoxylin and eosin. The latter method reveals pleomorphic eosinophilic intracytoplasmic inclusions peculiarly
of a spindle form; other inclusions are bipolar, or surround the nucleus.
The virus can be specifically identified by immunofluorescence staining, which may be positive as early as 24
48 hours post-inoculation when no CPE has yet become evident. Conjugates for direct immunofluorescence may
be prepared from hyperimmune mouse ascitic fluids, and from immune rabbit or sheep antisera by standard
methods. Some cross-fluorescence may occur with other Nairoviruses at low dilutions of the conjugate, but these
viruses are not normally associated with disease in sheep or goats.
The agar gel immunodiffusion test (AGID) can be a valuable primary diagnostic tool for the detection of NSD
antigen in tissues. The test can be carried out in laboratories without tissue culture facilities and at field
investigation laboratories. The spleen and mesenteric lymph nodes are the tissues of choice to be used in the
test. Aliquots of 0.51 g should be homogenised with sterile sand in a pestle or a homogeniser to give 1020%
suspensions in phosphate buffered saline (PBS) or saline. The suspension should be centrifuged for 10
15 minutes at approximately 1000 g and the supernatant fluid is used in the test. This test can also be used for
the identification of NSD virus antigen in mouse brain harvested from experimentally infected mice (see above).
Rabbit hyperimmune serum against NSD can be prepared by repeated inoculation of NSD-infected mouse brain.
A mouse brain suspension at 25% (w/v) is prepared as above and centrifuged at 35000 g for 15 minutes.
Aliquots are then mixed with an equal volume of Eg Titremax adjuvant. Various inoculation regimes may be used
but 1-ml volumes may be given subcutaneously and/or intramuscularly at 7-day intervals for 35 weeks, or at
multiple inoculation sites in 0.1-ml volumes for a similar period. Serum should be collected 57 days after the last
injection and stored in aliquots at 20C.
1167
Difco Noble or other suitable agar may be used in the test, using 0.85% sodium chloride at pH 7.2. Slides are
prepared to give an agar layer approximately 2 mm in depth. Six wells should be placed hexagonally around a
central well. The hyperimmune rabbit serum is placed in the central well and positive control antigen in wells
1 and 4. The tissue under test is placed in wells 2 and 5. Negative control tissue is placed in wells 3 and 6. Wells
containing test tissue that give a precipitin line of continuity with the line formed between the positive antigen and
the hyperimmune serum are considered to be positive.
Mouse brain suspensions or infective tissue culture fluids can be used as antigens for complement fixation (CF)
tests for virus identification. Both have proved satisfactory after partial purification with fluorocarbon; the mouse
brain can also be used in the form of a suspension in a borate buffer solution.
An enzyme-linked immunosorbent assay (ELISA) antigen for virus identification purposes can be prepared from
an infected tissue culture in a bottle. The cells are removed using a pipette fitted with a rubber bulb when
approximately 20% of the monolayer is showing CPE. They are sedimented and washed three times in borate
saline buffer, pH 9. The cells are then lysed and solubilised with SDS (sodium dodecyl sulphate) and 1% Triton
X100, diluted approximately 1/5 in borate saline buffer and sonicated to provide an antigen for the ELISA. A
control negative antigen is prepared in the same manner from uninfected cells. These are adsorbed directly on to
ELISA plates and the test is carried out with NSD immune and normal serum with both antigens.
2.
Serological tests
These include haemagglutination inhibition (HI), CF and virus neutralisation (VN) tests and ELISA.
Cache Valley virus
a)

VN tests for CVV used to be done by a plaque reduction neutralisation method but are now usually
performed using inhibition of CPE on Vero cells in microtitre plates (Chung et al., 1990).
Test procedure
i)
Inactivate test sera at 56C for 30 minutes in a water bath.
ii)
Make serial twofold dilutions of the sera in MEM from 1/2 to 1/16 and incubate at 37C for 60 minutes
with an equal volume of 100 TCID50 (tissue culture infective dose) per ml of virus. Standard controls
are prepared in a similar manner.
iii)
Discard the medium in a 96-well flat-bottomed cell-culture grade microtitre plate containing a preformed
24-hour Vero monolayer.
iv)
Add the serum/virus mixtures to the plate, 50 l per well, using three wells per dilution.
v)
Back titrate the virus used in the test, making three tenfold dilutions using 50 l per well and four wells
per dilution.
vi)
Cover the plates and incubate for a further 60 minutes at 37C.
vii)
Add 50 l MEM maintenance medium to each well.
viii) Incubate the plates at 37C for 6 days in a humidified CO2 incubator.
b)
ix)
Read the plates microscopically, evaluate the CPE and determine the 50% end points.
x)
The virus control should give a value of 100 TCID50 and there should be no neutralisation by the
negative control serum at the lowest dilution tested. The positive control should give a titre within an
expected range of its predetermined mean.

An ELISA, modified and based on the one for Rift Valley fever described by Meegan et al. (1987), has been
used for CVV serological surveys. Modifications include a 1/400 dilution of mouse ascitic fluid to coat the
plates, followed by a 1/25 dilution of a sucrose/acetone mouse brain antigen in a sandwich ELISA format.
The diluent used is PBS with 0.5% Tween 20, 5% equine serum and 500 g dextran sulphate per ml. A
horseradish peroxidase conjugate detection system and an ABTS (2,2-azino-di-[3-ethyl-benzthiazoline]-6sulphonic acid) substrate are used (Meegan et al., 1987).
c)
Other tests
Not all members of the Bunyamwera group produce haemagglutinins, but a HI test has been described for
CVV using a sucrose/acetone suckling mouse brain antigen and goose erythrocytes at pH 6.2. The test is
1168
said to lack sensitivity compared with a VN test, providing only 50% detection of antibodies. The CF test is
little used because of extensive cross-reactivity within the Bunyamwera group.
Akabane virus
a)

The HI test is modified after Clarke & Casals (1958) and better haemagglutination is achieved with an
increased NaCl molarity. The test is also pH dependant. Sera are pretreated with kaolin or acetone and then
heat inactivated at 56C for 30 minutes. The test is performed using four units of sucrose/acetone-extracted
mouse brain antigen, 0.3% red blood cells and borate buffer, pH 9 (Goto et al., 1978).
b)

VN tests have been described using HmLu-1 cells in tube cultures or Vero and BHK cells in flat-bottomed
96-well microtitre plates (Da Costa Mendes, 1984; Weir, 2003). Two techniques have been described with a
serum/virus incubation period of 1 hour or incubation overnight before the addition of the cells.
Test procedure
i)
Inactivate the test sera at 56C for 30 minutes in a water bath.
ii)
Prepare serial twofold dilutions of the sera in Eagles medium from 1/2 to 1/16 in a 96-well flat-bottomed
microtitre plate using duplicate wells and 25 l per well. Standard controls are prepared in a similar
manner.
iii)
Add 25 l per well of virus in Eagles medium diluted to provide 200 TCID50 per 50 l.
iv)
Cover and incubate at room temperature for 1 hour.
v)
Include a back titration of virus in triplicate, making three tenfold dilutions using 25 l per well.
vi)
Add 100 l per well Vero cells in Eagles medium with 2% serum at 5 105 cells/ml.
vii)
Incubate the plates at 3437C for 5 days in a humidified CO2 incubator.
viii) Read the plates microscopically and calculate the titre as the reciprocal of the highest serum dilution
completely inhibiting the CPE.
ix)
The virus and serum controls should give the expected results.
Where overnight incubation is used, duplicate twofold serial dilutions of inactivated serum are mixed with
100 TCID50 of virus using 100-l volumes in each case. Following incubation for 1 hour at 37C and
overnight at 4C, 50 l BHK cells is added to the test. The plate is examined at 3 and 5 days incubation at
37C and checked for CPE.
c)

Akabane virus ELISAs, using both IgG and IgM, have been described. Coating antigen is 106 TCID50 per ml
of virus grown on HmLu-1 cells diluted in a 0.05 M carbonate/bicarbonate buffer, pH 9.6. The wash medium
is PBS containing Tween 20 and alkaline phosphatase. Rabbit anti-bovine IgG and IgM conjugates are used
(Ungar-Waron et al., 1989).
A similar ELISA using horseradish peroxidase rabbit anti-bovine IgG conjugate has also been described.
A competitive ELISA with a 98% specificity has also been developed (Tsuda et al., 2004).
d)

The CF test is not described here as it is a group specific test and is rarely used anymore.
a)

The indirect fluorescent antibody test (FAT) is the most suitable test for use with members of the Nairovirus
serogroup. There are, however, some cross-reactions, particularly with Dugbe virus and also with other
members of the group, such as CongoCrimean haemorrhagic fever virus (Davies et al., 1978). The NSD
1169
antibody titres by this method range from 1/640 to 1/10,240, and such titres are not obtained with immune
sera to other members of the group (Davies et al., 1976).
The method has been used in epidemiological studies and to study the response to experimental vaccines.
There do not appear to be any serological differences among the 4050 isolates that have been examined.
An NSD I-34 strain2 was the virus usually used to prepare antigen, and this has been adapted to grow in
BHK-21-C13 cells, after a series of passages.
The virus antigen in the cell substrate of choice may be grown in loose cover-slips, multiwell slides, Tefloncoated slides or microtitre plates for the test. A method using Teflon-coated slides is described.
Preparation of antigen slides
i)
Wash and sterilise Teflon-coated slides. This is done briefly with a hot detergent that is used for tissue
culture glassware in the laboratory, then three rinses in tap water for 30 minutes, each followed by
similar rinses in distilled/deionised water. Slides are then placed in 70% alcohol for 10 minutes,
removed with a sterile forceps and wrapped in greaseproof paper. They will then be found to be sterile,
but further sterilisation in a microwave for two cycles of 5 minutes each is recommended.
ii)
Place these slides in sterile dishes using a sterile forceps; a square polystyrene type is better than the
round variety.
iii)
Mix a suspension of BHK cells containing approximately 25,000 cells/ml in BHK growth medium
(usually Eagles for BHK cells), and add 1000 TCID50 of NSD I-34 strain per ml. Mix by pipetting.
Prepare some uninfected negative control slides.
iv)
Add the infected cells in 50 l volumes (for the 12-well size) or as appropriate to the size of the Teflon
wells. Replace the cover on the dishes and put into a humidified CO2 incubator or anaerobic container.
v)
Leave overnight for the monolayer to form. Then remove the plates from the incubator to a laminar flow
cabinet, and flood with maintenance medium using a pipette to cover the slides to a depth of 23 mm.
Return to the incubator.
vi)
Harvest the antigen slides just as foci of CPE become detectable. This will be in 3656 hours (more
accurate determination of the optimal harvesting time may be made by fixing and staining one slide
after 24, 36 and 48 hours).
vii)
The slides are washed three times in PBS and dried. They are then fixed with dry heat (minimum 80C)
or with ice-cold acetone for 10 minutes. The slides are wrapped and may be stored at 4C for 2
3 months, or at 20C for 12 years. Slides stored at 20C must be brought to 4C overnight before
use.
Similar procedures may be followed to prepare antigen on flying cover-slips or multiwell culture slides. When
using Nunc tissue culture multiwell plates, however, fixation should be with 75% acetone.
Test procedure
i)
Hydrate the slides by adding a drop of PBS to the wells with a Pasteur pipette. Number the slides
according to the number of sera to be tested. Include in the series control positive and negative sera
with infected and uninfected cell cultures.
ii)
Discard the PBS and add the serum dilutions 1/801/2560 in a predetermined manner to wells 1 to 6. It
is preferable to duplicate each dilution on the same side.
iii)
Place the slides in dishes and hold at 37C in a humid incubator for 40 minutes.
iv)
Wash the slides in racks in three changes of PBS, 5 minutes per wash.
v)
Add the fluorescein-conjugated anti-species conjugate (usually anti-sheep or anti-goat) at a

predetermined working dilution; one drop can be added to each well with a Pasteur or other pipette.
vi)
Incubate as before for 30 minutes.
vii)
Wash three times in PBS and dry the slides.
viii) Examine the slides by fluorescent microscopy. NSD virus antigen is found in the cell cytoplasm, and
foci of bundles of fluorescing BHK cells will be seen. The antigen is seen mainly in fine fluorescent
particles, but larger irregularly shaped antigen clumps occur, often surrounding the nucleus, or in
The I-34 strain was a virulent NSD isolate made in Kenya that was used extensively as a reference strain at the Kabete
Laboratory Kenya Agriculture Research Institute, P.O. Box 58137, Kabete, Nairobi, Kenya.
1170
spindle-like masses filling the cytoplasm to the pole of the cells. These particles will not be seen with
negative sera or in the uninoculated control culture.
ix)
b)
Sera that show this fluorescence at dilutions of 1/640 or 1/1280 are indicative of recent infection with
NSD (Davies et al., 1976).
Other tests
CF tests are complicated by the marked anticomplementary activity of many sheep sera.
Immunodiffusion tests have been used successfully with crude antigens prepared from infected sheep
tissue, tissue culture fluids or mouse brain material. Hyperimmune sera can be prepared in sheep, mice or
rabbits for use in the test, using infected spleen taken from sheep dying from NSD as the source of antigen
for immunisation.
An ELISA using a partially purified tissue culture antigen has been described for antibody testing and is
suitable for use in serological surveys. The indirect FAT test should, however, be used to check doubtful
results (Munz et al., 1984).
Monoclonal antibodies to the antigens of NSD virus strain I-34 have been developed and are being
evaluated for their application as diagnostic reagents.
RNA probes have also been developed from the S (small) and M (medium) genome segments of Dugbe
virus and have been used to demonstrate that the NSD serogroup of the genus Nairovirus is more closely
related to the CrimeanCongo haemorrhagic fever serogroup than any of the remaining serogroups (Marriott
et al., 1990; Ward et al., 1992). These probes also have the potential to be applied as diagnostic tools.
Cache Valley virus
Due to the sporadic nature of disease outbreaks, no vaccine has been developed.
Akabane virus
Major epizootics of Akabane virus disease have only been reported from Japan and Australia, albeit at irregular
intervals, but vaccination is seen to have merit in preventing fetal loss.
Vaccines for domestic use have been produced in Japan and Australia.
An inactivated vaccine is used in Japan for immunising cattle and goats. It is a formalin-inactivated intramuscular
preparation with an aluminium phosphate gel adjuvant. Two 3-ml doses are given at a 4-week interval and yearly
boosters are recommended. It is safe for use in pregnant animals. In field trials 88% of animals developed high
VN antibodies after the first inoculation and there was a 100% response after the second dose (Kurogi et al.,
1978). Similarly, in Australia an inactivated vaccine has been produced for intramuscular use giving two doses at
a 4-week interval just before mating.
In Japan, a live Akabane virus vaccine is commercially available. A 1-ml dose is administered subcutaneously to
cattle before the haematophagous arthropod vectors become active. Pregnant cattle and calves have been
inoculated subcutaneously, intramuscularly and intracerebrally; no leukopaenia, viraemia or pyrexia were
observed and a good VN antibody response was produced. A live Akabane virus vaccine, safe in cattle, was
tested in pregnant ewes. During the trials, some ewes became viraemic and virus was found in the organs of
several fetuses. Although no fetal deformities were produced, the vaccine is deemed unsuitable for use in sheep.
Epidemiological investigations have shown that in a state of enzootic stability, no problems are encountered with
NSD. The disease arises from animal movements from free areas into endemic areas and can be avoided when
such areas have been defined. Ecological changes that permit spread of the vector tick will result in extensions of
these areas.
Experimental vaccines have been prepared for such situations. One vaccine consisted of virus attenuated by
35 passages in adult mice, but such vaccines can produce severe reactions in some breeds of sheep, and are not
1171
considered to be safe for general use. A similar vaccine was developed in Entebbe by further mouse brain
passages, but this has not been further developed for use in the field in Uganda or elsewhere.
A tissue-culture-adapted strain of NSD virus has been grown to high titre in cultures grown in roller bottles. When
precipitated with methanol, inactivated, and administered with an adjuvant, this was found to give good protection
following two inoculations given at an interval of 14 days. Neither of these vaccines is routinely produced, for
there has been little demand for their use from the field (Davies et al., 1974; 1977b).
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Off. int. Epiz., 19, 7991.
*
* *
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CHAPTER 2.9.2.
CAMELPOX
SUMMARY
Camelpox is a wide-spread infectious viral disease of Old World camelids. New World camelids are
also susceptible. It occurs throughout the camel-breeding areas of Africa, north of the equator, the
Middle East and Asia, causing economic impact through loss of production and sometimes death.
The camelpox virus belongs to the family Poxviridae, subfamily Chordopoxvirinae, genus
Orthopoxvirus. The disease is characterised by fever, local or generalised pox lesions on the skin
and in the mucous membranes of the mouth, respiratory and digestive tracts. The clinical
manifestations range from inapparent infection to mild, moderate and, less commonly, severe
systemic infection and death. The disease occurs more frequently and more severely in young
animals. Transmission is by either direct contact between infected and susceptible animals or
indirect infection via a contaminated environment. The role of insects in transmission has been
suspected because the disease is often observed after rainfall. Camelpox virus is very host specific
and does not infect other animals. Only one suspected case of human camelpox involving mild skin
lesions has been described, underlining that camelpox is of no public health importance.
Identification of the agent: The presumptive diagnosis of camelpox infection is based on clinical
signs. However, infections of camels with contagious ecthyma (orf), papilloma virus and reaction to
insect bites are considered differential diagnoses in the early clinical stages and in mild cases of
camelpox. Several diagnostic methods are available and, where possible, more than one should be
used to make a confirmatory diagnosis of disease.
The fastest method of laboratory confirmation of camelpox is by the demonstration of the
characteristic, brick-shaped orthopox virions in skin lesions, scabs or tissue samples using
transmission electron microscopy (TEM). Camelpox virus is distinct from the ovoid-shaped parapox
virus, the aetiological agent of the principle differential diagnosis: camel orf. However, both viruses
may be seen simultaneously on TEM as infection with both viruses has been reported to occur.
Camelpox can be confirmed by demonstration of the camelpox antigen in scabs and pox lesions in
tissues by immunohistochemistry. It is a relatively simple method that can be performed in
laboratories where TEM is not available. In addition, the paraffin-embedded samples can be stored
for a long period of time, enabling future epidemiological, retrospective studies.
Camelpox virus may be propagated on the chorioallantoic membrane (CAM) of embryonated
chicken eggs. After 5 days, characteristic lesions can be observed on the CAM. Camelpox virus
shows typical cytopathic effect on a wide variety of cell cultures. Intracytoplasmic eosinophilic
inclusion bodies, characteristic of poxvirus infection, may be demonstrated in infected cells using
haematoxylin and eosin staining. The presence of viral nucleic acid may be confirmed by
polymerase chain reaction, and different strains of camelpox virus may be identified using DNA
restriction enzyme analysis. An antigen-capture enzyme-linked immunosorbent assay (ELISA) for
the detection of camelpox virus has been described.
Serological tests: A wide range of serological tests are available to identify camelpox. The tests
used for the detection of the antibodies against camelpox virus include neutralisation, agar gel
precipitation, haemagglutination, haemagglutination inhibition, complement fixation, fluorescent
antibody and antibody-capturing ELISA.
Requirements for vaccines and diagnostic biologicals: Both attenuated and inactivated
vaccines are commercially available. Vaccination with live attenuated vaccine provides protection
1175
Chapter 2.9.2. Camelpox
for at least 6 years and with inactivated vaccine for 12 months. There are no standardised
requirements for diagnostic biologicals.
A. INTRODUCTION
Camelpox occurs in almost every country in which camel husbandry is practised apart from the introduced
dromedary camel in Australia and tylopods (llama and related species) in South America. Outbreaks have been
reported in the Middle East (Bahrain, Iran, Iraq, Oman, Saudi Arabia, United Arab Emirates and Yemen), in Asia
(Afghanistan and Pakistan), in Africa (Algeria, Egypt, Ethiopia, Kenya, Mauritania, Morocco, Niger, Somalia and
Sudan) (Mayer & Czerny, 1990; Wernery et al., 1997b) and in the southern parts of Russia and India. The
disease is endemic in these countries and a pattern of sporadic outbreaks occurs with a rise in the seasonal
incidence usually during the rainy season.
Camelpox is caused by Orthopoxvirus cameli virus which belongs to genus Orthopoxvirus within family
Poxviridae. Based on sequence analysis, it has been determined that the camelpox virus is the most closely
related to variola virus, the aetiological agent for small pox. Camels have been successfully vaccinated against
camelpox with vaccinia virus strains. The average size of the virion is 265295 nm. Orthopoxviruses are
enveloped, brick-shaped and the outer membrane is covered with irregularly arranged tubular proteins. A virion
consists of an envelope, outer membrane, two lateral bodies and a core. The nucleic acid is a double-stranded
linear DNA. Virus replicates in the cytoplasm of the host cell, in so-called inclusion bodies. Camelpox virus
haemagglutinates cockerel erythrocytes. However, the haemagglutination may be poor (Davies et al., 1975).
Camelpox virus is ether resistant and chloroform sensitive (Davies et al., 1975; Tantawi et al., 1974). The virus is
sensitive to pH 35 and pH 8.510 (Davies et al., 1975). Poxviruses are susceptible to various disinfectants
including 1% sodium hypochlorite, 1% sodium hydroxide, 1% peracetic acid, formaldehyde, 0.51% formalin and
0.5% quaternary ammonium compounds. The virus can be destroyed by autoclaving, boiling for 10 minutes and is
killed by ultraviolet rays (245 nm wave length) in a few minutes (Coetzer, 2004).
The incubation period is usually 913 days (varying between 3 and 15 days). Clinical manifestations of camelpox
range from inapparent and mild local infections, confined to the skin, to moderate and severe systemic infections,
possibly reflecting differences between the strains of camelpox (Wernery & Kaaden, 2002). The disease is
characterised by fever, enlarged lymph nodes and skin lesions. Skin lesions appear 13 days after the onset of
fever, starting as erythematous macules, developing into papules and vesicles, and later turning into pustules.
Crusts develop on the ruptured pustules. These lesions first appear on the head, eyelids, nostrils and the margins
of the ears. In severe cases the whole head may be swollen. Later, skin lesions may extend to the neck, limbs,
genitalia, mammary glands and perineum. In the generalised form, pox lesions may cover the entire body. Skin
lesions may take up to 46 weeks to heal. In the systemic form of the disease, pox lesions can be found in the
mucous membranes of the mouth, respiratory and digestive tracts (Kritz, 1982; Wernery & Kaaden, 2002).
The animals may show salivation, lacrimation and a mucopurulent nasal discharge. Diarrhoea and anorexia may
occur in the systemic form of the disease. Pregnant females may abort. Death is usually due to secondary
infections and septicaemia (Wernery & Kaaden, 2002).
Histopathological examination of the early skin nodules reveals characteristic cytoplasmic swelling, vacuolation
and ballooning of the keratinocytes of the outer stratum spinosum. The rupture of these cells produces vesicles
and localised oedema. Perivascular infiltration of mononuclear cells and variable infiltration of neutrophils and
eosinophils occurs. Marked epithelial hyperplasia may occur in the borders of the skin lesions (Yager et al., 1991).
There are only a few detailed pathological descriptions of internal camelpox lesions. The lesions observed on
post-mortem examination of camels that die following severe infection with camelpox are multiple pox-like lesions
on the mucous membranes of the mouth, respiratory and digestive tract. The size of the lesions in the lungs may
vary in diameter between 0.5 and 1.3 cm, occasionally up to 45 cm. Smaller lesions may have a haemorrhagic
centre. The lung lesions are characterised by hydropic degeneration, proliferation of bronchial epithelial cells, and
infiltration of the affected areas by macrophages, necrosis and fibrosis (Kinne et al., 1998; Pfeffer et al., 1998a;
Wernery et al., 1997a).
The morbidity rate of camelpox is variable and depends on whether the virus is circulating in the herd. Serological
surveys taken in several countries reveal a high prevalence of antibodies to camelpox (Wernery & Kaaden, 2002).
The incidence of disease is higher in males than females, and the mortality rate is greater in young animals than
in adults (Kritz, 1982). The mortality rate in adult animals is between 5% and 28% and in young animals between
25% and 100% (Mayer & Czerny, 1990).
Transmission is by either direct contact between infected and susceptible animals or indirect infection via a
contaminated environment. The infection is usually achieved by inhalation or through skin abrasions. Virus is
secreted in milk, saliva, and ocular and nasal discharges. Dried scabs shed from the pox lesions may contain live
virus for at least 4 months and contaminate the environment. The role of an arthropod vector in the transmission
1176
of the disease has been suspected. Camelpox virus has been detected by transmission electron microscopy
(TEM) and virus isolation from the camel tick, Hyalomma dromedarii, collected from animals infected with
camelpox virus. The increased density of the tick population during the rainy season may be responsible for the
spread of the disease (17). However, other potential vectors may be involved, such as biting flies and mosquitoes.
It has been suggested that different strains of camelpox virus may show some variation in their virulence
(Wernery & Kaaden, 2002). Restriction enzyme analysis of viral DNA allows isolates to be compared. However,
no major differences from the vaccine strain have so far been demonstrated (Wernery et al., 1997a).
Immunity against camelpox is both humoral and cell mediated. The relative importance of these two mechanisms
is not fully understood, but it is believed that circulating antibodies do not reflect the immune status of the animal
(Wernery & Kaaden, 2002). Life-long immunity follows after natural infection. Live, attenuated vaccine provides
protection against the disease for at least 6 years, probably longer (Wernery & Zachariah, 1999). Inactivated
vaccine provides protection for 1 year only.
The camelpox virus is very host specific and does not infect other animal species, including cattle, sheep and
goats. Field reports of mild skin lesions in humans associated with camel pox have been made (Coetzer, 2004),
but it appears that only one suspected case of human camelpox has been described (Kritz, 1982), underlining
that camelpox is of no public health importance.
During the viremic stage of the disease (within the first week of the occurrence of clinical signs) camelpox virus
can be isolated in cell culture from heparinised blood samples, or viral DNA can be detected by the polymerase
chain reaction (PCR) from blood in EDTA (ethylene diamine tetra-acetic acid). The blood samples should be
collected in a sterile manner by venepuncture. Blood samples, with anticoagulant for virus isolation from the buffy
coat, should be placed immediately on ice and processed as soon as possible. In practice, the samples can be
kept at 4C for up to 2 days prior to processing, but should not be frozen or kept at ambient temperatures.
Blood obtained for serum samples should be collected in plain tubes with no anticoagulant. The blood tubes
should be left to stand at room temperature for 12 hours until the clot begins to contract, after which the blood is
centrifuged at 1000 g for 1015 minutes. Separated serum can be collected with a pipette and held at 4C for a
short period of time or stored at 20C.
A minimum of 2 g of tissue from skin biopsies and organs should be collected for virus isolation and
histopathology. For the PCR, approximately 3050 mg of tissue sample should be placed in a cryotube or similar
container, kept at 4C for transportation and stored at 20C until processed. Tissue samples collected for virus
isolation should be placed in a virus transport medium, such as Tris-buffered tryptose broth, kept at 4C for
transportation and stored at 80C until processed. Material for histology should be placed immediately after
collection into ten times the sample volume of 10% formalin. The size of the samples should not exceed 0.5 cm
12 cm. Samples in formalin can be transported at room temperature.
1.
a)
Transmission electron microscopy

TEM is a rapid method to demonstrate camelpox virus in scabs or tissue samples. However, a relatively high
concentration of virus in the sample is required for positive diagnosis and camelpox virus cannot be
differentiated from other orthopoxvirus species. However, currently, TEM is the best method for
distinguishing clinical cases of camelpox and orf caused by camelpox and parapox viruses respectively,
although the viruses can be differentiated by serological techniques and by PCR (Mayer & Czerny, 1990).
The size of a sample should be at least 3050 mg. Mince the scabs or tissue sample with a disposable
blade or sterile scissors and forceps. Grind the sample in a five-fold volume of phosphate buffered saline
(PBS) with antibiotics (such as 105 International Units [IU] penicillin and 10 mg streptomycin per ml) using a
mortar and pestle with sterile sand. Transfer the sample into a centrifuge tube and freeze and thaw two to
three times to release the virus from the cells. Vortex the samples while thawing. Place the tubes on ice and
sonicate once for 30 seconds at 80 Hz. Centrifuge at 1000 g for 10 minutes to remove the gross particles
and collect the supernatant (Pfeffer et al., 1996; 1998b).
Test procedure
Place 10 l of above-mentioned supernatant on poly-L-lysine-covered grids and incubate at room

temperature for 5 minutes. Remove the fluid with a chromatography filter paper. Add one drop of 2%
phosphotungstic acid (diluted in sterile water and pH adjusted to 7.2 with NaOH) to the grid, incubate at
room temperature for 5 minutes and air dry. Examine the grid by TEM (Pfeffer et al., 1996; 1998b).
1177
Camelpox virus has a typical brick-shaped appearance with irregularly arranged, tubular surface proteins.
Parapoxviruses are slightly smaller, ovoid-shaped and the surface proteins are regularly arranged.
b)
Virus isolation in cell cultures

Camelpox virus can be propagated in a large variety cell cultures including the following cell lines: Vero, MA104 and MS monkey kidney, and, baby hamster kidney (BHK) and the following primary cell cultures: lamb
testis, lamb kidney, camel embryonic kidney, calf kidney, and chicken embryo fibroblast (Davies et al., 1975;
Tantawi et al., 1974).
The samples are prepared for virus isolation as described above in Section B.1.a.
Test procedure
Incubate 400 l of the supernatant for 1 hour at room temperature and overnight at 4C. Filter the
supernatant through a 0.45 m filter and inoculate into a 25 cm2 flask of confluent cells. Flush the filter with
0.5 ml of the maintenance medium used in the cell culture and incubate the flasks at 37C for 1 hour. Add 6
7 ml of fresh medium into the flask and continue the incubation for about 10 days. If there is any reason to
suspect fungal contamination, the contaminated medium must be discarded and 5 g/ml of amphotericin B
added to a new medium. The flasks must be monitored daily for 1012 days.
Characteristic, plaque-type cytopathic effect (CPE) showing foci of rounded cells, cell detachment, giant cell
formation and syncytia may appear as soon as 24 hours post-inoculation. Syncytia may contain up to 20
25 nuclei (Tantawi et al., 1974). The growth of camelpox virus on a cell culture can be confirmed by TEM,
PCR or antigen-capture enzyme-linked immunosorbent assay (ELISA) (Johann & Czerny, 1993).
c)
Virus isolation on chorioallantoic membrane of embryonated chicken eggs

Camelpox virus can be isolated on the chorioallantoic membrane (CAM) of 1113-day old embryonating
chicken eggs. The eggs should be incubated at 37C degrees and after 5 days, the eggs containing living
embryos are opened and the CAM examined for the presence of characteristic pock lesions: dense, greyishwhite pocks. Camelpox virus does not cause death in inoculated embryonated chicken eggs. The maximum
temperature for the formation of pock lesions is 38.5C degrees. If the eggs are incubated at 34.5C the
pocks are flatter and a haemorrhagic centre may develop (Tantawi et al., 1974).
d)
Immunohistochemistry for the detection of the infectious agent of camelpox is a relatively fast method and
can be used instead of electron microscopy for establish a tentative diagnosis (Nothelfer et al., 1995).
Almost any polyclonal antibody against vaccinia virus is likely to produce reasonable results in this test
because of the wide homology between vaccinia and camelpox viruses (Nothelfer et al., 1995).
Test procedure
The following procedure for immunohistochemistry is described by Kinne et al. (1998) and Pfeffer et al.
(1998). The entire skin pustule should be collected for the immunohistochemical examination. Fix the tissue
in 10% formalin, dehydrate through graded alcohols and embed in paraffin wax according to standard
histopathological procedures. Cut approximately 3 m sections and place on the glass slides. Treat the
deparaffinised and dehydrated sections with 3% H2O2, prepared in distilled water, for 5 minutes and wash
with PBS. Incubate the slides for 60 minutes at 37C with anti-vaccinia virus monoclonal antibody 5B4,
diluted 1/500. Remove the monoclonal antibody by washing twice with cold PBS. Incubate the slides for
30 minutes with anti-mouse antibodies labelled with biotin (ABC-kit, Dako, Glostrup, Denmark). Wash with
PBS for 5 minutes and incubate with streptavidin-peroxidase for 30 minutes. Wash again with PBS for
5 minutes and add diaminobenzidine as chromogen for 10 minutes.
e)

The PCR is a fast and sensitive method for the detection of orthopoxviral DNA. A generic PCR assay,
described by Meyer et al. (1994), allows the detection and differentiation of species of the genus
Orthopoxvirus because of the size differences of the amplicons. Using the primer pair: 5-AAT-ACA-AGGAGG-ATC-T-3 and 5-CTT-AAC-TTT-TTC-TTT-CTC-3, the gene sequence encoding the A-type inclusion
protein (ATIP) will be amplified. The size of the PCR product, specific for the camelpox virus, is 881 bp.
Test procedure
Suspend a small aliquot of crusted scabs in 90 l of lysis solution (50 mM Tris/HCl, pH 8.0, 100 mM
Na2 EDTA, 100 mM NaCl, 1% sodium dodecyl sulphate) and add 10 l of proteinase K (20 mg/ml,
Invitrogen). Digest the sample for 10 minutes at 37C prior to the disruption of the scab or tissue with a
1178
microfuge tube pestle. Add another 350 l lysis solution and 50 l of proteinase K, mix gently and incubate
for 3 hours at 37C. Extract the lysed suspension with an equal volume of phenol/chloroform/isoamylalcohol
(25/24/1) and centrifuge at 8000 g at 4C for 1 minute. Collect the upper aqueous phase and mix it again
with an equal volume of phenol/chloroform/isoamylalcohol (25/24/1). Centrifuge at 8000 g at 4C for
1 minute and transfer the upper, aqueous phase to a new tube. Precipitate the DNA by adding 1/10 volume
of 3 M sodium acetate and two volumes of ice-cold absolute ethanol. Place the mixture at 70C for
30 minutes or 20C overnight. Centrifuge at 15,000 g for 5 minutes at 4C. Discard the supernatant and
wash the pellet with 0.5 ml of 70% ethanol. Centrifuge at 15,000 g for 5 minutes. Discard the supernatant
and air-dry the pellets. Resuspend the pellets in 10 l of nuclease-free water.
DNA amplification is carried out in a final volume of 50 l containing 2 l of each dNTP (10 mM), 5 l of 10
PCR buffer, 1.5 l of MgCl2 (50 mM), 1 l of each primer, 2.5 U Taq DNA polymerase, 1 l DNA template
and an appropriate volume of nuclease-free water.
Incubate the samples in a thermal cycler: first cycle: 5 minutes at 94C (initial denaturation step), second
cycle: 1 minute at 94C, 1 minute at 45C, 2.5 minutes at 72C. Repeat the second cycle 29 times. Last
cycle: 10 minutes at 72C (final elongation step) and hold at 4C until analysis.
Mix 10 l of a sample with loading dye solution and load in 1% agarose gel in TBE (Tris/Borate/EDTA) buffer
containing ethidium bromide. Load a parallel lane with a 100 bp DNA-marker ladder. Separate the products
at 100 V for 3040 minutes and visualise using an UV transilluminator. Confirm the positive reactions
according to the size.
A commercial PCR kit has been developed that allows detection of orthopoxvirus-DNA and contains a
second conventional amplification system, consisting of primers to the haemagglutinin (HA) gene of the
orthopoxvirus. The amplicon can be sequenced and identified by comparison with already existing
orthopoxvirus sequences.
2.
Serological tests
All the viruses in the genus Orthopoxvirus cross-react serologically. However, within the genus only camelpox
virus can cause pox-like lesions in camels. Parapox and camelpox viruses do not cross-react and so infections of
camelpox and camel orf can be distinguished serologically. Most of the conventional serological tests are very
time- and labour-consuming, which makes them not suitable for primary diagnosis. However, serological tests are
a valuable tool for secondary confirmatory testing and retrospective epidemiological studies in those areas where
vaccination against camelpox is not practised.
a)
Serum neutralisation test

In this method the test sera are titrated against a constant titre of camelpox virus (100 TCID50 [50% tissue
culture infectious dose]).
Test procedure
i)
Mark the microtitre plates.
ii)
Dilute the test sera, positive and negative serum controls 1/5 in Dulbeccos Modified Eagles Medium
(DMEM).
iii)
Inactivate the sera at 56C for 30 minutes.
iv)
Add the growth medium (DMEM containing 510% of fetal calf serum and antibiotics) into the wells:
100 l into rows A to H, columns 2 to 6 (test serum, positive and negative serum controls) and
rows A to D, columns 7 to 12 (virus control rows),
200 l into rows G to H, columns 7 to 12 (cell control rows).
v)
Add the diluted and inactivated test sera, positive and negative control sera: 200 l/well into rows A to
H, column 1 (duplicate rows for each sample).
vi)
Collect 100 l from the test serum wells (1/5 dilution) and prepare twofold dilutions (1/10, 1/20, 1/40,
1/80, 1/160) of the serum samples on the microtitre plate using a multichannel pipette. Discard 100 l
from the last wells.
vii)
Prepare 100 TCID50/100 l dilution of the virus suspension with a known titre of over log10 6 TCID50 to
be used as a working virus seed.
viii) Prepare tenfold dilution series from working virus seed (101, 102, 103, 104).
1179
ix)
Add 100 l of each virus dilution into the virus control rows:
Working virus seed in rows A to D, columns 7 and 8,
101 dilution of the working virus seed into rows A to D, column 9,
104 dilution of the working virus seed into rows A to D, column 12.
x)
Add 100 l of working virus seed into each well in test serum, positive and negative serum control
rows.
xi)
Incubate the microtitre plate at 37C with 5% CO2 for 1 hour.
xii)
Add 80 l of suitable cell suspension, such as lamb testis cells, at a concentration of 480,000 cells/ml
into each well.
xiii) Incubate at 37C with 5% CO2.

xiv) Read the results using an inverted microscope. Examine the microtitre plates daily. Serum antibody
titres are calculated and recorded when CPE is evident in the virus control wells. The test is valid if the
virus titre is approximately 100 TCID50. The antibody titre is determined by the SpearmanKrber
method.
b)
Enzyme-linked immunosorbent assay for the detection of antibodies against camelpox virus
The following procedure for antibody-detecting ELISA test for Orthopoxvirus cameli is described by Azwai et
al. (1996) and Pfeffer et al. (1998b). The following description gives general guidelines for the test
procedure.
Preparation of the antigen
i)
Harvest the cell culture when 100% infected with camelpox virus. Freeze and thaw two to three times.
Sonicate for 30 seconds at 80 Hz on ice to release the virus from the cells.
ii)
Centrifuge at 1000 g for 10 minutes and collect the supernatant.
iii)
Centrifuge the supernatant at 45.000 g at 4C for 1 hour. Re-suspend the pellet in PBS.
iv)
Add NaCl to a final concentration of 330 mM and polyethylene glycol (PEG 6000) to a final
concentration of 7%.
v)
Stir overnight at 4C, centrifuge at 3000 g at 4C for 10 minutes and wash the pellet twice with 15 mM
NaCl.
vi)
Freeze and thaw, and treat with 1% non-ionic detergent (Nonidet P40, Sigma) at 37C for 3 hours.
vii)
Freeze and thaw and centrifuge at 3000 g for 10 minutes at 4C.
viii) Collect the supernatant and dialyse at least three times against PBS.
ix)
Measure the protein concentration as described by Lowry et al. (1951).
x)
Store the aliquots at -20C.
Preparation of rabbit anti-camel IgG horseradish-peroxidase conjugate
Unfortunately rabbit anti-camel IgG horseradish-peroxidase conjugate is not commercially available. The
method for producing monoclonal antibodies for camel IgM and IgG has been described by Azwai et al.
(1995). However, rabbit anti-camel IgG horseradish-peroxidase can be replaced with commercially available
product in which the antibodies are raised against IgG of the New World camelids and conjugated with
fluorescein (Fluorescein Conjugated Camelid IgG, Kent Laboratories, Triple J Farms, USA).
i)
Precipitate camel sera twice adding saturated ammonium sulphate to a final concentration of 40% (v/v)
(29.6% ammonium sulphate [w/v]) at room temperature. Centrifuge at 12,000 g for 15 minutes and
dissolve in PBS, pH 7.2. Dialyse against several changes of PBS overnight.
ii)
Separate the immunoglobulins using gel filtration chromatography: an ACA-34 (LBK) column (2.6
100 cm) can be used to separate the salt precipitated immunoglobulins (IgM and IgG) by size. Elution
can be effected with PBS at 20 ml/hour and 6 ml fractions can be collected. Determine the protein
concentrations by absorbance at 280 nm.
1180
Antiserum production
Immunise rabbits with a subcutaneous injection of camel IgG emulsified in appropriate adjuvant. The
animals should be immunised three times to booster antibody production. Collect the serum and store at
20C until used.
Test procedure
Non-activated, 96-well, microtitre ELISA plates, such as Immulon 2 supplied by Dynatech can be used.
i)
Coat the microtitre plates with prepared antigen at 1 g/ml in carbonate/bicarbonate buffer, 0.05 M,
pH 9.6 (100 l per well).
ii)
Incubate the ELISA plates in a humid chamber (100% humidity) at 37C for 1 hour and then overnight
at 4C.
iii)
Wash off the unbound antigen with PBS containing 0.05% Tween 20 (PBS/Tween) three times.
iv)
Add 100 l of test and control serum at a predetermined optimal dilution in blocking buffer (PBS
containing 0.05% Tween 20, and 1% fat-free milk powder) in duplicate wells.
v)
Incubate the plates for 30 minutes at 37C.
vi)
Wash the plates three times with PBS/Tween.
vii)
Dilute rabbit anti-camel IgG horseradish-peroxidase conjugate or fluorescein-conjugated camelid IgG

at a predetermined working dilution in blocking buffer and add 100 l into the wells.
viii) Incubate the plates at 37C for 30 minutes.

ix)
Wash the plates three times with PBS/Tween.
x)
Make the reaction visible using 100 l chromogen 3.3 5.5-tetramethylbenzidine (TMB) per well and
incubate 15 minutes at 37C with shaking.
xi)
Stop the reaction 10 minutes later by 2 M H2SO4 at a volume of 50 l/well.
xii)
Measure the values with a photometer at a wave length of 450 nm. Seropositivity can be calculated as
values above the mean +2 standard deviations from negative control sera.

A live attenuated vaccine, Ducapox, is manufactured by Highveld Biologicals, Onderstepoort, South Africa and an
inactivated vaccine by Biopharma, Rabat, Morocco. A live attenuated vaccine gives long-term protection against
camelpox (Wernery & Zachariah, 1999). However, a booster vaccination is recommended for young animals
vaccinated before the age of 69 months. When inactivated vaccine is used, the animals must be vaccinated
annually. Guidelines for the production of the veterinary vaccines are given in Chapter 1.1.6 Principles of
veterinary vaccine production.
ACKNOWLEDGEMENT
The authors would like to thank Dr U. Wernery, Scientific Director of the Central Veterinary Research Laboratory,
Dubai, United Arab Emirates (UAE) and Ms R. Zachariah, Central Veterinary Research Laboratory, Dubai, UAE
for their valuable advice in the selection of the appropriate tests for this chapter and for the description of the
antibody-detecting ELISA assay.
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KRITZ B. (1982). A study of camelpox in Somalia. J. Comp. Pathol., 92, 18.
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Ruminants, Dinter Z. & Morein B., eds. Elsevier Science Publisher B.V., Amsterdam, Oxford, New York, Tokyo,
Chapter 4, 1922.
MEYER H., PFEFFER M. & RZIHA H.-J. (1994). Sequence alterations within and downstream of the A-type inclusion
protein genes allow differentiation of Orthopoxvirus species by polymerase chain reaction. J. Gen. Virol., 75,
19751981.
NOTHELFER H.B., WERNERY U. & CZERNY C.P. (1995). Camel pox: antigen detection within skin lesions
Immunohistochemistry as a simple method of etiological diagnosis. J. Camel Pract. Res., 2 (2), 119121.
PFEFFER M., MEYER H., WERNERY U. & KAADEN O.-R. (1996). Comparison of camelpox viruses isolated in Dubai.
PFEFFER M., NEUBAUER H., WERNERY U., KAADEN O.-R. & MEYER H. (1998a). Fatal form of camelpox virus infection.
Vet. J., 155, 107109.
PFEFFER M., WERNERY U., KAADEN O.-R. & MEYER H. (1998b). Diagnostic procedures for poxvirus infections in
camelids. J. Camel Pract. Res., 5 (2), 189195.
TANTAWI H.H., SABAN M.S., REDA I.M. & EL-DAHABY H (1974). Camelpox virus in Egypt I Isolation and
Characterization. Bull. Epizoot. Dis. Africa, 22 (4), 315319.
WERNERY U. & KAADEN O.-R. (2002). Camel pox. In: Infectious Diseases in Camelids, Second Edition, Wernery U.
& Kaaden O.-R., eds. Blackwell Science Berlin, Vienna, 176185.
WERNERY U., KAADEN O.-R. & ALI M. (1997a). Orthopox virus infections in dromedary camels in United Arab
Emirates (U.A.E.) during winter season. J. Camel Pract. Res., 4 (1), 5155.
WERNERY U., MEYER H. & PFEFFER M. (1997b). Camel pox in the United Arab Emirates and its prevention. J.
Camel Pract. Res., 4 (2), 135139.
WERNERY U. & ZACHARIAH R. (1999). Experimental camel pox infection in vaccinated and unvaccinated
dromedaries. J. Vet. Med. [B] Infect. Dis., 46 (2), 131136.
YAGER J.A., SCOTT D.W. & WILCOCK B.P. (1991). Viral diseases of the skin. In: Pathology of Domestic Animals,
Fourth Edition, Jubb K.V.F., Kennedy P.C. & Palmer N., eds. Academic Press, San Diego, USA, 629644.
*
* *
NB: There is an OIE Reference Laboratory for Camelpox
diagnostic tests, reagents and vaccines for camelpox
1182
CHAPTER 2.9.3.
CAMPYLOBACTER JEJUNI AND

CAMPYLOBACTER COLI
SUMMARY
Definition of the disease: Campylobacter jejuni and C. coli can colonise the intestinal tract of most
mammals and birds and are the most frequently isolated Campylobacter species in humans with
gastro-enteritis. Transmission from animals to humans is mainly through consumption and handling
of animal food products but also direct contact with colonised animals may contribute to human
campylobacteriosis. This chapter focuses on C. jejuni and C. coli in primary livestock production
with regard to food safety.
Description of the disease: Campylobacter jejuni and C. coli do not cause clinical disease in adult
animals except for sporadic cases of abortion in ruminants and very rare cases of hepatitis in
ostriches. The faecal contamination of meat (especially poultry meat) during processing is
considered to be a major source of human food-borne disease. In humans, extraintestinal
infections, including bacteraemia, can occur and some sequelae of infection, such as
polyneuropathies, though rare, can be serious.
Identification of the agent: In mammals and birds, detection of intestinal colonisation is based on
the isolation of the organism from faeces, rectal swabs and/or caecal contents. Campylobacter
jejuni and C. coli are thermophilic, Gram-negative, highly motile bacteria that, for optimal growth,
require microaerobic environment and incubation temperatures of 3742C. Agar media containing
selective antibiotics are required to isolate these bacteria from faecal/intestinal samples.
Alternatively, their high motility can be exploited using filtration techniques for isolation. Enrichment
techniques to detect intestinal colonisation are not routinely used. Preliminary confirmation of
isolates can be made by light microscopy. The organisms in the log growth phase are short and Sshaped in appearance, while coccoid forms predominate in older cultures. Under phase-contrast
microscopy the organisms have a characteristic rapid corkscrew-like motility. Phenotypic
identification is based on reactions under different growth conditions. Biochemical and molecular
tests can be used to confirm various Campylobacter species. Polymerase chain reaction assays
also can be used for the direct detection of C. jejuni and C. coli.
Serological tests: serological assays are not routinely in use for the detection of C. jejuni/C. coli
colonisation.
Requirements for vaccines and diagnostic biologicals: There are no effective vaccines
available for the prevention of enteric Campylobacter infections in birds or mammals.
A. INTRODUCTION
1.
Disease
Campylobacter jejuni and C. coli are generally considered commensals of livestock, domestic pet animals and
birds. Large numbers of Campylobacter have been isolated from young livestock, including piglets, lambs and
calves, with enteritis, but the organisms are also found in healthy animals. Outbreaks of avian hepatitis have been
reported, but the pathogenic role of Campylobacter spp. is unclear. One possible exception is ostriches where
Campylobacter-associated death and enteritis occurs in young birds. Campylobacter are the main cause of
human bacterial intestinal disease identified in many industrialised countries (Tauxe, 1992). Over 80% of cases
are caused by C. jejuni and about 10% of cases are caused by C. coli. In humans, C. jejuni/coli infection is
associated with acute enteritis and abdominal pain lasting for 7 days or more. Although such infections are
1183
Chapter 2.9.3. Campylobacter jejuni and Campylobacter coli
generally self-limiting, complications can arise and may include bacteraemia, GuillainBarr syndrome, reactive
arthritis, and abortion (Skirrow & Blaser, 2000). The primarily source of C. jejuni/coli infections in human is
believed to be the handling and/or consumption of contaminated meat, especially poultry meat. However, contact
with pets and livestock, the consumption of contaminated water or raw milk and travelling in high prevalence
areas are also considered risks factors in human disease (Friedman et al., 2000). The control of Campylobacter in
the food chain has now become a major target of agencies responsible for food safety world-wide.
2.
Taxonomy
In 1991 a revision of the taxonomy and nomenclature of the genus Campylobacter was proposed. According to
Bergeys Manual, the genus Campylobacter comprises sixteen species and six subspecies. More recently, two
additional species have been proposed (Vandamme, 2000). Members of the genus are typically Gram-negative,
non-spore-forming, S-shaped or spiral shaped bacteria (0.20.8 m wide and 0.55 m long), with single polar
flagella at one or both ends, conferring a characteristic corkscrew-like motility. These bacteria require
microaerobic conditions, but some strains also grow aerobically or anaerobically. They neither ferment nor oxidise
carbohydrates. Some species, particularly C. jejuni, C. coli and C. lari, are thermophilic, growing optimally at
42C. They can colonise mucosal surfaces, usually the intestinal tract, of most mammalian and avian species
tested. The species C. jejuni includes two subspecies (C. jejuni subsp. jejuni and C. jejuni subsp. doylei) that can
be discriminated on the basis of several phenotypic tests (nitrate reduction, selenite reduction, sodium fluoride,
and safranine) and growth at 42C (subsp. doylei does not grow at 42C) (Garrity, 2005). Subspecies jejuni is
much more frequently isolated then subspecies doylei.
1.
Isolation and identification of the agent
Two ISO (International Organization for Standardization) procedures for detection of Campylobacter exist, a
horizontal method for detection of thermotolerant Campylobacter in food and animal feeding stuffs (ISO, 2006)
and a procedure for the isolation of Campylobacter from water (ISO, 2005). However, neither of these standard
methods may be optimal for the isolation of campylobacters from live animals. An appendix to ISO 10272 on this
topic is currently being developed.
a)
i)
Poultry at the farm

Poultry is frequently colonised with C. jejuni (6595%) less often with C. coli and rarely with other
Campylobacter species (Newell & Wagenaar, 2000). Colonisation rates in broiler chickens are agerelated. Most flocks are negative until 2 weeks of age. Once Campylobacter colonisation occurs in a
broiler flock, transmission, via coprophagy, is extremely rapid and up to 100% of birds within a flock
can become colonised within 72 hours. Samples from live birds, destined for the food chain, should
therefore be taken as close to slaughter as possible (Newell & Wagenaar, 2000). The majority of birds
shed large numbers of organisms (>106 colony-forming units/g faeces). Campylobacters can be
isolated from fresh faeces/caecal droppings or cloacal swabs. For reliable detection of Campylobacter
by culture, freshly voided faeces (preferably without traces of urine) should be collected. Such samples
must be prevented from drying out before culture. When swabs are used, a transport medium such as
Amies, Cary Blair or Stuart must be used.
ii)
Cattle, sheep and pigs at the farm

Campylobacters are frequent colonisers of the intestine of livestock such as cattle, sheep and pigs
(Atabay & Corry, 1998; Weijtens, 1996; Wesley et al., 2000). Cattle and sheep are found to be
colonised mainly with C. jejuni, C. coli, C. hyointestinalis, and C. fetus, whereas pigs are predominantly
colonised by C. coli. In young animals, the numbers are higher than in older animals. In older animals,
the organisms can be intermittently detected in faeces, probably due to low numbers or due to
intermittent shedding. Fresh samples have to be taken (rectal samples if possible) and they should be
prevented from drying out. When swabs are used, a transport medium (like Amies, Cary Blair or Stuart)
must be used.
iii)
At slaughter
In poultry, the caeca are usually used for the detection of Campylobacter. They can be cut with sterile
scissors from the remaining part of the intestines and submitted intact to the laboratory in a plastic bag
or Petri dish.
1184
Samples from cattle, sheep and pigs are collected from the intestines by aseptically opening the gut
wall or by taking rectal swabs.
b)
Transportation and treatment of samples

i)
Transport
Campylobacters are remarkably sensitive to environmental conditions, including dehydration,
atmospheric oxygen, sunlight and elevated temperature. Transport to the laboratory and subsequent
processing should therefore be as rapid as possible, preferably the same day, but within at least
2 days. The samples must be protected from light.
No recommendation on the ideal temperature for transportation can be made, but it is clear that
freezing or high temperatures can reduce viability. High temperatures (>20C), low temperatures
(<0C) and fluctuations in temperature must be avoided. When the time between sampling and
processing is longer, storage at 4C (2C) is advised.
ii)
Transport media
Swabs: When samples are collected in swabs, the use of commercially available transport tubes,
containing a medium, such as Amies, is recommended. This medium may be plain agar or charcoalbased. The function of the medium is not for growth of Campylobacter spp., but to protect the swab
contents from drying and the toxic effects of oxygen.
When only small amounts of faecal/caecal samples can be collected and transport tubes are not
available, shipment of the specimen in transport medium is recommended. Several transport media
have been described: Cary-Blair, modified Cary-Blair, modified Stuart medium, Campythioglycolate
medium, alkaline peptone water and semisolid motility test medium. Good recovery results have been
reported using Cary-Blair (Luechtefeld et al., 1981; Sjogren et al., 1987).
iii)
Maintenance of samples
On arrival at the laboratory, samples should be processed as soon as possible, preferably on the day
of arrival but no longer than 3 days after collecting the samples. To avoid temperature variation,
samples should only be refrigerated when they cannot be processed on the same day, otherwise they
should be kept at room temperature. When samples are submitted or kept in the laboratory at 4C, they
should be allowed to equilibrate to room temperature before processing to avoid temperature shock.
c)
Isolation of Campylobacter
For the isolation of Campylobacter from faecal/caecal or intestinal samples, no pretreatment is needed;
samples can be plated on to selective medium or the filtration method on non-selective agar can be used. In
the case of caecal samples, caeca are aseptically opened by cutting the end with a sterile scissors and
squeezing out the material to be processed. Enrichment is recommended to enhance the culture sensitivity
of potentially environmentally stressed organisms or in the case of low levels of organisms in faeces, for
example from cattle, sheep or pigs. However, enrichment from the latter samples is not carried out routinely
and in research setting only.
i)
Selective media for isolation

Many media can be used in the recovery of Campylobacter spp. Modified charcoal, cefoperazone,
desoxycholate agar (mCCDA), is the recommended medium, .although alternative media may be used.
A detailed description on Campylobacter detection by culture and the variety of existing media is given
by Corry et al. (Corry et al., 1995; 2003). The selective media can be divided into two main groups:
blood-containing media and charcoal-containing media. Blood components and charcoal serve to
remove toxic oxygen derivatives. Most media are commercially available. The selectivity of the media
is determined by the antibiotics used. Cefalosporins (generally cefoperazone) are used, sometimes in
combination with other antibiotics (e.g. vancomycin, trimethoprim). Cycloheximide (actidione) and more
recently amphotericin B are used to inhibit yeasts and molds (Martin et al., 2002). The main difference
between the media is the degree of inhibition of contaminating flora. All the selective agents allow the
growth of both C. jejuni and C. coli. There is no medium available that allows growth of C. jejuni and
inhibits C. coli or vice versa. To some extent, other Campylobacter species (e.g. C. lari, C. upsaliensis,
C. helveticus, C. fetus and C. hyointestinalis) will grow on most media, especially at the less selective
temperature of 37C.
Examples of selective blood-containing solid media:
Preston agar
Skirrow agar
1185
Butzler agar
Campy-cefex
Examples of charcoal-based solid media:
ii)
mCCDA (modified charcoal cefoperazone deoxycholate agar), slightly modified version of the
originally described CCDA) (Bolton et al., 1984; 1988)
Karmali agar or CSM (charcoal-selective medium) (Karmali et al., 1986)
CAT agar (cefoperazone, amphotericin and teicoplanin), facilitating growth of C. upsaliensis
(Aspinall et al., 1993).
Passive filtration
Passive filtration, a method developed by Steele & McDermott (1984) obviates the need for selective
media; thus it is very useful for the isolation of antimicrobial-sensitive Campylobacter species. As the
method does not use expensive selective media, it may be used in laboratories with fewer resources.
For passive filtration, faeces are mixed with PBS (approximately 1/10 dilution) to produce a
suspension. Approximately 100 l of this suspension are then carefully layered on to a 0.45 or 0.65 m
filter, which has been previously placed on top of a non-selective blood agar plate. Care must be taken
not to allow the inoculum to spill over the edge of the filter. The bacteria are allowed to migrate through
the filter for 3045 minutes at 37C or room temperature. The filter is then removed, the fluid that has
passed through the filter is spread with a sterile glass or plastic spreader, and the plate is incubated
microaerobically at 42C.
iii)
Incubation
Atmosphere
Microaerobic atmospheres of 510% oxygen, 510% carbon dioxide are required for optimal growth
(Corry et al., 2003; Vandamme, 2000). Appropriate atmospheric conditions may be produced by a
variety of methods. In some laboratories, (repeated) gas jar evacuations followed by atmosphere
replacement with bottled gasses are used. Gas generator kits are available from commercial sources.
Variable atmosphere incubators are more suitable if large numbers of cultures are undertaken.
Temperature
Media may be incubated at 37C or 42C, but it is common practice to incubate at 42C to minimise
growth of contaminants and to select for optimal growth of C. jejuni/C. coli. The fungistatic agents
cycloheximide or amphothericin are added in order to prevent growth of yeasts and mould at 37C
(Bolton et al., 1988). In some laboratories, incubation takes place at 41.5C to harmonise with
Salmonella and Escherichia coli O157 isolation protocols (ISO, 2006).
Time
Campylobacter jejuni and C. coli usually show growth on solid media within 2448 hours at 42C. As
the additional number of positive samples obtained by prolonged incubation is very low, 48 hours of
incubation is recommended for routine diagnosis (Bolton et al., 1988).
d)
Confirmation
A pure culture is required for confirmatory tests, but a preliminary confirmation can be obtained by direct
microscopic examination of suspect colony material.
i)
Identification on solid medium: On Skirrow or other blood-containing agars, characteristic

Campylobacter colonies are slightly pink, round, convex, smooth and shiny, with a regular edge. On
charcoal-based media such as mCCDA, the characteristic colonies are greyish, flat and moistened,
with a tendency to spread, and may have a metal sheen.
ii)
Microscopic examination of morphology and motility: material from a suspect colony is suspended in
saline and evaluated, preferably by a phase-contrast microscope, for characteristic, spiral or curved
slender rods with a corkscrew-like motility. Older cultures show less motile coccod forms.
iii)
Detection of oxidase: take material from a suspect colony and place it on to a filter paper moistened
with oxidase reagent. The appearance of a violet or deep blue colour within 10 seconds is a positive
reaction. If a commercially available oxidase test kit is used, follow the manufacturers instructions.
iv)
Microaerobic growth at 25C: Inoculate the pure culture on to a non-selective blood agar plate and
incubate at 25C in a microaerobic atmosphere for 48 hours.
v)
Aerobic growth at 41.5C: Inoculate the pure culture on to a non-selective blood agar plate and
incubate at 41.5C in an aerobic atmosphere for 48 hours.
1186
vi)
e)
Latex agglutination tests for confirmation of pure cultures of C. jejuni/C. coli (often also including C. lari)
are commercially available.
Identification of Campylobacter to the species level

Among the Campylobacter spp. growing at 42C, the most frequently encountered species from samples of
animal origin are C. jejuni and C. coli. However, low frequencies of other species have been described.
Generally, C. jejuni can be differentiated from other Campylobacter species on the basis of the hydrolysis of
hippurate as this is the only hippurate-positive species isolated from veterinary or food samples. The
presence of hippurate-negative C. jejuni strains has been reported (Steinhauserova et al., 2001). Table 2
gives some basic classical phenotypic characteristics of the most important thermophilic Campylobacter
species (ISO, 2006). Sensitivity to nalidixic acid used to be one of the most commonly tested characteristics,
but nowadays may give difficulties in interpretation, both due to an increase in nalidixic acid-resistant strains
of C. jejuni and C. coli and to the isolation of nalidixic acid-sensitive genogroups of C. lari. More extensive
speciation schemes have been described in the literature (On, 1996; Vandamme, 2000). Speciation results
should be confirmed using defined positive and negative controls.
The confirmatory tests for the presence of thermophilic campylobacters and the interpretation (ISO, 2006)
are given in Table 1. Confirm results of confirmation tests using positive and negative controls.
Table 1. Confirmatory tests for thermophilic Campylobacter
Confirmatory test
Result for thermophilic Campylobacter
Morphology
Small curved bacilli
Motility
Characteristic (highly motile and cork-screw like)
Oxidase
Aerobic growth at 41.5C
Microaerobic growth at 25C
Table 2. Basic phenotypic characteristics of selected thermophilic Campylobacter species

Characteristics
C. jejuni
C. coli
C. lari
Hydrolysis of hippurate
Hydrolysis of indoxyl acetate
Key: + = positive; = negative.
i)
Detection of hippurate hydrolysis: Suspend a loopful of growth from a suspect colony in 400 l of a 1%
sodium hippurate solution (care should be taken not to incorporate agar). Incubate at 37C for 2 hours,
then slowly add 200 l 3.5% ninhydrin solution to the side of the tube to form an overlay. Re-incubate
at 37C for 10 minutes, and read the reaction. Positive reaction: dark purple/blue. Negative reaction:
clear or grey. If commercially available hippurate hydrolysis test disks are used, follow the
manufacturers instructions.
ii)
Detection of indoxyl acetate hydrolysis: Place a suspect colony on an indoxyl acetate disk and add a
drop of sterile distilled water. If indoxyl acetate is hydrolysed a colour change to dark blue occurs within
510 minutes. No colour change indicates hydrolysis has not taken place. If commercially available
indoxyl acetate hydrolysis test disks are used, follow the manufacturers instructions.
Biochemical speciation may be supplemented or even replaced with molecular methods. A variety of DNA
probes and polymerase chain reaction (PCR)-based identification assays has been described for
Campylobacter species (On, 1996; Vandamme, 2000). On & Jordan (2003) evaluated the specificity of
11 PCR-based identification assays for C. jejuni and C. coli.
f)
Molecular detection of Campylobacter

PCR-based methods for the detection of Campylobacter in animal faecal samples and enriched meat
samples have been previously described in the literature (Olsen et al., 1995). One of these assays is in use
in Denmark for routine screening of cloacal swabs from broilers at the slaughterhouse (Bang et al., 2001;
Lund et al., 2003).
1187
g)
Antigen-capture-based tests
Enzyme immunoassays are available for the detection of Campylobacter in human stool samples only.
2.
Serological tests
There are no serological assays in routine use for the detection of colonisation of C. jejuni/C. coli in livestock.

There are no vaccines specifically developed for C. jejuni or C. coli in animals or birds.
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campylobacters including Campylobacter upsaliensis. J. Clin. Pathol., 46, 829831.
ATABAY H.I. & CORRY J.E.L. (1998). The isolation and prevalence of campylobacters from dairy cattle using a
variety of methods. J. Appl. Microbiol., 84, 733740.
BANG D.D., PEDERSEN K. & MADSEN M. (2001). Development of a PCR assay suitable for Campylobacter spp. mass
screening programs in broiler production. J. Rapid Methods Autom. Microbiol., 9, 97113.
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CORRY J.E.L., ATABAY H.I., FORSYTHE S.J. & MANSFIELD L.P. (2003). Culture media for the isolation of
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Edition, Corry J.E.L., Curtis G.D.W. & Baird R.M. eds. Elsevier, Amsterdam, The Netherlands, 271315.
CORRY J.E.L., POST D.E., COLIN P. & LAISNEY M.J. (1995). Culture media for the isolation of campylobacters. Int. J.
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FRIEDMAN C.R., NEIMANN J., WEGENER H.C. & TAUXE R.V. (2000). Epidemiology of Campylobacter jejuni infections
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LUND M., WEDDERKOPP A., WAINO M., NORDENTOFT S., BANG D.D., PEDERSEN K., & MADSEN M. (2003). Evaluation of
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*
* *
NB: There is an OIE Reference Laboratory for Campylobacteriosis
diagnostic tests and reagents for campylobacteriosis
1189
CHAPTER 2.9.4.
CRYPTOSPORIDIOSIS
SUMMARY
Cryptosporidiosis is caused by protozoan parasites of the genus Cryptosporidium, in which there
are 18 valid species. In livestock, C. parvum, C. andersoni, C. baileyi, C. meleagridis and C. galli
have been reported to cause morbidity and outbreaks of disease. Laboratory identification is
required to confirm diagnosis. Cryptosporidium parvum cryptosporidiosis causes scour in young,
unweaned mammalian livestock, however, weaned and adult animals can also become infected.
Signs range from a mild inapparent infection to severe scouring, and the young, old or
immunocompromised are most susceptible. Mortality is low. Weaned and adult animals normally do
not exhibit signs of disease, but will excrete oocysts that may contaminate the environment.
Cryptosporidium andersoni cryptosporidiosis affects the digestive glands of the abomasum of older
calves and adult cattle. Some infected animals exhibit reduced weight gain, but do not develop
diarrhoea. Cryptosporidium baileyi, C. meleagridis and C. galli cryptosporidiosis are diseases of
birds. Cryptosporidium baileyi affects primarily the bursa of Fabricius and cloaca of gallinaceous
birds, C. meleagridis affects primarily the ileum of turkey poults and C. galli infects the surface,
ductal, and glandular epithelium of the proventriculusof adult hens and some wild birds.
Identification of the agent: There is no prescribed test for cryptosporidium infection. The
demonstration of cryptosporidium species oocysts or cryptosporidium antigen in a properly
collected and submitted sample is sufficient for a positive diagnosis. Diagnosis is established
microscopically, with the acid-fast ZiehlNeelsen or auramine phenol methods using
unconcentrated or concentrated faecal smears. Microscopy-based methods for detecting oocysts
and enzyme-linked immunosorbent assays for detecting cryptosporidium antigens are relatively
insensitive, but are sufficiently sensitive for detecting clinical cases. Neither tinctorial- nor
fluorescence-based stains can determine the species of cryptosporidium present if the oocysts fall
within the size range of 46 m. These methods can detect oocysts in clinically ill animals, but
sometimes are not sufficiently sensitive to detect infection in clinically normal animals. Nucleic acid
detection tests have a greater sensitivity. The polymerase chain reaction (PCR) and restriction
fragment length polymorphism (RFLP) and/or sequencing can be used to determine some or all
cryptosporidium species/genotypes or subtypes. Those typing and subtyping systems used for
veterinary (and human) samples should also be used for environmental samples, to avoid any
confusion arising from using different systems during the investigation of disease outbreaks with
both veterinary and public health implications. However the sensitivity of subtyping systems will
need to be increased so that they can be used with clinical and environmental samples containing
small numbers (<10) of oocysts. Current limitations of discriminatory species subtyping systems are
that they are only applicable to C. parvum and C. hominis. Discriminatory subtyping systems for
non-parvum and non-hominis pathogens (including the majority of livestock pathogens) have yet
to be developed.
Specimens for primary diagnosis should be collected during acute infection, and should be
processed as soon as possible, ideally, within 24 hours. Transportation to the laboratory should be
in accordance with the International Air Transport Association regulations, which are summarised in
Chapter 1.1.1 Collection and shipment of diagnostic specimens.
Demonstration of Cryptosporidium oocysts or Cryptosporidium-specific antigen in faecal samples is
the most appropriate test for most applications. Most infections that cause morbidity and/or
mortality in mammalian livestock are likely to be caused by C. parvum cryptosporidiosis.
Cryptosporidium bovis is a highly prevalent species that infects primarily post-weaned calves.
Cryptosporidium baileyi, C. meleagridis and C. galli cryptosporidiosis cause morbidity and/or
1190
Chapter 2.9.4. Cryptosporidiosis
mortality in avian livestock. The species of Cryptosporidium responsible can be determined by

PCR-RFLP and/or sequencing of cryptosporidium oocyst DNA. There are no international
standards for the preparation of purified oocysts, antisera, antigens, monoclonal antibodies or
hybridomas, although a variety of purified oocysts and coproantigen detection kits using
monoclonal antibodies are available commercially.
Serological tests: Cryptosporidiosis is often a disease of the newborn and unless there has been
exclusion of exposure to infectious oocysts, serological tests do not offer any benefit.
Requirements for vaccines and diagnostic biologicals: There is no control programme for
cryptosporidiosis, neither is there a rigorously tested and accepted vaccine available.
A. INTRODUCTION
Originally described in 1907, Cryptosporidium spp. were regarded as commensals until their association with
diarrhoea in young turkeys (C. meleagridis) in the 1950s, and with large outbreaks of diarrhoea in calves
(C. parvum) in the 1970s. Cryptosporidium is an important pathogen of livestock and human beings, and since the
1980s, C. parvum cryptosporidiosis has been recognised as a common cause of acute self-limiting gastroenteritis
in immunocompetent hosts. Fayer (2008) provides a good account of the biology of Cryptosporidium.
Cryptosporidiosis is caused by protozoan parasites of the genus Cryptosporidium (family Cryptosporidiidae, order
Eucoccidiorida, subclass Coccidiasina, class Sporozoasida, phylum Apicomplexa). Although more than
20 species of this coccidian parasite have been described on the basis of the animal hosts from which they were
isolated, host specificity as a criterion for speciation appears to be ill-founded as some species lack such
specificity. Species definition and identification of this genus is constantly changing, with the addition of new
species based primarily on molecular criteria. Currently, there are 18 valid species (Table 1) namely: C. hominis
found primarily in humans (previously known as C. parvum Type 1), C. parvum, found in humans and other
mammals (previously known as C. parvum Type 2), C. andersoni and C. bovis in cattle, C. canis in dogs, C. muris
in mice, C. felis in cats, C. wrairi in guinea-pigs, C. suis in pigs, C. fayeri in red kangaroo (Ryan et al., 2008),
C. macropodum in grey kangaroo (Power & Ryan, 2008), C. meleagridis in turkeys and humans, C. baileyi in
chickens, C. galli in adult hens and some wild birds (Pavlasek, 1999; 2001), C. varanii in emerald monitor lizards,
C. serpentis in snakes and lizards, and C. molnari in fish (Fayer, 2008). In livestock, C. parvum, C. andersoni,
C. baileyi and C. meleagridis have been reported to cause morbidity and outbreaks of disease.
Cryptosporidium bovis has been described recently (Fayer et al., 2005). Previously identified as Cryptosporidium
genotype Bovine B (GenBank AY120911), C. bovis oocysts are morphologically indistinguishable from C. parvum
oocysts (Table 1). Cryptosporidium bovis is a highly prevalent species that infects primarily post-weaned calves
(Fayer et al., 2005; Feng et al., 2007). Cryptosporidium bovis oocysts were not infectious for neonatal BALB/c
mice or for two experimentally exposed lambs (<1 week of age), but were infectious for two calves that were
previously infected with C. parvum. Cryptosporidium bovis was detected in calves, 27 months of age, none of
which had diarrhoea; C. bovis has also been detected in a 2-week-old lamb.
In addition to the 18 valid species, there are over 40 Cryptosporidium genotypes (Fayer, 2008; Xiao et al., 2008).
Some of these are likely to become recognised as species as further research is carried out. The Cryptosporidium
cervine, skunk, chipmunk genotype I and the C. hominis monkey genotypes (Table 2) have been described in
humans. Laboratory identification is required to confirm diagnosis.
In human and non-human hosts, molecular methods including the polymerase chain reaction (PCR), restriction
fragment length polymorphism (RFLP) and DNA sequencing have demonstrated a broader range of
cryptosporidium species than previously thought. Cryptosporidium meleagridis, C. canis, C. muris, C. felis and
C. suis has been described in immunocompetent and immunocompromised human patients, as well as
C. hominis and C. parvum. For example, C. meleagridis oocysts purified from human faeces are indistinguishable
from C. parvum by conventional methods (described in this chapter), but show genetic identity to C. meleagridis
from turkeys at a variety of separate genetic loci. There is some evidence that C. andersoni can also infect
humans. The most significant zoonotic threat for humans is from C. parvum and C. meleagridis.
The discovery of sequence-based differences within various genes (ribosomal RNA [rRNA], Cryptosporidium
heat-shock protein 70, actin, Cryptosporidium oocyst wall protein [COWP], Cryptosporidium thrombospondinrelated adhesive protein-1 & -2) and between individual isolates within a previously valid species has resulted in
revision of the taxonomy of the genus. Some of the 40+ Cryptosporidium genotypes currently described may
represent different species (Fayer, 2008; Xiao et al., 2008). Therefore, as for Cryptosporidium species, the current
classification of Cryptosporidium genotypes will be subject to change.
1191
Table 1. Some differences among species within the genus Cryptosporidium

Species
Oocyst dimensions (m)
Site of infection
Major host
Infectious
to humans
C. hominis
4.5 5.5
Small intestine
Humans
Yes
C. parvum
4.5 5.5
Small intestine
Neonatal mammalian
livestock, humans
Yes
C. suis
5.05 4.41
Small intestine
Pigs
Yes
C. felis
4.5 5.0
Small intestine
Cats
Yes
C. canis
4.95 4.71
Small intestine
Dogs
Yes
4.54.0 4.65.2
Intestine
Turkeys
Yes
5.5 7.4
Stomach
Rodents
Yes
C. meleagridis
C. muris
5.6 7.4 (5.06.5 8.16.0)
Stomach
Cattle
No
C. wrairi
4.05.0 4.85.6
Small intestine
Guinea-pigs
No
C. bovis
4.75.3 4.24.8
Small intestine
Cattle
No
C. baileyi
4.6 6.2
Trachea, bursa of
Fabricius, cloaca
Poultry
No
C. fayeri
4.55.1 3.85.0
(mean 4.9 4.3)
Intestine
Red kangaroo
(Macropus rufus)
No
Grey kangaroo
(Macropus giganteus)
No
C. andersoni
C. macropodum
C. galli
8.08.5 6.26.4
Proventriculus
Finches, chicken
No
C. serpentis
5.66.6 4.85.6
Stomach
Reptiles
No
C. varanii
6.3 x 5.5
Intestine
Emerald monitor lizard

(Varanus prasinus)
No
C. molnari
4.72 4.47
Intestine
Fish (gilthead
seabream)
No
3.75.0 3.04.7
(mean 4.44 3.91)
Intestine, very seldom

in the stomach
Fish (turbot)
No
C. scophthalmi
For many of the Cryptosporidium species in Table 1, oocyst size and shape are similar. This makes species
identification based on oocyst morphometry at the light microscope level difficult if not impossible, due to size
overlap.
Table 2. Some Cryptosporidium genotypes, infection site, and their infection status relative to humans
Cryptosporidium genotypes
Intestinal
Bear
Cervine* (3)
Deer
Deer-like
Deer mice
Duck
Ferret
Fox (2)
Goose (2)
Horse
Marsupial (2)
Mongoose
Monkey*
Mouse
Muskrat (2)
Opossum (2)
Ostrich
Ovine
Pig II
Rabbit
Raccoon
Seal (2)
Sheep novel genotype (2)
Skunk*
Snake
C. canis Coyote
C. canis Dog
C. canis Fox
Tortoise
Woodcock
Gastric
Caribou
Lizard
C. galli Finch
C. muris Japanese field

mouse genotype
Key: * infectious to humans. (Data from the following references: Fayer, 2008; Xiao et al., 2004; 2008).
1192
1.
Clinical signs
Cryptosporidium parvum cryptosporidiosis is a cause of scour in young, unweaned farmed livestock including
calves, lambs, goat kids and alpaca. Endogenous stages infect enterocytes of the distal small intestine, caecum
and colon. Villous atrophy, shortening of microvilli and sloughing of enterocytes are the major pathological
changes associated with disease, and affected animals usually recover within 2 weeks of showing signs of illness.
Scouring is most common in young animals but weaned and adult animals can also become infected. Signs can
range from a mild to inapparent infection in older animals to severe scouring in young animals. Mortality is low
unless occurring as a mixed infection with other enteric pathogens such as rotavirus. Older animals can remain
infected and excrete oocysts that can be transmitted to other susceptible hosts.
Cryptosporidium parvum infections of cattle can cause varying degrees of dehydration, dullness, anorexia, fever
and loss of condition. Mortality may be high. Rarely do they cause the acute dehydration, collapse and high
mortality seen with enterotoxigenic Escherichia coli or rotavirus, which can occur at a similar time. Oocysts can be
detected in clinically normal and clinically ill hosts. Scouring calves and lambs can excrete between 106 and
108 oocysts per g of faeces. Infected adult cattle excrete far fewer oocysts, yet subclinical infections of adult cattle
can generate similar numbers of oocysts over a 12-month period.
Cryptosporidium andersoni colonises the digestive glands of the abomasum of older calves and adult cattle. The
microvilli of peptic glands are destroyed by endogenous stages, which may account for the elevated
concentrations of plasma pepsinogen detected in infected hosts. Some infected animals exhibit reduced weight
gain compared with uninfected controls. Infected cattle do not develop diarrhoea, but can excrete oocysts for
several months.
Cryptosporidium is a primary pathogen in chicken, turkeys and quail, causing respiratory and/or intestinal disease,
leading to morbidity and mortality. Currently, two named species (Morgan et al., 2001) infect chicken and turkeys
(C. baileyi and C. meleagridis), and a third, unnamed species infects quail (Cryptosporidium sp.). Cryptosporidium
spp. are common intestinal infections in broiler chicken in the USA and Japan. Cryptosporidium baileyi
cryptosporidiosis is a disease of the epithelial lining of the bursa of Fabricius and cloaca of chicken, although the
trachea and the conjunctiva are lesser sites of infection. Cryptosporidium baileyi intestinal cryptosporidiosis of
chicken does not normally result in gross lesions or result in overt signs of disease. Villous atrophy, shortening of
microvilli and enterocyte detachment are the major pathological changes associated with disease.
Cryptosporidium baileyi respiratory cryptosporidiosis of chicken can result in severe morbidity and, on occasion,
mortality. Initially, severe disease is accompanied by sneezing and coughing, followed by head extension to
facilitate breathing. Epithelial cell deciliation and hyperplasia, mucosal thickening and discharge of mucocellular
exudate into the airways are major pathological changes associated with disease in young broilers. Severe signs
of respiratory disease can last up to 4 weeks post-infection (Current, 1997). Cryptosporidium baileyi
cryptosporidiosis of turkeys is similar to that of chicken. Chicken isolates of C. baileyi cause infection in other
birds.
Cryptosporidium meleagridis cryptosporidiosis is a disease of the ileum of turkey and other poults and human
beings. Cryptosporidium meleagridis cryptosporidiosis can cause severe diarrhoea in turkey poults. Villous
atrophy, crypt hyperplasia and shortening of microvilli are major pathological changes associated with disease
(Current, 1997). Transmission of a turkey isolate of C. meleagridis to chicken and domestic ducks has been
reported.
Cryptosporidium galli cryptosporidiosis is a disease of adult hens and some wild and exotic birds (Pavlasek, 1999;
2001; Ryan et al., 2003). Unlike the life cycle stages of either C. meleagridis or C. baileyi, the life cycle stages of
C. galli are limited to the epithelial cells of the proventriculus. Histology reveals Cryptosporidium parasites in the
proventriculus (surface, ductal, and glandular epithelium) (Blagburn et al., 1990; Pavlasek, 1999; 2001; Ryan et
al., 2003). Clinical signs include puffed plumage with head held under the wing, responsiveness to external
stimuli, and failure to thrive (Ryan et al., 2003). Histopathology of haematoxylin and eosin stained sections from
finches demonstrated necrosis and hyperplasia of proventricular glandular epithelial cells, and a mixed
inflammatory cell infiltration into the lamina propria of the proventriculus associated with large numbers of
Cryptosporidium oocysts attached to the surface of glandular epithelial cells (Ryan et al., 2003). Cryptosporidium
galli oocysts (8.08.5 6.26.4 m) are larger than those of C. baileyi.
Respiratory and intestinal cryptosporidiosis have been reported in commercially grown quail caused by
Cryptosporidium of an inadequately described species (Cryptosporidium sp.) whose oocysts are smaller than
those of C. baileyi and are not infectious to chicken or turkeys. Pathological changes are similar to those
described for C. baileyi respiratory and intestinal cryptosporidiosis of chicken (Current, 1997).
2.
Infectious dose
The infectious dose for C. parvum varies from isolate to isolate and from host species to host species. For mouse
(CD-1 strain) neonates the ID50 (median infectious dose) is between 87 and 60 oocysts (Korich et al., 2000). Ten
1193
oocysts produced infection in two out of two primates tested, and five oocysts produced clinical disease in
gnotobiotic lambs. The infectious dose for cattle is not known, but is thought to be small. Whether C. parvum
isolates vary in their ability to colonise different host species is unknown. In healthy adult human volunteers, the
ID50 is also dependent on both the isolate and the host immune status. In human volunteer infectivity studies,
C. parvum isolates differ in their ID50, their attack rate, and the duration of diarrhoea they induce. The ID50 of the
C. parvum UCP isolate (Ungar Cryptosporidium parvum, human derived, from Dr B. Ungar, USA) is 1042 oocysts;
the C. parvum IOWA isolate (bovine derived, from Ames, Iowa, USA) is 132 oocysts; and the C. parvum TAMU
isolate (equine derived, from Texas A & M University, USA) is 9 oocysts (Okhuysen et al., 1999). Oral infection
with 100 C. baileyi oocysts can result in intestinal cryptosporidiosis (Current, 1997).
3.
Transmission
Transmission can occur via any route by which material contaminated with viable oocysts excreted by infected
individuals can be ingested. Practices likely to enhance the spread of cryptosporidiosis include indoor calving and
lambing and the communal feeding and husbandry of neonates, where young susceptible animals are in close
contact with each other and the faeces of infected animals. Similarly, the disposal of faeces, farmyard manure or
other contaminated waste in land-based dumps, when followed by periods of heavy rainfall or melting snow can
lead to C. parvum oocyst contamination of water courses. These courses may be used as a source of drinking
water for other animals and for potable water for human consumption. Contaminated waste includes both the
liquid and solid by-products of animal husbandry.
4.
Maintenance of infection
A variety of wild mammals act as hosts to C. parvum (Fayer, 2008; Sturdee et al., 1999; Xiao et al., 2004),
particularly neonates, but little is known of the importance of their involvement in transmitting infection to, or
maintaining infection, in domesticated species in farmyard environments. Their role in on farm epidemiology in
domesticated species is also uncertain. The methods used for diagnosing infection in small mammals and wildlife
are the same as those described for farm animals. Oocysts are environmentally robust and can survive for long
time periods (>6 months) in moist, cool microclimates. Evidence exists for transmission of cryptosporidiosis from
clinically normal dams to suckling neonates, but, in general, the duration of the carrier state remains unknown. A
variety of bird species act as hosts to C. baileyi.
1.
There is no prescribed test for diagnosing Cryptosporidium infection. The demonstration of Cryptosporidium
species oocysts or Cryptosporidium antigen in a properly collected and handled sample is sufficient for a positive
diagnosis, and the methods of choice for collection of the samples are non-invasive. There are no reproducible invitro culture techniques available to amplify parasite numbers prior to identification, therefore the detection of the
oocyst (the transmissive stage), Cryptosporidium antigen and/or DNA from faeces, or other suitable body fluids
are the methods of choice. In addition to these tests, haematoxylin and eosin can be used for histological
confirmation of the diagnosis on post-mortem. Haematoxylin and eosin histology is useful for confirming
diagnosis, is commonplace world-wide, and will not be described in this chapter.
Further analyses including species and/or C. parvum subtype identity, can be performed on Cryptosporidium DNA
using molecular techniques, such as the PCR-RFLP and/or sequencing of products amplified from defined
genetic loci. This not only confirms diagnosis, but also provides discrimination beyond that possible with
morphology and morphometry using light microscopy.
For Cryptosporidium species that infect the gastrointestinal tract (Table 1), primary diagnosis is based on the
demonstration of oocysts in faeces by conventional tinctorial stains, fluorescent/immunofluorescent stains or
Cryptosporidium antigen(s) (copro-antigens) in faeces by enzyme-linked immunosorbent assay (ELISA) or
immuno-chromatographic (IC) methods. The majority of diagnostic methods have been developed using
C. parvum because of its commercial importance and availability. There is anecdotal evidence indicating that in a
minority of samples, the methods described below may not detect all isolates. The methods described below are
expected to detect most C. parvum infections, but their usefulness for detecting non-parvum species from clinical
material is less well understood.
Demonstration of Cryptosporidium oocysts or Cryptosporidium-specific antigen in faecal samples is the most
appropriate test for most applications. Most infections that cause morbidity and/or mortality in mammals are likely
to be due to C. parvum cryptosporidiosis. The species of Cryptosporidium responsible can be determined by
PCR-RFLP or sequencing of Cryptosporidium DNA isolated from oocysts, later. There are no international
standards for the preparation of purified oocysts, antisera, antigens, monoclonal antibodies (MAbs) or
1194
hybridomas, although a variety of purified oocysts and coproantigen detection kits using MAbs are available
commercially.
a)
Laboratory diagnosis
The diagnostic features of C. parvum oocysts viewed in suspension using Nomarski Differential Interference
Contrast (DIC) microscopy are as follows. Oocysts are smooth, thick walled, colourless, have spherical or
slightly ovoid bodies containing, when fully developed (sporulated), four elongated, naked (i.e. not within a
sporocyst[s]) sporozoites and a cytoplasmic residual body. The modal size measurement of C. parvum
oocysts is 4.5 5.0 m (range 46 m).
Diagnosis is normally established by conventional microscopic methods, and the modified ZiehlNeelsen
(mZN) or auramine phenol (AP) methods using unconcentrated faecal smears are frequently used
(Casemore, 1991; Casemore et al., 1985; Smith, 1992; 2008). Where low oocyst numbers are expected in
samples, or purified oocysts are required for molecular investigations, concentrating oocysts in faecal
samples can increase the sensitivity of detection. Sugar (e.g. Sheather), salt, zinc sulphate or formalin-ether
(formalin-ethyl acetate) solutions or specific concentration techniques, such as immuno-magnetic
separation, are the best options for concentrating oocysts from faeces (Smith, 1992; 1998; 2008).
b)
Demonstration in faeces
Stool samples from most clinically ill cases will contain large numbers of thick-walled oocysts and sufficient
Cryptosporidium antigen, therefore, the use of standard staining and immunological techniques should result
in a positive diagnosis. The numbers of clinically normal excretors is not known, and, given the insensitivity
of conventional methods, low oocyst excretors may not be diagnosed using conventional techniques, as
oocyst numbers may be below the limit of detection of these methods (Smith, 2008; Weber et al., 1991;
Webster et al., 1996). In clinically ill animals, oocysts can normally be demonstrated in unconcentrated stool
smears. The use of an oocyst concentration method can enhance the detection rate. Both flotation and
sedimentation methods are suitable for concentrating Cryptosporidium spp. oocysts, and oocyst antigens
can still be sought in faeces following these oocyst concentration procedures.
Oocysts might not be detectable in clinical samples from all cryptosporidiosis cases, and the absence of
oocysts in repeated submissions of samples from symptomatic hosts does not necessarily indicate the
absence of infection. In these instances, and particularly when clinical suspicion is high, oocyst negative
stool samples should be subjected to antigen and/or PCR-based detection, as sufficient Cryptosporidium
antigen or DNA from asexual life cycle forms should be present in faeces (Smith, 2008). This is a major
advantage of copro-antigen detection immunoassays. Nucleic acid detection tests such as the PCR are
being used increasingly as they offer both improved sensitivity and species/genotype/subtype identity. For
PCR-based methods, nested PCR methods, being more sensitive than direct PCR methods, are likely to
have a higher diagnostic index (Smith, 2008).
Cryptosporidium spp. positive faecal samples should be available to personnel who are familiarising
themselves with staining and concentration techniques, and smears from positive faecal samples should be
included each time a test is performed. Stool samples containing C. parvum oocysts can be stored at 4C in
either 2.5% K2Cr2O7 or 10% formalin for reference purposes. Similarly, oocyst-positive faecal smears, air
dried and fixed in absolute methanol, can be prepared in advance from previous positive samples for use as
positive controls. Where bronchio-pulmonary involvement is suspected, similar tests can be performed on
bronchial and pleural exudates or lavages.
Note that 2.5% K2Cr2O7 can be inhibitory to PCR. Oocyst positive stool samples or partially purified oocysts
stored in 2.5% K2Cr2O7 and intended for nucleic acid amplification by PCR should be washed in deionised
water to remove residual 2.5% K2Cr2O7 prior to DNA extraction. A series of three washes each followed by
centrifugation (3000 g for 10 minutes), removal of the supernatant and resuspension of the pellet in
deionised water should minimise PCR inhibition by 2.5% K2Cr2O7. It should be noted that inhibitory factors
can still be present even after long-term (>6 months) storage.
c)
Laboratory staff and operator safety

Cryptosporidium is included in laboratory risk Hazard Group 2 and all laboratory procedures that can give
rise to infectious aerosols must be conducted in a biosafety cabinet. Specimens for Cryptosporidium
analyses can contain other pathogenic organisms and should be processed accordingly. In order to
safeguard the health of laboratory workers, the safety procedures outlined in Chapter 1.1.3 Biosafety and
biosecurity in the veterinary microbiological laboratory and animal facilities must be followed.
The laboratory should have an internal and external quality assurance programme in place as outlined in
Chapter 1.1.4 Quality management in veterinary testing laboratories.
1195
d)
Collections and submission of samples

Where possible, specimens for primary diagnosis should be collected during acute infection, and should be
processed as soon as possible. Ideally, transportation systems should be selected to ensure that specimens
arrive at the laboratory within 24 hours. If prompt examination for Cryptosporidium cannot be carried out, the
deterioration of protozoan morphology and their overgrowth by other microorganisms, particularly yeasts,
can be reduced by the addition of 10% aqueous (v/v) formalin, although 10% formalin can interfere with PCR
tests. Both oocyst morphology for microscopic identification and sporozoite DNA for PCR testing can usually
be preserved for long periods at 4C without formalinisation. Faeces can be stored frozen for over 2 years
without affecting the ability to extract Cryptosporidium DNA for molecular analysis. In some instances, this
can enhance DNA extraction rates, possibly due to the softening of the outer oocyst wall. Repeated freeze
thawing of frozen faecal samples is not recommended as the released DNA will degrade.
The procedures used for collection and transport of specimens are critically important for successful
laboratory analyses. Specimens should be collected in a suitable leak-proof sample container and should be
enclosed in secure primary and secondary packaging. Procedures for packaging and shipping of specimens
must be as outlined in the International Air Transport Associations Dangerous Goods Regulations (IATA,
2003). These regulations are summarised in Chapter 1.1.1 Collection and shipment of diagnostic
specimens.
e)
Threshold of detection in faeces

Most tinctorial and fluorescence methods for detecting oocysts, and ELISA and IC for detecting
Cryptosporidium antigens, are relatively insensitive. These methods can detect oocysts in clinically ill
animals, but may not be sufficiently sensitive to detect infection in clinically normal animals. Anusz et al.
(1990) reported a detection limit of 106 oocysts per ml of faeces using the Kinyoun modification of mZN on
unconcentrated faecal smears. Concentrating oocysts in the sample can increase the sensitivity of detection.
In oocyst-positive human stool samples, between 1 104 and 5 104 oocysts per g of unconcentrated stool
are necessary to obtain a 100% detection efficiency using the Kinyoun mZN staining method (Weber et al.,
1991). Variations in faecal consistency influence the ease of detection, with oocysts being more easily
detected in concentrates made from watery, diarrhoeal specimens than from formed stool specimens
(Weber et al., 1991). In addition to microscopic techniques, a number of antigen-capture ELISAs and ICs
have been reported in the literature with detection limits in the region of 3 105106 oocysts per ml (Anusz
et al., 1990; Robert et al., 1990; Smith, 2008), which indicates that they do not appear to offer increased
sensitivity over microscopical methods.
In bovine faecal samples, oocysts were not detected in samples seeded with 10,000 C. parvum oocysts
per g following formolether sedimentation and examination using AP or immunofluorescence (IF) staining.
When oocysts were concentrated using sucrose flotation, the threshold of detection was 4000 oocysts per g
for both staining methods. After salt flotation, 4000 oocysts per g could be reliably detected by AP staining,
but the detection limit was increased to 6000 oocysts per g using IF staining (Weber et al., 1991). Webster et
al. (1996) also compared microscopy with PCR and found that PCR coupled with immunomagnetic particle
separation (IMS) of oocysts from faecal samples detected five oocysts per ml of diluted faeces, which
corresponds to 8090 oocysts per g. Even allowing for the dilution of formed faecal samples required for
IMS, this represented an increase in sensitivity of several orders of magnitude over the conventional
coprodiagnostic methods. Currently, a variety of sensitive, PCR-based tests are available (see Section B.1
Nucleic acid recognition methods).
1
2
Preparation of unconcentrated faecal (or appropriate body fluid) smears (include a positive
control slide each time this procedure is performed)
Test procedure
i)
Wear protective clothing and disposable gloves. Score the reference number of the specimen on a
microscope slide with a diamond marker1, and use separate microscope slides for each specimen.
Place 1 drop of saline (about 50 l) in the centre of the slide.
ii)
Remove a small sample of faeces (about 2 mg) with the tip of a clean applicator stick2 (or pipette after
mixing thoroughly, if liquid) and emulsify the sample in saline by thorough mixing. For liquid stools (or
other appropriate body fluid) dispense one drop directly on to the slide. In liquid stools, mucus strands
and exudates or pus can be mixed with saline on the microscope slide. Liquid stools can be diluted
with a drop of 150 mM saline solution.
Alternatively, a lead pencil can be used to mark the etched (frosted) portion of a frosted glass microscope slide.
For formed stools, the sample should include portions from the surface and from within the stool.
1196
f)
iii)
Prepare a medium to thick smear with areas of varying thickness. Ensure that the smear is of the
correct transparency3.
iv)
Air dry the smear at room temperature.
v)
Fix the smear4 in methanol for 3 minutes.
Preparation of faecal (or appropriate body fluid) smears following concentration by flotation or
sedimentation
No flotation or sedimentation method is specific for Cryptosporidium spp. oocysts. Flotation fluids are denser
than the parasites to be concentrated and are formulated to a defined specific gravity using a suitable
hydrometer available from most large laboratory suppliers. Parasites concentrated by flotation or
sedimentation methods can be identified by all the methods described in this chapter. Flotation/
sedimentation fluids can sometimes interfere with diagnostic tests. Excess sucrose can reduce both oocyst
attachment to glass slides and subsequent antibody binding, prolonged exposure to NaCl can distort
morphology and morphometry, and formalin can reduce the sensitivity of PCR reactions. When oocysts are
concentrated, excess flotation/sedimentation fluid can be removed by washing the concentrate in water and
re-centrifuging. The supernatant is then aspirated and discarded, care being taken not to disturb the pellet.
These concentration methods are suitable for any appropriate body fluid that could contain oocysts.
1.
Flotation
The flotation principle uses a liquid suspending medium, which is denser than the oocysts to be
concentrated. Therefore, when mixed with flotation fluid, the oocysts rise to the surface and can be
skimmed out of the surface film and detected using the chosen method. For a flotation fluid to be useful
in diagnostics, when morphology and morphometry are the critical factors, the suspending medium
must not only be heavier than the object to be floated but must not produce shrinkage sufficient to
render the object undiagnosable (Ryan et al., 2008). Sucrose flotation, zinc sulphate flotation and
saturated salt flotation methods are all suitable for concentrating cryptosporidium oocysts. The
following is a description of the methods used to prepare flotation solutions and to concentrate oocysts.
Sucrose flotation
Sucrose solution (specific gravity 1.18) is prepared in a glass beaker by adding 256 g of sucrose to
300 ml of deionised water. The solution is gently heated (<60C) and continuously stirred with the aid
of a magnetic stirrer on a hot plate stirrer, until the sucrose has dissolved completely. The sucrose
solution is either placed on ice or in a refrigerator until its temperature has adjusted to 4C. The cold
sucrose solution is transferred to a 500 ml measuring cylinder and its specific gravity is adjusted to
1.18 by adding sufficient cold, deionised water (4C). The sucrose solution is transferred to a screwcap glass bottle, labelled, dated, initialled and stored at 4C until used.
Zinc sulphate flotation
Zinc sulphate solution (specific gravity 1.18) is prepared in a glass beaker by adding 100 g of zinc
sulphate to 300 ml of deionised water. The solution is gently heated (<60C) and continuously stirred
with the aid of a magnetic stirrer on a hot plate stirrer, until the zinc sulphate had dissolved completely.
The zinc sulphate solution is transferred to a 500 ml measuring cylinder and its specific gravity is
adjusted to 1.18 by adding sufficient cold, deionised water (4C). The zinc sulphate solution is
transferred to a screw-cap glass bottle, labelled, dated, initialled and stored at 4C until used.
Salt flotation
Saturated salt solution (specific gravity 1.2) is prepared by adding approximately 200 g of sodium
chloride to 200 ml of deionised water. The solution is gently heated (<60C) and continuously stirred
with the aid of a magnetic stirrer on a hot plate stirrer. Small amounts of sodium chloride
(approximately 10 g) are added at 10-minute intervals until the solution becomes saturated. The
saturated salt solution is then decanted into a clean glass bottle and either placed on ice or in a
refrigerator until its temperature had adjusted to 4C. The cold saturated salt solution is then
transferred into a 500 ml measuring cylinder and its specific gravity is adjusted to 1.2 by adding cold,
deionised water (4C). The saturated salt solution is transferred to a screw-cap glass bottle and
labelled, dated, initialled and stored at 4C until used.
3
4
Moderately thick smears are recommended for this procedure. If the smear is too thin or thick, oocysts will be missed. An
acceptable thickness can be achieved when either the hands of your watch or the print on this page can just be read
when viewed through the preparation.
Air-dried, methanol-fixed smears can be kept at room temperature for >6 months before staining.
1197
Brine is a concentrated aqueous NaCl solution, which has a specific gravity between 1.120 and 1.200
depending on the impurity of the salt used. While suitable for concentrating Cryptosporidium spp.
oocysts, some protozoan cysts can become badly shrivelled or open up in this flotation fluid. The
optimal time to examine specimens obtained from brine flotation is between 5 and 20 minutes after
their recovery following flotation.
Centrifugal flotation has also been used to recover Cryptosporidium oocysts (and a various other
parasite cysts and ova) from faeces. Most centrifugal flotation methods are based on modifications of
the Clayton-Lane technique, whereby oocysts are concentrated by flotation and collected as a hanging
drop on the underside of a glass cover-slip placed onto the positive meniscus of the floatation fluid.
Centrifugation is used to separate particles which are denser than the flotation fluid from oocysts and
particulates which will float on the surface of the flotation fluid. The inclusion of a centrifugation step
speeds up separation of oocysts from other particulates (and hence, the concentration of oocysts) and
minimises the risk of the flotation fluid adversely affecting the morphometry or morphology of the
oocysts. The operator should note the health and safety issues, including lacerations and puncture
wounds, associated with handling cover-slips.
Concentration of Cryptosporidium spp. oocysts by flotation
Test procedure
i)
Wear protective clothing and disposable gloves. Transfer approximately 12 g of faeces5 with an
applicator stick to 3 ml of flotation fluid in a 12 ml test tube and mix thoroughly. If the stool is liquid, mix
thoroughly, and dispense approx. 12 ml fluid into the test tube.
ii)
Add, with gentle stirring, sufficient flotation fluid to form a positive meniscus at the rim of the test tube.
Remove any large particles from the surface and if necessary, add more flotation fluid to maintain this
positive meniscus.
iii)
Leave for 20 minutes, then, taking great care not to disturb the positive meniscus gently remove the
meniscus with a disposable pipette and dispense gently on to a microscope slide6.
iv)
Air dry the smear at room temperature.
v)
Fix the smear7 in methanol for 3 minutes.
Concentration of Cryptosporidium spp. oocysts by centrifugal flotation
Test procedure
i)
Wear protective clothing and disposable gloves. Transfer approximately 12 g of faeces8 with an
applicator stick to 3 ml of flotation fluid in a 12 ml centrifuge tube and mix thoroughly. If the stool is
liquid, mix thoroughly, and dispense approx. 12 ml fluid into the centrifuge tube.
ii)
Add, with gentle stirring, sufficient flotation fluid to form a positive meniscus at the rim of the centrifuge
tube. Remove any large particles from the surface and if necessary, add more flotation fluid to maintain
this positive meniscus.
iii)
Place the centrifuge tube in a bench top centrifuge with swing out buckets, and place a 22 mm
22 mm glass cover-slip onto the rim of the centrifuge tube, so that it flattens the positive meniscus. Add
a balance tube if necessary, and centrifuge at 1100 g9 for 5 minutes.
iv)
Once the centrifuge stops, pick up the glass cover-slip between index finger and thumb at opposing
corners of the cover-slip. A hanging drop will be present on the underside of the cover-slip. Carefully
place the cover-slip, with the hanging drop lowermost, onto a glass microscope slide.
2.
Centrifugal sedimentation
Parasites will settle more rapidly if the stool suspension is subjected to centrifugation, however, food
particles will also sediment more rapidly and can mask the presence of parasites in the film examined.
To overcome this potential problem, larger food particles can be removed prior to centrifugation by
5
6
7
8
9
Moderately thick smears are recommended for this procedure. If the smear is too thin or thick, oocysts will be missed. An
acceptable thickness can be achieved when either the hands of your watch or the print on this page can just be read
when viewed through the preparation. Score the reference number of the specimen on a microscope slide with a diamond
marker, and use separate microscope slides for each specimen. Alternatively, a lead pencil can be used to mark the
etched (frosted) portion of a frosted glass microscope slide.
Air-dried, methanol-fixed smears can be kept at room temperature for >6 months before staining.
Centrifugation at speeds higher than 1100 g for longer (>5 minutes) periods of time is not advised as some parasites may
deform and/or rupture and collapse.
1198
filtering the emulsified stool through a sieve with an aperture size large enough for parasites to pass
through, but which retains the larger food particles. As this process is more efficient than sedimentation
by gravity, a smaller faecal sample (500 mg1 g: the size of a pea) is sufficient for examination.
Although centrifugation concentrates the material more quickly, faecal debris, which can obscure
parasites, remains present. The efficiency of detection is increased by adding formalin for fixation and
preservation of parasites, and ether to remove fats and oils. Both 10% formalin and ether are
bactericidal. After centrifugation, a fatty plug, which may adhere to the inner walls of the tube, can be
seen at the interface of the two liquids. The ether layer, the fatty plug and the formalin below it are
discarded and the whole pellet is retained for examination.
Many modifications to this procedure have been advocated, and the following protocol is typical of the
method used in diagnostic laboratories. Less distortion of protozoan cysts occurs with this method than
with zinc sulphate flotation. This method achieves a concentration of 1550-fold, dependent on the
parasite sought, and provides a good concentrate of protozoan cysts and helminth eggs, which are
diagnostically satisfactory. All steps that can generate aerosols (excluding centrifugation) should be
performed in an operator protection safety cabinet.
Concentration of Cryptosporidium spp. oocysts by (formol/ether) centrifugal sedimentation
Test procedure
i)
Wear protective clothing and disposable gloves. Sample approximately 500 mg1 g faeces10 with an
applicator stick11 and place in a clean 1215 ml centrifuge tube containing 7 ml of 10% formalin. If the
stool is liquid, dispense about 750 l into the centrifuge tube.
ii)
Break up the sample thoroughly and emulsify using the applicator stick.
iii)
Filter the resulting suspension through a sieve into a beaker, then pour the filtrate back into the same
centrifuge tube12.
iv)
Add 3 ml of diethyl ether (or ethyl acetate13) to the formalinised solution, seal the neck of the tube with
a rubber bung (or a gloved thumb over the top of the tube) and shake the mixture vigorously for
30 seconds. Invert the tube a few times during this procedure and release the pressure developed
gently by removing the rubber bung (or your thumb) slowly.
v)
Centrifuge the tube at 1100 g14 for 2 minutes.
vi)
Loosen the fatty plug with a wooden stick by passing the stick between the inner wall of the tube and
the plug. Discard the plug and the fluid both above and below it by inverting the tube, allowing only the
last one or two drops to fall back into the tube. Discard this fluid, containing diethyl ether and formalin,
into a marked re-sealable liquid waste container.
vii)
Re-suspend the pellet15 by agitation. Pour the whole, or the majority of the re-suspended pellet on to a
microscope slide, or transfer the re-suspended contents on to a microscope slide with a disposable
pipette, and air dry.
A commercial device for concentrating helminth ova, larvae and protozoan cysts and oocysts using the
formalin-ether method is available. Sold as the Fecal Parasite Concentrator16 it is an enclosed system, and
consists of two polypropylene tubes, a flat-bottomed tube used for emulsifying the stool, and a conical tube
used for centrifugation, with an interconnecting sieve. The comprehensive method states that both fresh and
preserved (10% formalin, merthiolate-iodine-formalin, polyvinyl alcohol, and sodium acetate-formalin) stool
specimens may be used.
g)
Conventional staining methods

Both mZN and AP are effective for detecting Cryptosporidium oocysts in faeces (Casemore, 1991;
Casemore et al., 1985; Ryan et al., 2008; Smith, 2008). mZN-stained slides should be screened under the
40 objective lens and putative oocysts confirmed and measured under the 100 objective lens (morphology
10
11
12
13
14
15
16
This is the size of a pea.

The sample should include portions from the surface and from within a formed stool.
425 m aperture size, 38 mm diameter is equivalent to 36 mesh British Standard (BS 410-86) or 40 mesh American
Standard (ASTM E11-81). The skirt of the sieve should fit neatly into the rim of the beaker. Debris trapped on the sieve is
discarded by inverting the sieve and passing a stream of tap water through the mesh. Both the sieve and the beaker
should be washed thoroughly in running tap water between each sample.
Ethyl acetate, although less flammable than diethyl ether is nevertheless flammable, therefore the procedure should be
performed in well ventilated areas, ensuring that they contain no naked flames. Avoid prolonged breathing or skin contact.
Centrifugation at speeds higher than 1100 g for longer (>5 minutes) periods of time is not advised as some parasites may
deform and/or rupture and collapse.
Too large a pellet is indicative of one or more of the following: centrifuging above the recommended speed and/or time,
insufficient shaking (step iv), taking too large a faecal sample.
FPC, Evergreen Scientific, 2300 East 49th Street, P.O. Box 58248, Los Angeles, California 90058, USA.
1199
and morphometry) using a bright-field microscope with a 10 eyepiece. AP-stained slides require to be read
using an epifluorescence microscope equipped with a fluorescein isothiocyanate (FITC) filter set (excitation
490 nm; emission 510 nm). A UV filter set (excitation 355 nm, emission 450 nm) can assist in visualising APstained sporozoites. AP-stained slides can be screened under the 20 objective lens and oocysts with
typical morphology can be confirmed under the 40 objective lens. The 100 objective lens must be used for
all morphometric (size) measurements. AP-stained oocysts visualised under either the FITC or UV filters can
be measured by slowly increasing the voltage (light intensity) of the bright-field light source so that both
fluorescent and bright-field images can be seen concurrently. The object can then be measured with the
eye-piece graticule.
Calibration of size using the eye-piece and stage micrometers
Diagnosis of intact organisms often necessitates the measurement of the size and shape (morphometry) of
the organism in question, in order to ensure that it falls within the accepted range of standard parameters
(e.g. size and shape) for the species in question. At the light microscope level, measurement of objects
<1 mm is achieved by means of a stage micrometer used in conjunction with an eye-piece micrometer.
Objects are measured in Systeme International (SI) units, and the standard unit of measurement for
conventional microscopy is the micron ( = 0.001 mm).
The stage micrometer consists of a 76 26 mm glass slide that has a millimetre scale, graduated in microns
permanently mounted on it. The eye-piece micrometer is a disc of transparent glass or plastic bearing a
graduated scale, which is placed in one of the eye-pieces of a binocular microscope. The scale is usually
1 cm in length and is subdivided into millimetre intervals. When the microscope is focused on the object to
be measured, both the scale on the eye-piece micrometer and the image of the object are seen
simultaneously in focus. The standard scale on the stage micrometer is usually 1 or 2 mm.
When measurements are to be made, the appropriate objective lens, which is dependent on the
magnification required, is chosen, and the number of divisions corresponding to the length or breadth of the
image of the object is read on the scale of the eye-piece micrometer. The observed measurement is
translated into real length (which corresponds to the number of eye-piece micrometer divisions representing
the chosen parameter to be measured) by substituting the stage micrometer for the object and determining
the number of divisions on the eye-piece micrometer corresponding to a definite number of divisions of the
millimetre scale on the stage micrometer, under the same magnification.
Remember that your calculation, in real length, of the value of the division on the eye-piece micrometer
scale will only be valid for the magnification of the objective chosen. You will have to recalculate the value of
a division on the eye-piece micrometer for each objective of differing magnification on the microscope.
Because morphometry is a significant component of diagnostic parasitology, repetitive measurements of
similar objects present in a single sample, or of various objects of varying sizes in sequential samples,
necessitating the use of a variety of magnifications, will have to be undertaken. By determining the
micrometer value of the eye-piece scale for each objective used, the constant interchange of objects and
stage micrometer can be overcome. This enables rapid calculation of morphometry, in millimetres, or
fractions thereof, to be undertaken with any of the objective lenses available.
The graticule is placed in the eye-piece by unscrewing the lower component of the eye-piece and placing it
into the open tube. It must be seated correctly before the lower component is screwed back on to the eyepiece. Ensure that the diameter of the graticule is similar to the internal diameter of the lower lens tube. Do
not touch the surface of the graticule hold it by its edges. Make sure it is dust and grease free. The eye
lens is focused on to the graticule by adjusting it until the scale on the graticule is critically sharp.
The determination is carried out as follows:
i)
Insert the eye-piece micrometer, with its scale already in focus, into the microscope, making sure that
the graticule scale is the right way up.
ii)
Select the lowest power objective lens (e.g. 10 objective) and focus the microscope on the stage
micrometer, rotating the eye-piece and positioning the stage micrometer until the scales of the eyepiece micrometer and the stage micrometer lie parallel and close to or overlapping each other.
iii)
Count the number of intervals on the stage micrometer that correspond exactly to a whole number of
divisions on the eye-piece micrometer.
iv)
Divide the value observed on the stage micrometer by the number of divisions counted on the eyepiece micrometer scale to determine the value of each division on the eye-piece micrometer scale.
1200
v)
Repeat the above for each objective lens17.
vi)
Keep a permanent record of the calculation of the value for each of the divisions on the eye-piece
micrometer for each objective lens in close proximity to or attached on to the body of the microscope
(e.g. a piece of cardboard stuck to the front of the microscope).
Example
For each objective lens:

x eye-piece divisions = y m (on the stage micrometer)
1 eye-piece division = y/x m
Microscopical examination of a sample
The sample must be examined in a systematic manner. Observation should commence using the lowest
suitable objective, ensuring that the entire sample is viewed. A suggested scheme is as follows: commence
in the upper left-hand corner of the sample, working across the slide from left to right, one field width at a
time, until the upper right-hand edge of the sample is reached. Move down one field height and continue
working across the slide from right to left, field by field, until the left-hand edge of the sample is reached.
Continue in this manner until the end of the sample (lower right-hand corner) is reached. During this period
of observation, the fine focus should be adjusted continuously so that the depth of the sample is also
scanned. When a suspicious object is located, it is inspected under high power magnification and either
verified or disregarded. If the magnification of the image of the object is insufficient to be able to visualise
definitive morphological characteristics under the high (dry) objective, the immersion lens (100) must be
used. Wet mounts can be sealed with nail varnish or a proprietary permanent sealant.
Neither tinctorial- nor fluorescence-based stains can determine the species of cryptosporidium present if the
oocysts fall within the size range of 46 m (see Table 1). For mammalian livestock, the consensus opinion
is that the majority of infections are likely to be due to C. parvum, therefore, a preliminary diagnosis of
C. parvum cryptosporidiosis can be made. However, the presence of oocysts of the size range 46 m does
not necessarily indicate that the infectious species is C. parvum. Similarly, for birds, a preliminary diagnosis
of C. baileyi, C. meleagridis or C. galli cryptosporidiosis can be made depending on infection site and oocyst
size. Molecular identification of species/genotype/subtype can be performed later.
Reporting results of microscopical examination
Negative specimens should be reported as NO Cryptosporidium oocysts seen.

Positive specimens should be reported as Cryptosporidium oocysts seen.
A scoring system for positive samples can be used, based on the number of oocysts observed under the
40 objective lens. However, microscopic examination cannot be considered as a quantitative determination
as oocyst numbers vary considerably during the course of infection.
+
++
+++
=
=
=
less than 5 oocysts per slide

1 to 10 oocysts per field of view
11 or more oocysts per field of view
Modified ZiehlNeelsen (mZN)
Strong carbol fuchsin: Dissolve 20 g basic fuchsin in 200 ml absolute methanol and mix on a magnetic stirrer
until dissolved. Add 125 ml liquid phenol (GPR [80% w/w in distilled water]) carefully until well mixed, and
make up to the final volume with 1675 ml deionised water. Mix thoroughly. Filter before use through
Whatman No.1 filter paper to remove debris and store in a stock reagent bottle. Label, date and initial. Store
the stock reagent in a dark cupboard at room temperature.
Commercial supplies are also available. Often the concentration of basic fuchsin can vary within the
acceptable range of 13%.
1% acid methanol: Carefully add 20 ml hydrochloric acid (GPR/SLR) to 1980 ml of absolute methanol and
mix. Transfer to a stock reagent bottle, and label, date and initial.
0.4% malachite green: Add 2 g malachite green to 480 ml deionised water and mix on a magnetic stirrer.
Filter through Whatman No.1 filter paper into a stock reagent bottle, label, date and initial.
17
Notice that the value calculated in millimetres for each of the divisions on the eye-piece micrometer will be different for
objectives of different magnifications, with values calculated for real length being smaller for each of the divisions of the
eye-piece micrometer with increasing magnification.
1201
Test procedure
Include a positive control slide each time you perform this procedure.
i)
Wear protective clothing and disposable gloves. Fix the air-dried smear or concentrate18 in methanol
for 3 minutes.
ii)
Immerse the slide in cold strong carbol-fuchsin and stain for 15 minutes.
iii)
Rinse the slide thoroughly in tap water.
iv)
Decolourise in 1% acid methanol for 1015 seconds19.
v)
Rinse the slide in tap water.
vi)
Counterstain with 0.4% malachite green for 30 seconds.
vii)
Rinse the slide in tap water.
viii) Air-dry the slide. (The smear can be examined with or without a cover-slip. A little immersion oil is
spread over the smear which is then viewed with either dry or oil immersion lenses, without the addition
of a cover-slip. An alternative method is to add a cover-slip and mounting medium and then examine
the smear.)
ix)
Examine for the presence of oocysts by scanning the slide using the 40 objective lens of a bright-field
microscope. Confirm the presence of oocysts under the oil immersion objective lens.
x)
Measure the size and shape of the red-stained bodies20.
Diagnostic features of Cryptosporidium spp. oocysts stained with mZN
Cryptosporidium spp. oocysts stain red on a pale green background. The degree and proportion of staining
varies with individual oocysts. In addition, the internal structures take up the stain to varying degrees. Some
may appear amorphous while others may contain the characteristic crescentic forms of the sporozoites.
Cryptosporidium parvum oocysts appear as discs, 46 m in diameter. Yeasts and faecal debris stain a dull
red. Some bacterial spores may also stain red, but these are too small to cause confusion.
Auramine-phenol
Auramine phenol (AP): Dissolve 3 g phenol in 100 ml deionised water and slowly add 0.3 g Auramine O.
Filter through Whatman No. 1 filter paper into a stock reagent bottle. Label, date and initial the stock
reagent. Store at room temperature in a light-proof glass bottle with an airtight stopper. Commercially
available stains, such as Lemperts reagent, are also acceptable.
3% Acid methanol: Carefully add 60 ml hydrochloric acid (GPR/SLR) to 1940 ml absolute methanol and mix.
Transfer to a stock reagent bottle, and label, date and initial.
0.1% potassium permanganate: Add 0.5 g potassium permanganate to the 499.5 ml deionised water and
mix using a magnetic stirrer. Filter through Whatman No. 1 filter paper into a stock reagent bottle, and label,
date and initial.
Test procedure
Include a positive control slide each time you perform this procedure.
18
19
20
21
i)
Wear protective clothing and disposable gloves. Fix air-dried smears or concentrate21 in absolute
methanol for 3 minutes.
ii)
Immerse the slides in AP stain for 10 minutes.
iii)
Rinse in tap water to remove excess stain.
iv)
Decolourise with 3% acid alcohol for 5 minutes.
v)
Counterstain in 0.1% potassium permanganate for 30 seconds.
Moderately thick smears are recommended for this procedure.

Over-destaining must be avoided.
Isospora spp. oocysts stain red and appear as large elongated ovoid bodies, tapered at the end and containing either a
granular zygote or two sporoblasts. Cyclospora spp. oocysts stain pinkish red and appear as circular discs (810 m in
diameter) containing a central morula. The degree and proportion of staining varies with individual oocysts. The
unsporulated oocyst is seen generally in stool samples.
Moderately thick smears are recommended for this procedure.
1202
vi)
Air dry slide at room temperature22 (see Modified ZiehlNeelsen (mZN) step viii, above).
vii)
Examine for the presence of oocysts, using an epifluorescence microscope equipped with FITC filters,
by scanning the slide under the 20 objective lens. Confirm the presence of oocysts under the
40 objective lens.
viii) Measure the size and shape of the fluorescent bodies23.
Diagnostic features of Cryptosporidium spp. oocysts stained with AP
Cryptosporidium spp. oocysts appear ring or ovoid shaped and exhibit a characteristically bright apple-green
fluorescence against a dark background. Cryptosporidium parvum oocysts are ring or doughnut shaped,
measuring 46 m in diameter. If available, view the preparation under a UV filter (excitation 355 nm,
emission 450 nm), as sporozoites are more readily seen under the UV rather than the FITC filter set. Under
the UV filter, oocysts appear light green and sporozoites appear yellow green.
Culture
There is no reproducible method for culturing cryptosporidium from body fluids. In-vitro cell culture systems have
been described for semi-purified, infectious oocysts, but they have not been tested in sufficient depth with enough
isolates or inhibitory materials to be recommended for routine purposes.
Three approaches to the immunological detection of Cryptosporidium oocysts have proven useful, and a variety of
commercial kits are available. Each has a similar level of sensitivity, and either unconcentrated or concentrated
stool samples can be used depending on the likely number of oocysts in the sample. Immunofluorescence-based
kits, using a fluorescein isothiocyanate-conjugated anti-Cryptosporidium MAb that recognises surface exposed
epitopes of oocysts (FITC-C-MAbs) are more specific for, and can be more sensitive at, detecting
Cryptosporidium oocysts in faecal smears than conventional tinctorial stains. Compared with conventional
tinctorial stains, antibody-based detection kits (immunofluorescence, ELISA and immunochromatography) appear
to be expensive, considering that many report a similar detection threshold.
a)
Direct immunofluorescence
In direct immunofluorescence, a FITC-labelled MAb reactive with genus-specific, surface-exposed epitopes
on cryptosporidium oocysts binds to oocysts present in the sample. UV excitation using a FITC filter system
(maximum excitation wavelength 490 nm, mean emission wavelength 530 nm) causes the labelled oocysts
to exhibit a bright apple-green fluorescence. Materials provided with commercial kits vary but C. parvum
oocyst positive and negative controls, FITC-labelled anti-cryptosporidium MAb (provided at the working
dilution), and glycerol-based mounting medium containing a photo-bleaching inhibitor are normally included.
Known negative and positive samples must always be included in each test.
Air-dried faecal smears or faecal concentrates are fixed in absolute methanol (or acetone, depending on the
manufacturers instructions) and air dried. Often the slides provided in the kit are welled slides into which
both sample and kit reagents are dispensed and retained. The manufacturers instructions must be followed.
It is false economy to dilute kit reagents to increase the testing volume. The FITC-labelled antiCryptosporidium genus-specific MAb (Cryptosporidium MAb), at the predetermined working dilution, is
applied over the fixed, air-dried specimen(s) and the slide(s) are incubated horizontally in the dark in a
humidified chamber. Excess antibody is removed by gentle rinsing, and the excess moisture drained.
Mounting medium is placed over the specimen and a cover-slip is applied to the sample, ensuring that no air
bubbles are trapped over the specimen. If mounting medium is not supplied, a mixture of 50% nonfluorescent glycerol: 50% phosphate buffered saline (PBS) (v/v) is suitable. Samples are scanned using
20 objective lens, oocysts are confirmed using the 40 objective lens, and the number of oocysts present is
determined. Numbers can be recorded as identified previously. In the absence of a manufacturers method,
the following method will produce satisfactory results.
The nuclear fluorogen, 46-diamidino-2-phenyl indole (DAPI; [C16H15N5.2HCl, FW 350.2]), can be used to
highlight sporozoite nuclei within fluorescent oocysts providing further confirmatory morphological
information (Grimason et al., 1994; Smith et al., 2002). DAPI is a non-specific DNA intercalator, therefore
DNA of other cellular interferents including bacteria and yeasts will also be stained. DAPI at a working
strength of 0.4 g/ml is particularly useful when oocysts are sought in non-faecal samples (e.g. water and
22
23
Do not blot slides dry, as some blotting papers contain fluorescent fibres.
Putative oocysts are measured by slowly increasing the voltage (light intensity) of the bright-field light source so that both
fluorescent and bright-field images can be seen concurrently. Objects can then be measured with the eye-piece graticule.
1203
foodstuffs). DAPI intercalates with the nuclei of the sporozoites within viable or non-viable oocysts and
causes them to fluoresce sky blue.
A blue filter block (excitation 490 nm; emission 510 nm) is used to visualise FITC-C-mAb localisation and an
ultra-violet (UV) excitation (excitation 355 nm, emission 450 nm) is used to determine the presence of DAPI
stained sporozoite nuclei.
Test procedure
Include a positive and negative control slide each time this procedure is performed. Positive and negative
slides are supplied with the majority of immunofluorescence kits, and C. parvum oocysts can be purchased
from commercial suppliers24.
Sources other than C. parvum may be available from diagnostic veterinary laboratories or research institutes
or facilities. Local distributors may also supply oocysts obtained from commercial sources.
i)
Wear protective clothing and disposable gloves. Fix air-dried faecal smears or faecal concentrates in
absolute methanol for 5 minutes25.
ii)
Dispense 50 l of anti-cryptosporidium MAb at its working dilution on to the well of each slide. Ensure
complete coverage of the well.26
iii)
Place the prepared slide(s) in a humidity chamber with the slide(s) elevated above the absorbent
material used to generate humidity. Ensure that the absorbent material is moist.
iv)
Place the humidity chamber in an incubator at approximately 37C for the period of time prescribed by
the manufacturer (normally 3060 minutes).22
v)
Using a Bchner-type aspirator gently aspirate excess MAb from each well. Tilt the slide to an angle of
about 45C from the horizontal towards the operator, and aspirate the fluid that collects at the bottom of
the well(s) by placing the tip of an aspirator close to, but not touching, the fluid. The suction at the
aspirator tip will draw the fluid to waste. Repeat this procedure for each slide well containing a sample.
vi)
Dispense 50 l of PBS to each well and allow to stand for 2 minutes at room temperature.22
vii)
Gently aspirate the PBS from each well as described in step v. Apply a further 50 l of PBS to each
well and allow to stand for a further 2 minutes, before gently aspirating the PBS as described.22
viii) Apply 50 l of a 1/5000 DAPI in PBS solution to each well and allow to stand for 2 minutes at room
temperature. The working solution of DAPI is prepared by diluting a 2 mg/ml DAPI stock solution by
1/5000 in PBS (150 mM, pH 7.2). The working solution should be prepared for each day it is required.
A stock solution of DAPI (2 mg/ml in methanol) can be stored at 4C in the dark, indefinitely.
ix)
Gently aspirate off the DAPI solution from each well as described in step v.
x)
Apply 50 l of deionised water to each well and leave to stand for 13 seconds at room temperature,
then gently aspirate the deionised water from each well as described in step v.
xi)
Dispense 50 l of mounting medium to the centre of each well of each slide, then gently apply a coverslip on to the microscope slide.22
xii)
Allow the cover-slip to settle into place before scanning the slide27.
xiii) Scan the preparation for oocysts under the 20 and confirm under the 40 objective of an
epifluorescence microscope equipped with an FITC filter set. Measure oocysts under the 100
objective28. If necessary, slides can be stored at room temperature, in the dark, until read.
24
25
26
27
28
For example from Kate Miller, Sterling Parasitology Laboratory, University of Arizona, Dept of Veterinary Science and
Microbiology, Building 90, Room 308, Tucson, Arizona, 85721, USA; millerk@email.arizona.edu or Geoff and Sue
Pritchard, Bunch Grass Farm, 1301 Drury Road, Deary, Idaho, 83823, USA. pritchard@turbonet.com
Alternatively, allow the methanol to evaporate to dryness at room temperature.
Volumes, times, etc. can vary according to manufacturers instructions. Always follow the manufacturers instructions.
These temporary mounts can be made semi-permanent by sealing around the edges of the cover-slip with clear nail
varnish. Allow the cover-slip to settle into place for approximately 30 minutes to 1 hour before sealing with nail varnish.
Using the brush supplied with the nail varnish, carefully apply the nail varnish around the perimeter of the settled coverslip, using the width of the brush as the guide to the width of nail varnish applied. Ensure even coverage around the
cover-slip perimeter. Do not leave any gaps. Allow the nail varnish to dry at room temperature before labelling the slide(s)
appropriately with their unique identifier number. If required, use a scalpel to carefully shave/scrape any excess dried and
hardened nail varnish.
Putative oocysts are measured by slowly increasing the voltage (light intensity) of the bright-field light source so that both
fluorescent and bright-field images can be seen concurrently. Objects can then be measured with the eye-piece graticule.
1204
Diagnostic features of Cryptosporidium spp. oocysts stained by FITC-labelled anti-cryptosporidium MAb
Cryptosporidium spp. oocysts are round or slightly ovoid objects that exhibit a bright apple-green
fluorescence under the FITC filter set. Their measurements (measured length breadth) are presented in
Table 1. Often the fluorescence has an increased intensity around the entire circumference of the oocyst,
with no visible breaks in oocyst wall staining. If Evans blue, which reduces nonspecific fluorescence, is
included in the kit, the background fluorescence will be red. Nonspecific fluorescence is yellow. Always refer
to the positive control to ensure that the size, shape and colour of the putative oocyst is consistent with
those of the positive control. DAPI intercalates with the nuclei of the sporozoites within viable or non-viable
oocysts and causes them to fluoresce sky blue. Under the 100 oil immersion lens, a sporozoite nucleus is
spherical to subspherical, measuring approximately 1 micron in diameter. In the event of an oocyst being
distorted, the demonstration of up to four fluorescent nuclei in an object of a comparable size to an oocyst
will assist in its identification (Grimason et al., 1994; Smith et al., 2002).
b)
Detection of Cryptosporidium antigens by enzyme linked immunosorbent assay

In the ELISA, the presence of Cryptosporidium antigens in faeces (coproantigen) is sought. Depending on
the commercial kit, Cryptosporidium coproantigens are captured and developed using a mixture of
monoclonal and polyclonal antibodies. With the exception of increased throughput and automation,
coproantigen detection kits do not offer increased sensitivity beyond the methods described.
Commercially available sandwich ELISA antigen detection kits contain anti-Cryptosporidium-coated well
strips for capturing Cryptosporidium coproantigens, anti-Cryptosporidium antibodies for developing the
reaction that is conjugated to an enzyme (frequently horseradish peroxidase), substrate, chromogen/
substrate development system and stopping solution (which inhibits further enzyme catalysis when added to
the reaction mixture). These have been developed to detect C. parvum antigens in stool samples, but they
may also be capable of detecting common epitopes from non-C. parvum infections. Known negative and
positive samples are included in commercial kits. Commercial kits normally contain all the necessary
reagents to perform the analysis and the manufacturers instructions must be followed. It is false economy to
dilute kit reagents to increase testing capacity. A comprehensive method and a formula for calculating the
cut-off value and assigning positive or negative status to samples are usually included. Kit reagents are
normally stored at 4C when not in use. All reagents should reach room temperature before being used.The
diagnostician should always determine whether any contraindications apply to the use of a commercial test
and any stool/sample fixative used.Because of the variation in the methods described for different
commercial kits, no method for ELISA or IC coproantigen detection is included in this chapter.
c)
Detection of Cryptosporidium antigens by immunochromatography

Rather than relying on molecular diffusion to dictate the rate of antigen binding by the capture antibody as in
the ELISA format, which normally takes about an hour per reaction, in lateral flow immunochromatography
(IC), the speed of antigen binding to the solid phase-bound capture antibody is increased by a wicking
action. This draws all fluids rapidly through a membrane enclosed in the immunochromatography cassette
and reduces the time required for analysis from hours to minutes or seconds. Soluble Cryptosporidium
antigens in the test sample are drawn through the membrane and come into contact with, and bind to,
immobilised antibodies raised against Cryptosporidium antigens, which dramatically increases the speed of
antigenantibody interaction. Positive reactions are qualitative and are seen as a band of colour at a specific
location on the membrane, normally identified by a line on the cassette. The assay format can vary between
commercial kits. As for antigen detection by ELISA, the diagnostician should always determine whether any
contraindications apply to the use of a commercial test and any fixative used.
IC is a convenient alternative method for detecting Cryptosporidium antigen in stool samples and specificity
is reported to be high (98100%). Debate continues as to whether IC (or indeed ELISA) has reduced, equal
or better sensitivity than oocyst-staining methods. As with ELISA, IC assays can be invaluable in cases of
infection in the absence of detectable oocysts. Because of the variation in the methods described for
different commercial kits, no method for IC coproantigen detection is included in this chapter.
PCR is more sensitive than conventional and immunological assays for detecting oocysts in faeces, although the
sensitivity of published methods can range between 1 and 106 oocysts. These techniques are often restricted to
specialist laboratories. Care is necessary when choosing primers, as some primers amplify genus-specific DNA
whereas others amplify species-specific DNA. Prior to routine adoption in clinical laboratories, both the variability
between methods and the recognised difficulties in amplifying nucleic acids from faecal specimens by PCR must
be overcome.
1205
Faecal samples can contain many PCR inhibitors. In addition to bilirubin and bile salts, complex polysaccharides
are also significant inhibitors. For Cryptosporidium, boiling faecal samples in 10% polyvinylpolypyrrilidone (PVPP)
before extraction can reduce inhibition.
The most robust information regarding species/genotype/subtype information has been derived from the study of
three genetic loci (two 18S rRNA [Johnson et al., 1995; Nichols & Smith, 2004; Xiao et al., 1999b; 2001] and the
Cryptosporidium Oocyst Wall Protein [COWP] [Homan et al., 1999; Spano et al., 1997]) gene fragments by PCRRFLP and/or sequencing amplicons.
PCR amplification of Cryptosporidium DNA using the 18S rRNA primers (CPB-DIAGF/R) of Johnston et al. (1995)
yields products that vary in length from 428 bp to 455 bp. The Johnson et al. primers are included because they
have been evaluated for cross reactions against a total of 23 microorganisms and the primers have been shown
to work in a variety of matrices. The Ward et al. (2002) modification of the Johnson et al. (1995) reverse primer
(substitution of CPB-DIAGR with PW99R [TAA-GGA-ACA-ACC-TCC-AAT-CTC], which produces an amplicon of
approximately 420 bp) was shown by the authors to be more sensitive than CPB-DIAGF/R (Johnson et al., 1995)
in both direct and nested PCR assays. Further corroboration is required in different matrices before PW99R
(Ward et al., 2002) can be fully recommended as a replacement for CPB-DIAGR. The nested (NicholsJohnson;
Nichols & Smith, 2004) 18S rRNA assay has also been shown to be sensitive. A multi-locus approach to
characterising Cryptosporidium isolates is essential. A multiplex allele-specific PCR (MAS-PCR) based on the
dihydrofolate reductase gene sequence differentiates C. hominis (357 bp) from C. parvum (190 bp) in a one-step
reaction, which can be distinguished on agarose gel, without the requirement for endonuclease digestion and
RFLP analysis, thus reducing assay time considerably (Giles et al., 2002). MAS-PCR is as sensitive as other
diagnostically used assays targeting the 18S rRNA gene for C. hominis and C. parvum detection, and can detect
more mixed (C. hominis and C. parvum) species infections than 18S rRNA gene assays (Giles et al., 2002).
However, MAS-PCR failed to detect the DNA of C. felis, C. canis, C. muris and Cryptosporidium pig genotype I in
human samples (Jiang & Xiao, 2003) and its usefulness for determining the range of pathogenic species found in
livestock samples remains to be determined.
Currently, there is no standard genetic locus recommended for species identity, but RFLP or sequencing of 18S
rRNA gene loci are widely used (Xiao et al., 1999a) and provide information about more species than the COWP
gene locus. For detecting small numbers of oocysts (<100) consistently, a nested PCR is required. Two 18s rRNA
PCRs have been tested extensively in the authors laboratory (Nichols & Smith, 2004; Xiao et al., 1999b; 2001)
and the NicholsJohnson primers (Nichols & Smith, 2004) appear to be more sensitive, particularly with small
numbers (10) of C. parvum, C. hominis, C. felis, and C. muris oocysts (Nichols & Smith, 2004). Although less
sensitive, the Xiao et al. assay (Xiao et al., 1999b; 2001) has the benefit of being able to detect all species by
RFLP, particularly C. parvum and C. hominis, which the Nichols-Johnson assay (Nichols & Smith, 2004) cannot.
Table 3 contains the Cryptosporidium spp. and genotypes determined by RFLP of the amplicon defined by the
CPB-DIAGR/F primers following simultaneous digestion with restriction enzymes VspI/DraI (Nichols & Smith,
2004), which can identify five major species. Table 4 contains the RFLP result of the same amplicons separately
digested with the restriction enzymes VspI/AseI, SspI and DdeI according to available GenBank complete/partial
sequences of the 18S rRNA gene (Nichols & Smith, 2004 and unpublished data). Table 5 contains the
Cryptosporidium spp. and genotypes determined by RFLP of the amplicon defined by the XR2/XF2 primers
following digestion with enzymes AseI and SspI (Xiao et al., 1999b).
Not all Cryptosporidium species/genotypes can be identified by PCR-RFLP of the 18S rRNA loci; however most of
the species that are currently known to be commercially important for livestock can be identified by PCR-RFLP.
Sequencing has been used to distinguish C. parvum from C. bovis or the Cryptosporidium deer-like genotype, but
they can also be readily differentiated by digestion with MboII (Feng et al., 2007). Sequencing of the amplicon can
offer better information than PCR-RFLP, but sequencing is more expensive and takes longer than PCR-RFLP.
Currently, sequencing availability varies in different parts of the world and, for the more common species that
infect livestock, PCR-RFLP has an important role to play.
A single-tube nested PCR-RFLP assay (Homan et al., 1999) amplifying a fragment of the gene coding for COWP
distinguishes between C. hominis and C. parvum. This assay is recommended over and above that in Spano et
al., 1997 as it is more sensitive and offers a solution to the contamination frequently experienced in nested PCRs
due to re-amplification of PCR products. In this single-tube nested PCR, the inner and outer primers are added to
the initial reaction mixture. Optimisation of primer set concentrations and annealing temperatures result in the
preferential amplification of one product size only, defined by the inner primers.
No recommended method for extracting Cryptosporidium DNA from oocysts exists, and the sensitivity of most
methods described has not been addressed fully. Cryptosporidium DNA can be extracted either following partial
purification of oocysts using one of the flotation/sedimentation techniques described above, or from oocysts in
faeces following zirconia bead extraction (McLauchlin et al., 1999). If concentration by formolether sedimentation
is the routine laboratory test, oocyst concentrates suitable for lysis and amplification by PCR can be made by
substituting deionised water for the 10% formalin used in the method described. DNA loss can be a consequence
1206
of subsequent DNA purification using commercial purification columns, but normally there should be an adequate
number of oocysts present in the sample to extract sufficient Cryptosporidium DNA for PCR-RFLP/sequencing
analysis. The selection of a suitable DNA extraction technique is the most important step in determining the final
sensitivity of oocyst DNA detection.
Table 3. Structural analysis of the 18S rRNA gene defined by CPB-DIAGR primers after simultaneous digestion with
the restriction enzymes VspI or AseI and DraI (reproduced from Nichols & Smith, 2004)
Cryptosporidium species
(amplicon length in bp)
Number of
VspI/AseI sites
(ATTAAT)
Number of
DraI sites
(TTTAAA)
Fragments
length in bp
C. hominis (438)
None
222; 112; 104
C. parvum (435)
None
219; 112; 104
C. muris (431 or 432)
None
319; 112
C. felis (455)
189; 112; 104; 50
C. baileyi (428)
128; 112; 104; 84
C. meleagridis (434)
None
171; 112; 104; 47
Table 4. Cryptosporidium spp. and genotypes determined by RFLP of the amplicons defined by the CPB-DIAG
primers following digestion with enzymes VspI or AseI, SspI and DdeI, according to available GenBank
complete/partial sequences of the 18S rRNA gene
(amplicon length in bp)
VspI / AseI
SspI
DdeI
GenBank
Accession No.
C. hominis (438)
222, 104,112
264,111, 40,12, 11
204, 166,68
L16997
C. parvum (435)
219, 104,112
264, 108, 40,12, 11
201, 166, 68
L16996
C. muris (432)
320, 112
395, 37
224,166, 42
AF093498
C. andersoni (431)
319, 112
394, 37
265, 166
L19069
C. felis (455)
239, 104,112
401, 40,14
221, 166, 68
AF087577
C. baileyi (428)
212, 104,112
264, 164
262, 166
L19068
C. meleagridis (434)
171, 104,112, 47
264, 119, 40,11
200, 166, 68
AF112574
C. serpentis (430)
318, 112
380, 36,14
264, 166
AF093502
C. wrairi (435)
219, 104,112
264, 109, 40,11,11
201, 166, 68
AF115378
Cryptosporidium pig (435)
219, 104,112
375, 40, 11, 9
201, 166, 68
AF108861
C. saurophilum (432)
216, 108,112
264, 109, 40,19
198, 166, 68
AF112573
Cryptosporidium mouse (439)
175, 104,112,48
264, 112, 40,12,11
205, 166, 68
AF108863
Cryptosporidium ferret (438)
174, 103,113, 48
264, 111, 40,23
204, 166, 68
AF112572
Cryptosporidium dog (429)
213, 104,112
264, 105, 40,20
195, 166, 68
AF112576
Cryptosporidium koala (436)
220, 104,112
264, 109, 63
202, 166, 68
AF108860
Cryptosporidium kangaroo
(436)
220, 104,112
373, 63
202, 166, 68
AF112570
Cryptosporidium monkey (436)
220, 104, 112
264, 109, 52, 11
202, 166, 68
AF112569
1207
Table 5. Cryptosporidium spp. and genotypes determined by RFLP of the amplicons defined by the XR2 / XF2 primers
following digestion with enzymes AseI and Ssp1 (Reproduced from Xiao et al., 1999b)
VspI
SspI
C. hominis
561, 104, 102, 70
449, 254, 111, 12, 11
C. parvum gene type A
628, 104, 102
449, 254, 108, 12, 11
C. parvum gene type B
625, 104, 102
449, 254, 119, 9
C. muris
731, 102
448, 385
C. andersoni
731, 102
448, 385
C. felis
476, 182, 104, 102
426, 390, 33, 15
C. baileyi
620, 104, 102
572, 254
C. meleagridis
456, 171, 104, 102
449, 254, 108, 11, 11
C. serpentis
729, 102
414, 370, 33, 14
C. wrairi
628, 104, 102
449, 254, 109, 11, 11
Cryptosporidium pig
632, 104, 102
453, 365, 11, 9
C. saurophilum
628, 104, 102
418, 255, 109, 33, 19
Cryptosporidium mouse
457, 175, 104, 102
449, 254, 112, 12, 11
Cryptosporidium ferret
457, 174, 104, 102
449, 254, 111, 12, 11
Cryptosporidium kangaroo, koala
631, 104, 102
441,254, 109, 33
Cryptosporidium dog
633, 102, 94
417, 254, 105, 33, 20
Cryptosporidium monkey
559, 104, 102, 70
461, 254, 109, 11
The following method is effective in extracting DNA from small numbers (~10+) of partially purified oocysts, and is
used in the authors laboratory (Nichols & Smith, 2004; Nichols et al., 2003; 2006). Partially purified oocysts are
suspended in 100 l of lysis buffer (50 mM Tris/HCl, pH 8.5, 1 mM ethylene diamine tetra-acetic acid, pH 8, 0.5%
sodium dodecyl sulphate [SDS], Sigma-Aldrich) and subjected to 15 freezethaw cycles (1 minute in liquid
nitrogen; 1 minute 65C). Samples are then transferred to a 55C water bath, proteinase K (at a final
concentration of 200 g/ml) is added, and the samples are incubated for 3 hours. Proteinase K is heat denatured
(90C, 20 minutes), samples are chilled on ice for 1 minute, centrifuged (16,000 g, 5 minutes) then 70 l of
supernatant is removed for PCR amplification. SDS is inhibitory to Taq polymerase at concentrations as low as
0.01%, therefore, neutralisation of SDS in the extracted DNA is necessary. The addition of 2% Tween 20 will
neutralise up to 0.05% SDS.
Reagents for PCR reactions are dispensed in 0.5 ml thin-walled tubes. Each tube contains 90 l of pre-mixed
reagents (200 M of each of the four dNTPs, 200 nM each of primers CPB-DIAGR and CPB-DIAGF, bovine
serum albumin at a final concentration of 400 g/ml, MgCl2 at 3.5 mM, 2.5 U of Taq polymerase in PCR buffer
and Tween 20 at a final concentration of 2% to inactivate 0.05% SDS). Finally, 10 l of DNA template is
introduced below approximately 40 l of mineral oil. Samples are subjected to 39 amplification cycles, and
products are visualised following ethidium bromide staining of 1.4% agarose gels (Nichols & Smith, 2004).
The primers and step cycle protocols for amplifying either 18S rRNA gene fragment (Nichols & Smith, 2004; Xiao
et al., 1999b; 2001) or the COWP gene fragment (Homan et al., 1999) are given in Table 6.
Reporting results of PCR-RFLP/sequencing examination

Negative specimens should be reported as NO Cryptosporidium DNA detected.
Positive specimens should be reported as Cryptosporidium DNA detected inserting the species/
genotype(s)/subtype(s) identified (see Tables 35) after identifying the respective species from the RFLP
profiles presented in Table 2.
1208
Table 6. Step cycle PCR protocols for 18S rRNA (Nichols & Smith, 2004; Xiao et al., 1999b; 2001)
and single-tube nested COWP (Homan et al., 1999) PCR
Primers
Step cycle protocol
Ref.
CPB-DIAGF
AAG-CTC-GTA-GTT-GGA-TTT-CTG
CPB-DIAGR
TAA-GGT-GCT-GAA-GGA-GTA-AGG
80C, 5 minutes; 98C, 30 seconds

55C, 30 seconds; 72C, 1 minute; 94C, 30 seconds
72C, 10 minutes
4C
1 cycle
39 cycles
1 cycle
soak
XF1 (outer)
TTC-TAG-AGC-TAA-TAC-ATG-CG
XR1 (outer)
CCC-ATT-TCC-TTC-GAA-ACA-GGA
XF2 (inner)
GGA-AGG-GTT-GTA-TTT-ATT-AGA-TAA-AG
XR2 (inner)
AAG-GAG-TAA-GGA-ACA-ACC-TCC-A
94C, 3 minutes
94C, 35 seconds; 55C, 45 seconds; 72C, 1 minute
72C, 7 minutes
4C
1 cycle
35cycles
1 cycle
soak
oocry3 (outer)
AGA-TTA-ACA-GAA-TGC-CCA-CCA-GGT-A
oocry4 (outer)
CCA-TGA-TGA-TGT-CCT-GGA-TTT-TGT-A
oocry1 (inner)
CCT-GGA-TAT-CTC-GAC-AAT
Oocry2 (inner)
GCG-AAC-TAA-TCG-ATC-TCT-CT
94C, 5 minutes
94C, 1 minute; 67C, 1 minute; 72C, 1 minute
94C, 1 minute; 54C, 1 minute; 72C, 1 minute
72C, 10 minutes
4C
1 cycle
20cycles
35 cycles
1 cycle
soak
a: Nichols & Smith, 2004; b: Xiao et al., 1999b, 2001; c: Homan et al., 1999
a)
Quantitative real-time PCR

Quantitative real-time PCR methods use patented technologies (e.g. TaqMan, Applied Biosystems,
LightCycler, Roche Molecular Biochemicals). For both technologies, fluorogenic dyes are incorporated into
the amplicon during PCR, thus amplicon fluorescence increases as more PCR product is generated. Both
systems can detect PCR products during the initial cycles of the PCR reaction when amplification is
exponential, thus enabling quantitative analysis of fluorescent product. Quantitative real-time PCR methods
have been used to identify different Cryptosporidium species by exploiting the genetic polymorphism of the
18S rRNA gene for devising probes with differing melting temperatures, and for the quantitative detection of
cryptosporidium oocysts in environmental water samples and sewage. The increased sensitivity of real-time
PCR guarantees increased speed of detection and qualitative diagnosis while the quantitative nature of the
assay will be invaluable in estimating levels of contamination. The closed-tube assay format reduces the
danger of contamination from carry over (Smith & Nichols, 2007). Quantitative real-time PCR should be a
useful tool for the future, once issues surrounding matrix inhibition are overcome. Currently, there are no
standardised quantitative real-time PCR methods.
b)
Typing and subtyping for disease and source tracking

The value of characterising the genetic diversity of Cryptosporidium at different levels of specificity and the
importance of appropriate nucleic acid analysis cannot be over-emphasised. Molecular tools for inter- and
intra-species discrimination differ (Cacci et al., 2005) and the publication of complete genomic sequences
of C. parvum (Abrahamsen et al., 2004) and C. hominis (Xu et al., 2004) has assisted in developing
appropriate typing and subtyping tools (Cacci et al., 2005). Genetic markers differ in their information
content and the nature of the DNA fragment selected for detecting and characterising cryptosporidium and
Giardia should be considered carefully (see Table 4 below). In order to detect species, analysis of highly or
moderately conserved coding regions (e.g. 18S ribosomal DNA, structural and house-keeping genes) is
necessary, whereas investigations into the transmission of genotypes and subtypes, identifying sources of
infection and risk factors, requires more discriminatory fingerprinting techniques, which can identify individual
isolates or clonal lineages (Cacci et al., 2005; Smith et al., 2006).
Typing and subtyping systems used for veterinary (and human) samples should also be used for
environmental samples, particularly for source and disease tracking in order to avoid any confusion arising
from using different systems for human, non-human hosts and environmental samples for veterinary and
public health investigation of disease outbreaks (Cacci et al., 2005; Smith et al., 2006). Species
determination should be based on the analysis of at least two loci since this provides more robust
information. At least one locus should be 18S rRNA, and, where possible, another should be suitable for
both species identification and further subtyping analysis. For Cryptosporidium, mini- and micro-satellite
typing, GP60 sequencing and analysis of a double stranded RNA element have been used to subtype
1209
C. parvum and C. hominis, and may offer sufficient sub-species discrimination to address veterinary and
public health investigations, either separately or in combination (reviewed in Cacci et al., 2005; and Smith
et al., 2006). Further development of these subtyping systems is required e.g. gp60 sequencing shows
variability within C. parvum populations but not C. hominis. The goal would be to find common isolate
identifiers. There is also evidence that mixed species infections occur in both the human and animal
populations. It is therefore important that species and subtyping systems be capable of identifying mixed
infections to give accurate diagnosis and information. Recent work has confirmed the utility of mini- and
micro-satellite markers in the study of the population structure of Cryptosporidium, and in understanding
transmission dynamics of infection (reviewed in Cacci et al., 2005; Enemark et al., 2002; and Smith et al.,
2006). The most commonly used PCR assays for detecting and typing Cryptosporidium are listed in Table 7.
Table 7. List of targets, type of assay and main use of amplification-based
techniques for Cryptosporidium (Cacci et al., 2005; Smith et al., 2006)
Amplification target
Assay type
Main application
PCR, nested PCR, sequencing, PCR-RFLP, Real-time

PCR , microarray
Species & genotype identification
Hsp70
PCR, nested PCR, sequencing, Real-time PCR,

microarray
COWP
PCR, nested PCR, sequencing, PCR-RFLP,

microarray
PCR, nested PCR, sequencing,
PCR, nested PCR, sequencing, PCR-RFLP
PCR, nested PCR, sequencing,
Subgenotype identification
Microsatellites
PCR, nested PCR, sequencing, fragment typing
Minisatellites
PCR, nested PCR, sequencing, fragment typing
Reverse transcriptase, PCR, sequencing,

heteroduplex mobility assays
18S rDNA
Actin
-tubulin
GP60
Extra-chromosomal double
stranded RNA
Key: RFLP = restriction fragment length polymorphism; Hsp70 = heat shock protein 70;
COWP = Cryptosporidium oocyst wall protein; GP60 = glycoprotein 60.
2.
Serological tests (and/or tests for cellular immunity where relevant)
Cryptosporidiosis is often a disease of the newborn and unless there is sufficient evidence to exclude exposure to
infectious oocysts, serological tests do not offer any benefit. Serological tests can be used for seroepidemiological
surveys of exposure: most are ELISA based, using various aqueous extracts of C. parvum oocysts (e.g. Hill et al.,
1990). Tests for cellular immunity do not appear to offer specific benefit, and are not available.

There is no control programme for cryptosporidiosis, neither is there a rigorously tested and accepted vaccine
available.
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*
* *
1213
CHAPTER 2.9.5
CYSTICERCOSIS*
SUMMARY
Cysticercosis of farmed and wild animals is caused by the larval stages (metacestodes) of cestodes
(tapeworms), the adult stages of which occur in the intestine of humans and dogs or wild Canidae.
Bovine cysticercosis (primarily in muscle) and porcine cysticercosis (primarily in muscle, the central
nervous system and the liver) are caused by the metacestodes (cysticerci) of the human cestodes
Taenia saginata and T. solium, respectively. Cysticerci of T. solium also develop in the central
nervous system and musculature of humans. Cysticerci of T. asiatica occur in the liver and viscera
of pigs. Cysticercosis and coenurosis of sheep and goats (in the muscles, brain, liver and peritoneal
cavity) are caused by T. ovis, T. multiceps and T. hydatigena, adults of which occur in the intestines
of dogs and wild canids.
Most adult and larval tapeworm infections cause little or no disease. Exceptions are severe,
potentially fatal human neurocysticercosis (NCC) caused by T. solium, and occasionally neurocoenurosis caused by T. multiceps in humans. These parasites are also occasional causes of
muscle or ocular signs in humans. Gid caused by T. multiceps in ruminants can require slaughter
of the animal. Acute T. multiceps coenurosis and T. hydatigena cysticercosis in sheep and goats is
rare but may be fatal. Cysticercosis causes economic loss through condemnation of infected meat
and offal.
Identification of the agent: Adult Taenia tapeworms are dorsoventrally flattened, segmented and
large, reaching from 20 to 50 cm (species in dogs) to several metres (species in humans).
Anteriorly, the scolex (head) has four muscular suckers and may have a rostellum, often armed with
two rows of hooks, the length and number of these being relatively characteristic of a species. A
neck follows the scolex, and this is followed by immature and then by mature reproductive
segments, and finally gravid segments filled with eggs. Segment structure, although unreliable, can
aid in the identification of the species. Taenia species cannot be differentiated by egg structure.
Metacestodes consist of a fluid-filled bladder with one or more invaginated protoscoleces. These
bladderworms are each contained within a cyst wall at the parasitehost interface. This structure
comprises the cysticercus or coenurus.
Adult Taenia are recognised at post mortem or by passage of segments or eggs. Metacestodes are
grossly visible at post mortem and meat inspection, but light infections are often missed. NCC can
be diagnosed by imaging techniques.
Immunological tests: Adult Taenia infections can be recognised by detection of Taenia
coproantigen in faeces using an antigen-capture enzyme-linked immunosorbent assay, but the test
does not differentiate species and is not commercially available. Use of species-specific probes
remains experimental.
Serological tests: Tests for antibodies in serum are not used currently for the diagnosis of
cysticercosis in animals except for epidemiological purposes. Diagnosis is by meat inspection.
Antigens have been identified for the serological diagnosis of NCC in humans.
Requirements for vaccines and diagnostic biologicals: Vaccine antigens have been identified
for the metacestodes, but not for the adult stages of T. ovis, T. multiceps, T. saginata and T. solium.
A T. ovis vaccine is registered in New Zealand, but is not commercially available. A T. solium
vaccine is undergoing the steps for practical production.
1214
Chapter 2.9.5. Cysticercosis
A. INTRODUCTION
The metacestodes (or larval cestodes) of Taenia spp. tapeworms are the cause of cysticercosis in various farmed
and wild animals and in humans. Adult tapeworms are found in the small intestine of carnivore definitive hosts:
humans, dogs, and wild canids. Taenia saginata of humans causes bovine cysticercosis, which occurs virtually
world-wide, but particularly in Africa, Latin America, Caucasian and South/Central Asian and eastern
Mediterranean countries and the infection occurs in many countries in Europe. Taenia solium of humans causes
porcine cysticercosis and human neurocysticercosis (NCC). It is found principally in Mexico, Central and South
America, sub-Saharan Africa, non-Islamic countries of Asia, including India and China where there are freeranging, scavenging pigs. The cysticerci of T. asiatica of humans in South-East Asia occur in the liver of pigs.
Dogs and wild canids are the definitive hosts of metacestodes of sheep, goats and other ruminants, which occur
throughout most of the world, although T. multiceps has disappeared from the United States of America (USA)
and New Zealand. Taenia ovis occurs in the muscles of sheep, T. multiceps in the brain (occasionally in the
muscles) of sheep, goats, sometimes other ruminants and rarely humans, and T. hydatigena is found in the
peritoneal cavity and on the liver of ruminants and pigs. Diagnosis in animals is usually based on the host and the
location of the metacestode when identified at meat inspection or necropsy. Adults in definitive hosts are acquired
by the ingestion of viable metacestodes in meat and offal that has not been adequately cooked or frozen to kill the
parasite.
Gravid segments that are shed by the adult tapeworms migrate spontaneously from the anus to fall to the ground
and release eggs on the ground, or the segments and free eggs are passed in the faeces. Eggs may also be
disseminated from these sites by physical means or transport hosts, particularly flies. Taenia solium segments,
however, are often passed in chains. Eggs are immediately infective when passed. Animals acquire infection from
ingestion of segments and eggs contaminating herbage or in faeces. It is possible that pigs acquire T. solium by
coprophagy of the faeces of pigs that have eaten segments. Humans may be infected with T. solium by eggs on
vegetables, etc., that have been contaminated by faeces or dirty hands, by faecooral transmission or through
retro-peristalsis and hatching of eggs internally. Routine diagnosis continues to be mainly based on the
morphology of the adult tapeworm and the presence of eggs or segments in the faeces of infected definitive
hosts.
1.
Taenia saginata (the beef tapeworm): The adult is large, 48 metres long and can survive many years, usually
singly, in the small intestine of humans. The scolex (or head) has no rostellum or hooks. Useful diagnostic
features are presented in Table 1 (Khalil et al., 1994; Kyvsgaard & Murrell, 2005; Loos-Frank, 2000; Soulsby,
1982; Verster, 1969). Gravid segments usually leave the host singly and often migrate spontaneously from the
anus.
The eggs are typical taeniid eggs that cannot be differentiated from other Taenia or Echinococcus spp. eggs.
Taeniid eggs measure about 3045 m in diameter; contain an oncosphere (or hexacanth embryo) bearing three
pairs of hooks; have a thick, brown, radially striated embryophore or shell composed of blocks; and there is an
outer, oval, membranous coat, the true egg shell, that is lost from faecal eggs.
Metacestodes (Cysticercus bovis) of T. saginata usually occur in the striated muscles of cattle (beef measles), but
also buffalo, reindeer and deer. They are oval, about 0.51 0.5 cm long, translucent and contain a single white
scolex that is morphologically similar to the scolex of the future adult tapeworm. They are contained in a thin,
host-produced fibrous capsule. Cysts occasionally are found in the liver, lung, kidney, fat and elsewhere.
Taenia solium (the pork tapeworm) is smaller than T. saginata being up to 35 metres. The scolex has an armed
rostellum bearing two rows of hooks; the number and size of hooks can aid differentiation of Taenia spp. (Table
1). Gravid segments have 716 (<17) uterine branches and do not usually leave the host spontaneously, but
passively in chains with the faeces.
Metacestodes (C. cellulosae) occur in the muscles and central nervous system of pigs (pork measles), bear and
dogs and in the muscles, subcutaneous tissues and central nervous system of humans. Cysts are grossly similar
to those of T. saginata, but may be larger than the T. saginata cyst. They have a scolex bearing a rostellum and
hooks similar to the adult. Occasionally, in the brain of humans, they develop as racemose cysts up to 2 cm or
more across that lack a scolex.
1215
Table 1. Useful features for identification of scoleces and segments of Taenia spp.
Parasite
species
Number
of hooks
Length of hooks (m)

Large
hooks
Small
hooks
No.
testes
Layers
of
testes
Cirrus sac
extends to
longitudinal
vessels
No.
uterine
branches
T. hydatigena
2836
(2644)
191218
(170235)
118143
(110168)
600
700
Yes
610 that
re-divide
Lobes of ovary
unequal in size. No
vaginal sphincter.
Testes extend to
vitellarium, but not
confluent behind.
T. ovis
3034
(2438)
170191
(131202)
111127
(89157)
350
750
No
1120 that
re-divide
Lobes of ovary
unequal in size. Well
developed vaginal
sphincter. Testes
extend to posterior
edge of ovary.
T. multiceps
2230
(2034)
157177
(120190)
98136
(73160)
284
388
Yes
1420 that
re-divide
Lobes of ovary
equal in size. Pad of
muscle on anterior
wall of vagina.
Testes extend to
vitellarium, but not
confluent behind.
T. saginata
765
1200
No
1432 that
re-divide
Lobes of ovary
unequal in size with
small Well
developed vaginal
sphincter. Testes
extend to vitellarium,
but not confluent
behind.
without
rostellum
Ratio of
uterine
twigs to
branches
2.3
T. solium
2236
139200
93159
375
575
T. asiatica
with
rostellum
on some
868
904
Yes
716 that
re-divide
Lobes of ovary
unequal in size with
small accessory
lobe. No vaginal
sphincter. Testes
confluent behind
vitellarium
No
1632 that
re-divide
Ovary, vaginal
sphincter and extent
of testes as
T. saginata.
Posterior
protuberances on
some gravid
segments
Ratio of
uterine
twigs to
branches
4.4
Taenia asiatica (Asian Taenia): Closely related to but genetically distinguishable from T. saginata (Eom, 2006),
the adult in humans has an ovary, vaginal sphincter muscle and cirrus sac like those of T. saginata, but T. asiatica
has a rostellum and posterior protuberances on segments and 1632 uterine buds with 5799 uterine twigs on
one side. Segments are passed singly and often spontaneously.
The metacestodes (C. viscerotropica) are small, about 2 mm, and have a rostellum and two rows of primitive
hooks, those of the outer row being numerous and tiny. They occur mainly in the parenchyma and on the surface
of the liver of domesticated and wild pigs; they may be found on the omentum and, rarely, on the lungs and
colonic serosa. Occasionally they are found in cattle, goats, and monkeys.
Taenia ovis: Adults in the intestine of dogs and wild canines reach 12 metres in length and have an armed
rostellum (Table 1). Metacestodes (C. ovis) that occur in the musculature (skeletal and cardiac) of sheep and less
commonly goats reach 3.51.0 0.20.4 cm. Commonly, the cysticerci are degenerate with a green or cream,
1216
caseous or calcified centre. A similar parasite occurs in wild canines and dogs and the muscles of reindeer and
deer in northern areas.
Taenia hydatigena: Adults are 15 metres long, are found in the intestine of dogs and wild canines, and have an
armed rostellum (Table 1). Metacestodes (C. tenuicollis) are large, from 1 cm up to 67 cm, and the scolex has a
long neck. They are found attached to the omentum, mesentery and occasionally on the liver surface, particularly
of sheep, but also of other domesticated and wild ruminants and pigs. A wolf and reindeer/deer cycle exists in
northern latitudes, in which the metacestodes are found in the liver of the intermediate host; dogs are also
infected as definitive hosts.
Taenia multiceps: Adults are 40100 cm long in the intestine of canines and have an armed rostellum (Table 1).
The metacestodes (Coenurus cerebralis) are large, white fluid-filled cysts that may have up to several hundred
scoleces invaginated on the wall in clusters. Coenuri grow to 5 cm or so in size in the brain of sheep, the brain
and intermuscular tissues of goats, and also the brain of cattle, wild ruminants and occasionally humans. In neural
tissue the cysts are not encapsulated. The cysts induce neurological signs that in sheep are called gid, sturdy,
etc.
a)
Diagnosis of adult parasites in humans or canine carnivores

All parasite or faecal material from humans with possible T. solium infections must be handled with suitable
safety precautions to prevent accidental infection with the eggs. Taenia multiceps and Echinocccus spp. also
infect humans and, as taeniid eggs in dogs cannot be differentiated to species or genus level, in areas
where these are endemic, the same safety precautions apply. In addition to Taenia spp., humans and canine
carnivores may be infected by Diphyllobothrium and Hymenolepis spp., while six other cestode genera are
recorded occasionally in humans. These are described by Lloyd (1998b) and all can be differentiated from
Taenia spp. by egg/proglottid morphology. Recently however, T. taeniaeformis with morphologically
indistinguishable taeniid eggs was recorded in a child. In canids, Echinococcus spp. eggs cannot be
distinguished from Taenia spp. eggs, but the presence of the former can be determined by tapeworm size
and, more recently, Echinococcus species-specific antigen-capture enzyme-linked immunosorbent assay
(AG-ELISA) (Allan et al., 1992). Other worms, Dipylidium, Diplopylidium, Mesocestoides and
Diphyllobothrium spp. have morphologically distinct eggs and proglottids (Lloyd, 1998b; Soulsby, 1982).
Adult cestodes can be expelled from humans using an anthelmintic followed by a saline purgative and are
identified on the basis of scolex and proglottid morphology. A self-detection tool has been developed and
tested in Mexico (Flisser et al., 2005); medical staff in health centres are supplied with preserved tapeworm
segments in a bottle and a manual of questions to ask patients to try to identify carriers. In animals,
arecoline purgation has been useful; again, the recovered tapeworms are identified morphologically.
Arecoline is no longer available as an anthelmintic, but can be obtained from chemical supply companies. As
it has side-effects, old, infirm and pregnant animals should be excluded from treatment. A dose of 4 mg/kg
should result in purgation in under 30 minutes, provided food has been withheld for several hours (i.e.
administer to dogs with empty stomachs). Walking and abdominal massage of recalcitrant cases or enema
for constipated dogs may avoid the use of a second dose (2 mg/kg), which should be given only sparingly.
Fortunately, arecoline purgation is being replaced rapidly by the copro-antigen ELISA for Echinococcus spp.
and perhaps in the future this will also be the case for Taenia spp. Tapeworms can be recovered after
anthelmintic treatment, and require appropriate disposal.
Verster (1969) and Loos-Frank (2000) have given descriptions of parasitic diagnosis of all the Taenia spp. of
humans and animals, their hosts and geographical distributions. Keys for identification are given by Khalil et
al. (1994). Mayta et al. (2000) and Loos-Frank (2000) give methods for mounting, embedding, sectioning
and staining the proglottids. The following staining technique is that of Loos-Frank (2000). Worms, after
relaxation in water, can be stained directly, although small worms should be fixed in ethanol for a few
minutes. Alternatively, worms can be fixed and stored in 70% ethanol containing 10% lactic acid, the scolex
and worm being stored separately. The rostellar hooks of scoleces or protoscoleces should be cut off and
mounted en face in Berleses fluid (made by dissolving 15 g gum arabic in 20 ml distilled water and adding
10 ml glucose syrup and 5 ml acetic acid, the whole then being saturated with chloral hydrate, up to 100 g).
The stain is lactic acid carmine: 0.3 g carmine is dissolved at boiling point in 42 ml lactic acid and 58 ml
distilled water, 5 ml of 5% iron chloride solution (FeCl2.4H2O) is added after cooling and can be used again
to refresh older solutions. Specimens are allowed to sink in the stain in a vial and then are left in the stain for
some more minutes to allow the stain to penetrate. Specimens are then washed in 1-day-old tap water until
blue in colour. They are then fixed in 5070% ethanol and dehydrated under the slight pressure of plastic foil
keeping the segments flat. Salicylic acid methyl ester is used as clearant.
When segments break from the end of the worm, some eggs are expelled in the intestine and can be found
in the faeces. Spontaneous migration of T. saginata or T. asiatica from the anus is likely to be noticed by
the patient (>95%). When the segments migrate, the sticky eggs are deposited in the perianal area and
might be detected by application and examination of sticky tape. These signs are far less likely for chains of
1217
T. solium (Allan et al., 1996). Segments of all three may be found on the faeces, but are passed
intermittently. In dogs, approximately 50% of the segments migrate spontaneously from the anus. These
segments, when they fall to the ground, will migrate, shedding eggs. The remainder of the segments are
passed in the faeces, but commonly, the segments migrate and void the majority of their eggs in trails on the
surface of the faeces and surrounding area. Even if a migrating segment sheds all its eggs, it can be
identified as a cestode by the many concentric calcareous corpuscles contained within its tissues. Faeces,
after mixing to reduce aggregation, can be examined for eggs. Various techniques are used throughout the
world and include ethyl acetate extraction and flotation. For the latter, NaNO3 or Sheathers sugar solution
(500 g sugar, 6.6 ml phenol, 360 ml water), with their higher specific gravities, are superior to saturated NaCl
as flotation media for taeniid eggs. Flotation can be carried out in commercially marketed qualitative or
quantitative flotation chambers or by centrifugal flotation that includes a modified Wisconsin technique
(faeces, diluted in water, are sieved and centrifuged, the pellet is resuspended in sugar or Sheathers
solution and centrifuged at 300 g for 4 minutes). Eggs adhering to the cover-slip can then be detected.
Faecal egg examination will be less sensitive for T. solium than the other species. Species cannot be
determined by egg morphology, but DNA probes, the polymerase chain reaction (PCR) and PCR restriction
fragment length polymorphism (RFLP), have proved useful for differentiation in the laboratory. These have
been largely used experimentally to differentiate faecal eggs of T. solium, T. saginata and T. asiatica
(Gasser & Chilton, 1995; Gonzalez et al., 2004; Harrison et al., 1990). While equally applicable to
differentiation in dogs, the same examinations have not been done.
An AG-ELISA to detect Taenia coproantigen in faeces is no longer available commercially, but can be
developed if laboratory facilities are available (Allan et al., 1992; 1996). Information on availability for
epidemiological studies or collaborative use can be obtained from Professor P.S. Craig, OIE Reference
Expert on Echinococcosis (see Table given in Part 4 of this Terrestrial Manual). This AG-ELISA was
developed experimentally by Allan et al. (1992) to detect coproantigen in dogs, and so, with appropriate
controls, could be used to detect Taenia infection in this species. The technique, however, is only Taenia
species specific. The test is a solid-phase, microwell assay with wells coated with polyclonal, rabbit antiTaenia-specific antibody (TSA). The following is the basic technique:
b)
i)
Faecal supernatants are recovered from fresh, frozen or formalinised (5% formalin at 4C) faecal
samples. The sample is vigorously shaken forming a slurry in an equal weight/volume of 0.15 M
phosphate buffered saline (PBS) containing 0.3% Tween 30. The supernatant is recovered by
centrifugation at 2000 g for 30 minutes.
ii)
A soluble aqueous extract of non-gravid proglottids from Taenia are obtained following emulsification in
PBS and centrifugation.
iii)
Hyperimmune rabbit antiserum is produced against the soluble proglottid extract and the IgG fraction is
isolated by passage into and elution of the bound IgG from Protein A-Sepharose CL 4B (Pharmacia).
Some of the IgG fraction is conjugated to peroxidase type VI (Sigma). The sera are stored in small
quantities frozen at 20C. Sera may need to be absorbed by packed normal dog faeces in a ratio 2/1
with mixing for 1 hour and recovered by centrifigation.
iv)
Flat-bottomed microtitre plates (Dynatech) are coated with rabbit anti-Taenia IgG (protein content 5
25 g/ml determined by UV spectrophotometry) using 100 l/well, the antisera are diluted in 0.05 M
NaHCO3/Na2CO3 buffer, pH 9.6. Plates are incubated overnight at 4C, the wells are washed three
times with PBS/0.1% Tween, blocked with PBS/0.3% Tween for 1 hour and washed again. 100 l of
faecal supernatant containing 50% fetal calf serum is added and the plates are incubated for 1 hour
and then washed three times. 100 l of the peroxidase-conjugated anti-Taenia IgG (diluted 1/100 or
1/200) is added and the plates are incubated for 1 hour before washing three times. Substrate solution
(100 l of 5-amino-salacylic acid [Sigma] and 0.005% H2O2 in 0.1 M phosphate buffer containing 1 mM
Na2EDTA [ethylene diamine tetra-acetic acid] at pH 6.0) is added for 25 minutes and the result is read
at 450 nm. Cut-off values are the mean plus 3 standard deviations of values for normal dog faeces.
Diagnosis of metacestodes
In live animals, T. solium or T. saginata metacestodes might be palpable in the tongue but, both in the living
animal and on post-mortem examination or meat inspection, tongue palpation is of diagnostic value only in
pigs or cattle heavily infected with metacestodes; these will also be difficult to differentiate from large
sarcocysts.
Meat inspection the main diagnostic procedure
Metacestodes are visible first as very small, about 1 mm, cysts, but detection of these requires thin slicing of
tissues in the laboratory. Many young cysts are surrounded by a layer or capsule of inflammatory cells
(mononuclear cells and eosinophils being prominent histologically). Cysts may later degenerate, but the
parasites abilities to evade the immune response mean that later in infection, as the cyst matures, few
1218
inflammatory cells are present in its vicinity and the cysticercus in its intermuscular location is surrounded by
a delicate fibrous tissue capsule.
In theory, cysts can be visualised or felt in tissues such as the tongue of heavily infected animals as early as
2 weeks after infection. Cysts are readily visible by 6 weeks and, when mature, are usually oval, about 10
5 mm or larger, with a delicate, fairly translucent, white parasite membrane and host capsule; pale fluid
within the cyst and the scolex, visible as a white dot within the cyst, usually invaginates midway along the
long axis of the cyst.
At meat inspection many of the cysts detected, often as many as 85100%, are dead. The rate at which
cysts age and die and so degenerate varies with the parasite species and also with the tissue within which
the cyst is embedded. Death usually occurs within 9 months of infection of adult cattle with T. saginata.
However, cysts may remain viable for several years. Cysts of T. hydatigena in the peritoneal cavity of sheep
and those of T. solium also have been described as surviving in sheep and pigs for long periods. Taenia
solium cysts survive for many years in the brain of humans, and frequently symptoms begin only as the cyst
begins to degenerate. In general, cysts tend to die more rapidly in the muscular predilection sites, such as
heart. The preferential distribution of parasites in these areas may be because of greater blood circulation to
these muscles. Conversely, the higher rate of activity in these muscles may damage the parasites, allowing
leakage of fluid and perhaps disrupting the parasites ability to evade the immune response. Cysts at
different stages of viability and degeneration can be found in the same host.
Degenerating cysts vary in appearance. The hosts fibrous tissue capsule thickens and becomes opaque,
but initially the cyst within remains apparently normal. The fluid gradually becomes colloid and inflammatory
cells infiltrate. The cyst cavity becomes filled with greenish (eosinophilic) and then yellow, caseous material
and is very unaesthetic, usually being larger in size and certainly more obvious in meat than the original
viable cyst. Later the cyst may calcify. While PCR assays have been used largely for the differentiation of
adult taeniids in humans, they could be usefully applied to unambiguously identify a metacestode; in a
recent study though, PCR identified only 50% of degenerate presumed T. saginata cysts (Abuseir et al.,
2006). Where very young (without a scolex) or degenerate cysts need to be differentiated from other lesions,
compression of the cyst, smears of the caseous contents and histological examination of haematoxylin and
eosin (H&E) stained sections are used. Microscopic examination may reveal the calcareous corpuscles
(concentric concretions of salts that are around 510 m in size). These indicate a cestode origin of the
tissue and differentiate, for example, an immature or degenerate cyst from a cyst of another aetiology. The
presence of hooks and their length together with knowledge of the host and tissue may aid in identification of
cestode species. Experimentally, immunohistochemical staining has differentiated T. saginata cysts from
non-Taenia structures (Ogunremi et al., 2004). PCR would be useful, but is experimental, in the finding of a
new cestode in a host species or geographical area from which, historically, the parasite was absent
(Abuseir et al., 2006).
After treatment of T. saginata and T. solium in cattle and pigs with drugs such as albendazole and
oxfendazole, the cysts may lose their fluid and collapse. The resultant lesion is much smaller than lesions
observed following natural death. However, cysts that have died before treatment of the animal will remain
large and visible. Treatment of pigs with oxfendazole (30 mg/kg) 3 months before slaughter has been
suggested as a control measure.
Meat inspection procedures vary with the parasite and the host involved, i.e. zoonosis or not, the tissue
involved, and regulations within a country. Examinations tend to be more extensive with the zoonotic
infections T. saginata and T. solium.
In general, meat inspection procedures consist of:
i)
Visual inspection of the carcass, its cut surfaces and the organs within it. This may reveal T. saginata,
T. solium and T. ovis in the muscles, T. hydatigena on the liver or mesenteries and omentum, or
T. multiceps in the brain.
ii)
The external and internal masseters and the pterygoid muscles each must be examined and one or
two incisions made into each, the cuts being parallel to the bone and right through the muscle.
iii)
The freed tongue is examined visually and palpated, particularly for T. solium
iv)
The pericardium and heart are examined visually. The heart usually is incised once lengthwise through
the left ventricle and interventricular septum so exposing the interior and cut surfaces for examination.
Incisions may go from the base to the apex and regulations also may require additional, perhaps four,
deep incisions into the left ventricle. Alternately, the heart may be examined externally and then
internally after cutting through the interventricular septum and eversion.
v)
The muscles of the diaphragm, after removal of the peritoneum, are examined visually and may be
incised.
1219
vi)
The oesophagus is examined visually.
vii)
In African countries in particular, the triceps brachii muscle of cattle is incised deeply some 5 cm above
the elbow. Additional cuts into it may be made. The gracilis muscle also may be incised parallel to the
pubic symphisis. These cuts are usually also undertaken for T. solium in pigs. Such incisions into the
legs are made in Africa as it is suspected that more parasites lodge in these muscles in working or
range animals walking long distances due to the exercise and consequent increased blood flow to
these muscles. Other countries may also require such incisions into the legs. However, as this
devalues the meat, such incisions are made most commonly once one or more cysts have been found
at the predilection sites so as to determine the extent of the infection.
Overall, the initial incision into any tissue is the most important, but additional incisions may be required by
the regulations or are required if cysts are found on the initial incision(s). Details on meat inspection are
supplied by Herenda (2000) and by Kyvsgaard & Murrell (2005).
Additional or fewer procedures may be required for specific parasites and the judgements on the carcass,
viscera, offal and blood will vary dependent on Taenia species and regulations within a country. Judgement
on infected carcasses will fall into three main categories: i) approve for human consumption; ii) partially
condemn and pass the remainder of the carcass, but in the case of the zoonoses, T. saginata and T. solium,
the carcass, meat and viscera must be treated; and iii) totally condemn heavily infected carcasses or
emaciated diseased ones.
Taenia saginata: Calves under 6 weeks or <32 kg are not usually examined. Predilection sites are the heart,
tongue, masseters and diaphragm, presumably because they receive the greatest circulation. Nonetheless,
cysts may be found in any muscle of the body. If one carcass in a lot is found to be infected, all carcasses
from the same lot can be held until laboratory confirmation is obtained. If T. saginata infection is confirmed,
additional incisions are usually made in the carcasses in the lot; all suspicious lesions found in the rest of the
lot are considered to be T. saginata without laboratory confirmation. Lesions of T. saginata may need to be
differentiated from Sarcocystis sarcocysts and other lesions. In recent PCR studies in Germany, Switzerland
and New Zealand up to 20% of viable, presumed T. saginata cysts could not be positively identified; this
suggests there may be an unidentified cestode infecting cattle (Abuseir et al., 2006). Conversely, aberrant
T. saginata oncospheres can lead to cerebral oncospheral lesions and thus need to be taken into account
for differential diagnosis of neurological disorders (Fatzer et al., 2002).
Judgement: If a carcass is considered to be heavily infected then the carcass, meat, offal and blood all are
condemned. The description of a heavy infection varies, but generally it is the detection of cysts at two of the
predilection sites plus two sites in the legs. In the case of a lesser infection, the infected parts and
surrounding tissues are removed and condemned. Even a single dead cyst requires that the carcass and
edible viscera must then be treated and this is justifiable as about 10% of lightly infected carcasses were
found on dissection to have both dead and viable parasites within them. Treatment varies with country and
facilities available and includes: i) freezing at lower than 10C for >10 or 14 days, or lower than 7C for
21 days; ii) boxes of boned meat are frozen at less than 10C for >20 days; iii) heated to above 60C
throughout; iv) steamed at moderate pressure (0.49 kg/cm2); v) heated at 95100C for 30 minutes; or
vi) pickled in salt solution for 21 days at 812C. Blast freezing needs examination; generally a 30 kg box is
reported to require 2 24-hour cycles at 30.9C followed by 72 hours cold storage at 23.3C for death of
scoleces. Often no treated meat can be exported although in some countries it can be exported in canned
form.
Taenia solium: The predilection sites are as for T. saginata although there are reports of higher prevalence
in shoulder and thigh. Commonly one or more cuts are required 2.5 cm above the elbow joint. This is said to
detect some 13% of infected carcasses that would otherwise have been missed.
Judgement: In some countries, any lightly or heavily infected pigs and their viscera and blood are
condemned. In areas where infection is common, lightly infected carcasses can be passed for cooking and
pickling and occasionally freezing.
Taenia hydatigena: The parasite migrating in the liver leaves haemorrhagic tracks that then become
green/brown with inflammation and later white due to fibrosis. For the records, these must be differentiated
from those of liver flukes, if possible, by identification of the cysticerci or adult flukes. White spot from
Ascaris infection is differentiated as the lesions appear as pale to white, small, isolated foci. Some cysts
remain trapped below the liver capsule. These usually are small and degenerate early and then calcify into
cauliflower-like lesions. Taenia hydatigena cysts usually mature in the omental or mesenteric fat. Those that
are retained at the liver surface are usually superficial and subserosal, while Echinococcus granulosus
hydatid cysts tend to be deeper in the parenchyma. If viable, the former has a long-necked single scolex in a
virtually translucent fluid-filled cyst. Fertile hydatid cysts have thicker white outer membranous walls from
which brood capsules containing numerous protoscoleces. These appear as a sandy deposit with the cysts.
1220
Differentiation can be important in the implementation and monitoring of hydated disease control measures
for which histology may be required. H&E-stained sections will reveal the laminated membrane of very
young hydatid cysts as indicated by Lloyd et al. (1991). Its presence or absence can be confirmed by
periodic acidSchiff staining when the highly glycosylated proteins in the laminated membrane stain red.
Taenia hydatigena lesions in cattle and pigs can be similar to tuberculosis. However, the portal and
mesenteric lymph nodes are not involved, the contents of parasite cysts are more easily shelled-out and
remainders of hooks and calcareous corpuscles may be seen or ZiehlNeelsen staining may reveal bacteria.
Judgement: Usually only a few cysts or tracks are present and these can be trimmed. Heavily infected livers
and omentum are condemned. Rarely, acute infections are seen with large numbers of migrating parasites
producing traumatic hepatitis, ascites, oedema, etc., and would result in secondary condemnation of the
carcass.
Taenia multiceps: The parasites have a predilection site for the brain and spinal cord.
Early migrating parasites can cause reddish haemorrhagic and later grey purulent tracks in the brain, and in
heavy infections, the sheep may have a meningoencephalitis. Clinical signs due to the mature cyst relate to
pressure atrophy of adjacent nervous tissue and vary according to location in the brain. There may be
impaired vision or locomotion if cysts are in the cerebral hemispheres and the sheep gradually may be
unable to feed and will become emaciated. Cerebellar cysts may precipitate more acute and severe signs of
ataxia or opisthotonus. In heavy infections, parasites migrate and begin development in other tissues, but
they die early. These produce small lesions, 1 mm or so in size, that first contain an encapsulated cyst, then
eosinophilic, caseous material that later may calcify.
Judgement: Initially only the head is condemned or occasional cysts in intermuscular or subcutaneous sites
are trimmed. With chronic infection, the animal may have been unable to feed, resulting in condemnation
due to emaciation, etc.
Taenia ovis: The predilection sites are as for T. saginata. Cysts may be confused with large Sarcocystis
gigantea sarcocysts.
Judgement: Commonly detection of up to 25 cysts results in trimming and the carcass is passed. This does
not prevent the unaesthetic presence of live or degenerate parasites in other tissues. Ultrasound and X-rays
are being tested for detection of these. Some authorities may require that the meat be boned, trimmed and
frozen or cooked. In heavy infections the carcass is condemned.
In general, meat inspection procedures detect only about 2050% of the animals that are actually infected.
Light infections are easily missed on palpation and meat inspection in one study, 78% of carcasses
infected with >20 cysts were detected compared with those detected following dissection and slicing, while
only 31% of those with fewer cysts were detected (Walther & Koske, 1980). Meat inspection efficacy will
vary with the number and location of incisions (and the skill and experience of the inspector). For example,
in Zimbabwe, 58% of cattle were positive in the head only, 20% in the shoulder only and 8% in the heart
only, although overall 81% were found to be infected if all three organs were included. Walther & Koske
(1980) in Kenya also found that the predilection sites were not necessarily infected in 57% of the cattle found
positive on dissection. They also confirmed the importance of the shoulder incisions in detection of infection
in Africa as 20% of the cattle found to be infected were positive in the shoulder only. Wanzala et al. (2003),
also in Kenya, described the insensitivity of meat inspection in detecting cysticerci: only 50% of naturally or
artificially infested cattle were identified. Their observations indicated that a number of viable cysticerci may
be missed.
In humans, the most common presenting sign in T. solium NCC is seizures followed by headache, but a
range of signs, such as vomiting, psychoses, etc., are seen depending on the number, location and viability
or level of degeneration of the cysticerci (viable, transitional dying, calcification) (Carpio et al., 1994; Lloyd,
1998a; Nash, 2005). In humans, clinical evaluation and either computerised tomography (CT) scan (best for
calcified cysts) or magnetic resonance imaging (MRI) (detects cysts in both parenchymal and
extraparenchymal locations and can follow the progression of the lesion) are used to detect the exact
locations and viability of T. solium and T. multiceps metacestodes. These remain the most efficient means of
diagnosis, but access to imaging facilities may not be available in endemic areas. Calcified cysts in tissues
are detected by radiography. Serology now is available for NCC.
2.
Serological tests
The development of an automated sensitive and specific diagnostic test would greatly reduce the costs of
damage to the carcass and also the costs of labour. Serological tests for animals have not reached the stage
where commercialisation for individual diagnosis or large-scale detection of infected carcasses in slaughter
1221
houses is possible. All assays tested AG-ELISA, antibody ELISA, enzyme-linked immunoelectro transfer blot
(EITB) and tongue inspection show low sensitivity in rural pigs infected naturally with low levels of T. solium
(Dorny et al., 2005; Sciutto et al., 1998a). This contrasts with their high sensitivity and specificity when applied to
commercially reared pigs free from infection and such pigs experimentally infected with T. solium (Sciutto et al.,
1998a; 1998b). This finding is also true for T. saginata infections in cattle (Onyango-Abuje et al., 1996; Van
Kerckhoven et al., 1998). Thus, only a small percentage (1322%) of cattle carrying fewer than 3050 viable
cysticerci is detected by AG-ELISA. Conversely, antibody has proven most useful for detecting cysts that are no
longer viable. Nonetheless, AG-ELISAs do have a use in field-based epidemiological studies for indicating
transmission. For example, the detection of viable infections in cattle or pigs could indicate point sources of
infection, season of transmission and age of animals at risk. The development of more sensitive and specific
assays with recombinant antigens for diagnosis of NCC should improve immunodiagnosis of T. solium in pigs.
A number of EITB and ELISA assays for antibodies to T. solium in humans are now widely available commercially
(i.e. Immunetics, USA; Cypress Diagnostics, USA; Diagnostic Automation, USA; United Biotech, USA, Arup
Laboratories, USA; Biopharm, Germany). An AG-ELISA using polyclonal or monoclonal antibody (used to detect
antigen in cerebrospinal fluid) has a specificity and sensitivity of up to 86% in selected patients. The specificity of
these tests tends to be very high but sensitivity is lower, this is in part related to cyst number. The hierarchy of
clinical symptoms, imaging studies and serological tests has been presented by del Brutto et al. (2001).

Immunochemical identification of protective antigens and their production by recombinant DNA technology has
been uniquely successful in the Taeniidae compared with other eukaryotes, and is described by Lightowlers &
Gauci (Lightowlers, 2006; Lightowlers & Gauci, 2001; Lightowlers et al., 2003). Vaccination with the resultant
products has been highly effective. Overall the success was advanced by the fact that a strong protective
immunity occurs after natural infection, high levels of protective immunity are induced by antigens in oncospheral
extracts, there is good cross-immunity between Taenia species, and immunity is largely antibody mediated as
evidenced by passive and maternal transfer of immunity so that antibody could be used to probe for protective
antigens. Initially the T. ovis vaccine was developed. The T. solium 45W antigen was isolated as a recombinant
protein from Escherichia coli. Potency control is by AG-ELISA and in vivo immunogenicity, and the vaccine has
provided protection for a period of at least 1 year in field trials. Two other antigens (To16K and To18K) have been
isolated and cloned and each individual T. ovis protein is protective. Using the benefits of cross-reaction and
probing with the T. ovis cDNA, homologues of the T. ovis antigens, TSA9 and TSA18, equivalent to To45W and
To18, were identified in T. saginata and cloned from T. saginata oncosphere mRNA. In contrast to the T. ovis
individual antigens, immunisation with both T. saginata antigens was required to produce high level protection.
Homologues of the T. ovis antigens also were cloned from T. solium mRNA and TSO45 and TSOL18 antigens of
T.solium. Both the T. ovis and T. saginata vaccines have given >94% and 98% protection in cattle and sheep,
respectively. Comparable antigens now have been identified for T. multiceps. The T. ovis vaccine was registered
in 1994 by the New Zealand Animal Remedies Board. However, due to market changes in New Zealand, the
vaccine is not available commercially. Costs of large-scale production of antigens, processing conditions and
potential variants in expressed antigens have been outlined by Lightowlers & Gauci (2001). The T. solium
antigens, in particular TSOL18, have given high levels of protection experimentally (99%) (Lightowlers, 2006); the
antigen is undergoing the steps similar to the T. ovis antigens to develop a practical vaccine. Other avenues for
vaccine antigens are being explored. Synthetic peptides from the sequences of the recombinant Taeniidae
antigens induced antibody but not protection, indicating that the protective epitopes seemed conformational.
Reasonable levels of protection were induced experimentally in piglets exposed to natural T. solium challenge
using synthetic peptides based on protein sequences of the murine parasite T. crassiceps (Huerta et al., 2001).
The S3Pvac T. solium subunit vaccine has had some field efficacy in protecting against natural infection but
requires further examination (de Aluja et al., 2005; Sciutto et al., 2007). It is possible that costbenefit analyses
concerning the use of the T. saginata vaccine could obviate its use in many countries, as cost of the vaccine is
very important to the livestock industries. The importance of T. solium in humans increases the costs of the
disease, but it remains to be seen whether the significance of the disease in endemic countries will be sufficient to
push commercial production of the vaccine for use in pigs. Sensitive immunodiagnosis for metacestodes still
requires development to access vaccine efficacy.
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*
* *
1224
CHAPTER 2.9.6.
HENDRA AND NIPAH VIRUS DISEASES
SUMMARY
Hendra virus (HeV) and Nipah virus (NiV) emerged in the last decade of the twentieth century as
the causes of outbreaks of respiratory and neurological disease that infected a number of animal
species. In 1994, HeV caused severe respiratory disease and the death of 13 horses and a horse
trainer at a stable in Brisbane, Australia. Between September 1998 and April 1999, after spreading
unrecognised as a respiratory or encephalitic infection in Malaysian pigs, NiV appeared in the
human population there and was the cause of fatal encephalitis. Over one million pigs were culled
to stop spread of the disease. HeV has caused the death of four of seven infected people while it
has been reported that there have been 400 cases of NiV in humans, with approximately
200 deaths, in Malaysia, Singapore, Bangladesh and India. Fruit bats (flying foxes) in the genus
Pteropus are natural hosts of both viruses.
HeV infection of horses is characterised progressively by high fevers, facial swelling, severe
respiratory difficulty and, terminally, copious frothy nasal discharge. Some horses display
neurological signs. The most common post-mortem observations are dilated pulmonary lymphatics,
severe pulmonary oedema and congestion. The underlying lesion is generalised degeneration of
small blood vessels in a range of organs. Syncytial endothelial cells containing viral antigen are
common in capillaries and arterioles. HeV infection of horses is not uniformly fatal and some horses
manifesting clinical signs survive infection. Hendra virus does not appear to be highly contagious
among horses, and close contact seems to be necessary for it to spread. Infected horses on
pastures have rarely transmitted the virus. However, transmission appears to occur more readily in
closed environments such as stables.
NiV infection of pigs is highly contagious, but it was not initially identified as a new disease because
morbidity and mortality were not marked and clinical signs were not significantly different from other
known pig diseases. Observations made during the outbreak investigation and during experimental
infections confirmed that NiV infection of pigs is characterised by fever with respiratory involvement.
In animals showing disease, nervous signs have been frequently reported, but many infections are
subclinical. Some infected animals display an unusual barking cough. Abortion is reported in sows.
Immunohistochemical lesions are found in either or both the respiratory system (tracheitis and
bronchial and interstitial pneumonia) and the brain (meningitis) of infected animals. Syncytial cells
containing viral antigen are seen in small blood vessels, lymphatic vessels and the respiratory
epithelium.
Both viruses affect companion animals. HeV causes pulmonary disease in cats similar to that
observed in horses. Natural infection of dogs with NiV causes a distemper-like syndrome with a
high mortality rates; there is serological evidence that some dogs survive infection. Experimentally
NiV causes a similar disease to HeV in cats. Syncytial endothelial cells containing viral antigen
were demonstrated in both HeV and NiV infections in cats and in NiV infection in dogs.
Infection of humans is from animal contact, usually from an amplifier host rather than directly from
the natural, reservoir host: NiV from swine and HeV from horses. However investigations of
outbreaks of human NiV in Bangladesh have indicated infection from Pteropid bats. Human-tohuman transmission has not been seen with HeV or with NiV in Malaysia and Singapore, but
human-to-human transmission is suspected in recent outbreaks of NiV in Bangladesh.
HeV and NiV are closely related members of the family Paramyxoviridae. Differences between
them and other family members have led to their classification in a new genus, Henipavirus, in the
subfamily Paramyxovirinae. HeV and NiV are biosafety level 4 agents and it is important that
1225
Chapter 2.9.6. Hendra and Nipah virus diseases
samples from suspect animals be transported to authorised laboratories only under biologically
secure conditions according to international regulations.
Identification of the agent: Both HeV and NiV may be propagated in a range of cultured cells.
Virus isolation from unfixed field samples should be attempted, but only in situations where operator
safety can be assured. Identification procedures following virus isolation include immunostaining of
infected cells, neutralisation with specific antisera and molecular characterisation. Real-time
polymerase chain reaction (PCR) is now available as a diagnostic test.
Viral antigen is present in vascular endothelium, and in the case of NiV in pigs, the respiratory
epithelium. A wide range of formalin-fixed tissues can be examined to detect HeV and NiV
antigens. Submissions for immunohistochemistry should include samples of brain at various levels
including meninges, lung, spleen and kidney. In pregnant animals or in cases of abortion, uterus,
placenta and fetal tissues should be included as appropriate. Specimens for virus isolation and
molecular detection of virus should be fresh tissues from the same organs, or urine or throat or
nasal swabs.
Serological tests: Virus neutralisation tests (VNT) and enzyme-linked immunosorbent assay
(ELISA) are available. VNT is currently accepted as the reference procedure. The ability of antisera
to HeV and NiV to cross-neutralise to a limited degree means that a single VNT using either virus
does not provide definitive identification of antibody specificity. Neutralising antibodies to HeV and
NiV can be differentiated by the greater capacity to neutralise the homologous compared with the
heterologous virus. This may not be a major impediment in outbreak situations where the causative
agent is known, but serum samples from suspect cases or from areas of the world other than
Australia and Malaysia should be subjected to VNT analyses with both HeV and NiV. The
serological relationship between HeV and NiV ensures that ELISAs using HeV or NiV antigen can
be used to detect antibodies to both viruses.
Requirements for vaccines and diagnostic biologicals: There are no vaccines currently
available for either HeV or NiV.
A. INTRODUCTION
Hendra virus (HeV) and Nipah virus (NiV) occur naturally as viruses of fruit bats commonly known as flying
foxes, members of the genus Pteropus. Antibodies to HeV are found in approximately 50% of the four Australian
Pteropus species (Young et al., 1996). Serological surveys of antibodies to NiV show seroprevalances up to 20%
in Malaysian pteropid bats (Epstein et al., 2006; Johara et al., 2001). Antibodies to NiV or putative closely related
viruses have subsequently been detected in pteropid bats in Bangladesh (Hsu et al., 2004), Cambodia (Olson et
al., 2002; Reynes et al., 2005), Indonesia (Sendow et al., 2006), Madagascar (Lehle et al., 2007) and Thailand
(Wacharapluesadee et al., 2005). HeV has been isolated from Australian flying foxes (Halpin et al., 2000), and
NiV from flying foxes from Malaysia and Cambodia (Chua et al., 2002; Reynes et al., 2005). NiV RNA has been
detected by polymerase chain reaction (PCR) in pteropid bat urine, saliva and blood in Thailand
(Wacharapluesadee & Hemachudha, 2007; Wacharapluesadee et al., 2005).
HeV disease emerged in Brisbane, Australia, in September 1994 in an outbreak of acute respiratory disease that
killed 13 horses and a horse trainer (Murray et al., 1995). The virus was initially called equine morbillivirus, but
subsequent genetic analyses indicated that it did not resemble morbilliviruses closely enough to merit inclusion in
that genus. There have been other instances of fatal HeV infection of horses in northern Queensland and further
instances of infection of people. Two horses developed an acute disease and died almost 1 month before the
Brisbane outbreak, but HeV was determined to be the cause of death only after the horse owner, who probably
acquired HeV during necropsy of the horses, died 13 months later with HeV-mediated encephalitis (Rogers et al.,
1996). A third horse died in January 1999 with no associated human disease (Field et al., 2000). Two further
equine cases occurred in 2004, one confirmed and the other unconfirmed, the latter identified by an associated
human infection (Hanna et al., 2006). In 2006 Australia reported two further cases in horses, one in Southern
Queensland and one in northern New South Wales. Detailed reports of the three most recent human cases have
not been published, but all three people were infected by contact with horses. One of the two cases reported in
2008, as a well as a case the occurred in August 2009, were fatal.
In Malaysia, retrospective studies of archival histological specimens indicate that NiV has caused low mortality in
pigs since 1996, but remained unknown until 1999 when it emerged as the causative agent of an outbreak of
encephalitis in humans that had commenced in 1998 (Chua et al., 2000; Nor et al., 2000). Unlike respiratory
disease caused by HeV in horses, which was frequently fatal but characterised by poor transmissibility
1226
(Williamson et al., 1998), respiratory disease caused by NiV in pigs was often subclinical but highly contagious,
properties that led to rapid virus dispersal through the Malaysian pig population and forced authorities to choose
culling as the primary means to control spread (Nor et al., 2000). Over one million pigs were destroyed; 106 of
267 infected humans, mostly pig farmers in Malaysia and abattoir workers in Singapore who had direct contact
with live pigs, died of encephalitis (Chua et al., 2000; Paton et al., 1999).
New foci of human NiV disease have subsequently been identified in Bangladesh and India. In outbreaks in 2001
and 2003 an animal source of the human infections was not identified (Hsu et al., 2004), but pteropid bats,
Pteropus giganteus, were present and had antibodies capable of neutralising Nipah virus. Clustering of cases and
timesequence studies indicated that there is human-to-human transmission but at low levels (Hsu et al., 2004).
In another outbreak in 2004 in which 27 of 36 infected humans died, epidemiological evidence indicated personto-person transmission and serological studies identified seropositive fruit bats at the location (Anonymous, 2004).
Drinking fresh date palm sap contaminated by fruit bat saliva, urine or excreta has been identified as one possible
route of transmission from the wildlife reservoir to humans (Luby et al., 2006). As a result of these ongoing
outbreaks it is estimated that across Malaysia, Singapore, Bangladesh and India there have now been up to
400 cases of NiV in humans, with approximately 200 deaths.
NiV and HeV are classified taxonomically as paramyxoviruses in the subfamily Paramyxovirinae, and have been
grouped in a separate and new genus, the henipaviruses (Eaton et al., 2006).
Diagnosis of disease caused by Henipaviruses is by virus isolation, detection of viral RNA in clinical or postmortem specimens or demonstration of viral antigen in tissue samples taken at necropsy (Daniels et al., 2001).
Detection of specific antibody can also be useful particularly in pigs where NiV infection may go unnoticed.
Identification of HeV antibody in horses is less useful because of the high case fatality rate of infection in that
species. Human infections of both HeV and NiV have been diagnosed retrospectively by serology. Demonstration
of specific antibody to HeV or NiV in either animals or humans is of diagnostic significance because of the rarity of
infection and the serious zoonotic implication of transmission of infection.
1.
a)
Virus isolation and characterisation

HeV and NiV are classified as biosafety level 4 (BSL4) agents, as they are dangerous human pathogens with a
high case fatality rate and for which there is no vaccination or effective antiviral treatment. BSL4 is similar to
containment group 4 as described in the OIE biosafety guidelines in Chapter 1.1.3 Biosafety and biosecurity in the
veterinary microbiology laboratory and animal facilities, provide additional information. However, due to the high
risk consequences of human infection in the laboratory, BSL4 requirements surpass the OIE containment level 4
requirements. Virus isolation greatly facilitates identification procedures and definitive diagnosis should be
undertaken where operator safety can be guaranteed. Isolation is especially relevant in any new case or outbreak,
particularly in countries or geographical areas where infection by HeV or NiV has not been previously
documented. Implication of wildlife species as natural hosts of the viruses requires positive serology, PCR or virus
isolation from wild-caught animals (Daniels et al., 2007).
i)
Sampling and submission of samples

Diagnostic samples should be submitted to designated laboratories in specially designed containers.
The International Air Transport Association (IATA), Dangerous Goods Regulations (DGR) for shipping
specimens from a suspected zoonotic disease must be followed (International Air Transport
Association, 2002). The requirements are summarised in Chapter 1.1.1 Collection and shipment of
diagnostic specimens.
The range of tissues yielding virus in natural and experimental cases has been summarised (Daniels et
al., 2000). Brain, lung, kidney and spleen should always be submitted. Samples should be transported
at 4C if they can arrive at the laboratory within 48 hours; if shipping time will be over 48 hours, the
samples should be sent frozen on dry ice or nitrogen vapours should be used. Samples should not be
held at 20C for long periods.
ii)
Isolation in cultured cells

Virus propagation should be conducted under BSL4 conditions. Strict adherence to this guideline would
limit the handling of diagnostic specimens where the presence of HeV or NiV may be suspected but not
confirmed to laboratories with BSL4 facilities. Primary virus isolation from suspect samples may of
necessity be conducted under BSL3 conditions. However, if this is to be attempted, stringent local
guidelines must be developed to ensure operator safety and applied if a paramyxovirus-like cytopathic
1227
effect (CPE) develops in infected cultures. Such guidelines will emphasise good laboratory practice,
the use of class II safety cabinet with appropriate personal protective equipment or a class III cabinet
and may require acetone fixation of infected cells, to destroy infectious virus, followed by
immunofluorescent detection of Henipavirus antigen. The culture medium from Henipavirus-positive
cells should be transferred to a BSL4 laboratory.
At the recipient laboratory tissues are handled under sterile conditions, and 10% (w/v) suspensions are
generated by grinding the tissues in a closed homogenisation system, e.g. stomacher/bag mixer using
plastic bag or mixer mills using autoclavable steel balls in closed metal cylinders. All processes should
be carried out in a Class III cabinet or a Class II cabinet with appropriate personal protective
equipment, with the stomacher operated in the cabinet and the centrifuge pots, with aerosol covers,
loaded and unloaded in the cabinet. Following clarification of the homogenate by centrifugation at
300 g, the supernatant is added to cultured cell monolayers. Virus isolation is aided by the fact that
HeV and NiV grow rapidly to high titre in many cultured cells. African green monkey kidney (Vero) and
rabbit kidney (RK-13) cells have been found to be particularly susceptible. HeV also replicates in
suckling mouse brain and in embryonated hens eggs, and although the former may represent a viable
method of primary isolation, there are no data on the relative susceptibility of in-vivo systems such as
these compared with the more convenient cell culture systems. A CPE usually develops within 3 days,
but two 5-day passages are recommended before judging the attempt unsuccessful. After low
multiplicity of infection, the CPE is characterised by formation of syncytia that may, after 2448 hours,
contain over 60 or more nuclei. Syncytia formed by NiV in Vero cell monolayers are significantly larger
than those created by HeV in the same time period. Although the distribution of nuclei in NiV-induced
syncytia early in infection resembles that induced by HeV, with nuclei aggregated in the middle of the
syncytia, nuclei in mature NiV-induced syncytia are distributed around the outside of the giant cell
(Hyatt et al., 2001).
iii)
Methods of identification
Immunostaining of fixed cells
The speed with which HeV and NiV replicate and the high levels of viral antigen generated in infected
cells make immunofluorescence a useful method to rapidly identify the presence of Henipaviruses
using either anti-NiV or anti-HeV antiserum. At present the Henipavirus genus consists of HeV and NiV
and there are no known antigenically related viruses.
The serological cross reactivity between HeV and NiV means that polyclonal antiserum to either virus
or mono-specific antisera to individual proteins of either virus, will fail to differentiate between HeV and
NiV. Monoclonal antibodies (MAbs) are currently being generated and tested to fulfil this function both
in primary identification of the virus upon isolation and for use in immunohistochemical examination of
tissues from suspect cases.
Test procedure
Under BSL4, monolayers of Vero or RK-13 cells grown on glass cover-slips or chamber slides are
infected with the isolated virus, and the monolayers are examined for the presence of syncytia after
incubation for 2448 hours at 37C. It is recommended that a range of virus dilutions (undiluted, 1/10,
1/100) be tested because syncytia are more readily observed after infection at low multiplicity. Once
visible syncytia are detected, infected cells are fixed by immersion in a vessel filled completely with
acetone. The vessel is sealed and surface sterilised prior to removal to a less secure laboratory
environment, for example BSL2, where the slides are air-dried. Viral antigen is detected using anti-HeV
or anti-NiV antiserum and standard immunofluorescent procedures. A characteristic feature of
Henipavirus-induced syncytia is the presence of large polygonal structures containing viral antigen.
These are observed most readily with monospecific and MAbs to the nucleocapsid protein N and
phosphoprotein P.
Immunoelectron microscopy
The high titres generated by HeV and NiV in cells in vitro permits their visualisation in the culture
medium by negative-contrast electron microscopy without a centrifugal concentration step. Detection of
virus antibody interactions by immunoelectron microscopy provides valuable information on virus
structure and antigenic reactivity, even during primary isolation of the virus. Other ultrastructural
techniques, such as grid cell culture (Hanna et al., 2006), in which cells are grown, infected and
visualised on electron microscope grids, and identification of replicating viruses and inclusion bodies in
thin sections of fixed, embedded cell cultures and infected tissues complement the diagnostic effort.
The details of these techniques and their application to the detection and analysis of HeV and NiV have
been described (Hyatt et al., 2001).
b)
Virus neutralisation: differentiation of HeV and NiV

Neutralisation tests rely on quantification methods and three procedures are available to titre HeV and NiV.
In the traditional plaque and microtitre assay procedures, the titre is calculated as plaque forming units
1228
(PFU) or the tissue culture infectious dose capable of causing CPE in 50% of replicate wells (TCID50),
respectively. In an alternative procedure, the viruses are titrated on Vero cell monolayers in 96-well plates
and after 1824 hours, foci of infection are detected immunologically in acetone-fixed cells using anti-viral
antiserum (Crameri et al., 2002). The virus titre is expressed as focus-forming units (FFU)/ml.
Neutralisation assays using these three methods are described below. A virus isolate that reacts with antiHeV and/or anti-NiV antisera in an immunofluorescence assay is considered to be serologically identical to
either HeV or NiV if it displays the same sensitivity to neutralisation by anti-HeV and anti-NiV antisera as
HeV or NiV. Anti-HeV antiserum neutralises HeV at an approximately four-fold greater dilution than that
which neutralises NiV to the same extent. Conversely, anti-NiV antiserum neutralises NiV approximately four
times more efficiently than HeV (Chua et al., 2000). Virus quantification procedures should be conducted at
BSL4. A new version of the differential neutralisation test has been recently described, which avoids the use
of infectious virus by the use of ephrin-B2-bound biospheres (Bossart et al., 2007). Although the test has yet
to be formally validated, it appears to have the potential to be a screening tool for use in countries without
BSL4 facilities.
i)
Plaque reduction
Stock HeV and NiV and the unidentified Henipavirus are diluted in media and replicates of each virus
containing approximately 100 PFU in 50100 l are mixed with an equal volume of either Eagles
minimal essential media (EMEM) or a range of dilutions of anti-HeV or anti-NiV antiserum in EMEM.
The virusantiserum mixtures are incubated at 37C for 45 minutes, adsorbed to monolayers of Vero
cells at 37C for 45 minutes and the number of plaques determined by traditional plaque assay
procedures after incubation at 37C for 3 days.
ii)
Microtitre neutralisation
Stock HeV and NiV and the unidentified Henipavirus are diluted and replicates of each virus containing
approximately 100 TCID50 in 50 l are added to the test wells of a flat bottom 96-well microtitre plate.
The viruses are mixed with an equal volume of either EMEM or a range of dilutions of anti-HeV or antiNiV antiserum in EMEM. The mixtures are incubated at 37C for 45 minutes and approximately 2.4
104 cells are added to each well to a final volume of approximately 200 l. After 3 days at 37C, the
test is read using an inverted microscope and wells are scored for the degree of CPE observed. Those
that contain cells only or cells and antiserum should show no CPE. In contrast, wells containing cells
and virus should show syncytia and cell destruction. A positive well is one where all or a proportion of
cells in the monolayer form large syncytia typical of henipavirus infection.
iii)
Immune plaque assay

Vero cells (2 104 in 200 l medium/well) are added to flat-bottom microtitre plates and grown
overnight at 37C. Stock HeV and NiV and the unidentified Henipavirus are diluted and replicates
containing about 60 FFU/50 l are mixed with an equal volume of either EMEM or a range of dilutions
of anti-HeV and anti-NiV antisera diluted in EMEM. Virusantiserum mixtures are incubated for
45 minutes at 37C and adsorbed to Vero cell monolayers for 45 minutes at 37C with 5% CO2. Virus
antiserum mixtures are removed, 200 l EMEM is added to each well and incubation is continued at
37C. After 1824 hours the culture medium is discarded and plates are immersed in cold, absolute
acetone for 10 minutes and then placed in plastic bags, which are filled with acetone, heat-sealed and
surface sterilised with 4% (v/v) lysol during removal from the BSL4 laboratory. Gluteraldehyde can also
be used for sterilisation at concentrations as low as 0.1% for 24 hours. It is recommended that each
laboratory determine the concentration of gluteraldehyde required for sterilisation within the time frame
required. Acetone-fixed plates are air-dried, the wells are blocked with phosphate buffered saline (PBS)
containing 0.05% Tween 20 and 2% skim milk powder, and incubated for 30 minutes at 37C with
antiserum to either HeV or NiV or a monospecific antiserum to a virus protein. Anti-viral antibody
binding to syncytia can be detected using alkaline phosphatase-conjugated species-specific antibody
and the substrate 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium substrate
(NBT/BCIP; Promega, Catalog number S3771). When purple plaques appear against a clear
background (1030 minutes), the substrate is removed and the plates are rinsed with distilled water
and air-dried. Plaques are counted using a magnifying glass.
c)
Nucleic acid based recognition methods

The complete genomes of both HeV and NiV have been sequenced (Wang et al., 2001), and PCR-based
methods have been used to detect virus and are being validated in a number of laboratories.
A particularly sensitive and useful approach to the detection of henipavirus genome in specimens is realtime PCR. This method has the biosafety advantage of not propagating live infectious virus. Test methods
and primers used depend on the technology platform and associated chemistry being used in individual
laboratories (Mungall et al., 2006; Wacharapluesadee & Hemachudha, 2007). The virus-specific reagents
used in one such assay (Mungall et al., 2006) based on Taqman chemistry are as follows:
1229
For Hendra virus detection:

Primer # 1
(HENDRA-N1433F)
5-ATC-TCA-GAT-CCA-GAT-TAG-CTG-CAA-3
Primer # 2
(HENDRA-N1572R)
5-ATC-ATT-TTG-GGC-AGG-TTT-GG-3
TaqMan Probe
(HENDRA-N1510T-FAM)
5-6FAM-AAC-CGC-CCT-CAG-GCA-GAC-TCA-GGA-TAMRA-3
For Nipah virus detection:

Primer # 1
(Nipah-N1198F)
5-TCA-GCA-GGA-AGG-CAA-GAG-AGT-AA-3
Primer # 2
(Nipah-N1297R)
5-CCC-CTT-CAT-CGA-TAT-CTT-GAT-CA-3
TaqMan probe
(Nipah-1247comp-FAM):
5-6FAM-CCT-CCA-ATG-AGC-ACA-CCT-CCT-GCA-G-TAMRA-3
Laboratories wishing to establish molecular detection methods should refer to published protocols or consult
the OIE Reference Laboratory.
d)
Henipavirus antigen detection in fixed tissue immunohistochemistry

Immunohistochemistry is also a useful test in HeV and NiV detection. Performed on formalin-fixed tissues or
formalin-fixed cells, it is safe and has allowed retrospective investigations on archival material. As virus
replication and the primary pathology occur in the vascular endothelium (Hooper et al., 2001), there is a wide
range of tissues in which HeV and NiV antigen can be detected (Daniels et al., 2001). It is thought that HeV
antigens may be cleared from lung tissue early in the course of infection and so the sample submitted
should include a range of tissues, not just lung. HeV antigen has been detected in the kidney of a horse
21 days post-infection (Williamson et al., 1998) and so this organ should always be submitted. Ideally a
submission for immunohistochemistry would include samples of the brain at various levels, lung, mediastinal
lymph nodes, spleen and kidney. In pregnant animals the uterus, placenta and fetal tissues should be
included.
A range of antisera to HeV and NiV may be used in immunohistochemical investigations of HeV- and NiVinfected tissues, but rabbit antisera to plaque-purified HeV and NiV have been found to be particularly
useful. Some MAbs are also available. The Nipah Virus Pathology Working Group has described a detection
system (Wong et al., 2002). A biotinstreptavidin peroxidase-linked detection system has also been used
successfully (Hooper et al., 2001). The following detection system is an anti-rabbit/anti-mouse dextran
polymer-linked reagent conjugated with alkaline phosphatase.
Test procedure
i)
Dewax slides containing formalin-fixed, paraffin-embedded test material and positive and negative
control tissue sections by immersion three times in xylene for 1 minute. Hydrate sections through two
changes of 98100% ethanol, one change of 70% ethanol and running tap water to remove residual
alcohol.
ii)
Rinse slides in distilled water, immerse in 0.01 M CaCl2 (adjusted to pH 7.8 with 0.1 M sodium
hydroxide) containing 0.1% (w/v) trypsin (Difco Trypsin 250) for 20 minutes at 37C and wash in
distilled water.
iii)
Lay slides flat in a humid chamber and rinse with PBS for 5 minutes. Add 200 l 3% aqueous H2O2 to
each slide for 20 minutes at room temperature to block endogenous peroxidase. Rinse slides in PBS
for 5 minutes.
iv)
Add 200 l of an appropriate dilution of rabbit anti-Nipah or anti-Hendra antibody in PBS containing
0.1% (w/v) skim milk powder to test tissue slides and positive and negative control slides. To a
duplicate set of test and positive and negative control slides add rabbit antibody to an unrelated
pathogen. Cover the slides and incubate at 37C for 1 hour.
v)
Rinse slides in PBS for 5 minutes and apply 23 drops of EnvisionTM solution (anti-rabbit Ig conjugated
to peroxidase-labelled dextran polymer [DAKO Corporation, 6392 Via Real, Carpinteria, CA 03013]).
Incubate at 37C for 20 minutes.
vi)
Prepare the substrate by dissolving 2 mg 3-amino-9-ethylcarbazole (AEC) in 200 l dimethyl

formamide (Merck) and add to 10 ml 0.02 M acetate buffer, pH 5.0. Add 5 l H2O2 (30% w/v) and mix.
1230
Check the positive control slide for sufficient staining, usually 25 minutes, and stop the reaction by
rinsing in distilled water. The substrate solution should be made fresh prior to use.
vii)
Counterstain the slides in haematoxylin for 13 minutes, rinse in tap water, add Scotts solution (0.04 M
sodium bicarbonate, 0.3 M magnesium sulphate), and wash well in running tap water. Rinse the slides
in distilled water and mount with a cover-slip using aqueous mounting medium.
viii) Read the slides for cytoplasmic deposition of the chromogen indicating the presence of viral antigen.
Brown/red granular staining will be seen in the cytoplasm of positive cells. The cell nuclei are blue and
this facilitates identification of tissue morphology and assists in the location of viral antigen within the
tissue.
2.
Serological tests
In laboratories doing serological testing, particularly in outbreak situations, several strategies have been adopted
to reduce the risk of exposure of laboratory personnel to HeV and NiV. Sera may be gamma-irradiated
(6 kiloGreys) or diluted 1/5 in PBS containing 0.5% Tween 20 and 0.5% Triton-X100 and heat-inactivated at 56C
for 30 minutes. The process used will be based on a risk assessment. Specimens for surveillance testing and
testing for animal movement certification may be considered a lesser biosafety risk than those for disease
investigation. In some circumstances heat inactivation may be adopted as a sufficient precaution. However there
is value in having a standardised approach for all samples in managing a test, rather than be maintaining multiple
test methods.
a)
Virus neutralisation tests

Henipaviruses can be quantified by plaque, microtitre or immune plaque assays and these assays can be
modified to detect anti-virus antibody (see above). The virus neutralisation test (VNT) (Kaku et al., 2009;
Tamin et al., 2009) is accepted as the reference standard. In the most commonly used microtitre assay,
which is performed under BSL4 conditions, sera are incubated with virus in the wells of 96-well microtitre
plates prior to the addition of Vero cells. Sera are screened starting at a 1/2 dilution although this may lead
to problems with serum-induced cytotoxicity. Where sample quality is poor or sample volumes are small, as
may be the case with flying fox or microbat sera, an initial dilution of 1/5 may be used. Cultures are read at
3 days, and those sera that completely block development of CPE are designated as positive. If cytotoxicity
is a problem the immune plaque assay described above has merit because the virus/serum mixtures are
removed from the Vero cell monolayers after the adsorption period, thereby limiting their CPE.
b)

Henipavirus antigens derived from tissue culture for use in the enzyme-linked immunosorbent assay (ELISA)
are irradiated with 6 kiloGreys prior to use, a treatment that has negligible effect on antigen titre. In the
indirect ELISA developed in response to the initial outbreak at Hendra in 1994, antigen was derived from
HeV-infected cells subjected to several cycles of freezing and thawing and treatment with 0.1% (w/v) sodium
dodecyl sulphate (P. Selleck, unpublished data). In the national swine surveillance programme in Malaysia in
1999 (Daniels et al., 2000) a similar indirect ELISA format was used in which antigen was derived by nonionic detergent treatment of NiV-infected cells. Subsequently, to control for high levels of nonspecific binding
activity in some porcine antisera, a modified ELISA was developed based on the relative reactivity of sera
with NiV antigen and a control antigen derived from uninfected Vero cells. At the Centers for Disease Control
(CDC), Atlanta, USA, the approach has been to not only have an indirect ELISA for detection of IgG but also
to use a capture ELISA for detection of IgM. For NiV, an ELISA using a recombinant nucleocapsid antigen
has also been described (Yu et al., 2006), which is also configured to detect either IgG or IgM.
The specificity of the indirect NiV ELISA (98.4%) (Ong et al., 2000) means that in surveillance programmes,
the test will yield false positives. This may not be a significant problem in the face of a NiV outbreak where a
high proportion of pigs are infected and the purpose of the surveillance is to detect infected farms. However,
this level of test specificity creates a problem in the absence of an outbreak or if the number of samples to
be tested is limited. If a positive ELISA result was indicative of a bona fide infection, failure to respond may
lead to virus spread and human fatalities. In contrast, initiating control measures in response to a falsepositive ELISA result would be wasteful of resources (Daniels et al., 2001). The current approach is to test
all ELISA reactive sera by VNT, with sera reacting in the VNT considered to be positive. Confirmatory VNT
should be done under BSL4 conditions and this may entail sending the samples to an internationally
recognised laboratory.
The following procedure for the NiV ELISA has been developed at Australian Animal Health Laboratory
(AAHL) for porcine sera and standardised after collaborative studies in the Veterinary Research Institute,
Ipoh, Malaysia.
1231
Test procedure
Preparation of NiV antigens
i)
Grow Vero cells until confluent in roller bottles in EMEM containing 10% (v/v) fetal calf serum (FCS).
To infect with virus, pour off all but 5 ml of the medium from each roller bottle and, in a BSL4
laboratory, add low passage, plaque-purified NiV to a multiplicity of infection of 0.1 TCID50/cell.
ii)
Rotate roller bottles for 30 minutes at 33C to adsorb virus, add 60 ml EMEM containing 10% FCS to
each bottle and roll for a further 48 hours at 33C. The multiplicity of infection, incubation time and
temperature are chosen so that although the majority of cells become infected and are incorporated
into syncytia within 48 hours, few cells detach into the culture medium. The culture medium of cells
infected under these conditions is an excellent source of virus for further purification.
iii)
Wash monolayers of virus-infected cells once with cold 0.01 M PBS and, using a large scraper, scrape
cells from each roller bottle into 510 ml ice-cold PBS.
iv)
Pool scraped cells into 50 ml tubes kept in ice and pellet the cells at 300 g for 5 minutes at 4C. Pour
off PBS and resuspend cells in ice cold TNM (10 mM Tris, 10 mM NaCl, 1.5 mM MgCl2, pH 7.2),
approximately 0.5 ml TNM per roller bottle.
v)
Add NP40 (non-ionic detergent, Nonidet P40) to 1% (by addition of 1/10 volume of 10% [v/v] NP-40 in
water) and lyse cells using 510 strokes of a Dounce homogeniser. This also releases from the
cytoskeleton viral antigen that would otherwise be removed by centrifugation (step vi).
vi)
Pellet the nuclei at 600 g for 10 minutes at 4C. The nuclei will not lyse under these conditions and
should form a tight white pellet.
vii)
Gently remove the supernatant cytoplasmic extract into a clean tube and add ethylene diamine tetraacetic acid to 1.5 mM. Make up to 10 ml with TNE, aliquot in small amounts, freeze at 80C and
gamma-irradiate with 6 kiloGreys. Store aliquots at 80C.
Preparation of control, uninfected Vero cell antigen
viii) Grow Vero cells in roller bottles in EMEM containing 10% FCS. When confluent, wash monolayers
once with cold PBS and scrape the cells from each roller bottle into 510 ml ice-cold PBS. Proceed as
described for virus-infected cells in steps ivvii above.
Preparation of test sera
ix)
In a biological class II safety cabinet with appropriate personal protective equipment or a class III
cabinet, dilute test serum 1/5 in PBS containing 0.5% (v/v) Triton X-100 and 0.5% (v/v) Tween 20 in the
wells of a 96-well microtitre plate. Seal the microtitre plate. Laboratory personnel should wear gowns
and gloves and spray both their hands and the sealed microtitre plate with 1% Virkon before removing
the microtitre plate from the biosafety cabinet to heat at 56C for 30 minutes.
x)
Mix 22.5 l heat-inactivated serum with an equal volume of uninfected Vero cell antigen diluted 1/100
in PBS. Mix thoroughly and incubate at 1822C for 30 minutes.
xi)
Add 405 l blocking solution (PBS containing 5% chicken serum and 5% skim milk powder) to give a
final serum dilution of 1/100 and incubate at 1822C for 30 minutes. Aliquots of 100 l are added to
two wells containing NiV antigen and two wells containing uninfected Vero cell control antigen as
described in step xiv.
ELISA procedure
xii)
Dilute Vero cell control and NiV antigens in PBS to ensure that control and virus antigen wells are
coated with a similar concentration of protein. Antigen is usually diluted 1/1000 to 1/4000, but a specific
dilution factor must be determined for each batch of antigen. Add 50 l virus and cell control antigen to
the wells of a Nunc Maxisorp 96-well microtitre plate as follows: virus antigen in columns 1, 3, 5, 7, 9
and 11 and cell control antigen in columns 2, 4, 6, 8 10 and 12 (Fig. 1). Incubate at 37C for 1 hour with
shaking. Plates can be also incubated at 4C overnight.
xiii) Wash ELISA plates three times with PBS containing 0.5% Tween 20 (PBST) (250 l/well) and block
with PBS containing 5% chicken serum and 5% skim milk powder (100 l/well) for 30 minutes at 37C
on a shaker.
xiv) Wash plates three times with PBST and add 100 l of inactivated, absorbed sera from step xi to each
well as indicated in the format below. Add 100 l PBS containing 5% chicken serum and 5% skim milk
powder to conjugate and substrate control wells. Incubate the plates without shaking for 1 hour at 37C
and wash three times with PBST.
1232
xv)
Dilute protein A/G-horseradish peroxidase conjugate (Protein-A/G-Conjugate Supplied by Pierce,

through Progen Biosciences Product No. 32490) in PBST containing 1% (w/v) skim milk powder. The
dilution factor is approximately 1/50,000. Mix well and add 100 l protein A-conjugate to all wells
except the substrate control wells. Add 100 l PBST containing 1% skim milk powder to the substrate
control wells. Incubate the plates for 1 hour at 37C without shaking and wash four times with PBST.
xvi) Prepare the substrate (3,3,5,5-tetramethylbenzidine; TMB; Sigma, catalogue number T 3405) by
dissolving one tablet (1 mg) in 10 ml of 0.05 M phosphate citrate buffer, pH 5.0, and add 2 l of fresh
30% (v/v) H2O2. Add 100 l of the TMB substrate to each well. Incubate for 10 minutes at 1822C and
stop the test by adding 100 l 1 M sulphuric acid to each well.
xvii) Read plates after blanking on a substrate control well. The optical density (OD) at 450 nm on NiV
antigen and control Vero cell antigen are used to calculate an OD ratio for each serum (OD on NiV
antigen/OD on Vero control antigen).
Interpretation of results
xviii) An OD ratio >2.0 with an OD on NiV antigen >0.20 is considered positive.
xix) An OD ratio >2.0 with an OD on NiV antigen <0.20 is considered negative.
xx)
Sera displaying an OD ratio between 2.0 and 2.2 should be considered doubtful.
xxi) Doubtful and positive sera should be tested by VNT.
Fig. 1. ELISA plate format and result sheet.

Ni
Ni
Ni
Ni
Ni
Ni
10
11
12
Test serum #1
Test serum #1
Test serum #1
Test serum #1
Test serum #2
Test serum #2
Test serum #2
Test serum #2
Test serum #3
Test serum #3
Test serum #3
Test serum #3
Test serum #4
Test serum #4
Test serum #4
Test serum #4
Test serum #5
Test serum #5
Test serum #5
Test serum #5
H+
H+
H+
H+
Test serum #6
Test serum #6
Test serum #6
Test serum #6
L+
L+
L+
L+
Test serum #7
Test serum #7
Test serum #7
Test serum #7
Test serum #8
Test serum #8
Test serum #8
Test serum #8
CC
TMB
CC
TMB
Nunc Maxisorp 96 well plate; Ni: Nipah virus infected cell antigen; U: uninfected Vero antigen (control antigen);
H+: High positive control sera e.g. LAF pig 6 sera; N: Negative control sera e.g. negative pig sera;
L+: Low Positive control sera e.g. LAF Pig 6 sera 1:800

Veterinary vaccines for henipaviruses
1.
Background
a)

There is no commercially produced vaccine against Hendra and/or Nipah virus available. Development of
veterinary vaccines against henipaviruses is important both to protect susceptible domestic animal species
(i.e. porcine, equine, feline, and canine) and to reduce transmission from domestic animals to humans. The
original outbreak of NiV in Malaysia and Singapore was linked to transmission of the virus from pigs to
humans, and all of the human infections with Hendra virus in Australia have been linked to contact with sick
horses.
1233
A vaccine that protects against both NiV and HeV, and could be used in a number of species (i.e. equine,
swine, feline, canine) would be desirable. In addition, a vaccine for wildlife may be advantageous to assist in
outbreak control. Henipaviruses are considered to be bioterrorism and agroterrorism threats, which increase
the need for development and production of safe and effective vaccines for domestic animals.
2.
The desired profile for henipavirus vaccines includes a manufacturing process that is safe under low containment
conditions, yielding a large number of doses at a reasonable cost. The vaccine should be highly efficacious with a
quick onset of immunity following a single dose. The vaccine should ideally cross-protect against both NiV and
HeV and be safe in a wide range of species, and across all ages. Vaccination should prevent virus transmission
to susceptible animals and people, and should prevent virus entry into the brain. The vaccine should allow
detection of vaccinated animals which become infected (differentiation of infected from vaccinated animals
[DIVA]) and should have duration of immunity of at least 1 year.
Vaccine production techniques which require growing large quantities of henipaviruses have not been considered
to date because of the requirement for biosafety level 4 containment, and subsequent extensive safety testing.
This precludes the development of conventional killed, split, split-subunit or live attenuated vaccines.
3.
a)

The use of biotechnology presents opportunities for production of safe and effective henipavirus vaccines.
Live vectored vaccines expressing the fusion (F) and/or attachment proteins (G) and subunit vaccines
containing recombinant F and/or G proteins have been produced and shown to be safe and effective under
experimental conditions.
The vaccine currently under development for commercialisation is a recombinant canarypox virus
expressing Nipah virus F and/or G proteins. The ALVAC canarypox vector has been successfully used to
produce licensed, commercially available live vectored vaccines in equine, feline, and canine species. This
vector will infect mammalian cells and produce viral-encoded protein, but does not replicate in mammalian
hosts or cells. These properties provide an acceptable safety and efficacy profile and provide a vaccine that
is DIVA compatible.
b)

Live recombinant vectored vaccines must meet all requirements for safety, efficacy, potency, and purity
required of all vaccines. In addition they must be shown to stably express the recombinant proteins upon
passage in vitro and growth in cell culture to produce vaccine virus. A risk assessment should be conducted
before biotechnology-derived vaccines are released into the environment. The risk assessment should
include information on the design, construction, and testing of the biotechnology-derived vaccine. Detailed
information should be provided about the documented genetic characteristics and history of the organisms
used to construct the final recombinant biological agent and its survivability in the environment.
c)
Experimental vaccines based on biotechnology

The data on experimental vaccines published by mid-2009 indicate that experimental henipavirus vaccines
can prevent clinical disease, elicit systemic and mucosal immunity, and prevent viral replication in target
tissues in several mammalian animal species In addition there are indications that the HeV vaccine
formulations can cross protect against NiV.
Canarypox-vectored NiV vaccines
The ALVAC canarypox virus-based recombinant vaccine vector was used to construct two experimental NiV
vaccines, carrying the gene for NiV glycoprotein (ALVAC-G) or the fusion protein (ALVAC-F). The efficacy of
both the ALVAC-G and ALVAC-F were tested in 10-week old pigs, either as monovalent vaccine or in
combination (ALVAC-G/F) in a pilot protection study (Weingartl et al., 2006).
The vaccination regimen was two doses administered intramuscularly 14 days apart, each of them
containing 108 PFU. Both non-vaccinated controls and vaccinated pigs were challenged intra-nasally with
105.4 PFU of NiV 2 weeks later.
The combined ALVAC-F/G vaccine induced the highest levels of neutralising antibodies, and stimulated both
type 1 and type 2 cytokine responses. Virus was not isolated from the tissues of any of the vaccinated pigs
1234
post-challenge, and no virus shedding was detected in vaccinated animals, in contrast to challenge control
pigs. Histopathological findings indicated that there was no enhancement of lesions in the challenged
vaccinates. Based on the data generated in this one study, the combined ALVAC-F/G vaccine appears in
particular to be a very promising vaccine candidate. The canarypox vaccine vector has been licensed for
commercially available vaccines for dogs, cats, and horses. The canarypox (ALVAC) vaccine vectors induce
antibody and cytotoxic T cell responses in a range of mammalian species, and the replication of canarypox
viral vectors is abortive in mammalian cells, eliminating some of the safety concerns.
Vaccinia-vectored NiV vaccine
The NYVAC vaccinia virus-based recombinant vaccine vector was used to construct experimental NiV
vaccines where the vaccinia virus expresses either NiV G or F glycoprotein (Guillaume et al., 2004). The
recombinant vaccines were used for subcutaneous immunisation of hamsters, either individually or in
combination, using 107 PFU/animal in two doses (1 month apart). Both of the NiV glycoproteins G and F
vaccinia virus recombinants induced an immune response in hamsters that protected against a lethal
intraperitoneal challenge with 103 PFU of NiV/animal. This team also demonstrated that passive transfer of
antibody induced by the glycoproteins protected the animals against NiV, and also against HeV (Guillaume
et al., 2009).
Although NYVAC vaccinia-based vector is highly attenuated, it still has the potential to infect people,
creating safety concerns for use of the vaccine in domestic animals (or humans). However vaccinia vectors
were successfully used in wildlife rabies vaccination campaigns in Europe with the advantage for lending
themselves to oral immunisation.
Soluble G henipavirus vaccine
In preliminary evaluation of experimental subunit vaccine formulations containing either soluble forms of HeV
sG or NiV sG glycoprotein in a NiV challenge study in cats, good crossreactivity was demonstrated, with
HeV sG possibly providing better protection (Mungall et al., 2006). In a subsequent study, a subunit
formulation containing CpG as an adjuvant and HeV sG with its cytoplasmic tail and transmembrane
domains replaced by an immunoglobulin kappa leader sequence coupled with an S-peptide tag to facilitate
purification, was evaluated as a potential NiV vaccine. Intramuscularly vaccinated cats developed varying
levels of NiV-specific Ig systemically and importantly, all vaccinated cats possessed antigen-specific IgA on
the mucosa. Upon oronasal challenge with NiV (104.7 TCID50), all vaccinated animals were protected from
disease although virus was detected on day 21 post-challenge in one animal. However shedding was
detected at 6 and 8 days based on virus genome detection at about the same levels in both vaccinated and
control challenged animals (McEachern et al., 2008). In addition NiV genome was detected in the brain of
several vaccinated challenged animals compared to challenge control animals raising some concerns about
significance of this phenomenon in the light of the observed late henipavirus encephalitis in humans
(Paterson et al., 1998; Tan et al., 2002). Beside the potential efficacy concerns, cost of production may
prohibit development of this vaccine for veterinary application.
ACKNOWLEDGEMENTS
Many individuals at the Australian Animal Health Laboratory contributed to the development of the tests described
here, specifically Peter Hooper, Gail Russell and Megan Braun (immunohistochemistry), Paul Selleck, Chris
Morrissy, Brenda van der Heide, Greer Meehan and John White (ELISA) and Gary Crameri (immune plaque
assay).
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*
* *
NB: There is an OIE Reference Laboratory for Hendra and Nipah virus diseases
diagnostic tests, reagents and vaccines for Hendra and Nipah virus diseases
1238
CHAPTER 2.9.7.
LISTERIA MONOCYTOGENES
SUMMARY
A wide variety of animal species can be infected with Listeria monocytogenes, but clinical listeriosis
is mainly a ruminant disease, with occasional sporadic cases in other species. The main clinical
manifestations of animal listeriosis are encephalitis, septicaemia and abortion, and the disease is
often associated with stored forages, usually silage. Post-mortem findings and histopathology
depend on the clinical presentation.
Listeriosis is one of the most important food-borne diseases of humans. The disease manifestations
include septicaemia, meningitis (or meningoencephalitis) and encephalitis, usually preceded by
influenza-like symptoms including fever. In pregnant women, intrauterine or cervical infections may
result in spontaneous abortion or stillbirths. Listeria monocytogenes has also been associated with
gastroenteric manifestations with fever. Although the morbidity of listeriosis is relatively low, the
mortality of the systemic/encephalitic disease can be very high, with values in the vicinity of 30%.
The elderly, pregnant women, newborns and the immunocompromised are considered to be at high
risk of contracting the disease.
A number of molecular and cellular determinants of virulence have been identified for this
intracellular pathogen, and although there is evidence of polymorphism among different strains of
L. monocytogenes for some of these virulence determinants, this heterogeneity cannot be
correlated with the ability or inability of the organism to produce disease. Therefore, all
L. monocytogenes strains are considered to be potentially pathogenic.
Identification of the agent: A variety of conventional and rapid methods are available for the
detection and identification of L. monocytogenes in food samples and specimens from animal
listeriosis. Conventional methods remain the gold standard with which other methods are
compared. They are usually very sensitive. These methods use selective agents and enrichment
procedures to reduce the number of contaminating microorganisms and allow multiplication of
L. monocytogenes.
Although not required for regulatory purposes, different levels of subtyping L. monocytogenes
strains are available, including serotyping, phage typing, multilocus enzyme electrophoresis, DNA
restriction enzyme digestion patterns (conventional and pulse-field gel electrophoresis), nucleic acid
sequence-based typing and random amplification of polymorphic DNA.
Serological tests: Serological tests for the detection of antibodies have not been traditionally used
for the diagnosis of listeriosis. A number of formats have been tried and they have all been found to
be largely unreliable, lacking sensitivity and specificity. Experimental serological assays based on
the detection of anti-listeriolysin O have been used in some epidemiological investigations and as
support for the diagnosis of culture-negative central nervous system infections.
Immunohistochemical detection of L. monocytogenes antigens is a useful tool for the diagnosis of
the encephalitic form of the disease.
Requirements for vaccines and diagnostic biologicals: It has proven very difficult to
develop effective vaccines against L. monocytogenes which, as an intracellular organism, requires
effector T cells for an effective immune response. Experimental vaccines in laboratory animals are
being explored to confer protection to L. monocytogenes infection by a number of different
approaches, including immunisation with plasmid DNA, CD40 signalling along with heat-killed
L. monocytogenes, listeriolysin O-deficient mutants inoculated along with liposome-encapsulated
listeriolysin O, and immunisation with listerial antigens and IL-12.
1239
Chapter 2.9.7. Listeria monocytogenes
A. INTRODUCTION
A wide variety of animal species can be infected by Listeria monocytogenes, including mammals, birds, fish and
crustaceans, although most of the clinical listeriosis occurs in ruminants; pigs rarely develop disease and birds
are generally subclinical carriers of the organism. Most infections in animals are subclinical, but listeriosis can
occur either sporadically or in epidemic form. In addition to the economic impact of listeriosis in animals, there is a
link between animals and their role as a source of infection for humans primarily from consumption of
contaminated animal products. Infection can be as a result of direct contact with infected animals, especially
during calving or lambing (Wesley, 1999); however, these infections are very rare. The relative importance of the
zoonotic transmission of the disease to humans is not clear, and contamination from the food processing
environment is apparently more relevant to public health (Roberts & Wiedemann, 2003).
The clinical manifestations of listeriosis in animals include encephalitis, septicaemia and abortion, especially in
sheep, goats and cattle. The septicaemic form is relatively uncommon and generally, but not invariably, occurs in
the neonate. It is marked by depression, inappetence, fever and death. The encephalitic form is sometimes
referred to as circling disease because of a tendency to circle in one direction, and it is the most common
manifestation of the disease in ruminants. The signs include depression, anorexia, head pressing or turning of the
head to one side and unilateral facial paralysis. Abortion is usually late term (after 7 months in cattle and
12 weeks in sheep) (Hird & Genigeorgis, 1990; Walker, 1999). Only one clinical form of listeriosis usually occurs
in a particular group of animals. Bovine and ovine ophthalmitis have also been described (Walker & Morgan,
1993). Rarely mastitis of ruminants has been associated with L. monocytogenes infection. Gastro-intestinal
infections can occasionally occur in sheep (Clark et al., 2004). When listeriosis occurs in pigs, the primary
manifestation is septicaemia, with encephalitis reported less frequently and abortions rarely. Although birds are
usually subclinical carriers, sporadic cases of listeriosis have been reported, most frequently septicaemia and far
less commonly meningoencephalitis. Avian listeriosis may be the result of a secondary infection in viral disease
conditions and salmonellosis (Wesley, 1999).
The post-mortem findings and histopathology, in animal listeriosis, depend on the clinical presentation. In the
encephalitic form, the cerebrospinal fluid may be cloudy and the meningeal vessels congested. Gross
pathological lesions of the brain are rare. On occasion, the medulla shows areas of softening. However, the
histopathology is characteristic of the disease, consisting of foci of inflammatory cells with adjacent perivascular
cuffing, predominantly of lymphocytes and histiocytes, plasma cells and occasional neutrophils. The
microabscesses in the brain stem often more severally affect one side of the brain. More extensive malacic
pathology may occur. The medulla and pons are most commonly involved. In the septicaemic form, multiple foci
of necrosis in the liver and, less frequently the spleen, may be noted. Aborted fetuses of ruminants show very little
gross lesions, but autolysis may be present if the fetus was retained before being expelled (Low & Donachie,
1997; Walker, 1999).
The evidence indicates that animal listeriosis is predominantly associated with stored forage and with the
environment as the main source of contamination. Silage is the most frequent source (Fenlon, 1985; Wiedmann
et al., 1997). The intestinal mucosa is the main route of entry, after oral ingestion, in the case of
septicaemic/abortive listeriosis. The incubation period can be as short as 1 day. The incubation period for the
encephalitic form is usually 23 weeks, and the course of the disease is usually short in sheep and goats; 1
4 days (Roberts & Wiedemann, 2003), although it can be more protracted in cattle.
Although Listeria monocytogenes has been recognised as an animal pathogen for many years, its significant role
as a food-borne human pathogen became evident only in the 1980s, when documented reports of listeriosis
outbreaks, traced to contaminated food, started to appear in the literature (Schlech et al., 1983). Today,
L. monocytogenes is considered to be one of the most important agents of food-borne disease. Possible
explanations for the emergence of human food-borne listeriosis as a major public health concern include major
changes in food production, processing and distribution, increased use of refrigeration as a primary preservation
means for foods, changes in the eating habits of people, particularly towards convenience and ready-to-eat foods,
and an increase in the number of people considered to be at high risk for the disease (elderly, pregnant women,
newborns, immunocompromised) (Rocourt & Bille, 1997; Swaminathan, 2001).
The primary manifestations of listeriosis in humans include septicaemia, meningitis (or meningoencephalitis) and
encephalitis, usually preceded by influenza-like symptoms including fever. Gastroenteric manifestations with fever
also occur. Although the morbidity of listeriosis is relatively low, the mortality can reach values around 30%. In
pregnant women, infection may result in abortion, stillbirth or premature birth (Rocourt & Bille, 1997; Slutsker &
Schuchat, 1999).
Listeria monocytogenes is a Gram-positive rod and is responsible for almost all infections in humans; although
rare cases of infection due to L. ivanovii and L. seeligeri have been reported. In animals, L. monocytogenes is
responsible for the majority of infections, but L. ivanovii and L. innocua infections have also been recorded.
Listeria invanovil has been associated with abortions and has been reported to very occasionally cause
menigoencephaliis in sheep.
1240
Although L. monocytogenes has definite zoonotic potential, it is also an important environmental contaminant of
public health significance.
Subtyping of L. monocytogenes strains by a variety of methods is available for epidemiological investigations, but
the fundamental question of whether all strains of L. monocytogenes are capable of causing disease remains
unanswered (Graves et al., 1999; Jacquet et al., 2002; Lopez et al., 1992; 1993).
Several molecular virulence determinants have been identified that play a role in the cellular infection by
L. monocytogenes and the unravelling of their mechanism of action has made of L. monocytogenes one of the
most exciting models of host-pathogen interaction at the cellular and molecular levels. These virulence
determinants include, among others, the internalins, listeriolysin O (LLO), ActA protein, two phospholipases, a
metalloprotease, Vip protein, a bile exclusion system (BilE) and a bile salt hydrolase (Cabanes et al., 2005;
Cossart & Portnoy, 2000; Dussurget et al., 2002; Farber et al., 1993; Sleator et al., 2005). Although there is
polymorphism among different strains of L. monocytogenes for some of these virulence determinants, it cannot be
correlated with the ability or inability of the organism to produce disease (Jacquet et al., 2002).
1.
There is a variety of conventional and rapid methods currently available for the detection and identification of
L. monocytogenes in food samples and specimens from animal listeriosis. Conventional bacteriological methods
are important for various reasons: Their use results in a pure culture of the organism, which is useful for
regulatory purposes. They remain the gold standards against which other methods are compared and validated.
These methods are usually very sensitive and they do not require sophisticated and expensive equipment. Some
of the disadvantages of this group of methods include the relatively long period of time that the protocols require
for completion, several hands-on manipulations, the requirement for many different chemicals, reagents and
media, the possibility of contaminating microorganisms masking the presence of the target ones, including
overgrowth, the potential overlook of atypical variants of the target organism and the relative subjectivity involved
when interpreting bacterial growth on selective and differential agar plates (Andrews, 2002).
The isolation and identification of L. monocytogenes from food, environmental samples and animal specimens
require the use of selective agents and enrichment procedures that keep the levels of contaminating
microorganisms to reasonable numbers and allow multiplication of L. monocytogenes to levels that are enough for
detection of the organism. In the early days of listerial clinical bacteriology, cold enrichment (Gray et al., 1948)
was regularly used to this end, exploiting the ability of the organism to multiply at refrigeration temperatures,
whereas contaminating bacteria would not multiply under these conditions. However, this procedure requires very
long incubation times, often months, making it unacceptable for current investigations of food-borne outbreaks
and sporadic cases, as well as for the implementation of effective hazard analysis critical control points (HACCP)
programmes in food production and processing establishments. A number of selective compounds that allow
growth of L. monocytogenes at normal incubation temperatures have been incorporated into culture media, thus
shortening the time required for selective growth of the organism. Examples of these selective compounds include
cycloheximide, colistin, cefotetan, fosfomycin, lithium chloride, nalidixic acid, acriflavine, phenylethanol,
ceftazidime, polymixin B and moxalactam (AFNOR, 1997; 2000; Association of Official Analytical Chemists
[AOAC] Official Method 993.12, 2000; Hitchins, 1998; International Organization for Standardization [ISO], 1996;
USDA-FSIS, 2002).
Bacteriological diagnosis of animal listeriosis has traditionally involved direct plating of specimens on blood agar
or other enriched media and concomitant use of the cold enrichment technique, with weekly subculturing for up
to 12 weeks (Gray et al., 1948; Quinn et al., 1999; Walker, 1999). Immunohistochemical detection of
L. monocytogenes antigens in formalin-fixed tissue has proven to be more sensitive than direct plating and cold
enrichment bacterial culture for the diagnosis of the encephalitic form of the disease in ruminants (Campero et al.,
2002; Johnson et al., 1995). The introduction of alternative enrichment procedures and selective agents for the
isolation of L. monocytogenes from food and environmental samples has opened up the possibility of using some
of these techniques for the bacteriological analysis of samples from animal listeriosis.
In spite of advances made in the selective isolation of L. monocytogenes from food, there is still room for
improvement in a number of areas. No single procedure can be credited with being sensitive enough to detect
L. monocytogenes from all types of food (Donnelly, 1999). In addition, sublethally injured L. monocytogenes cells
can be found in processed food due to freezing, heating, acidification and other types of chemical or physical
treatment. These sublethally injured bacteria require special culture conditions for damage repair, before being
able to be detected in culture.
1241
a)
Isolation methods
Conventional methods for the isolation of L. monocytogenes from food that have gained acceptance for
international regulatory purposes include the United States Food and Drug Administration (FDA) method
(Hitchins, 1998), the Association of Official Analytical Chemists (AOAC) official method (AOAC Official
Method 993.12, 2000), the ISO 11290 Standards (ISO, 1996; 1998; 2004), the United States Department of
Agriculture (USDA)-Food Safety and Inspection Service (FSIS) method (USDA-FSIS, 2002) and the French
Standards (AFNOR, 1997; 2000).
Depending on the nature of the sample, a particular method might be more suitable than others. The ISO
Technical Committee ISO/TC 34, Agricultural Food Products, Subcommittee SC 9, Microbiology, claims that
the ISO Standard 11290, parts 1 and 2 (ISO, 1996; 1998; 2004), can be used for the detection of
L. monocytogenes in a large variety of food and feed products. Although they recognise that this standard
might not be appropriate in every detail in certain instances, they recommend that every effort should be
made to apply this horizontal method as far as possible.
The FDA and AOAC methods can be used for milk and dairy products. The USDA-FSIS method is
recommended for red meat and poultry (raw or cooked ready-to-eat), eggs, egg products and environmental
samples.
The traditional procedure for the isolation of L. monocytogenes from animal tissues has been direct plating
of specimens on sheep blood agar or other rich culture media and concomitant use of the cold enrichment
technique, with weekly subculturing for up to 12 weeks (Gray et al., 1948; Quinn et al., 1999; Walker, 1999).
Isolation of the organism by direct plating is relatively easy when numbers are large in a normally sterile site,
like in the case of the septicaemic form of the disease, but isolation is difficult when the organism is present
in low numbers, as in the case of the encephalitic form or when samples are heavily contaminated. A
comparison of the efficiency of direct plating, cold enrichment and a slight modification of what became the
AOAC method, found the latter to be superior over the other two, for the isolation of L. monocytogenes from
a variety of animal necropsy material, both in terms of the time required for the isolation and identification of
the organism, and the isolation rates (Eld et al., 1993).
For the enumeration of L. monocytogenes, the ISO Standard 11290, part 2 (1998) applies, as well as
optional protocols mentioned in the FDA and USDA-FSIS methods (Hitchins, 1998; USDA-FSIS, 2002).
In the case of animal listeriosis, the samples should be chosen according to the clinical presentation of the
disease: material from lesions in the liver, kidneys and/or spleen, in the case of the septicaemic form; spinal
fluid, pons and medulla in the case of the encephalitic form; and placenta (cotyledons), fetal abomasal
contents and/or uterine discharges in the case of abortion. Refrigeration temperatures (4C) must be used
for handling, storing and shipping specimens. If the sample is already frozen, it should be kept frozen until
analysis.
The protocol recommended for isolation of L. monocytogenes from animal necropsy material is described
below as originally published (Eld et al., 1993). It is possible that this protocol could be improved by
incorporating the recent updates to the AOAC method and new developments for identification and
confirmation of Listeria isolates, but there is no published evidence to support this hypothesis yet.
Isolation procedure from animal necropsy material
i)
Inoculate 1025 g or ml of sample (depending on the amount of sample available) into 225 ml Listeria
enrichment broth. When dealing with samples from animal listeriosis, the size of the sample for
inoculation maybe limited and less than the recommended for food samples (25 g or ml). If that is the
case, as much sample material as possible (aiming at 1025 g or ml) should be inoculated (Eld et al.,
1993). (Listeria enrichment broth base: Oxoid tryptone soya broth, 30 g; Difco yeast extract, 6 g; water,
1 litre; selective agents: Acriflavine, 2.3 mg; nalidixic acid, 9.2 mg; cycloheximide, 11.5 mg; add
selective agents to 225 ml of the broth base).
ii)
Incubate broth at 30C for 48 hours.
iii)
Spread 0.1 ml of the enrichment broth culture onto Oxford agar plates.
iv)
Incubate plates at 37C. Examine bacterial growth after 24 and 48 hours.
v)
Test five colonies (or all when fewer available) with typical appearance of L. monocytogenes for cell
shape, Gram reaction, haemolytic activity on blood agar (defibrinated horse blood), tumbling motility at
20C, fermentation of glucose (+), rhamnose (+) and xylose (), hydrolysis of esculin and production of
catalase.
1242
All culture media prepared should be subjected to quality control and must support growth of the suspect
organism from a small inoculum. The reference strain should be cultured in parallel with the suspicious
samples to ensure that the tests are working correctly.
Food samples intended for analysis must be representative of the food, including the outer surface and the
interior. The conventional culture methods include an enrichment procedure based on the use of liquid
culture media containing selective agents. The nature of the media and the selective agents vary with the
method. Both the FDA (Hitchins, 1998) and the ISO methods include a pre-enrichment step that is intended
for the recovery of sublethally injured L. monocytogenes cells, whereas in the USDA-FSIS (2002) and the
AOAC methods (AOAC Official Method 993.12, 2000) the samples are processed directly into enrichment
broth. In the case of the FDA method, the pre-enrichment is carried out at 30C for 4 hours in trypticasesoy
broth containing yeast extract (TSB YE) without selective agents. The ISO protocol uses a primary
enrichment for 24 hours at 30C in the presence of selective agents, but at half the concentration (half
Fraser broth).
Samples are enriched for 2472 hours at 30C, 35C or 37C, depending on the method. The FDA method
uses TSB YE containing acriflavine, nalidixic acid and cycloheximide. The USDA-FSIS method uses two
enrichment steps: The primary enrichment is done in University of Vermont medium (UVM), containing
nalidixic acid and acriflavine; the secondary enrichment is carried out in Fraser broth, containing nalidixic
acid, lithium chloride and acriflavine. The ISO standard indicates Fraser broth for the secondary
enrichment, containing the selective agents at full concentration, whereas the primary enrichment is carried
out in half Fraser broth, as indicated above. The AOAC method calls for selective enrichment in tryptone
soy broth containing acriflavine, nalidixic acid and cycloheximide (selective enrichment medium).
After selective enrichment, cultures are then plated on to selective/differential agar plates for isolation of
presumptive colonies of L. monocytogenes. All methods, except that of the ISO standard, use Oxford agar or
a modification, MOX agar (USDA-FSIS). Oxford agar contains lithium chloride, cycloheximide, colistin,
acriflavine, cefotetan and fosfomycin as selective agents, and typical colonies of Listeria spp are small, black
and surrounded by a black halo. In addition to Oxford agar, the FDA includes lithium chloride/
phenylethanol/moxalactam (LPM) or PALCAM agar, which contains lithium chloride, polymixin B, acriflavine
and ceftazidime. The MOX agar, used in the USDA-FSIS method, contains lithium chloride, colistin and
moxalactam. The two selective plating media used in the ISO standard method are: agar Listeria according
to Ottaviani and Agosti (ALOA), which contains lithium chloride, nalidixic acid, ceftazidime, polymyxin B and
amphotericin B (or cycloheximide), and any other selective medium, of each laboratorys choice, such as
Oxford or PALCALM. Typical colonies of L. monocytogenes in ALOA agar are green-blue, surrounded by an
opaque halo (ISO, 2004).
Bio-Rad Laboratories have developed RAPIDL.Mono, a selective chromogenic medium for direct detection
and enumeration of L. monocytogenes, based on selective isolation, chromogenic detection of
phospatidylinositol-specific phospholipase C (PI-PLC) and xylose use. Listeria monocytogenes develops
blue colonies (PI-PLC positive) without yellow halo (xylose negative); L. ivanovii produces greenish-blue
colonies (PI-PLC positive) with yellow halo (xylose positive). Other Listeria spp. colonies are white (PI-PLC
negative.
b)
Conventional identification methods

Typical Listeria spp. colonies, on the above selective/differential agar plates, are then selected for further
identification to the species level, using a battery of tests. The tests include the Gram-staining reaction,
catalase, motility (both in a wet mount observed under phase-contrast microscopy and by inoculation into
motility test media), haemolysis and carbohydrate use. The ChristieAtkinsMunchPeterson (CAMP) test is
a very useful tool to help identify the species of a Listeria spp. isolate. It is used in the ISO and AOAC
protocols and it is considered to be optional in the FDA and USDA-FSIS methods. The test is simple to
perform and easy to read. It consists of streaking a -haemolytic Staphylococcus aureus (ATCC strain
49444 or 25923, NCTC strain 7428 or 1803) and Rhodococcus equi (ATCC strain 6939, NCTC strain 1621)
in single straight lines in parallel, on a sheep blood agar plate or a double-layered agar plate with a very thin
blood agar overlay. The streaks should have enough separation to allow test and control Listeria strains to
be streaked perpendicularly, in between the two indicator organisms, without quite touching them (separated
by 12 mm). After incubation for 2448 hours at 3537C (1218 hours if using the thin blood agar overlay),
a positive reaction consists of an enhanced zone of -haemolysis, at the intersection of the test/control and
indicator strains. Listeria monocytogenes is positive with the S. aureus streak and negative with R. equi,
whereas the test with L ivanovii gives the reverse reactions (Quinn et al., 1999).
1243
Table 1. Differentiation of Listeria species

Species
Haemolysis
Production of acid
CAMP test
Rhamnose
Xylose
S. aureus
R. equi
L. monocytogenes
L. innocua
L. ivanovii
L seeligeri
(+)
(+)
L. welshimeri
L. grayi subsp. Grayi
L. grayi subsp. Murrayi
V: variable; (+): weak reaction; +: >90% positive reactions; : no reaction.
Serology, lysogenic typing and the immunocompromised mouse pathogenicity assay are considered to be
optional methods.
c)
Rapid identification methods

The following protocols include conventional and nonconventional commercially available tests, e.g. Vitek,
API, MICRO-ID, enzyme-linked immunosorbent assay (ELISA) kits and nucleic acid assay kits, to help in the
identification of L. monocytogenes. Polymerase chain reaction (PCR), targeting the hly gene, has been
found to be a sensitive and rapid technique for confirmation of the identification of suspect
L. monocytogenes isolated on selective/differential agar plates (Gouws & Liedemann, 2005). In addition to
RAPIDL.Mono and ALOA agars, described above, other chromogenic media have been developed to
differentiate L. monocytogenes colonies from those of other Listeriae: Oxoid chromogenic Listeria agar
(OCLA), and CHROMagar Listeria, developed by CHROMagar Microbiology.
i)
MICRO-ID Listeria
MICRO-ID Listeria is a commercially available system (Organon Teknika Corp., 100 Akzo Ave., Durham, NC
27712, USA) that has been validated by the AOAC (Official Method 992.18, 2000) for the presumptive
identification of Listeria species isolated from food and environmental samples. It provides an alternative to
conventional biochemical testing of Listeria spp. isolates by the FDA and USDA-FSIS methods. It is based
on the principle that the test inoculum contains preformed enzymes that can be detected after 24 hours at
37C. Differentiation of Listeria species is based on an octal code derived after adding the numerical values
for each group of three tests and on the reactions obtained from the CAMP test and haemolysis
characteristics, which are assayed separately.
ii)
Vitek Automicrobic System
The Vitek Automicrobic System (bioMrieux Vitek, Inc., 595 Anglum Dr., Hazelwood, MO, USA) is an
automated microbial identification system that can be used for the presumptive identification of food-borne
Listeria species and for screening of non-Listeria isolates. It has been validated by the AOAC as method
992.19 (AOAC Official Method 992.19, 2000). The system uses an incubator chamber with an optical reader,
a filler/sealer unit for test kit inoculation and a computer. Gram-positive (GPI) and Gram-negative (GNI+)
identification cards each contain 30 biochemical tests. Changes are analysed by the computer, which then
assigns the test organism a genus and /or species. The identification of the Listeria species requires the use
of the GPI card and two reactions on the GNI+ card. However, for identification of some Listeriae, the
analyst must perform the CAMP, haemolysis and/or nitrate reduction tests as described under the FDA
method.
Organisms placed in the LM category are identified as L. monocytogenes or L. innocua; in the LI category,
as L. ivanovii or L. seeligeri; in the LW category, as L. welshimeri; and in the LG category, as L. grayi or
L. murrayi (a subspecies of L. grayi). Organisms in the O category are classified as non-Listeria species.
Further tests should be conducted to identify the species within each category according to the FDA method.
Other commercially available methods for the identification of Listeria species include the API LISTERIA
(bioMrieux), the MICROBACT 12L (Microgen), the MicroLog System (Biolog), the Sherlock Microbial
Identification System (MIS) (Microbial ID; based on fatty acid patterns) and the Walk/Away System
(MicroScan).
1244
iii)
Rapid immunological detection methods
A number of immunological methods have been developed to identify L. monocytogenes in foods and the
following commercially available methods have been validated by one or more recognised formal validation
systems (Dunbar et al., 2003; Sewell et al., 2003).
Colorimetric monoclonal enzyme-linked immunosorbent assay (Listeria-Tek)
The Listeria-Tek is AOAC official method (AOAC Official Method 994.03, 2000) and it is intended for the
detection of Listeria spp. in dairy products, seafood and meats. Because the monoclonal antibodies (MAbs)
used in the test may cross react with other Listeria spp., the test is not confirmatory for L. monocytogenes.
The kit is commercially available from Organon Teknika Corp., 100 Akzo Ave, Durham, NC 27704, USA.
Enrichment cultures found positive by this method are streaked on selective media and suspect colonies are
biochemically identified as L. monocytogenes according to the FDA method. A positive result is only valid
when the positive and negative controls give acceptable absorbance readings.
Colorimetric polyclonal enzyme immunoassay screening method (TECRA Listeria Visual Immunoassay
[TLVIA])
The TLVIA is AOAC official method 995.22 (AOAC Official Method 995.22, 2000) and it is intended for the
detection of Listeria spp. in dairy foods, seafood, poultry, meats (except raw ground meat), and leafy
vegetables. An optimised version, with enrichment protocols for additional foods and with omission of the
toxic antifungal agent cycloheximide, is AOAC official method 2002.09 (AOAC Official Method 2002.09,
2003), which is a screening procedure for the detection of Listeria spp. in raw meats, fresh
produce/vegetables, processed meats, seafood, dairy foods cultured/noncultured, fruit and fruit juices.
The commercial kit is available from TECRA International Pty Ltd, P.O. Box 788, Willoughby, NSW,
Australia.
Enrichment cultures that are positive must be inoculated on to selective media and suspect colonies
identified according to the criteria specified under the FDA and USDA methods.
Assurance polyclonal enzyme immunoassay method
The Assurance Listeria Enzyme Immunoassay is AOAC official method 996.14 (AOAC Official Method
996.14, 2003) and it can be used for the detection of Listeria spp., including L. monocytogenes, in dairy
foods, red meats, pork, poultry products, fruits, nutmeats, seafood, pasta, vegetables, cheese, bone meal,
chocolate, environmental surfaces, and eggs.
Readings above the cut-off value are considered presumptive positive and the enriched cultures are then
confirmed by culture and identification procedures as described under the FDA method.
The commercial kit is available from BioControl Systems, Inc., 12822 SE 32nd St., Bellevue, WA 98005,
USA.
Visual Immunoprecipitate assay (VIPTM)
TM
The VIP assay is AOAC official method 997.03 (AOAC Official Method 997.03, 2003). It can be used for
the detection of L. monocytogenes and other Listeria spp. in dairy foods, red meats, pork, poultry and poultry
products, seafood, fruits, vegetables, nutmeats, pasta, chocolate, eggs, environmental surfaces and bone
meal.
The test is performed with an enriched culture of the test samples. Presumptive positive tests must be
confirmed by culture and identification procedures as described under the FDA method.
The VIP units are commercially available from BioControl Systems, Inc., 12822 SE 32nd St., Bellevue, WA
98005, USA.
VIDAS LIS assay screening method
This enzyme-linked immunofluorescent assay (ELFA) is AOAC official method 999.06 (AOAC Official
Method 999.06, 2003). It has also been validated by the Association Franais de Normalisation (AFNOR)
and by the European Microbiological Methods Assessment Scheme (EMMAS) (Baylis, 2000). It is used for
screening dairy products, vegetables, seafood, raw meats and poultry, as well as processed meats and
poultry, for Listeria spp. antigens.
1245
This immunoassay is performed in the automated VIDAS instrument. The computer compares this value
with a standard and a positive or negative report is generated. Positive results must be confirmed by
standard culture methods as described under the FDA method. An enrichment modification to this protocol,
using demi Fraser and Fraser broths (AOAC official method 2004.06), was evaluated in a multilaboratory
study and no significant difference was found in its performance when compared to that of AOAC official
method 999.06 (Silbernagel et al., 2005).
The VIDAS Listeria monocytogenes 2 (LMO 2) method includes specific MAbs to capture L. monocytogenes
antigens and it has been validated by AFNOR, including its use with environmental samples.
The VIDAS system is available from bioMrieux, Inc., 595 Anglum Rd., Hazelwood, MO 63042, USA.
Other commercially available immunological methods that have been validated by formal systems include
the VIDAS Listeria Species Xpress (bioMrieux), validated by AFNOR; the Transia Plate Listeria ELISA
(Transia, Diffchamb Ltd), validated by AFNOR; the EIAFOSS Listeria automated ELISA (Foss Electric),
validated by the AOAC Research Institute; the immunochromatographic method REVEAL for Listeria
(Neogen Corporation), validated by the AOAC Research Institute; the immunochromatographic method
Clearview Listeria Rapid Test (Oxoid), validated by AFNOR, EMMAS and the AOAC Research Institute and
the immunomagnetic separation-based Listertest (Vicam), validated by the AOAC Research Institute (Baylis,
2000).
Other commercially available immunologically based methods include the Transia Plate Listeria
monocytogenes ELISA (Transia, Diffchamb Ltd), the immunomagnetic separation-based Dynabeads antiTM
Listeria (Dynal Ltd), the Listeria UniQue ELISA (TECRA), the Microscreen Listeria latex agglutination test
(Microgen BioProducts Ltd), and the Listeria Rapid Test EIA (Oxoid).
d)

A number of methods based on nucleic acid recognition have been developed to identify L. monocytogenes
in foods. A few of them have been validated by one or more recognised formal validation systems and are
commercially available (Norton, 2002). Novel target sequences for diagnostic purposes include the
metalloprotease gene (LightCycler foodproof Listeria monocytogenes Detection Kit, Roche Applied Science),
the prfA gene (Rossmanith et al., 2006) and the ssrA gene (OGrady et al., 2008) in a real-time PCR format.
i)
GENE-TRAK Listeria assay
The GENE-TRAK Listeria assay is a colorimetric DNA hybridisation method for the detection of Listeria
sequences, which has been validated by the AOAC as method 993.09 (AOAC Official Method 993.09, 2000)
for use with dairy products, meats and seafood. This assay has also been validated by AFNOR. Due to the
possibility of encountering false-positive reactions, positive samples must be confirmed by standard cultural
methods.
Test portions found positive by this DNA hybridisation assay must be confirmed by streaking a phosphate
buffered saline growth suspension on a Listeria selective plate and continuing with biochemical identification
of presumptive Listeria isolates as described under the FDA method.
The GENE-TRAK Listeria assay is commercially available from GENE-TRAK
Hopkinton, MA 01748, USA.
ii)
TM
Systems, 94 South Street,
BAX System
The USDA-FSIS has adopted the PCR-based BAX System (Qualicon) (USDA-FSIS, 2005) as their
screening method for L. monocytogenes in enriched meat and poultry samples. It reduces the report out
time for true negative samples by 24 hours and reduces false-positive results, with a detection limit better
that 1 cfu/g in a 25 g sample. All samples that are identified as presumptive positive for L. monocytogenes
are then subject to cultural confirmation by the conventional method.
iii)
GENE-TRAK test for Listeria monocytogenes
The GENE-TRAK test for L. monocytogenes (GENE-TRAKTM Systems) is a hybridisation probe-based

method validated by AFNOR (Baylis, 2000).
iv)
Gen-Probe (AccuProbe) Listeria monocytogenes confirmatory test
The Gen-Probe (AccuProbe) Listeria monocytogenes Confirmatory Test (Gen-Probe) is another

hybridisation probe-based method validated by AFNOR (Baylis, 2000).
1246
v)
AD713 method
The FDA uses a L. monocytogenes: a combination of invasion-associated protein and haemolysin (hly) gene
probes AD713 method (Hill et al., 2001). This method combines the detection of the L. monocytogenes
haemolysin (also called listeriolysin O) gene by use of the oligonucleotide probe AD13, and the detection of
the invasion-associated protein gene by a synthetic probe, AD07. Both probes are used in combination
(designated AD713) to avoid false-negative results because of silent mutations in the gene (nucleotide
changes that affect DNA probe binding but do not change the gene function). Positive samples must be
confirmed with conventional procedures as described under the FDA method.
Other commercially available methods based on nucleic acid recognition, include the Foodproof Listeria
TM
monocytogenes PCR assay (Biotecon Diagnostics) and the PROBELIA (L. monocytogenes) PCR assay
(Sanofi Diagnostics). The application of the real-time PCR as a quantitative detection method, specific for
L. monocytogenes, has also been developed (Cady et al., 2005; Hough et al., 2002) and it shows good
potential for routine analytical use. The IQ-Check Listeria monocytogenes kit (for all food products and
environmental samples), developed by Bio-Rad, and the GeneDisc (for all food, except milk products)
developed by GeneSystems, France, are based on real-time PCR detection and have been validated by
AFNOR (AFAQ-AFNOR Certification, [2005]).
Table 2. Some conventional and antibody-based commercial systems for rapid Listeria screening and confirmation1
Test
ID level
Principle
Approx.
test time2
MICRO-ID Listeria
L. monocytogenes/
innocua complex
Enzyme reaction
Vitek System
L. monocytogenes/
innocua complex
API Listeria
Company
Main use
24 hours
Organon
Teknika
Confirmation
Biochemical tests
24 hours
bioMrieux
Confirmation
L. monocytogenes
Biochemical tests
24 hours
bioMrieux
Confirmation
MicroLog System
L. monocytogenes
Carbon source
substrates
4 or 24 hours
Biolog
Confirmation
MICROBACT 12L
L. monocytogenes
Carbohydrate
useand micro
haemolysis test
46 or
24 hours
Microgen
Confirmation
Sherlock Microbial
Identification System
(MIS)
L. monocytogenes/
innocua complex
Fatty acid patterns
90 minutes
Microbial ID
Confirmation
Microscreen
Listeria spp.
Latex agglutination
1 minute
Microgen
BioProducts
Confirmation
VIP Listeria
Listeria spp.
Immunochromatography
2 minutes
(postenrichment)
BioControl
Systems
Screening
Dynabeads anti-Listeria
Listeria spp.
Immunomagnetic
separation
4872 hours
Dynal
Screening
REVEAL for Listeria
Listeria spp.
43 hours
Neogen
Screening
Clearview Listeria
(Oxoid Listeria Rapid
Test)
Listeria spp.
43 hours
Oxoid
Screening
Listertest
Listeria spp.
Immunomagnetic
separation
2448 hours
Vicam
Screening
1: Adapted and expanded from Baylis, 2000

2: When used for confirmation, the test time indicated is after enrichment and agar isolation
1247
Table 3. Some ELISA commercial systems for rapid Listeria screening

Test
ID level
Principle
Approx.
test time2
Listeria Tek
Listeria spp.
ELISA
TECRA Listeria
Visual Immunoassay
(TLVIA)
Listeria spp.
Assurance Listeria
EIA
Company
Main use
50 hours
Organon
Teknika
Screening
ELISA
50 hours
TECRA
Screening
Listeria spp.
ELISA
50 hours
BioControl
Systems
Screening
VIDAS Listeria (LIS)
Listeria spp.
ELISA
50 hours
bioMrieux
Screening
VIDAS Listeria
monocytogenes
(LMO)
L. monocytogenes
ELISA
50 hours
bioMrieux
Screening
Transia Plate
Listeria
Listeria spp.
ELISA
50 hours
Diffchamb
Screening
Transia Plate
Listeria
monocytogenes
L. monocytogenes
ELISA
50 hours
Diffchamb
Screening
EIAFOSS Listeria
Listeria spp.
Automated ELISA
48 hours
Foss Electric
Screening
Table 4. Some molecular commercial systems for rapid Listeria screening and confirmation1
Approx.
test time2
Company
Main use
Nucleic Acid
Hybridization Probe
30 minutes
Gen Probe
Confirmation
L. monocytogenes/
Listeria spp.
Nucleic acid
Hybridisation probe
3 hours
Vermicon
Confirmation
Foodproof Listeria
monocytogenes
L. monocytogenes
PCR
48 hours
Biotecon
Diagnostics
Screening
Gene Trak Listeria

Assay
Listeria spp.
Nucleic acid
Hybridisation probe
50 hours
Gene Trak
Screening
Gene Trak test for L.

monocytogenes
L. monocytogenes
Nucleic acid
Hybridisation probe
50 hours
Gene Trak
Screening
BAX for screening L.

monocytogenes
L. monocytogenes
PCR
48 hours
Qualicon
Screening
BAX for screening

Listeria Genus
Listeria spp.
PCR
48 hours
Qualicon
Screening
PROBELIA
L. monocytogenes
PCR
48 hours
Sanofi
Diagnostics
Pasteur
Screening
LightCycler
foodproof Listeria
monocytogenes
Detection Kit
L. monocytogenes
Real Time PCR
65 minutes
Roche Applied
Science
Screening
LightCycler
foodproof Listeria
Detection Kit
Listeria spp.
Real Time PCR
75 minuites
Roche Applied
Science
Screening
Test
ID level
Principle
Gen-Probe
(AccuProbe)
L. monocytogenes
VIT Listeria Kit

2: When used for confirmation, the test time indicated is after enrichment and agar isolation
3: After enrichment culture
e)
Subtyping methods
Regulatory identification of L. monocytogenes does not require any specific subtyping of the isolates.
However, subtyping schemes can be useful in outbreak investigations, environmental tracking and public
health surveillance.
1248
Listeria monocytogenes can be subtyped by a number of different approaches including serotyping, phage
typing, multilocus enzyme electrophoresis (MEE), DNA restriction enzyme analysis (either using highfrequency cutting enzymes and conventional gel electrophoresis to separate fragments, or using rare-cutting
enzymes and pulse-field gel electrophoresis [PFGE] to separate fragments), nucleic acid sequencing-based
typing, microarray analysis, amplified intergenic locus polymorphism (AILP), and random amplification of
polymorphic DNA (RAPD).
Because of the requirement for specific reagents, stringent quality assurance procedures and some
sophisticated equipment, it is recommended that subtyping of L. monocytogenes isolates be referred to the
appropriate reference centre. A list of international centres and subtyping methods can be found in Bille &
Rocourt, 1996 (pp. 258259).
i)
Serotyping
Strains of Listeria can be assigned to 13 different serotypes, based on their combination of somatic (O) and
flagellar (H) antigens. Although all of them are considered to be potentially pathogenic, most (>95%) human
clinical isolates belong to three serotypes 1/2a, 1/2b, and 4b. Compared with other subtyping methods,
serotyping has poor discriminatory power, but can provide valuable information to facilitate the ruling out of
isolates that are not part of an outbreak. Isolates from foods and from environmental sources are frequently
nontypable with standard typing antisera.
ii)
Phage typing
Bacteriophage typing is a technique with very good discriminatory power that can be used to subtype a large
number of isolates. However, the current available phage sets are unable to type a high proportion of strains
(2051% in the case of the international phage typing set). Because of stringent standardisation
requirements and the biological nature of the reagents, this technique is performed only at specialised
national and international reference laboratories and is subject to considerable experimental and biological
variability. In spite of these problems, phage typing remains the most practical and suitable method for
application in large acute outbreaks (Graves et al., 1999).
iii)
Multilocus enzyme electrophoresis
This technique takes advantage of nucleotide sequence differences, which result in distinct electrophoretic
mobilities of selected metabolic enzymes in starch gels, and can be applied to subtype-related bacterial
strains. However, MEE is only moderately discriminatory when used in epidemiological investigations
involving L. monocytogenes. Some strains may lack certain enzyme activities and therefore the technique
can get complicated. Because of its very nature, the interlaboratory results are highly variable (Graves et al.,
1999).
iv)
Chromosomal DNA restriction endonuclease analysis
Restriction endonuclease analysis (REA) of chromosomal DNA is a useful subtyping method for
L. monocytogenes. As these enzymes are highly specific in recognising nucleotide sequences, the resulting
DNA digestion fragments, of different size and electrophoretic mobility, reflect genomic differences, resulting
in specific fingerprints among otherwise related strains. Because of the restriction endonuclease specificity,
the method is highly reproducible. Of the restriction endonucleases tested on L. monocytogenes in a World
Health Organization (WHO) Multicentre study, HaeIII, HhaI and CfoI were the most useful (Graves et al.,
1999). However, because of a potentially large number of enzyme recognition sites in the bacterial genome,
sometimes complex fingerprints evolve, with overlapping or poorly resolved bands that are difficult to
interpret. The technique is therefore not adequate for comparing a large number of strain patterns or for
building dynamic databases (Graves et al., 1999).
By combining REA with Southern hybridisation, using chromosomal-labelled probes, only the particular
restriction fragments associated with the corresponding chromosomal loci are detected, thereby significantly
reducing the number of DNA fragments to be analysed. This technique is known as restriction fragment
length polymorphism (RFLP) analysis. When ribosomal RNA/DNA probes are used, only the particular
restriction fragments associated with the chromosomal loci for rRNA are detected. This technique is known
as ribotyping and it has been widely used for subtyping L. monocytogenes, mainly through the use of the
restriction endonuclease EcoRI. However, the technique was found to be less discriminating than phage
typing, REA or MEE. Qualicon has designed an automated ribotyping system, the RiboPrinter, which
generates, analyses and stores riboprint patterns of bacteria, including Listeria.
When restriction endonuclease enzymes that cut infrequently are used to digest unsheared chromosomal
DNA, such as ApaI, SmaI, NotI and AscI, very large fragments are obtained. Because of their size, these
large fragments do not separate when run under conventional agarose gel electrophoresis. However, by
1249
periodically changing the orientation of the electric field across the gel, through pulses, the large fragments
can crawl through the agarose matrix and are separated according to size differences. This technique is
known as pulsed-field gel electrophoresis (PFGE) and has revolutionised the precise separation of DNA
fragments larger than 40 kilobases. PFGE has been applied to the subtyping of L. monocytogenes and has
been found to be a highly discriminating and reproducible method. PFGE is particularly useful for subtyping
serotype 4b isolates, which are not satisfactorily subtyped by most other subtyping methods. The main
disadvantages of PFGE are the time required to complete the procedure (23 days), the large quantities of
expensive restriction enzymes required, and the need for specialised, expensive equipment (Graves et al.,
1999). The Centers for Disease Control and Prevention (CDC) in the United States of America has
established PulseNet, a network of public health and food regulatory laboratories that routinely subtype foodborne pathogenic bacteria by PFGE. PulseNet laboratories use highly standardised protocols and can
quickly compare PFGE patterns from different locations via the Internet. Listeria monocytogenes was added
to PulseNet in 1999 (Swaminathan, 2001).
v)
Nucleic acid sequence-based typing
Although there have been some reports on the sequence analysis of single genes as a means to type
L. monocytogenes strains, determination of allelic variation of multiple genes, has been recently introduced
as a very promising subtyping methodology for this microorganism. This approach has been reported for a
handful of other microorganisms and it is known as multi locus sequence typing (MLST) (Spratt, 1999).
Direct amplification and nucleotide sequencing (Cai et al., 2002; Salcedo et al., 2003), as well as an
alternative approach that targets the variable genetic changes directly in a DNA array format (Rudi et al.,
2003) have both been used with good discrimination between the strains analysed. Because MLST is based
on nucleotide sequence, it is highly discriminatory and provides unambiguous results.
Short repetitive sequence elements are widely distributed among bacteria and palindromic units, known as
repetitive extragenic palindromes (REP), are the best-characterised family of repetitive bacterial sequences.
REP elements are present in L. monocytogenes and a PCR based on the sequence of these elements (repPCR) has been used successfully to subtype strains of the organism. The four major strain clusters identified
by this method matched the origin of their isolation (Jersek et al., 1999).
vi)
Random amplification of polymorphic DNA
When arbitrarily selected primers are used in the PCR under low stringency conditions, with chromosomal
L. monocytogenes DNA as a template, amplicon patterns are generated that are useful for subtyping strains.
RAPD is a viable alternative to phage typing and is highly discriminating. However, despite its relative
simplicity and discriminating ability, its main drawback is the inconsistent reproducibility of patterns. The low
stringency conditions for primer annealing results in polymerisation with various efficiencies, and therefore
the quantities of DNA produced may be widely variable among the different amplicons from a given isolate,
which makes it difficult to compare and interpret the RAPD patterns. The technique requires a great deal of
standardisation and consistency to obtain reliable results (Graves et al., 1999).
Based on the results of the WHO multicentre L. monocytogenes subtyping study (Farber, 1996), which
compared several different subtyping methods using a well defined set of isolates, serotyping, phage typing,
REA, PFGE and RAPD were selected for standardisation in Phase II. This effort should eventually result in a
selected set of standardised L. monocytogenes subtyping methods (Graves et al., 1999).
2.
Serological tests
Serological tests for the detection of antibodies have not been traditionally used for the diagnosis of listeriosis.
They have been largely unreliable, lacking sensitivity and specificity. A number of formats, including ELISA,
complement fixation and microagglutination have been largely unsuccessful in the diagnosis of culture-proven
human listeriosis, even in the absence of immunosuppression. Considerable cross-reactivity with antigenic
determinants of other Gram-positive organisms has been observed. On the other hand, L. monocytogenes is a
ubiquitous organism, and regular exposure of animals and humans to this microorganism is very common. Many
healthy individuals are intestinal carriers (26%) and anti-L. monocytogenes serum antibody prevalence as high
as 53% have been reported in humans. Carriage rate for animals is similar to that of humans, with some
differences depending on the species and a little higher rate during indoor season, as compared to animals on
pasture (Husu, 1990; Iida et al., 1991).
The discovery that the L. monocytogenes haemolysin, listeriolysin O (LLO), is a major virulence factor and that it
can stimulate an antibody response, has recently renewed interest in the possibility of using serological tests for
the diagnosis of listeriosis, particularly in central nervous system patients, with sterile blood and cerebrospinal
fluid, and in perinatal listeriosis. An indirect ELISA based on the detection of anti-LLO was used for the diagnosis
of experimental listeriosis in sheep (Low et al., 1992). However, LLO is antigenically related to a number of
1250
cytolysins, including streptolysin O (SLO) from Streptococcus pyogenes, pneumolysin from S. pneumoniae and
perfringolysin from Clostridium perfringens. Problems of cross-reactivity of anti-LLO antibodies with these
cytolysins, particularly SLO and pneumolysin, have hampered the development of specific reliable serological
tests based on the detection of anti-LLO antibodies. In addition, anti-LLO antibodies have been found in a
proportion of healthy individuals and patients with other bacterial, fungal or viral infections (27%, all combined),
although at lower titres than in patients with listeriosis. Absorption of diagnostic antisera with SLO is only partially
effective in eliminating all cross-reactivity. These experimental assays have been used in some epidemiological
investigations and as support for the diagnosis of culture-negative central nervous system infections.
Recombinant forms of LLO have been explored as alternatives to wild LLO as a diagnostic antigen in Western
blot assays. This is currently an evolving field and we will have to wait for the development of reliable, validated
serological tests for the diagnosis of listeriosis.
Immunohistochemical detection of L. monocytogenes antigens in formalin-fixed tissue has proven to be more
sensitive than direct plating and cold enrichment bacterial culture for the diagnosis of the encephalitic form of the
disease in ruminants (Campero et al., 2002; Johnson et al., 1995).

It has proven very difficult to develop effective vaccines against L. monocytogenes which, as an intracellular
organism, requires effector T cells for an effective immune response. Experimental vaccines in laboratory animals
are being explored to confer protection to L. monocytogenes infection by a number of different approaches, but
these are still far from becoming available for human and farm animal use. These experimental approaches
include immunisation with plasmid DNA, CD40 signalling along with heat-killed L. monocytogenes, LLO-deficient
mutants inoculated along with liposome-encapsulated LLO, and immunisation with listerial antigens and IL-12.
Genetically modified L. monocytogenes is also being considered as an effective vaccine vector for the expression,
secretion and intracellular delivery of foreign antigens for the induction of potent immune responses against viral
antigens and tumour cells.
However, the most feasible and practical means to reduce the risk of listeriosis in humans is through dietary and
food preparation measures that not only decrease the risk of acquiring listeriosis, but also contribute to the
prevention of other common food-borne infections such as those caused by Escherichia coli O157:H7, Salmonella
and Campylobacter. These preventive measures include thorough cooking of raw food of animal origin, keeping
uncooked meats separate from vegetables, cooked foods, and ready-to-eat foods, thoroughly washing raw
vegetables before eating, washing hands, knives, and cutting boards after handling uncooked foods, and avoiding
unpasteurised milk or products made from it. Immunocompromised persons, pregnant women and other groups
at increased risk of listeriosis should avoid foods that have been epidemiologically linked to this disease, e.g. soft
cheeses and pt. These individuals should also avoid other ready-to-eat foods, unless they are heated until
steaming hot before being consumed.
The food industry and public health agencies play a pivotal role in the prevention of food-borne listeriosis by
developing and implementing effective HACCP programmes to reduce the presence of L. monocytogenes at all
critical points in the food production and distribution chain (from the farm to the market).
Likewise, the lack of well designed and tested vaccines for animal use, means that control of listeriosis in animals
is most feasible by preventing the environmental conditions that favour its presentation. There is a wellestablished linkage between silage feeding and listeriosis and, as L. monocytogenes is widely distributed in
nature, with animals and birds acting as carriers, contamination of silage is not uncommon. Emphasis should
therefore be placed on reducing the likelihood of the multiplication of the organism, which occurs more frequently
at pH values greater than 5, particularly where ineffective fermentation has occurred and where there is
concomitant growth of moulds. Every effort should be made to produce silage of good quality, with early cutting of
grass, minimal contamination with soil or faeces and ensuring optimal anaerobic fermentation, which will insure
that the pH falls below 5.0; at that level, growth of Listeria spp. is inhibited. The best silage for feeding should be
selected, especially in the case of sheep, discarding material that has obvious signs of contamination with mould.
Material a few centimetres from the top, front and sides of an opened bale or bag, should also be discarded.
Leftover silage should be removed (Low & Donachie, 1997).
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1
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AOAC: Association of Official Analytical Chemists
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*
* *
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CHAPTER 2.9.8.
MANGE*
SUMMARY
Mange is a contagious skin disease, characterised by crusty, pruritic dermatitis and hair/feather
loss, and caused by a variety of parasitic mites burrowing in or living on the skin. Some alternative
historical names for mange are la gale (in French), itch, scab, and scabies (a term that should
be reserved only for mange caused by Sarcoptes scabiei). Specifically, on domestic hosts (i.e.
livestock, poultry, companion and laboratory animals), about 50 mite species in 16 families and
26 genera may cause mange. A number of other skin conditions (e.g. dermatitis, wheals, blisters,
nodules) may be confused with mange and must be considered in differential diagnoses, including
those resulting from allergic reactions to other kinds of mites, various arthropod bites, fungal
diseases, or reactions to physical or chemical aspects of plants. Mange diagnosis in domestic
animals is based on clinical manifestations and the demonstration of mites or their developmental
stages in host skin scrapings.
Identification of the agent: Mange mites are mostly weakly sclerotised, slow-moving, very small
(100900 m), and live permanently on their hosts. Although the Acari is an extremely diverse and
ubiquitous group of arachnid arthropods, all of the major mange mite species fall within only two
acariform lineages, the Astigmata and the Prostigmata. Some economically important mange mite
genera are Cheyletiella, Chorioptes, Demodex, Knemidokoptes, Notoedres, Otodectes, Psorobia,
Psoroptes, and Sarcoptes. Specialised illustrated diagnostic keys, taxonomic descriptions, and
reference specimens should be consulted to properly identify the causative agents of mange.
Special collecting techniques and compound microscopy usually are necessary for diagnosis.
Certain identifying characteristics of each of the mange mite groups are highlighted in the following
discussion. Although availability is limited, serodiagnostic tests have been developed for certain
mange mites and are useful in some circumstances.
Requirements for vaccines and diagnostic biologicals: Currently, no commercial vaccines
against mange are available.
A. INTRODUCTION
Mange is a contagious skin disease, characterised by crusty, pruritic dermatitis and hair/feather loss, and caused
by a variety of parasitic mites burrowing in or living on the skin. The French term for mange is la gale (Pangui,
1994), and in English, it has been called itch, scab, or scabies (a term that should be reserved specifically for
mange caused by Sarcoptes scabiei).
Numerous species of mites cause mange in literally hundreds of species of wild and domestic birds and
mammals. In fact, approximately 60 mite families have members that live in or on the skin, hair, or feathers of
homoeothermic vertebrates and are potential mange mites. Specifically, on domestic hosts (i.e. livestock, poultry,
companion and laboratory animals), about 50 mite species in 16 families and 26 genera may cause mange.
Humans are host to the readily transmitted S. scabiei, and human scabies occurs most frequently in elderly
nursing homes and childrens day-care centres. Some other mange mites may cause transient disease in
humans, but infestations seldom persist.
Mites (Acari) are an extremely diverse, abundant, and ubiquitous group of arachnid arthropods with about 50,000
described species. Higher-level acarine classification is still an unsettled construct, but the following is a
consensus system encompassing the mange mites. Acari comprises three major evolutionary lineages,
Opilioacariformes, Parasitiformes, and Acariformes, but only certain acariform mites cause mange in domestic
animals. Two lineages within the Acariformes contain mange mites Trombidiiformes and Sarcoptiformes.
Trombidiiformes comprises the order Prostigmata and contains many families, five with mange mites. The
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Chapter 2.9.8. Mange
Sarcoptiformes contains two mite orders, the Oribatida and Astigmata, with many families each, but only
11 astigmatan families contain mange mites.
Some other mites may cause less serious dermatitis in animals or humans (Yunker, 1964). Certain Parasitiformes
(order Gamasida: e.g. Ornithonyssus, Dermanyssus) and Prostigmata (e.g. Trombicula [and other chiggers],
Pymotes) transiently bite a host while feeding, leaving itchy welts and wheals behind. Stored-products, animalnest, and house-dust mites (e.g. Acarus, Glycyphagus, Dermatophagoides) may cause contact dermatitis (e.g.
bakers itch, grocers itch) but no persistent infestation. Certain free-living bird-nest mites (Hypoderatidae) have a
parasitic nymphal stage (hypopus) that characteristically lives subcutaneously in the bird host (e.g. Hypodectes
propus in domestic pigeons), causing skin irregularities. Pediculosis or certain fungal diseases, such as ringworm,
can cause crusty dermatitis, and even the physical (e.g. awns, urticarial hairs) or chemical (e.g. urushiol) aspects
of some plants may cause host skin reactions that could be confused with mange.
Mange diagnosis in domestic animals is based on clinical manifestations and the demonstration of mites or their
developmental stages in host skin scrapings (Kettle, 1995). It is typified by hair loss, crusty or scaly skin lesions,
dermatitis, thickened skin, scurf, and pruritus.
1.
Detecting the agent
Hair loss and crusty or scaly skin are the most apparent clinical signs of mange. A number of other diseases must
be considered when one is confronted with a possible case of mange, including fungi, insect bites, irritating
plants, mechanical abrasion, etc. In most cases, scrapings should be taken from the edge of the lesion, from
obviously pruritic locations, and from where there are thick, crusty flakes. Take a skin scraping by holding a
scalpel blade or other sharp instrument at a right angle to the skin and scraping off the outer surface of the skin.
For those mite species that burrow into the skin, the scraping must be deep enough to cause a small amount of
blood to ooze from the scraping site. A drop of oil, such as glycerine, may be placed on the blade to help hold the
skin scrapings during the procedure. Skin scrapings should be placed in sealed containers (e.g. clean, empty
salve tins; stoppered glass/plastic test tubes; small, sealable plastic bags) and promptly taken or sent to a
laboratory for more thorough examination. An even more effective method of collecting mites from the skin
surface and hair is by using a vacuum cleaner fitted with an in-line filter (Klayman & Schillhorn van Veen, 1981).
The material collected, along with the filter, is then examined as a skin scraping would be. An otoscope can be
valuable in revealing the presence of ear mites. A cotton-tipped applicator can be used to swab the ear canal if
ear mites are observed or suspected; examine it in the same way as a skin scraping.
Perform an initial examination of the skin scraping under a dissecting microscope. Obviously visible mites,
especially those that are alive and moving, may be picked up with a dissecting needle dipped in glycerine or a
mounting medium and transferred to a drop of mounting medium on a glass slide. When the desired number of
mites has been collected, gently place a cover-slip on the drop of mounting medium, taking care to avoid air
bubbles. Hoyers medium, Berleses fluid, Vitzhums fluid, and Heinzes modified PVA medium are all acceptable
mounting media. If permanent mounts are desired, allow the slides to dry for at least 1 week at room temperature,
then ring the cover-slips with nail polish or other sealant to keep them from drying out.
Mites that are embedded in oil and exudate, Demodex for example, may be demonstrated by placing a small
amount of skin scraping directly on the slide with some glycerine or immersion oil and pressing a cover-slip on top
of it. The slide then can be examined directly with a compound microscope.
Skin scrapings that contain dead mites, large amounts of skin flakes or scabs, or large amounts of hair should be
processed further. Place the skin scraping (up to several grams of skin and hair) in a suitably sized beaker, then
add sufficient 10% potassium hydroxide to immerse the sample. Cautiously bring the solution to a gentle boil,
stirring frequently (a laboratory hot-plate with a magnetic stirrer works well for this), for 510 minutes or long
enough to digest most of the hair and skin. This step should be performed under a chemical fume hood to limit
exposure to caustic fumes. Do not boil for an extended period of time, or the mites may disintegrate. Transfer the
digested material to suitable test tubes, and centrifuge at 600 g for 10 minutes. Decant the supernatant.
Resuspend the pellet in a small amount of flotation medium (e.g. Sheathers solution or a mixture of 50% corn
syrup and 50% water); then, fill the tube completely with flotation medium, and place a cover-slip on top of the
tube, assuring that it makes contact with the flotation medium. Let stand for 1 hour, or centrifuge for 10 minutes.
Carefully remove the cover-slip by lifting straight up, so that a drop of fluid remains on the underside of the coverslip, and place on a glass slide. Any mites in the sample will have floated to the top and will be found in the drop
of fluid attached to the cover-slip. Another simpler but satisfactory technique, that is used in many laboratories, is
to re-suspend the pellet in a small amount of distilled water, drop onto a large (76 51 1 mm) glass slide and
cover with a 40 50 mm cover-slip. This is examined under a dissecting microscope (40 or 100) with
understage lighting. The slide then may be examined under a compound microscope for the presence of mites.
1257
DNA of Sarcoptes scabiei has been successfully amplified and detected by polymerase chain reaction (PCR)
from human cutaneous scales (Bezold et al., 2001). This technique holds promise as an additional procedure for
detecting specific, hard-to-find mange mites in skin scrapings.
In cases where mites are difficult to find in skin scrapings from small domestic pet animals, they sometimes may
be demonstrated by faecal flotation.
2.
Identifying the agent
Mange mites are mostly weakly sclerotised, slow-moving, very small (100900 m), and live permanently on their
hosts. The general life cycle of mange mites is brief (15 weeks) and includes four stages: egg, six-legged larva,
eight-legged nymph (one or more instars), and eight-legged adult (male and female.) Specialised illustrated
diagnostic keys (e.g. Baker et al., 1956; Gaud & Atyeo, 1996; Giesen, 1990; Kettle, 1995; Klompen, 1992;
Yunker, 1973), taxonomic descriptions, and reference specimens should be consulted to properly identify the
causative agents of mange. However, certain diagnostic characteristics of each of the mange mite groups are
highlighted in the following discussion.
Mange in domestic animals results from the hosts physiological, immunological and behavioural responses to
infestation by mites in any of eleven families of Astigmata or five families of Prostigmata.
a)
Astigmata
Astigmatan mange mites are generally small, globose or oval in outline, and thin-skinned. The somatic cuticle often
shows a pattern of fine, parallel striations (finger print patterns), with distinctively shaped and placed setae, spines,
pegs, or scales, and sometimes, lightly sclerotised plates or shields. Adults usually have eight legs and anterior
mouthparts that include paired palps and chelicerae used for cutting and feeding. The legs attach proximally to the
body through distinct cuticular epimeres (coxal apodemes) and terminate distally in a variety of setal forms or in a
pretarsal empodium that may be shaped like either a claw or a bell-like sucker (caruncle or ambulacrum.)
Astigmatan mites do not have true, paired pretarsal claws. Males sometimes bear somatic suckers or other
secondary sexual characteristics used in mating, but the form and placement of setae and empodia on the legs is
usually sufficient to separate the sexes as well as identify the various mange mite species. Fertilised eggs are
simple, soft, and translucent ovoids that are produced by mated females through a usually midventral ovipore.
i)
Sarcoptidae
Sarcoptid mites are all obligate, burrowing skin parasites of mammals, with over 100 described species
(Klompen, 1992). Survival time under moderate conditions for mites off the host is limited to about 10 days
or less (Arlian, 1989). Because of their activities in the epidermal layers of the skin, mange caused by these
mites is generally more severe than that caused by mites dwelling above the surface of the skin. The body
outline of sarcoptids is generally rounded, dorsoventrally flattened, and the cuticle is striated. The palps are
one-segmented, and the legs are usually short. Three genera contain domestic animal parasites of interest.
Sarcoptes scabiei
This mite causes sarcoptic mange (scabies) in humans and other mammals (Arlian, 1989). It is among the
most common, widespread, and serious types of mange extant. More than 100 known species of infested
hosts occur worldwide in at least 10 mammalian orders and 26 families (Bornstein et al., 2001). Domestic
hosts include camels, cattle, dogs, sheep, goats, horses, swine, llamas, and alpacas. Fain (1968) suggests
that humans were the original host of Sarcoptes, and all other hosts were secondarily infested. Despite
some dissention (Kutzer, 1970), scientific consensus generally has followed Fain (1968) in viewing all
Sarcoptes mites on all hosts as no more than host-adapted variants of a single, variable species.
Transmission between individuals within a host species or genus may occur easily by close contact, but
taxonomically unrelated hosts are not readily infested or infestations are self-limiting. For example,
S. scabiei var. canis easily transfers among dogs and can move to foxes, coyotes, and other canids
(Samuel, 1981), but humans serve as no more than transient hosts for this variant (Estes et al., 1983).
Recent molecular analyses support the conspecificity of all Sarcoptes variants (Zahler et al., 1999), and an
immune response has been demonstrated in Sarcoptes infected hosts (Arlian et al., 1994).
Mature female S. scabiei are approximately 500 m long, with fingerprint-like striations on the cuticle, short
and stubby legs, various characteristic setae and pegs, and with a dorsal patch of tooth-like spines. Males
are similar but smaller (about 275 m), and the tooth-like spines are reduced in size and number. The anus
is posterior in both sexes, and the first pair of epimeres is fused in a midventral Y-shape. Long-stalked,
unjointed pretarsal suckers occur on legs I and II in both sexes and on legs IV in males. The remaining legs
all terminate in long, hair-like setae. In addition, each tarsus bears at its tip one or two highly modified setae
in the form of short spurs. Nymphs resemble females but are smaller and lack an ovipore. Larvae are similar
but smaller still and have only six legs.
1258
Trixacarus caviae
This mite is a specific parasite of captive and laboratory guinea-pigs, Cavia porcellus, but it never has been
found on wild-caught animals (Klompen, 1992). Although these mites are a little smaller, the morphology and
life cycle are similar to S. scabiei. However, all dorsal setae in T. caviae are long and hair-like, unlike some
of those in Sarcoptes, which are short and broad or peg-like; males of Trixacarus also lack pretarsal suckers
on the fourth pair of legs, and the pedicels (stalks) of all suckers are a bit shorter than those typical of
Sarcoptes mites. This mite may cause a severe mange in host animals, especially in the laboratory setting.
A similar mite, T. diversus, rarely occurs on laboratory rats.
Notoedres spp.
Notoedres is a large genus comprising some 40 species, most of which are associated with bats
(Chiroptera) (Klompen, 1992). Four species are of some concern with respect to notoedric mange in
domestic animals. The cat mange mite, N. cati, is a cosmopolitan parasite of domestic cats, but it also
infests several wild cats (e.g. bobcat, cheetah, serval, snow leopard), palm civets, coatimundis, mongooses,
and domestic rabbits. These are highly contagious mites, and they cause intense mange, especially about
the hosts head and sometimes spreading to the legs, genital area, or even the tail. Laboratory rats are hosts
to N. muris, which burrows into the stratum corneum and causes thickening and cornification of the skin on
the pinnae, eyelids, nose, and tail. Additional hosts include other Rattus spp., several other rodents, two
marsupials, and a hedgehog (Klompen, 1992). The laboratory mouse may be infested by two Notoedres,
N. musculi and N. pseudomuris, but the latter primarily occurs in wild populations of this host. Each mite also
infests a few other murid rodent species. The mange caused is similar to that caused by N. muris in rats.
Notoedres mites are generally similar to Sarcoptes but about half the size, and they lack the mid-dorsal field
of tooth-like cuticular spines and peg-like setae, which may be replaced by a slight scale-like pattern in the
cuticular striations and short, stout setae. The anus is posterodorsal, the first pair of epimeres is not fused
medially, and the tarsi of legs I and II each end in three or four short, spur-like setae, not just two.
ii)
Psoroptidae
Psoroptid mites are obligate parasites of mammals. They dwell and feed on the surface of the hosts skin.
Survival time for some of these mites off the host may be two weeks or more (Otranto et al., 2004). The
generally oval-shaped body is dorsoventrally flattened, has a striate cuticle with scattered setae but no
spines, and bears longer legs and more prominent mouthparts than those of sarcoptid mites. The anus is
posteroventral. Males usually each have a pair of terminal posterior lobes bearing diagnostic setae and a
pair of ventral adanal suckers used in mating. The first pair of epimeres is not fused medially. More than
30 genera of psoroptid mites are known from at least seven mammalian orders, with the greatest number on
primates (OConnor, 1982). Three genera have veterinary importance for domestic animals.
Psoroptes ovis
For decades following Sweatman (Sweatman, 1958), conventional practice among acarologists has been to
distinguish several species of Psoroptes among the mites that cause psoroptic mange worldwide in wild and
domestic ungulates and rabbits, e.g. P. cuniculi in the ears of rabbits and various ungulates, P. equi on the
bodies of English equids, P. ovis on the bodies of sheep and other ungulates. Distinctions between the
species were based primarily on host and anatomical site infested and on morphology of the males.
Recently, several workers have invalidated these criteria and used genetic analysis to show conspecificity of
the traditionally different species (Bates, 1999; Ramey et al., 2000; Zahler et al., 1998; 2000). The earliest
published description for Psoroptes mites is that for P. ovis (Wall & Kolbe, 2006), making this the proper
designation for all such mange mites on all domestic hosts. Thus, the nomenclatural situation in Psoroptes
becomes similar to that in Sarcoptes, with one morphologically and genotypically variable species occurring
worldwide, albeit on a smaller spectrum of hosts and with a bit less stringent host specificity among the
variants. Psoroptic mange in both sheep and cattle seems to vary in its severity according to the variant of
P. ovis present, with the most severe form being a reportable condition caused by an especially virulent
genotype and known as sheep scab. This form has been eradicated from the USA, New Zealand, Canada,
and Australia, although it still persists in many other parts of the world. Thus, particularly for further
eradication efforts against psoroptic sheep mange, genotypic analysis of the involved mites may be an
especially valuable tool (Falconi et al., 2002).
Mature female Psoroptes are 550750 m long, with a striate cuticle and four long and 16 short dorsal
somatic setae (Sanders et al., 2000; Sweatman, 1958). A noticeable anterodorsal cuticular plate is present
behind the mouthparts, and the midventral ovipore is an inverted U-shape. Males are about one-fourth
smaller, and they have an additional, larger posterodorsal cuticular plate, a pair of posteroventral adanal
suckers, and two terminal posterior lobes, each equipped with four setae of varying lengths and structures.
Nymphs and larvae are somewhat similar to adults but progressively smaller, and all Psoroptes are pearly
white in colour. In all stages, the anterior two pairs of legs are thicker and more robust than the posterior
pairs, which are thinner, and in the male, shortened in the fourth pair. Legs I and II terminate in pretarsal
empodial suckers on long, segmented pedicels in both sexes, with similar structures on legs IV of the female
1259
and legs III of the males. The females third tarsus ends in two long, whip-like setae, and the male has a
single short seta on tarsus IV, plus a long, thin seta accompanying the empodial sucker on tarsus III.
Chorioptes spp.
This genus currently comprises five putative species of obligate ectoparasitic mites that may cause
chorioptic mange in domestic and wild mammals. Three of the species, collected rarely from wild animals,
are poorly known and may not be valid entities, but C. bovis and C. texanus, primarily from domestic
animals, have withstood modern biogenetic scrutiny and are accepted species (Essig et al., 1999; Zahler et
al., 2001). A number of allegedly host-specific varieties within these species are not separable from one
another (Sweatman, 1957). The two species are morphologically distinguishable only by differences in the
terminal posterior lobes and setae of males (Sweatman, 1957). Chorioptic mange, also called barn itch,
may be the most common form of mange in cattle and horses. It is a relatively mild condition that usually is
more localised and less intensely pruritic than psoroptic or sarcoptic manges. This is probably because
Chorioptes mites are able to feed and survive on host-produced epidermal debris at the skin surface, without
necessarily attacking the living parts of the hosts skin. Infestations tend to concentrate on the lower portions
of the host, especially the feet and legs, but may include the udder/scrotum, tailhead, and perineum. In some
cases, C. texanus infests the hosts ears (Sweatman, 1957). Chorioptes bovis has been known for more
than 160 years and occurs widely on cattle, goats, sheep, camelids, and possibly domestic rabbits.
Chorioptes texanus was not discovered until 1924, and for 50 years, it was recognised only from goats and
reindeer in the USA and Canada (Sweatman, 1957). Since 1975, it has been found on European elk, Alces
alces, and several times on cattle from Brazil, Germany, Israel, and the USA (Faccini & Massard, 1976;
Rosen et al., 1989; Zahler et al., 2001). Based on unpublished observations by the USDA, C. texanus may
now be the prevalent Chorioptes species on cattle in the USA.
Both Chorioptes species on domestic animals are nearly identical morphologically in all stages. The circular
body is dorsoventrally flattened, with a striate cuticle, and about 400 m long in the female; males are about
one-fourth smaller, and the somewhat similar nymphs and larvae are progressively smaller yet. Dorsally,
adults of both sexes have both anterior and posterior cuticular shields and a variety of mostly short, hair-like
setae. Ventrally, the female ovipore is a transverse slit with a pair of trailing apodemes. The mouthparts are
unremarkable, and the legs are moderately long and robust, except the fourth pair in the male are very short,
and the third and fourth pairs in the female are more slender. All legs in both sexes terminate distally in
empodial suckers with short, unjointed stalks, except for the females third pair, which end in two long, whiplike setae each. The male also has a long, whip-like seta on each third leg and a pair of adanal suckers. The
terminal posterior lobes of males bear five setae each. The lobes of C. bovis each have a nearly rectangular
margin, the seta at the external angle is long and whip-like, and the two spatulate setae are moderately
shorter (ca. 115 m) and broad. The lobes of C. texanus are each more angulate, almost bilobed, with a
very short hair-like seta at the external angle and two much longer (ca. 215 m) spatulate setae that seem
narrowed basally
Otodectes cynotis
Carnivores are the primary hosts for these highly contagious mites, which mainly infest the hosts ear canals
but sometimes spread to the pinnae and even beyond. Clinical signs of otodectic mange (otacariasis, ear
canker) may include rubbing and scratching the ears, vigourous head-shaking, depression, excessive
drainage, and haematoma of the ear. Worldwide, Otodectes is probably the most frequent mange mite
infesting carnivores, both wild and domestic. In addition to companion animals (e.g. dogs, cats, ferrets),
these mites also affect various farm-raised furbearers (e.g. foxes, mink) and occasionally may stray to
humans. As with other mange mites, past workers often have treated Otodectes mites from different
localities or different hosts as separate varieties, or even different species, but recent molecular and
phenotypic studies conclude that the genus is monobasic (Lohse et al., 2002).
Otodectes mites have a typical psoroptid morphology and life history mirroring those of P. ovis. The female
body is about 435 m long and oval-shaped; the male length is about 325 m. The female ovipore is a
transverse slit with trailing genital apodemes, and bilaterally, the epimeres of the first pair of legs are joined
to those of legs II. The terminal posterior somatic lobes of the male are only weakly produced, but adanal
suckers are present. Each lobe bears five hair-like setae of varying lengths. All of the legs are moderately
long and robust, except for the fourth pair, which is much reduced, especially in the female. Empodial
suckers with very short, simple pedicels occur distally on all legs except for the posterior two pairs in
females, which each end in a pair of long setae. The third tarsus of the male also bears a pair of long, whiplike setae in addition to its ambulacrum.
iii) Knemidokoptidae
This acarine family comprises seven genera and about two dozen species of mites that inhabit the same
microhabitats in birds that Sarcoptidae occupy in mammals (OConnor, 1982). As a result, possibly due to
convergence, the morphology of the two groups is similar. The body is generally globose, with cuticular
1260
striations that are sometimes modified into patches of scale-, furrow-, or tooth-like structures. The
mouthparts and legs are usually short and stubby. Pretarsal suckers may be present, incomplete, or absent
on all legs, and the tarsi may terminate in one or two chitinous spurs. Somatic setae are generally few,
unmodified, and quite short. Knemidokoptids have a distinctive anterior dorsal shield marked by a pair of
strongly sclerotised, longitudinal, paramedial apodemes running to the base of the mouthparts. Males (but
not females) also may have a median posterior dorsal shield, and their first pair of epimeres is fused into a
midventral Y-shape. The first epimeres in females (and immatures) may be free or joined by a transverse
apodeme into a V- or U-shape. The ovipore is a transverse slit or a three-valved, inverted Y-shape, and the
anus is terminal or posterodorsal. Males may or may not have adanal suckers. Most species occur,
sometimes worldwide, only on various wild birds in which they may cause clinical knemidokoptic mange;
however, species in three genera are of concern for domesticated and cage birds.
Knemidokoptes mutans commonly burrows in the epidermal layers of the skin on the feet and legs of
chickens, turkeys, and pheasants, causing a crusty mange known as scaly leg. If untreated, lameness,
distortion, or loss of digits may result. The first epimeres of female Knemidokoptes are free; legs I and II
each have two terminal spurs, but no ambulacrum occurs on any leg; the ovipore is transverse; the anus is
dorsal; and the body has a mid-dorsal patch of cuticular scales. Females are 350450 m, and males are
less than 240 m long. As in other knemidokoptids, legs of males are longer than those of females, and all
of them terminate in a small, long-stalked sucker. A second, similar species, K. pilae, infests the face, cere,
and legs of budgerigars, leading to a condition known as scaly face. These mites are slightly smaller than
K. mutans, and both species probably occur worldwide on their respective hosts.
Picinemidicoptes laevis infests columbid birds, including the domestic pigeon, sometimes leading to clinical
mange. In females, the first epimeres are fused in a U-shape; each leg has an empodial stalk only, and legs
I and II end in one spur each; the ovipore is transverse; the anus is terminal; and the dorsal cuticular striae
are unbroken by scales.
Neocnemidocoptes gallinae may infest the skin of the back, head, neck, abdomen, and upper legs of
chickens, geese, and pheasants, causing intense pruritus. Feathers in these areas may fall out, break, or be
plucked by the host, leading to a condition known as depluming itch. Affected skin, especially on the neck,
may become scaly, thickened, and wrinkly. Although depluming itch is less common worldwide than scaly
leg, it may be more damaging and even fatal. Female mites are 340440 m long, but males subtend about
210 m. The first epimeres of female Neocnemidocoptes are free; the tarsi each end an empodial stalk only,
and one spur terminates each of the anterior two pairs of legs; the ovipore is transverse; the anus is dorsal;
and the dorsal somatic cuticle is transversely striate but without scales. Two other, smaller
Neocnemidocoptes, N. columbicola and N. columbigallinae, infest columbiform birds in limited
circumstances and possibly might cause pathology in domestic pigeons.
iv)
Miscellaneous families
Eight remaining astigmatan fur and feather mite families contain a variety of mange mites that are generally
of minor significance due to their limited host ranges or relatively mild clinical effects on their hosts.
Three families of mammal parasites are worthy of note. Atopomelidae comprises nearly 50 genera of fur
mites with known hosts in five mammalian orders, mostly marsupials in the Southern Hemisphere. The body
plan is variable, but most are soft, slightly elongate, flattened or cylindrical, and the legs usually have some
flattened segments for grasping the hosts hairs to the mites ventral surface, which often is ridged in the
coxal areas of legs I and II. Chirodiscoides caviae probably occurs worldwide on guinea pigs, but it has been
reported commonly only in Asia and Europe, where it sometimes causes severe pruritis and alopecia to
laboratory animals. Listrophoridae is another family of fur mites comprising about 20 genera found on four
mammalian orders, mostly rodents and mostly in the Northern Hemisphere. These are somewhat soft,
elongate, cylindrical mites with various cuticular striae, spines, and punctate shields, including a sclerotised
tegmen dorsally covering the mouthparts. They cling to the host hair-shaft bases by means of a pair of
ridged flaps projecting ventrally from the area between the first pair of legs. Lepoacarus gibbus is a common
listrophorid that sometimes causes mange in domestic and laboratory rabbits, and Lynxacarus radovskyi
lives on several wild felines and the domestic cat, where mild, scurfy mange sometimes results.
Myocoptidae is a nearly cosmopolitan family containing six genera of skin-feeding, hair-clasping mites that
occur on rodents, insectivores, and marsupials. Myocoptids are generally oval-shaped and dorsoventrally
flattened. The cuticle may be extensively striate, scale-covered, or denticulate in females, whereas male
cuticles are generally less ornate and more heavily sclerotised. Host hairs are grasped by robust, highly
modified legs III and IV in females and legs III in males. Myocoptes musculinus is probably the most
ubiquitous ectoparasite of laboratory mice. Infestations are usually benign, but stressed or compromised
mice may suffer alopecia, erythema, pruritis, and traumatic dermatitis (myocoptic mange.) Another, smaller
myocoptid, Trichoecius romboutsi, occasionally occurs on laboratory mice, along with M. musculinus or
other mites, but its role in clinical mange is unclear.
1261
Five families of mites from the skin and feathers of birds deserve mention. These are classified among
36 astigmatan mite families in three superfamilies loosely known as feather mites (Gaud & Atyeo, 1996).
Thousands of species of feather mites live on or inside the feathers or skin of nearly every kind of bird
worldwide in generally commensal relationships. In rare and unexplained circumstances, the commensal
status of nearly any kind of feather mite may transition to that of a parasite, leading to negative
consequences for the host. Some entire families of nominal feather mites (e.g. Cytoditidae, Laminosioptidae)
have become true parasites with distinct associated pathologies, even mange (e.g. Knemidokoptidae). A few
species in other families are more prone than is usual to cause debilitation or injury to their hosts. In the
Analgidae, Megninia cubitalis, M. ortari, M. hologastra, and M. ginglymura occur on domestic chickens and
may cause depluming behavior and economic losses (Gaud et al., 1988). Dermoglyphus elongatus (family
Dermoglyphidae) occurs on caged canaries, and Dubininia melopsittaci (family Xolalgidae) occurs on
budgerigars, and excessive presence of each mite species may engender depluming and associated skin
lesions in the respective hosts. Members of the families Dermationidae and Epidermoptidae generally feed
on the skin or in the feather follicles of their bird hosts, placing them very close to being parasitic. Domestic
poultry are hosts to Rivoltasia bifurcata and Epidermoptes bilobatus from the two respective families, and
each mite has occasionally been associated with pityriasis (epidermoptic mange) in chickens (Baker et al.,
1956).
b)
Prostigmata
With nearly 15,000 named species classified into more than 130 families, prostigmatan mites as a group exhibit
tremendous morphological and biological diversity, making generalisations about them difficult. However, all of
the prostigmatan mange mites belong to the superfamily Cheyletoidea, which includes only about 1000 named
species in nine families. There are hundreds of undescribed species. The anterior mouthparts of cheyletoids may
be variously modified by palpal segment elaboration or reduction and by basal cheliceral fusion and extension into
elongate, needle-like stylets used to pierce the hosts tissues for feeding. Some cheyletoids have paired,
elongate, dorsal respiratory peritremes above the mouthparts. The body usually is elongate, sometimes very
much so, and usually soft and thin-skinned, but sometimes with sclerotised plates. Adults usually have eight legs
that vary in length and morphology according to the habits of the family, but they each usually terminate distally in
a pair of pretarsal claws and a linear empodium that often is equipped with numerous sticky hairs. Proximally, the
legs may articulate with simple coxal fields or sclerotised somatic apodemes. The ovipore is a longitudinal, usually
mid- or posteroventral slit, whereas, the genital pore in males is dorsal and sometimes equipped with a long
aedeagus.
i)
Demodecidae
The demodecids comprise more than 150 species of parasitic mites in seven genera from hosts in
11 mammalian orders. Demodex is the only genus of importance for domestic hosts, and it contains at least
67 named species plus many more that are unnamed and undescribed. Although other genera display their
own unique features, adult Demodex are elongate, spindle-shaped, or vermiform mites, 250850 m long,
that live in the host hair follicles, sebaceous glands, Meibomian glands, and occasionally in epidermal pits.
They have short anterior mouthparts with two-segmented palps and retractable needle-like stylets used to
puncture surrounding host tissues and feed on predigested cellular fluids. The normally four pairs of legs are
usually short, stumpy, composed of three segments each, and terminate distally in paired pretarsal claws,
usually with a linear empodium. Coxal fields occupy much of the anteroventral surface of the body where the
legs attach. The palps or one pair of legs of some stages of some species may be greatly elongated or
otherwise modified, primarily as holdfast organs. The very thin cuticle of the body and appendages is all but
devoid of setae, but the opisthosoma is usually transversely striate. Befitting the confines of their narrow
follicular or glandular habitats, the immature stages, including the eggs, of Demodex spp. are usually
spindle-shaped or elongate oval, sometimes extremely so. Demodex species are very host specific, only
rarely inhabiting more than one species of congeneric mammal host. However, it is not uncommon for a host
species to harbor two to four different species of parasitic Demodex. Transfer between hosts occurs only by
very close contact between individuals (most probably mother to neonate), making transmission between
animal species or from animals to humans very unlikely. Their very thin cuticles mean that demodecids
cannot survive away from their hosts for more than a few hours.
Although Demodex mites frequently infest the skins of 100% of the individuals of their respective host
species, their presence is usually without noticeable consequence for the hosts. On occasion, because of
stress or other poorly understood factors, resident mite populations explode in numbers that result in a
pathological condition known as demodectic mange. Healthy feral animals almost never suffer from
demodectic mange, and laboratory or domesticated hosts are the usual victims (Nutting, 1985). Clinical
signs may range from presence of small skin papules, to large nodules, to extensive hair loss. Although rare,
severe or generalised cases may lead to mites invading the host circulatory system, secondary bacterial skin
infection, and even death. Among domestic animals, clinical disease (sometimes called red mange) is most
often seen in dogs (Demodex canis and D. injai), but swine, (D. phylloides), goats (D. caprae), horses
(D. caballi), sheep (D. ovis), cats (D. cati and D. gatoi), cattle (D. bovis, D. tauri, and D. ghanensis), and
1262
rabbits (D. cuniculi) occasionally develop demodectic mange. Humans are normal hosts for two species of
Demodex (D. folliculorum and D. brevis).
ii)
Psorergatidae
Worldwide, fewer than 100 species of these small parasitic skin mites are described in three genera
(Giesen, 1990) (treated as subgenera of Psorergates by some authors). Known hosts are in eight
mammalian orders, mostly rodents and bats. Adult psorergatids are about 100200 m long, generally
circular in outline, and dorsoventrally flattened. The cuticle is very thin, finely striate, and a large, punctate,
lightly sclerotised shield covers most of the dorsum. The short anterior mouthparts have stylet-like chelicerae
and two-segmented palps, each of which ends in a stout, claw-like seta. There are no dorsal peritremes. The
four pairs of moderately long legs are radially attached ventrally, have five segments each, and terminate
distally in paired pretarsal claws but no empodium. The femur of each leg often bears a sturdy, retrorse spur
ventrally. Psorergatids have relatively few setae, including a few on the mouthparts, five or six pairs on the
dorsal shield, one small ventral pair, one or two long pairs on posteroventral body lobes, and less than 10 on
each leg. The eggs are almost round and large, nearly two-thirds the size of the mature female. They are
deposited in hair follicles or in epidermal pits made by the female. Immature stages are much like adults but
smaller, with only six legs for larvae and all legs greatly foreshortened. Transfer from host to host is
accomplished directly by motile adult mites, which then move selectively to less-keratinised areas of the host
skin, frequently about the head, neck, and the back. There, they invade the hair follicles or burrow bodysized pits into the epidermis, feed by puncturing cell walls with their stylets, and reproduce. Psorergatid
mites rarely survive off the host for more than a day.
Psorergatid infestations on healthy wild hosts and most domestic animals are generally low and of little
consequence. Sometimes, however, populations of a few species may explode, particularly on sheep and
laboratory mice, producing psorergatic mange. Skin damage from activities of adult mites usually is mild and
only slightly irritating, but their progeny, from egg nests cut into the dermis, may enlarge these pockets into
fluid- or keratin-filled papular lesions that may rupture and cause inflammation and other host immune
responses (Nutting, 1985). Psorergatid mange mites of concern occur in two genera, Psorobia (with four
pairs of marginal setae on the dorsal shield) and Psorergates (with three pairs of such marginal setae).
Infestations of Psorobia ovis, the sheep itch mite, are most troubling in older animals and cause the hosts to
rub, scratch, and bite at the wool in the most irritated areas, giving the fleece a ragged, tufted appearance.
Powdery scurf sometimes may be present, as well. The life cycle of P. ovis takes about five weeks, the
condition spreads slowly and inconsistently through a flock, and detection of infestations often is difficult. A
similar mite, P. bos, occurs widely on cattle, but it seems to have little pathological effect on hosts. Psorobia
cercopitheci, from Africa (and a similar undescribed Asian species), occasionally cause mange in colonies of
laboratory primates (Lee et al., 1981). The laboratory mouse is subject to papular lesions on the head and
neck and auricular mange caused by Psorergates simplex (Yunker, 1973). Incidence of these mites in some
mouse colonies may be as high as 80 per cent. Another Psorergates mite, P. muricola, has been found on
five different rodent species, including Mus musculus, and Psorergates rattus occurs on Rattus norvegicus;
whether either of these mites infests or damages laboratory rodents is unknown.
iii) Cheyletiellidae
This is a relatively small family of parasitic mites (about 100 species) that, until 1970, was part of
Cheyletidae, a larger family of mostly predacious prostigmatans. The species are arranged into
approximately 15 genera, about two-thirds on mammals and the rest on birds. Although a number of the
genera contain species capable of causing limited pathology in their hosts, only a few members of the genus
Cheyletiella are of concern as mange mites on domestic animals. Cheyletiella mites are 300530 m long,
elongate rhomboidal, and distinguished by a strongly striate cuticle with one (females) or two (males) large
dorsal shields. A number of moderately long, simple or barbed setae occur in distinctive patterns on the
body, mouthparts, and legs. The anterior mouthparts are large, with short piercing stylets and especially
robust, five-segmented palps, each of which terminates in a strong, curved claw-like seta that is lined with
weak, ridge-like teeth on the inner margin. Prominent M-shaped peritremes occur on the dorsal surface of
the mouthparts. The four pairs of legs are long and strong, and each terminates distally in a linear empodium
equipped with a double row of sticky hairs. Although almost all other cheyletiellids also have paired pretarsal
claws on each leg, none occurs in Cheyletiella. A small sensory organ (solenidion) occurs on the middle
segment (genu) of each leg I, and its shape is (statistically) distinctive for each species (Bronswijk & de
Kreek, 1976). Females lay their eggs singly and attach them to host hairs near the skin using a finely woven
mass of threads. Transmission between hosts is primarily by close contact, but phoresy on ectoparasitic
insects is a possibility, as well.
For many years, the identities of the various pathological Cheyletiella spp. were confused under the single
name C. parasitivorax (Smiley, 1970), and these mites were mistakenly thought of as predators on other
parasitic mites. However, C. yasguri (on dogs), C. blakei (on cats), and C. parasitivorax (on domestic
rabbits) are now separately known to be the cause of mange, and any of the three may sometimes afflict
humans in close contact with infested hosts, leading to severe dermatitis, pruritus, and other signs of
1263
cheyletiellosis for them, as well. The mites move easily among the host hairs on the keratin layer of the skin,
periodically attaching to the surface by means of the palpal claws and puncturing cells of the epidermis with
their stylets to engorge on predigested host fluids. The disease is similar in all three domestic hosts and
usually is most evident on the back, shoulders, and neck. However, clinical signs are generally mild and not
very distinctive or definitive. They may include scruffy hair coat, inflammation, occasional pruritus, alopecia,
and almost always, hyperkeratosis. The barely visible, moving mites in the fur of the host and the abundant,
powdery white scurf associated with cheyletiellic mange have engendered for it the alternative name
walking dandruff.
iv)
Myobiidae
Myobiids are small (to 900 m), soft, elongate rectangular, somewhat dorsoventrally flattened fur mites
known from five orders of mammals worldwide. More than 400 species of myobiids have been identified, at
least half of them from bats. The cuticle is generally transversely striate, without sclerotised shields, and
dorsally usually bears 12 to 16 pairs of setae, many of which are expanded, leaf-like, and longitudinally
striate. The anterior mouthparts are small, with simple two- or three-segmented palps, cheliceral stylets, and
dorsal peritremes. The legs, especially the first pair, are strong and highly modified for grasping host hairs,
one or two at a time. They terminate distally in large pretarsal claws but no empodium; sometimes one of the
two paired claws on a leg is greatly reduced or absent. The apparatus for clasping hairs are characteristic
and consist of various combinations of modified leg segments and setae in the form of spurs, hooks, bosses,
ridges, and grooved surfaces. Nymphal and larval myobiids generally resemble their respective adults
except for size. Myobiid eggs are usually attached by the females with an adhesive secretion to the bases of
the hosts hairs. Larvae may actually enter the hair follicles to feed on host fluids issuing from punctures
made with the stylets. Nymphs and adults feed at the surface of the host skin in the same way, sometimes
even puncturing capillaries and imbibing blood. The life cycles of myobiids are generally brief (ca. 14 days),
and the mites freely move between host individuals. Myobiid infestations on wild mammal hosts are usually
low in intensity and of little consequence (Nutting, 1985), but on laboratory rodents, they frequently expand
greatly and cause intense pruritus and hair loss known as myobiic mange.
Both Myobia musculi and Radfordia affinis occur on the laboratory mouse and its wild progenitor, the house
mouse, and each may cause pathology in laboratory animals. The two mites are superficially similar in
appearance, but differ in many minute details, the most readily observed of which is the number of pretarsal
claws present on the second leg; there are two in Radfordia and one in Myobia. Radfordia ensifera infests
the Norway rat and the laboratory rat, sometimes causing mange in the latter. Whereas, both pretarsal claws
on leg II in R. ensifera are of equal size, the posterior claw in R. affinis is smaller than the anterior one.
v)
Syringophilidae
Over 350 species of these very host-specific quill mites have been discovered on a wide variety of bird hosts
worldwide, but only a small proportion have been named and described. The body is elongate (about 500
950 m) and cylindrical in keeping with the infestation site within the quills of the host. The cuticle is thin,
striate, and without sclerotised plates, but a variety of usually long setae arise from its surface, particularly at
the posterior end. M-shaped peritremes arise above the mouthparts, which are equipped with stylets and
simple, linear palps. The legs are short, stubby, and terminate distally in paired claws and haired empodia.
Sclerotised epimeres occur in coxal fields I and II. While residing in the quill shafts, syringophilids puncture
the quill walls with their stylets to feed on fluids from the surrounding feather follicle tissues.
Two species of quill mites from domesticated hosts sometimes occur in large numbers and cause serious
irritation and severe feather loss that might be confused with knemidokoptic mange; Syringophilus columbae
parasitises domestic pigeons and S. bipectinatus occurs in the quills of chickens. Modern poultry production
methods that physically separate chick broods from laying hens have been very successful in breaking the
chain of passage for S. bipectinatus from one host generation to the next, all but eliminating the depluming
problem except in more traditional production settings. A second described (2001) quill mite from chickens,
Picobia polonica, has not yet been associated with host feather loss.
3.
Serological tests
Researchers have shown that Sarcoptes scabiei and Psoroptes ovis infestations cause measurable specific
antibody responses in hosts (Falconi et al., 2002; Lower et al., 2001); this makes possible serological detection of
sarcoptic and psoroptic manges. Enzyme-linked immunosorbent assays (ELISA) that detect antibodies to
Sarcoptes in pigs and dogs are commercially available in some countries and are used for serodiagnosis of
scabies (Lowenstein et al., 2004) in Sweden and Switzerland to support scabies eradication programs in swine.
Research efforts are currently in progress to develop a promising new immunodiagnostic assay for S. scabiei
using recombinant antigens (Walton & Currie , 2007). ELISA techniques for serodiagnosis and management of
Psoroptes infestations are under development and evaluation, but some issues with sensitivity and specificity
remain unresolved. Although the only unequivocal proof of mange is finding and identifying mites in skin
1264
scrapings, but in the future, this traditional (direct) method will be augmented by better and better biochemical
(indirect) methods.

There are no commercial vaccines for mange. Experimentally, inoculation with Psoroptes ovis antigen has
reduced the severity of mange. This introduces the future possibility of controlling the effects of mange without the
use of acaricides (Nesbet & Huntley, 2006; Smith et al., 2002).
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*
* *
1267
CHAPTER 2.9.9.
SALMONELLOSIS*
SUMMARY
Salmonellosis is an infectious disease of humans and animals caused by organisms of the two
species of Salmonella (Salmonella enterica, and S. bongori). Although primarily intestinal bacteria,
salmonellae are widespread in the environment and commonly found in farm effluents, human
sewage and in any material subject to faecal contamination. Salmonella organisms are aetiological
agents of diarrhoeal and systemic infections in humans, most commonly as secondary
contaminants of food originating from animals and the environment, usually as a consequence of
subclinical infection in food animals leading to contamination of meat, eggs, and milk or secondary
contamination of fruits and vegetables that have been fertilised or irrigated by faecal wastes.
Human salmonellosis is one of the most common and economically important zoonotic diseases.
Salmonella organisms may also be found in animal feedstuffs, causing subclinical gastro-intestinal
carriage or infectious disease in animals, particularly poultry and pigs. Salmonellosis has been
recognised in all countries, but appears to be most prevalent in areas of intensive animal
husbandry, especially in pigs and calves and some types of poultry reared in confinement. Reptiles
are commonly subclinical carriers of Salmonella. Several serovars are host specific (e.g.
S. Abortusovis in sheep or S. Typhi in humans) or host adapted (e.g. S. Choleraesuis in pigs and
S. Dublin in cattle).
The disease can affect all species of domestic animals; young animals and pregnant and lactating
animals are the most susceptible. Enteric disease is the commonest clinical manifestation, but a
wide range of clinical signs, which include acute septicaemia, abortion, arthritis and respiratory
disease, may be seen. Many animals, especially pigs and poultry, may also be infected but show
no clinical illness. Such animals may be important in relation to the spread of infection between
flocks and herds and as sources of food contamination and human infection.
Fowl typhoid and Pullorum disease, poultry diseases caused by Salmonella, are addressed in
chapter 2.3.11 of this Terrestrial Manual.
Identification of the agent: Diagnosis is based on the isolation of the organism either from tissues
collected aseptically at necropsy or from faeces, rectal swabs or environmental samples, food
products and feedstuffs; prior or current infection of animals by some serovars may also be
diagnosed serologically. When infection of the reproductive organs, abortion or conceptus occurs, it
is necessary to culture fetal stomach contents, placenta and vaginal swabs and, in the case of
poultry, embryonated eggs.
Salmonellae may be isolated using a variety of techniques that may include pre-enrichment to
resuscitate sublethally damaged salmonellae, enrichment media that contain inhibitory substances
to suppress competing organisms, and selective plating agars to differentiate salmonellae from
other enterobacteria.
Various biochemical, serological and molecular tests can be applied to the pure culture to provide a
definitive confirmation of an isolated strain. Salmonellae possess antigens designated somatic (O),
flagellar (H) and virulence (Vi), which may be identified by specific typing sera, and the serovar may
be determined by reference to the antigenic formulae in the KauffmanWhite scheme. Many
laboratories may need to send isolates to a reference laboratory to confirm the full serological
identity and to determine the phage type and genotype of the strain, where applicable.
Serological tests: Serological tests should be conducted on a statistically representative sample of
the population, but results are not always indicative of active infection. In poultry, the whole blood
test is used for rapid diagnosis of S. Pullorum/Gallinarum on the farm, being a relatively reliable
1268
Chapter 2.9.9. Salmonellosis
diagnostic test under certain circumstances. In the laboratory, the tube agglutination test is the
method of choice for export and diagnostic purposes for samples from all species of farm animals.
Enzyme-linked immunosorbent assays are available for some serovars and may be used for
serological diagnosis and surveillance, especially in poultry and pigs. Vaccination may compromise
the diagnostic value of serological tests.
Requirements for vaccines: Many inactivated vaccines are used against salmonellosis and some
live vaccines are available commercially. Owing to the low efficacy of inactivated vaccines, oil or
alhydrogel adjuvants are used to improve their immunogenic properties. Field efficacy data are
often lacking, although laboratory testing may provide a useful indication. Innocuity tests are
performed in laboratory animals and, in the case of inactivated vaccines, sterility tests using
bacteriological enrichment media are carried out. Further reassurances, such as environmental
impact and stability, are necessary for vaccines produced using genetic manipulation. Competitive
exclusion may be used to reduce Salmonella infections in poultry and other animal species.
A. INTRODUCTION
According to the latest nomenclature, which reflects recent advances in taxonomy (Grimont & Weill, 2007), the
genus Salmonella consists of only two major species: S. enterica and S. bongori. A third putative species,
S. subterranea, has also been proposed following the isolation of a single unusual environmental strain
(Shelobolina et al., 2004), but more recent unpublished data suggest that this organism does not actually belong
in the genus Salmonella (Grimont & Weill, 2007). Salmonella enterica is divided into six subspecies, which are
distinguishable by certain biochemical characteristics and susceptibility to lysis by bacteriophage Felix O1. These
subspecies are:
Original subgenera
Subspecies I
Subspecies II
Subspecies IIIa
Subspecies IIIb
Subspecies IV
Subspecies VI
=
=
=
=
=
=
Current nomenclature
subspecies enterica
subspecies salamae
subspecies arizonae
subspecies diarizonae
subspecies houtenae
subspecies indica
For the serovars of S. bongori, the symbol V was retained to avoid confusion with the serovar names of
S. enterica subsp. enterica. Strains of Salmonella are classified into serovars on the basis of extensive diversity of
lipopolysaccharide (LPS) antigens (O) and flagellar protein antigens (H) in accordance with the KauffmannWhite
scheme; currently over 2500 serovars are recognised (Grimont & Weill, 2007). This number is constantly being
increased. The most common serovars that cause infections in humans and food animals belong to subspecies
enterica. The serovars of the other subspecies are more likely to be found in poikilothermic (cold-blooded)
animals and in the environment, but are occasionally associated with human disease. Some serovars of
subspecies arizonae and subspecies diarizonae have been associated with disease in turkeys and sheep and
others may be carried by free-living or captive reptiles and amphibians.
Names are retained only for subspecies enterica serovars. These names must no longer be italicised. The first
letter is a capital letter. In clinical practice the subspecies name does not need to be indicated as only serovars of
subspecies enterica bear a name, e.g. Typhimurium, London or Montevideo are serovars of subspecies enterica.
The genus Salmonella followed by the serotype name may be used for routine practice (e.g. Salmonella
Typhimurium). Serovars of the other subspecies are designated by an antigenic formula, including subspecies
designated by Roman numerical (e.g. Salmonella IV 48:g.z51).
In this chapter, the abbreviated new conventions are followed, i.e. S. Typhimurium rather than the more complete
nomenclature S. enterica, subsp. enterica serovar Typhimurium. There are also regular changes to serovar
classifications as new evidence on genetic relatedness becomes available, e.g. S. Pullorum is now classified as
S. Gallinarum biovar Pullorum and S. Thomasville is designated as an antigenic variant of S. Orion (Grimont &
Weill, 2007).
Salmonellosis is an infectious disease of humans and animals caused by organisms of the two species of
Salmonella (Salmonella enterica and S. bongori). Although primarily intestinal bacteria, salmonellae are present in
the environment and may commonly be found in farm effluents, human sewage and in any material subject to
faecal contamination. Salmonellosis has been recognised in all countries, but appears to be most prevalent in
areas of intensive animal husbandry, especially of poultry or pigs.
1269
The disease can affect all species of domestic animals; young animals and pregnant animals are the most
susceptible. Enteric disease, often presenting as a bloody or profuse watery diarrhoea with pyrexia, is the
commonest clinical manifestation, but a wide range of clinical signs, which include acute septicaemia, abortion,
arthritis, necrosis of extremities and respiratory disease, may be seen. The signs and lesions are not
pathognomonic. Many animals, especially poultry and pigs, may also be infected but show no clinical illness
(Wray & Wray, eds, 2000). Such animals may be important in relation to the spread of infection between flocks
and herds and as causes of human food poisoning. In the latter case, this can occur when these animals enter
the food chain thus producing contaminated food products. Wild animals such as badgers and some types of
birds may carry specific strains of Salmonella (Wray & Wray, eds, 2000).
The course of infection, the clinical signs, the post-mortem findings and epidemiological patterns vary according
to the serovar and the animal species involved. Some serovars only affect certain hosts, e.g. S. Gallinarum in
poultry or S. Abortusovis in sheep, although most serovars may cause disease in a wide range of animal species
(Snoeyenbos, 1994). Many serovars, including some that are host adapted such as S. Choleraesuis and
S. Dublin, have been shown to cause serious disease in humans, and animal attendants, veterinarians and
abattoir workers may be infected directly during the course of their work, as may laboratory personnel.
Disease is usually referred to as salmonellosis, although the term paratyphoid may be used, e.g. swine
paratyphoid. In poultry, pullorum disease or bacillary white diarrhoea and fowl typhoid are often used to describe
infections caused by S. Pullorum and S. Gallinarum, respectively (Snoeyenbos, 1994). Fowl typhoid and Pullorum
disease are covered in detail in chapter 2.3.11 of this Terrestrial Manual.
For detailed epidemiological investigations, strain identification is necessary and such investigations have
traditionally relied on biochemical and serological methods, phage typing of some serovars, and antibiograms.
Genotypic analysis of the organism by use of real time-polymerase chain reaction (RT-PCR) and molecular
fingerprinting of DNA has been used to good effect. Plasmid profile analysis is a quick and relatively easy method
to fingerprint strains, and has been used in both human and veterinary medicine to study the spread of
Salmonella. This technique has limitations as not all strains of Salmonella harbour plasmids, and plasmids may
be readily acquired or may be of similar size but genetically different. The method has however proved to be
useful in outbreak investigations to supplement other methods. Alternative genetic techniques, such as PFGE
(pulsed-field gel electrophoresis), AFLP (amplified fragment length polymorphism), VNTR (variable number
tandem repeat), SNP (single nucleotide polymorphism) analysis and automated ribotyping, are increasingly used
and MLST (multi-locus sequence typing) can be used to investigate evolutionary pathways. Full sequencing is
increasingly used because low cost automated methods have been developed but analysis of results is complex
(Boxrud et al., 2007; Foley et al., 2007; Threlfall & Frost, 1990; Torpdahl et al., 2007). Genotyping is a rapidly
expanding field and many new methods have been developed in recent years. It should be remembered that a
single method may not work for all isolates and it may be necessary to evaluate a number of different techniques
to find a method or combination of methods that is satisfactory and capable of differentiating clones of a particular
serovar or phage type. The molecular techniques are often more discriminatory and rapid and are replacing the
phenotypic methods, such as serotyping and phage typing, for epidemiological investigations in some
laboratories. However, these molecular tools are not necessarily available in all laboratories or standardised to
give reproducible results in different laboratories, and isolates may need to be forwarded to a suitable Reference
Laboratory. There may also be problems with interpretation of minor genetic variations identified by highly
discriminatory techniques and it is usually necessary to consider such results in the context of specific
epidemiological data.
Genetic techniques such as micro-array and multiplex PCR analysis aimed at identifying specific serotypes as
well as providing additional information on virulence and antimicrobial resistance gene content have been
developed but are not yet fully validated for routine diagnostic use (Batchelor et al., 2008; Herrera-Leon et al.,
2004; Porwollik et al., 2004; Wattiau et al., 2008).
The isolation and subsequent identification of salmonellae depends not only on the quality of the sample but also
on the culture medium and growth characteristics of the serovar, particularly those adapted to a host species. A
comprehensive review of Salmonella infection in domestic animals is available (Wray & Wray, 2000). Increasing
resistance to important antimicrobials used for human therapy, e.g. cephalosporins and fluoroquinolones, related
to use in primary production in some parts of the world, as well as increasing multiple resistance, often linked with
virulence determinants, is of increasing concern (Garcia-Fernandez et al., 2009; Su et al., 2008).
National schemes have been implemented in many countries to control Salmonella infections in animals in order
to protect the consumer. In the European Union (EU), the Zoonoses Directive 2003/99/EC requires the monitoring
of breeding flocks of more than 250 birds and hatcheries for S. Enteritidis and S. Typhimurium and slaughter of
positive flocks (EU, 2003). In further legislation S. Virchow, S. Infantis and S. Hadar are also subject to special
controls (EU, 2003). Culture of day-old chicken delivery-box liners and dead or culled chickens is carried out on
the day of arrival. At 4 weeks of age and 2 weeks prior to laying, two pairs of boot swabs or pooled faeces of up to
60 samples are cultured. Subsequently, adults are sampled every 2 weeks. At the hatchery, hatcher basket liners,
broken egg shells or swabs from hatchers or hatcher baskets are taken or flocks can be monitored at the holding
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by taking five pairs of boot swabs per flock. New EU legislation controlling Salmonella in commercial laying flocks,
broilers, turkeys and pigs has been or is also being introduced. Serological monitoring is permitted as an
additional measure but can no longer replace bacteriological monitoring in poultry. In Denmark serological
monitoring for Salmonella is used for pig herds and commercial laying flocks. Several other countries also now
have serological monitoring programmes for finishing herds of pigs using meat-juice samples or serum taken at
slaughter. Baseline surveys have been carried out to determine the flock prevalence of Salmonella in laying hens,
broiler flocks, breeding and fattening turkey flocks, individual pigs at slaughter, herds of breeding pigs and
contamination of broiler carcases at slaughter (European Food Safety Authority [EFSA], 2009).
Standardised monitoring and control programmes have been defined for all categories of chicken and turkey
flocks. Monitoring of breeding flocks is as described above and commercial meat flocks are monitored by boot
swabs. In the case of cage laying flocks, naturally pooled faeces samples are collected and additional dust
samples are used for annual official monitoring of laying flocks by the competent authority. Operators are offered
the option of a voluntary confirmatory test, which may be five faecal samples and two dust samples, post-mortem
culture of carcases and ovary/oviduct of 3000 birds or culture of 4000 whole eggs in pools of 40. Eggs from laying
flocks where S. Enteritidis or S. Typhimurium is confirmed may not be sold as fresh table eggs. Details of these
complex control programmes can be found on the DG SANCO website (European Commission, 2009).
Salmonella infections of food animals play an important role in public health and particularly in food safety, as
food products of animal origin are considered to be the major source of human Salmonella infections. Special
programmes have been implemented for surveillance of poultry, swine and cattle and include the surveillance of
healthy animals that may be subclinical carriers of Salmonella organisms. Cross-contamination during food
processing is also monitored as contamination by healthy food handlers can occur (World Health Organization
[WHO], 1994).
Feed contaminated with Salmonella has been the most common original source of introduction of new strains of
Salmonella into livestock production networks, from whence it is further distributed by movement of carrier
animals and other routes. In many situations international or national trade in livestock or other animals may be
the major threat. Feed also may contain less pathogenic environmental serovars that may not be a cause of
disease or cycles of infection in animals. As feed contamination may occasionally be caused by Salmonella
serovars of relevance to public health, feedstuffs should be investigated for the presence of salmonellae (Anon,
2008). As feed is milled with ingredients of mixed global origin a wide range of exotic salmonellas, including
some serovars that are normally associated with reptiles, may be found in feed. Once established on a holding or
integration, spread between animals, environmental contamination and farm pests becomes more important in
perpetuating and disseminating the infection.
Samples of food and feed tested for Salmonella should be truly representative. Proper steps should be taken to
prevent contamination during transport or storage (International Organization for Standardization (ISO), 1996;
1999). Because of the large variety of food and feed products there is no single sampling method appropriate to
all products. Therefore different methods specific to the product should be used and it is usually more effective to
carry out process monitoring than mass testing of individual feed ingredients and finished feed (Anon, 2008).
The WHO provides information on the development of appropriate measures for the prevention and control of
food-borne diseases, including Salmonella infections, of humans. The most common vehicles of infection are
eggs and egg products, poultry meat and meat from other food animals, and meat products. Contaminated salad
crops, sprouted seeds, milk products and spices have also been involved in numerous outbreaks. Salmonella
Enteritidis and S. Typhimurium are the most widespread serovars in many European countries (although
Salmonella is rare in livestock in some EU countries because of their very strict control programmes). Salmonella
Typhimurium is the dominant serovar in North America (Saeed et al., 1999; Wray & Wray, eds, 2000). Recently a
monophasic variant of S. Typhimurium (S.4,5,12:i:-DT193 with resistance to ampicillin, streptomycin,
sulphonamides and tetracycline) has emerged in pigs and caused outbreaks of salmonellosis in humans in
several countries worldwide, and there are several other emergent monophasic S.4,5,12:i:- and S.4,12:i:- strains
with varying resistance patterns that have been recognised in various animal species and humans in many
countries in recent years (Soyer et al., 2009).
A Salmonella control policy for public health purposes should cover all stages from the stable to the table. It
should include the mandatory reporting of all outbreaks of the disease, and animal, food and feed testing (WHO,
1994). Feed monitoring includes sampling of compound feed and other feed materials which are fed
unprocessed, and ingredients, as well as sampling during feed processing. World-wide epidemiological
investigations should be done to monitor Salmonella transmission and support Salmonella control policies.
Health and hygiene controls at slaughter are essential, and special precautions should also be applied when
slaughtering potentially infected herds. In the EU, new regulations will require withdrawal from the market of
batches of meat where absence of Salmonella in 25 g of material cannot be demonstrated. Measures should be
implemented during processing to limit contamination, but chemical decontamination is not currently permitted in
the EU (European Commission, 2009). Vaccines are increasingly used to reduce Salmonella in poultry and it is
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essential that these can be distinguished from field infections for monitoring purposes and to ensure that the
vaccines do not persist long-term or spread beyond the vaccinated group of animals (Wallis, 2004).
Another essential element in the prophylaxis of human salmonellosis is retailer and consumer education, in
particular awareness of safe handling and storage of food, kitchen hygiene and proper cooking to limit the risk of
infection.
1.
The frequency of sampling and the type of samples obtained will depend largely on the objectives of the testing
programme (including any statutory requirements), clinical findings, level of detection or precision of prevalence
estimates required, cost and availability of sampling resources and laboratory facilities. A new ISO standard is
being prepared to provide guidance on sampling methods for primary production and preparation of samples in
the laboratory.
Individual samples for bacteriological tests are collected as aseptically as possible and in the case of clinical
disease or routine monitoring, samples should be collected before any antibiotic treatment has commenced.
Preferably clinical samples are collected during the acute phase of the disease or as soon as possible after death.
In the case of flocks of poultry or other avian species, environmental samples, such as naturally pooled faeces,
litter and dust or drag or boot swabs from floor surfaces (Carrique-Mas & Davies, 2008), may be the most costeffective way to identify infected flocks. Precautions should be taken to avoid cross-contamination of samples
during collection, transit and at the laboratory. Packages should be kept cool and accompanied by adequate
information. For smaller animal species, it may be preferable to submit a representative number of sick or recently
dead animals to the laboratory, if that is possible (WHO, 1994). Host-adapted serovars are usually more difficult
to isolate from faeces so if these are suspected, infected tissues should be cultured where possible.
Particular attention should be given to the isolation of salmonellae from animals with subclinical infection, as these
may only excrete bacteria intermittently and in low numbers. An increased sample size, increased number of
samples representing more individuals, combined in some cases with pooling of samples and repeat sampling
can provide an increased diagnostic sensitivity. In such situations bacteriological or serological methods may also
be used to identify infected flocks or herds, rather than to identify infected individual animals.
Culture
There are numerous methods for isolation of Salmonella in use world-wide (Ellis et al., 1976; Fricker, 1987;
Gerhardt, 1981; Harvey & Price, 1974; Reissbrodt, 1995). Some of the more common methods are described
below. The culture techniques and media that may work best in a particular diagnostic situation depend on a
variety of factors, including the Salmonella serovar, source and type of specimens, animal species of origin,
experience of the microbiologist, and availability of selective enrichment and selective plating media.
All culture media should be subjected to quality control and must support growth of the suspect organism from a
small inoculum in the presence of a relevant sample matrix. The routine use of a reference strain in parallel with
routine samples may lead to cross contamination of samples if careless techniques are used, so a rare serovar
with typical growth characteristics that are similar to the highest priority target strains should be used. It is also
possible to use strains with antimicrobial resistance or green fluorescent protein markers.
The increasing application of external quality assurance programmes has led to greater use of international
standard methods, such as ISO 6579:2002; even though this has not been validated for faecal and environmental
samples and was intended for foodstuffs and feedingstuffs. In recent years a standard method for detection of
Salmonella from primary animal production has been developed and evaluated, and an ISO-method (ISO
6579:2002 Annex D) has now been adopted (ISO, 2002). The core of the standard method is pre-enrichment in
buffered peptone water, enrichment on modified semi-solid RappaportVassiliadis (MSRV) and isolation on
xylose-lysine-deoxycholate (XLD) and an additional plate medium of choice. This method has also been shown to
be highly effective for animal feed and meat products, and is simpler and less expensive than the full ISO method.
a)
Pre-enrichment media
The number of salmonellae in faeces from asymptomatic animals, environmental samples, animal feed and
food is usually low, and it is usually necessary to use pre-enrichment media, such as buffered peptone water
or universal pre-enrichment broth, to assist isolation. This may allow the small numbers of salmonellae,
which may otherwise be killed by the toxic effect of selective enrichment media, to multiply, or it may help to
resuscitate salmonellae that have been sub-lethally damaged, e.g. by freezing, heating, exposure to
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biocides, organic acids, bacteriocins, phage or desiccation. Pre-enrichment may not be the best method for
isolating less vigorous Salmonella strains, such as the host-adapted strains, from faeces because of
overgrowth by competing organisms during non-selective pre-enrichment.
b)
Enrichment media
Enrichment media are liquid or semi-solid agar media that contain additives that selectively permit
salmonellae to grow while inhibiting the growth of other bacteria. Some, however, are also relatively toxic to
certain serovars of Salmonella, e.g. selenite inhibits S. Choleraesuis, and brilliant green is toxic to many
strains of S. Dublin. Elevated temperatures have also been used to increase the selectivity of enrichment
medium, and a temperature of 43C is used in some laboratories, although this may be inhibitory with some
media, e.g. tetrathionate and RappaportVassiliadis at 43C inhibit temperature-sensitive strains, especially
S. Dublin and 41.5C is now recommended for incubation of RappaportVassiliadis broth-based media.
Selective motility enrichment may also be used to increase the sensitivity of Salmonella isolation and semisolid enrichment media, e.g. MSRV or diagnostic semi-solid Salmonella medium (DIASALM), may provide
greater sensitivity (Voogt et al., 2001). The formulation of the medium, which may vary between suppliers, or
even between batches in some cases, temperature and duration of incubation, and the volume of the
samples used to inoculate the medium, may all serve to influence the isolation rate, and these variables
should always be taken into account. Examples of selective enrichment media are sodium tetrathionate, as
in MllerKaufman broth, selenite F, selenite cysteine, brilliant green broth and RappaportVassiliadis
broths, or semi-solid RappaportVassiliadis medium. In some cases it may be advantageous to use more
than one selective broth or to culture by both pre-enrichment and direct selective enrichment/direct plating,
although often the benefit does not justify the extra cost. Additions such as Ferrioxamine E may be added to
selective media to enhance isolation of Salmonella from iron or nutrient-limited samples such as eggs, water
or soil (Reissbrodt, 1995) or antibiotics such as novobiocin may be added to suppress most Gram-positive
organisms or other Gram-negative bacteria such as Proteus. Specific antibiotics can be added to enhance
the isolation of antimicrobial resistant Salmonella strains.
c)
Selective plating media

These are solid, selective agars that permit differential growth to varying degrees. They inhibit growth of
bacteria other than Salmonella and give information on some of the principal differential biochemical
characteristics usually non-lactose fermentation and hydrogen sulphide (H2S) production. The results are
read after 24 and 48 hours of culture at 37C. Salmonellae form characteristic colonies on such media that
are usually distinguishable from the colonies of other bacteria on the plate, with the possible exceptions of
Proteus, Pseudomonas, Citrobacter and Hafnia. Lactose-fermenting salmonellae may occasionally be
isolated and H2S production may be variable. Such atypical strains may be more effectively detected when
semi-solid selective media are used. DIASALM medium is particularly useful in this respect as presumptive
confirmation by slide agglutination testing using polyvalent O, H or specific antisera can be carried out on
liquid from the growth zone in the plate and plates such as desoxycholate-citrate agar (DCA) or bismuthsulphite agar (BS) can be used, but these are subject to a higher frequency of false-positive colonies.
Salmonella Abortusovis is a slow-growing serovar and it is usual to incubate plates for up to 72 hours and to
use the non-selective blood agar. Examples of selective media are brilliant green agar, xylose lysine
desoxycholate agar, deoxycholate/citrate agar, and bismuth sulphite agar, although many more will be found
in the literature and media catalogues. A wide range of chromogenic agars such as Rambach agar, SMID,
ASAP, are now available. Many of these may aid differentiation of suspect colonies, but must be validated
for the sample matrices, culture systems and serovar range used as sensitivity can be poor in some
circumstances.
Quantification methods
Salmonella from infected tissues can be enumerated by direct plating, but most probable number (MPN)
techniques are necessary for faecal, feed or environmental samples. Recently a miniaturised MPN method has
been adopted as the preferred method in the EU (Fravalo et al., 2003).
Identification of suspect colonies
Suspect colonies are subcultured onto selective and non-selective agars to ensure that possible contaminants,
such as Proteus spp., are absent. If there is an abundant pure growth, suspect colonies may be tested by slide
agglutination with polyvalent Salmonella-typing sera (Ellis et al., 1976). In some cases, the suspect colony may
not agglutinate or auto-agglutinate and it is necessary to use biochemical tests to confirm the identity. These tests
can be performed with peptone water sugars or commercial systems (such as the Analytical Profile Index [API]
system), OBIS test, or composite media (such as triple sugar iron agar [TSI]), can be used to screen organisms
(Ewing, 1986). It is particularly important to ensure that Salmonella cultures used for determination of
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antimicrobial resistance are not mixed with other organisms such as Pseudomonas that are more likely to be
multi-resistant.
The determination of the O factor(s) and the H antigen(s), and in special circumstances the Vi antigen (present in
S. Typhi, S. Paratyphi C and S. Dublin), is performed by direct slide agglutination or tube agglutination using
specific antisera. In the case of biphasic organisms, it is necessary to determine both phases, by the use of phase
inversion this involves passage through semi-solid agar containing antiserum to the known phase. Screening is
facilitated by the availability of antisera directed against several factors, which can be pursued further by the use
of monovalent typing sera. While many laboratories can identify the more common serovars, it is usually
necessary to use the facilities of a reference laboratory to confirm the identity of an isolate and possibly to obtain
information on the phage type, if there is a scheme available, and genetic profile. Colonial form variation may
occur in certain serogroup C2 serovars in which there is variable expression of minor antigens in different single
colony picks from the same strain. This may result in apparent recovery of two different serovars (e.g. S. Hadar
and S. Istanbul or S. Newport and S. Bardo) from the same isolate (Popoff, 2001).
Additional biochemical tests may be necessary to identify some serovar variants, e.g. d-tartrate fermentation,
which can be used to differentiate S. Paratyphi B var. Java from S. Paratyphi B. Isolates should also be tested for
their sensitivity to a range of antimicrobial agents as there is increasing concern about the emergence of new
multiple resistant strains harbouring transferable resistance genes to cephalosporins and fluoroquinolones
(Greene et al., 2008; Weill et al., 2004). Live vaccine strains are also commonly identified by antimicrobial
resistance markers, biochemical changes such as auxotrophism or roughness.
Immunological and nucleic acid recognition methods
Numerous alternative Salmonella detection methods are in use and are commercially available (AOAC
[Association of Official Analytical Chemists] International, 2009; Blackburn, 1993; De Boer & Beumer, 1999;
Ewing, 1986; Ricke et al., 1998; Rijpens & Herman, 2002; Swaminathan & Feng, 1994). These include electrical
conductance/impedance, immunomagnetic separation (IMS), enzyme-linked immunosorbent assay (ELISA), gene
probe PCR methods, real time PCR (Malorny et al., 2004), quantitative PCR (Piknova et al., 2005) or microarray
analysis (Rasooly & Herold, 2008). Many of these methods have not been fully validated for faecal and
environmental samples, although progress has been made (Jensen & Hoorfar, 2003; Malorny & Hoorfar, 2005),
and are more suited to analysis of human foodstuffs where inhibitors of the PCR reactions are not so problematic
(Kanki et al., 2009) although there is a role for rapid methods in test and release of batches of Salmonella-free
animal feedstuffs. The rapid methods are usually more expensive than conventional culture, but can be
economically viable for screening materials where a low prevalence of contamination is expected or where
materials, such as feedstuffs, are held pending a negative test. An enrichment/IMS method linked with ELISA or
PCR can identify most contamination within 24 hours but faecal and environmental samples can be problematic
for rapid methods. Currently none of the rapid methods has been shown to be suitable for direct detection of
Salmonella so non-selective or selective enrichment stages are required (Oliveira et al., 2003). Typically this
introduces more steps and operator time in the detection procedure. For DNA-based methods, inhibition of the
PCR reaction by elements of the test sample matrix, especially in the case of faeces, is problematic and requires
suitable DNA extraction techniques and controls to detect inhibition, which may reduce the sensitivity of the test in
some cases (Jensen & Hoorfar, 2003). Rapid isolation methodologies may also be linked with sophisticated
detection systems, such as biosensors (Olsen et al., 2003). There are many variations and developments in rapid
methods for Salmonella detection, but none has been shown to satisfactorily replace culture in all circumstances.
It is therefore not possible to provide details of all the methods in this chapter or to make recommendations, but
the review articles cited above will provide further information.
Example test procedures for isolation of Salmonella from food, feedstuffs, faecal and
environmental samples
i)
Add a 1025 g sample to 10 volume of buffered peptone water at ambient temperature. (NB: for many
host-adapted serovars and some arizonae serovars, it is preferable to add the sample to selective
enrichment medium, such as selenite cysteine broth, and to test tissue samples where possible
[including direct plating]; see culture method for S. Pullorum/Gallinarum in Chapter 2.3.11 Fowl typhoid
and Pullorum disease.)
ii)
Incubate buffered peptone water for 1620 hours at 37C.
iii)
Inoculate 20 ml modified semi-solid RappaportVassiliadis or DIASALM in a Petri dish with 0.1 ml

incubated buffered peptone water.
iv)
Inoculate 10 ml tetrathionate broth with 1 ml incubated buffered peptone water broth.
v)
Incubate modified semi-solid RappaportVassiliadis or DIASALM at 41.5C and tetrathionate broths at

37C (ensure that a reputable brand of tetrathionate suitable for use at 37C is used).
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vi)
After 24 and 48 hours of selective enrichment, plate out modified semi-solid RappaportVassiliadis or
DIASALM by taking 1 l loop of material from the edge of the turbid growth zone and streaking over
one plate of Rambach agar or brilliant green agar plus novobiocin and one plate of xylose lysine
desoxycholate agar .
vii)
Plate out 10 l of tetrathionate broth on Rambach agar or brilliant green agar plus novobiocin and
xylose lysine desoxycholate agar.
viii) Incubate plates at 37C for 24 hours.

ix)
Check up to five suspect colonies (red/pink with reddening of the media on brilliant green agar, crimson
with pale borders or orange/colourless on Rambach agar, red with black centre (or occasionally
translucent red in the case of H2S negative strains) on xylose lysine desoxycholate agar) using poly O
and poly H (phase 1 and phase 2) antiserum or composite biochemical media.
x)
Subculture strongly suspect colonies that do not agglutinate with poly H antisera on to non-selective
media then repeat testing. If a strong poly O and poly H agglutination can be obtained, this is
sufficient for presumptive confirmation. Such isolates can then be serogrouped. If agglutation results
are unclear then carry out further biochemical testing using composite media, such as TSI or use
ONPG (o-nitrophenyl-beta-d-galactopyranoside) and urea or commercial biochemical tests such as API
ID 32 E.
This method differs from ISO 6579:2002 and its Annex D method, but provides the possibility of additional
detection by combining elements of broth and semi-solid agar enrichment.
2.
Serological tests
Serological identification of infected animals, flocks and herds
A number of serological tests have been developed for the diagnosis of Salmonella infections in animals. In
poultry, the whole blood test, which uses a stained antigen, and the serum agglutination test (SAT) have been
used successfully for over 50 years for the identification of flocks infected with S. Pullorum/Gallinarum. Because
S. Enteritidis possesses the same group D somatic antigen as S. Pullorum/Gallinarum and is thought to originate
from it (Thomson et al., 2008), the whole blood test and related tests can be used for the diagnosis of
S. Enteritidis infection, but the sensitivity is low. In recent years, other tests, such as the ELISA (Barrow, 1994)
have been developed for the diagnosis of S. Enteritidis and S. Typhimurium infections in poultry and for other
serovars in farm animals. The ELISA has been used effectively to identify serologically S. Dublin carrier cattle and
can be applied to bulk milk for screening dairy herds. The mix-ELISA is used in Denmark, Germany, the
Netherlands, the United Kingdom, and some other countries on serum or tissue fluid released by freezing then
thawing muscle samples to detect Salmonella infections in pigs (Nielsen et al., 1998). A similar test can be used
to detect antibodies to S. Enteritidis and S. Typhimurium in egg yolk from unvaccinated commercial laying flocks.
Some ELISAs are now in routine use and a number are available commercially. There is a need for
standardisation of their use and to this end panels of control sera are available commercially from Denmark1 and
the Netherlands2. The purpose of this section is to consider the serological tests that have been fully evaluated
and are in routine use for the diagnosis of salmonellosis in animals. Other tests that are still in the development
stage will therefore not be considered. New tests for Salmonella diagnosis are developed frequently so an
Internet search is often the best means of obtaining current information.
Factors affecting serological diagnosis
1.
Serological methods should be used to identify infected flocks/herds rather than to identify infected individual
animals, although repeated herd tests can be used as an aid to selective culling of chronic carrier animals.
Serological tests are normally designed to detect a limited range of Salmonella serovars or serogroups.
2.
It is well recognised that some animals with a positive serological response may no longer be infected with
Salmonella organisms and in low prevalence countries specificity issues mean that most positive results will
be false. Animals that are actively excreting salmonellae may be serologically negative in the early stages of
disease and some individual infected animals never seroconvert. Similar considerations may also apply to
bacteriological culture methods, and negative faecal culture results may not necessarily indicate that an
animal is not infected. However, neither of these situations should be considered as a major problem if
enough tests are carried out. Animals that are serologically positive may have ceased to excrete
salmonellae although circulating immunoglobulin concentrations may remain high. Other animals on the
farm may still be infected. Serologically negative animals may result from a recent infection causing
1
2
Statens Serum Institut, Copenhagen, Denmark (www.ssi.dk)

GD, Deventer, the Netherlands (www.gddeventer.com)
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excretion before immunoglobulin production is maximal, or infection with less invasive serovars. Animals that
have been infected recently would, in all probability, eventually be detected serologically by an appropriate
monitoring programme throughout the life of the flock/herd but there are often cost limitations to the
application of effective monitoring programmes.
3.
Newborn animals are immunologically immature and do not respond serologically to the somatic LPS
antigen until 23 weeks of age. They do, however, produce a serological response to the flagellar protein
antigens. Cattle may be unresponsive until about 1012 weeks of age, and sucking pigs may fail to develop
an immune response or have an antibody response that reflects maternal immunity. Differential responses
involving different antibody classes (IgM, IgA, IgG) can be used in pigs to differentiate recent infection from
infection that occurred some time ago, but this is often not useful for herd testing where individuals are
usually at different stages of infection. Most tests are based on IgG and raised antibody levels typically
appear 13 weeks after infection and last 23 months.
Chickens may also acquire anti-Salmonella antibodies passively from their parents via the yolk sac; this may
indicate an infected parent flock. Mammals can acquire maternally derived antibodies via the colostrum.
4.
Immunisation has been used for many years to control certain Salmonella infections in farm animals, and if
diagnostic serology is to be used, it is necessary to differentiate the vaccine response from that of actual
infection. Many live vaccines given orally do not provoke a significant serum antibody response in the
majority of animals but there may be occasional exceptions that are difficult to interpret. Injectable killed
vaccines used for control of S. Enteritidis in chickens may produce a very prolonged antibody response. It
would be advantageous if a marked live vaccine could be produced which could be differentiated from field
challenge by serological testing.
5.
The effect of antibiotic therapy on the serological response remains unclear. Some workers found reduced
titres following therapy whereas others found no effect. Serology, however, may be a more useful diagnostic
technique for salmonellosis than culture if antimicrobial therapy has been used.
6.
Over 2500 different Salmonella serovars exist. Depending on the antigen and test used, serological crossreactions between different serovars may occur, e.g. S. Typhimurium, S. Pullorum and S. Enteritidis. In
some cases cross-reactions may also occur as a result of exposure to organisms other than Salmonella.
7.
In poultry, egg yolk may be tested for immunoglobulins to Salmonella, and eggs may provide a method to
screen flocks. This approach is used for monitoring commercial laying flocks in Denmark. In cattle, milk may
be tested for anti-Salmonella antibodies to screen dairy herds.
8.
The use of filter-paper discs for serum collection obviates the necessity to separate serum. The discs also
provide long-term storage and reduce transport costs to the laboratory. The sensitivity of the test may be
slightly reduced compared with tests carried out on fresh serum.
a)
The whole blood test

The whole blood test provides a rapid test for fowl typhoid and pullorum disease that can be used on the
farm. The sensitivity of the whole blood test is low and in inexperienced hands false-positive and falsenegative results may be recorded.
For a detailed description of the whole blood test, see chapter 2.3.11.
b)
Rapid slide agglutination test

Serum (0.02 ml) is mixed with polyvalent crystal-violet-stained antigen (0.02 ml). The tile is rocked gently for
2 minutes, after which the test is read. The test components are stored at 4C and must have reached room
temperature before being used.
Test sera should be free from contamination and haemolysis. It may be helpful to centrifuge serum samples
that have been stored for any period of time.
If nonspecific false-positive reactions are suspected, positive/suspicious sera may be retested after heatinactivation at 56C for 30 minutes.
c)
Serum agglutination test

The SAT is relatively insensitive, and many older animals have low levels of agglutinins in their sera caused
by enterobacteria other than Salmonella. Single samples are of little diagnostic value except for initial
screening on a herd basis. Paired samples are needed as the minimum requirement for confirmation of
active infection. The test is relatively inexpensive; the antigens can be readily prepared and expensive
1276
equipment is not necessary. The SAT can be adapted to the microtitre format and can be readily used to
determine somatic and flagellar titres. It is advisable to use standard sera and other confirmatory methods
for quality control of the purity and immungenicity of SAT antigen preparation(s) that are not dependant on
sera produced from those antigens. This method has been used for identification of exposure to various
Salmonella serovars, e.g. S. Typhimurium, S. Enteritidis, S. Dublin, S. diarizonae in turkeys, S. Abortusequi.
Preparation of somatic antigen
i)
Plate out the Salmonella culture from the appropriate stock culture onto a blood agar base (BAB) plate,
or other suitable medium, for single colony growth. Incubate overnight at 37C (2C).
ii)
Select a smooth colony and carry out a slide agglutination test to ensure that the required somatic
antigen is present.
iii)
Using a sterile loop, inoculate a nutrient agar slope in a universal container from the selected colony.
iv)
Incubate the culture for 812 hours at 37C (2C).
v)
Using a Pasteur pipette, wash off the culture, preferably inside a safety cabinet, with approximately
2 ml of absolute alcohol, and transfer into a sterile universal container.
vi)
Leave the antigen for 46 hours at room temperature to enable the alcohol to kill the bacteria and
detach flagellae.
vii)
Spin the universal container in a bench-top centrifuge for 5 minutes at 1000 g. Pour off the liquid and
add enough phenol saline to make the antigen up to an opacity equivalent to Browns tube No. 2
(approximately 108 colony-forming units/ml) or other appropriate standard.
viii) Carry out standard titration with known serum to ensure that the antigen is positive for the required
factor.
d)
ix)
Store in a refrigerator at 4C until required.
Preparation of flagellar antigens
i)
Plate out the appropriate Salmonella stock culture on to a BAB plate, or other appropriate medium.
Incubate overnight at 37C (2C).
ii)
Passage in semi-solid agar (about 0.3%) in a Craigies tube, or other suitable container, to induce
optimum expression of the appropriate flagellar antigen. If the serovar is biphasic, H antiserum
corresponding to the phase to be suppressed is added to the agar.
iii)
Use slide agglutination to check that the Salmonella is in the required phase. If this is correct, inoculate
a loop of culture into 20 ml of nutrient broth. Incubate for 1218 hours at 37C (2C) for optimum
growth. (If the phase is incorrect, repassage through semi-solid agar.)
iv)
Pipette 250 l of 40% formaldehyde into the antigen suspension (use gloves and preferably work in a
safety cabinet), and leave overnight.
v)
Test the antigen by SAT using the appropriate typing serum.
Test procedure
i)
It is easiest to screen the sera at a dilution of 1/20; 0.25 ml of antigen is added to 0.25 ml of serum
prediluted to 1/10 in normal saline.
ii)
The tests are incubated in a water bath at 50C for 24 hours in the case of somatic antigens and for
4 hours for the flagellar antigens. The dilution and time of incubation will vary depending on the
antisera that are used.
iii)
Sera that give a positive reaction are then diluted from 1/20 to 1/320 and retested with the appropriate
antigen.
Enzyme-linked immunosorbent assays for Salmonella Enteritidis

Two main basic systems are available for detection of IgG (IgY) specific for S. Enteritidis: the indirect ELISA
and the competitive sandwich type ELISA (Barrow, 1994).
The indirect ELISA involves the use of a detecting antigen coated on to the wells of a microtitre plate. After
the application of a blocking reagent to reduce nonspecific binding, test samples are applied to the wells.
Specifically bound antibody in the sample is detected by an antibody/enzyme conjugate. A variety of
antigens, including LPS, flagella, SEF14 fimbriae, outer membrane proteins and crude whole cell antigen
preparations have been used.
1277
The competitive sandwich ELISA employs a specific reagent a monoclonal antibody (MAb) or polyclonal
antibody for coating antigen to wells. This is then followed by a pure or crude antigen preparation. Test
samples are applied followed by conjugated antibody, which will not bind to the antigen if the sample
contained specific antibodies. The assay time can be shortened by adding both test sample and conjugate
together. MAbs have been prepared for LPS, flagella and SEF14 for S. Enteritidis.
There are advantages and disadvantages to both systems. The indirect assay is simpler and reagents are
available for all Salmonella serovars of chickens, turkeys, ducks and mammalian hosts. The competitive
ELISA can be applied to all animal species and in general shows higher specificity. However, reagents are
not available commercially for all serovars. There are also some affinity problems and it may be less
sensitive than the indirect assays. In the field, both systems have produced false-positive reactions and in
some cases screening with an indirect LPS ELISA may be followed by confirmation with a flagellar
competitive ELISA. This combination has been used to differentiate S. Enteritidis field infection from a
vaccinal response to S. Gallinarum 9R vaccine, which lacks flagellar antigens.
Both types of assay may be used with serum, egg yolk or reconstituted dried blood eluted from filter paper
discs. A mix-ELISA (or meat-juice ELISA), is used in Denmark and other countries to detect Salmonella
infections in pigs (Nielsen et al., 1998). This ELISA contains the O LPS antigens 1, 4, 5, 6, 7 and 12, from
S. Typhimurium and S. Choleraesuis, which enables it to detect serologically up to 95% of the Salmonella
serogroups found in pigs in most European countries. Group D antigens have also been added to some
ELISA kits. Serum is used to screen breeding and multiplying herds, whereas for pigs in the abattoir, the
assay is usually performed on the tissue fluid (meat-juice) that is liberated when a frozen 10 g muscle
sample is thawed.
With some ELISAs differentiation can be made between infections produced by Salmonella serovars from
different serogroups. Some cross-reaction occurs between groups B and D and other invasive serovars.
There is, however, usually a greater antibody response when LPS from the homologous serovar is used in
the ELISA. The optimal method for choosing a cut-off absorbance value, above which sera are designated
as having come from an S. Enteritidis-infected flock, without producing an unacceptable level of falsepositive tests, has not yet been decided on and agreed upon internationally.
ELISAs are readily adapted to automation and hence to large-scale testing programmes. A major problem is
that expensive equipment is necessary and many of the reagents are also expensive. Several commercial
ELISA kits for S. Enteritidis, S. Typhimurium and Group B/C mix-ELISAs are available. Ideally these should
be validated by international ring trials before adoption for surveillance purposes.
An example of a validated ELISA is the one developed at the OIE Reference Laboratory at VLA Weybridge
(see Table in Part 4 of this Terrestrial Manual for address). The requirements are given below.
Equipment
Falcon microtest III PVC plates; appropriate pipettes and measuring cylinders; ultrawash microtest plate
washer; ELISA plate reader; test filter of 405410 nm and reference filter of 630 nm.
Antigen
i)
Phenol-extracted S. Enteritidis LPS is available commercially (Sigma Cat. No. L6011). This is
reconstituted in 1 ml deionised water and stored at 20C in 100-l aliquots in phosphate buffered
saline (PBS), pH 7.2, at a concentration of 2.5 mg/ml. For use, the antigen should be thawed in coating
buffer at the appropriate concentration.
ii)
The LPS antigen can also be prepared by the technique of Westphal & Luderitz (1954) and
standardised as to its carbohydrate concentration by the method of Gerhardt (1981), and adjusted to
1000 g/ml.
Serum and conjugate diluent
Add bovine serum albumin (BSA) (2 g) and Tween 20 (0.05 ml) to PBS (100 ml). (Alternatively, powdered
milk [1 g] can replace the BSA.) Store at 4C and make fresh solutions every week.
Coating buffer: add sodium carbonate (1.59 g) and sodium bicarbonate (2.93 g) to deionised water (1 litre)
and adjust to pH 9.6. (Alternatively, dissolve one tablet of 0.05 M Sigma carbonate/bicarbonate buffer [Cat.
No. C-3041] in deionised water [100 ml].) Store at 4C and renew every 2 weeks.
Substrate buffer: make a 10% (v/v) solution of diethanolamine in deionised water. The diethanolamine
should be prewarmed to 37C before dispensing, and the pH of the solution should be adjusted to pH 9.8
with 1 M hydrochloric acid. Store at 4C and renew every 2 weeks.
1278
Enzyme conjugate: goat anti-chicken immunoglobulin conjugated to alkaline phosphatase (e.g. ICN
Immunobiologicals, alternative supply Sigma: A9171) or other species anti-chicken globulin. Store at 4C
diluted in diluent at the appropriate concentration and renew every week.
Enzyme substrate: dissolve one tablet of p-nitrophenyl phosphate disodium (5 mg) in substrate buffer (5 ml)
no earlier than 30 minutes before dispensing, and store in the dark.
Standards
i)
Positive control antiserum prepared by intramuscular inoculation of four 1-week-old specific pathogen
free (SPF) chickens with an inoculum containing 106 S. Enteritidis. The serum is subsequently
obtained 34 weeks later when antibody titres are maximal.
ii)
Negative control serum A from four 1-week-old SPF birds.
iii)
Negative control serum B from 58 1-week-old breeders known to be free from Salmonella infections.
Pool the sera and store in 100 l volumes at 20C.

1.
Background
a)

Many inactivated vaccines are used against salmonellosis caused by different serovars in various animal
species, including a combined S. Enteritidis and S. Typhimurium vaccine for use in poultry. Inactivation is
usually achieved by either heating or the use of formalin and an adjuvant, such as alhydrogel or mineral oil,
is usually used. Live vaccines have also been used in a number of countries; these include the semi-rough
strains, such as 9R for fowl typhoid and HWS51 for S. Dublin infections (Mastroeni et al., 2001). Other
attenuated vaccines include auxotrophic and metabolic drift mutants, which are used to prevent Salmonella
infections in farm animals in Germany and for S. Enteritidis and S. Typhimurium in poultry in the United
Kingdom and other countries. Mutant vaccines attenuated rationally by molecular biological gene-deletion
techniques have been developed for poultry and other species; these include aroA mutants and strains with
mutations in the genes encoding adenylate cyclase (cya) and the cyclic adenosine monophosphate receptor
protein (crp) (Cooper, 1994), which is available in the United States of America. In Europe genetically
modified organisms are not normally permitted for use as vaccines.
vaccine production. The guidelines given here and in chapter 1.1.6 are intended to be general in nature and
may be supplemented by national and regional requirements. Most vaccines are produced as highly
industrial commercial processes and are regulated by national veterinary medicines licensing authorities.
Smaller quantities of emergency herd vaccines or autogenous vaccines are produced by private laboratories
but each production has to be specifically licensed.
2.
a)

i)
For killed or live vaccines, the bacterial strain should be an organism as closely related to currently
circulating field strains as possible. It should be carefully chosen from cases of severe clinical disease,
and virulence and antigen production should be assessed. It is best to evaluate a panel of potential
strains in this way before testing the final selection. The final vaccinal strain should be identified by
historical records and characterised by stable phenotypic and/or genetic markers. Living vaccinal
strains should be marked by stable characters allowing distinction from wild strains. Markers, such as
resistance to antimicrobials, for example rifampicin or auxotrophism, may be used. Attenuation of
virulence should be stable and preferably obtained by two independent defined mutations. The stability
of live vaccine strains can be verified by regular checks using sensitive molecular fingerprinting and
micro-array techniques.
ii)

The vaccine strain must be checked as follows:
Staining of a smear of bacterial suspension on a glass slide using Gram stain.
Homogeneity of culture on non-selective media.
1279
Metabolic requirements as indicated by biochemical tests.

Detection of markers, and phage type.
Agglutination with specific antiserum.
The vaccine culture and any adjuvants, preservatives or other materials must be microbiologically
sterile and non-toxic at the concentrations used.
Safety
The LD50 (50% lethal dose) or ID50 (50% infectious dose) may be determined in mice or preferably
signs of more mild adverse reactions should be checked in the target species. Ten times the field dose
of live vaccine or twice the dose for killed vaccines must be given to the target species at the
recommended age and by the recommended route. The animals are observed for absence of adverse
reactions. Stability and non-reversion to virulence after serial passages in susceptible species should
be shown for live vaccines. It is also necessary to consider repeat vaccination. Live vaccine should be
shown not to persist for long in vaccinated animals or be transmitted to milk or eggs that may be
consumed, and the method of application should not present a hazard to operators.
Efficacy
Laboratory experiments and field trials should be used to show that the vaccine is effective. The
laboratory experiments consist of vaccinationchallenge tests in the target species at the
recommended dose and age. The efficacy data can also be used as the basis for a batch potency test.
Field trials are more difficult to undertake with respect to testing efficacy because of difficulties with
standardising the challenge and providing appropriate controls.
Environmental aspects
Live vaccine strains should be tested for their ability to persist in the environment and infect non-target
species such as rodents and wild birds that are likely to be exposed. Prolonged survival of some live
vaccines in faeces and litter may present an unacceptable environmental hazard when the material is
removed from the animal houses. Live vaccines should not be used in commercial laying flocks during
lay.
b)
i)
Procedure
The seed culture is propagated and maintained using suitable media, of which many have been
described (in textbooks) for growth of Salmonella. The media used must not contain serum or animal
tissues. Culture may be on solid medium, in Roux flasks, or in liquid medium, in which case large-scale
fermentation equipment may be used. Iron limitation or low temperature incubation on a minimal media
may enhance LPS antigen production by the vaccine strain.
Vaccine must be produced in suitable clean rooms to which only approved personnel have access.
Care must be taken to avoid cross-contamination between areas where live organisms are processed
and other areas. Contamination from operators and/or the environment must be avoided and vaccine
preparation should take place in a separate area from diagnostic culture work. Operators must not
work with vaccine whilst ill and must not be subject to immunosuppressive conditions or medications.
Personnel must be provided with protective clothing in production areas and in animal rooms.
Seed-lot cultures are prepared from the primary seed-lot, and the number of passages is dependent on
the validation of the process. The vaccine may be prepared by inoculation of a suitable medium, such
as nutrient broth, with a fresh culture and incubation on a shaker at 37C for 24 hours, with or without
aeration. The organisms are harvested by sedimentation or centrifugation. Alternatively, the organisms
may be grown on and harvested from a solid medium, such as nutrient agar. In the case of live
vaccines, the suspension is diluted in PBS, pH 7.0, and may be freeze-dried.
The time of inactivation of dead vaccines should be at least 33% more than that taken to reduce the
viable number to an undetectable level. The inactivation process must be applied to the whole volume
of the vaccine cell harvest.
Preservatives, excipient for lyophilisation, stabiliser for multidose containers or other substances added
to or combined with a vaccinal preparation must have no deleterious effect on the immunising potency
of the product.
ii)

All chemicals and growth media used should be guaranteed to be fit for purpose and checked by the
use of suitable controls.
1280
iii)
In-process controls
The following points require attention:
Visual control of the suspension, homogeneity by Gram stain, culture on non-selective medium.
Slide agglutination with specific antisera.
Titration of bacteria by turbidimetry and/or plate count.
Test of effective inactivation (killed vaccine) by plating on non-selective medium or use of a

medium that gives optimum chance of recovery e.g. production medium with neutralisation of the
inactivating compound.
Titration of viable bacteria (living vaccine) before and after lyophilisation.
iv)

Sterility/purity
Safety
A laboratory test that has previously shown a correlation with safety in the target species may be used
to determine the absence of deleterious effects on vaccinated animals. Each batch should be tested in
the target species at the recommended age and route, using at least twice the field dose for killed
vaccines and ten times the dose for live vaccines. Observations are made on any adverse effects on
the demeanour and health of the vaccinated animals and an assessment may be made of tissue
reactions at the injection site.
Batch potency
Potency is tested using vaccinationchallenge assay in mice and/or other species, including (if
practicable) the target species and immunological response in target species. Many Salmonella
vaccines are intended for use in poultry so these should be used for potency and safety tests.
c)

i)
Safety requirements
Certain killed vaccines may occasionally cause abortion in pregnant animals because of their LPS
content, and likewise live vaccines should be used with caution in pregnant animals. It is often
necessary, however, to vaccinate pregnant animals to provide maternal immunity for their offspring. It
may be useful to include endotoxin assay in the safety test programme so that the levels can be
compared with those shown to be safe in the double-dose tests. Vaccines may also cause swelling at
the site of injection, particularly if an oil-emulsion adjuvant is used.
Killed vaccines are assessed in a double-dose test, and live vaccines are assessed in a test using ten
times the dose, ideally in the target species. Live vaccines should be proven to be harmless in relevant
non-target species that could be exposed to vaccine excreted by vaccinated animals.
Reversion to virulence for attenuated/live vaccines
Live vaccines shall be shown in replication tests in target species to not revert to virulent strains during
a suitably large number of replications. Mutations, especially undefined mutations, should be shown to
be stable and checks on stability can be made by molecular fingerprinting methods or sequencing.
Although the risk is small it is wise not to use live vaccines in a country where the organism in the
vaccine has been eradicated.
Live vaccines should not be able to replicate in the environment or persist for more than a short period.
ii)
The duration of immunity is likely to vary considerably between products, vaccination regimes and
individual vaccinated animals. Immunity to Salmonella is normally serovar or serogroup specific.
Consultation among colleagues suggests that most killed vaccines will provide some protection for
6 months, while some live vaccines given by injection may elicit stronger immunity, which may persist
for 1 year or more. It should be remembered, however, that a strong challenge such as that associated
with continuously occupied farms or infected rodents may overwhelm vaccinal immunity and
commercial live vaccines may be attenuated to reduce environmental survival in a way that reduces
1281
the immune response. There may also be problems with ensuring effective oral administration with live
vaccines or accuracy of injection with killed and live injectable vaccines. The Salmonella vaccines are
intended to limit the extent of clinical disease in the case of ruminants, pigs and S. Gallinarum in
poultry. If possible, the potency test should relate to the efficacy of the vaccine in the target species,
and suitable criteria should be applied for passing batches. It may be possible to assess killed vaccines
by the O-H antibody response produced, although it should be remembered that serum antibodies are
only part of the hosts protective mechanism against Salmonella. Alternatively, the potency of the
vaccine may be assessed by its effect on challenged vaccinated animals compared quantitatively and
statistically with unvaccinated controls.
Vaccines for Salmonella are not capable of eradicating infection from herds or flocks but can increase
the threshold for infection, reduce the level of excretion of the organism and reduce vertical
transmission in poultry that results in contamination of hatching or table eggs. Vaccination is therefore
an aid to other eradication and control measures such as culling, all in-all out production, biosecurity
and farm hygiene.
iii)
Stability
Information is lacking on the stability of killed vaccines. Stability is affected by storage conditions and
by the presence of contaminating microorganisms growing in the product. Chemicals with antimicrobial
activity, such as thiomersal, phenol or crystal violet, are often included as preservatives in killed
bacterial vaccines. The stability is assessed by potency tests repeated at appropriate time intervals.
The stability of live vaccines can be assessed by performing counts of the number of viable organisms
repeated at appropriate time intervals, and genotyping tests to identify genetic changes during
fermentation production.
3.
a)

None.
b)

None.
D. COMPETITIVE EXCLUSION
Susceptibility to Salmonella infection in poultry can sometimes be substantially reduced by spray or oral treatment
prior to exposure (ideally in the hatchery) with an anaerobic culture of caecal microflora that inhibits colonisation
by Salmonella by occupying intestinal colonisation sites, producing acids and other inhibitory substances and
stimulating local and mucosal generalised immune responses. This treatment is widely used in some countries
but only used as an aid to decontaminating persistently infected farms in others. It is also useful to minimise
perturbation of intestinal flora following antimicrobial treatment or stress and to potentiate the effect of vaccines
given subsequently. As with any treatment, including vaccination or organic acid treatment, that may reduce the
prevalence or numbers of Salmonella organisms excreted by infected groups of animals, there may be some
interference with monitoring programmes and the sampling and test sensitivity may have to be adjusted to
compensate for this (Mead, 2000; Revolledo et al., 2006).
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*
* *
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diagnostic tests, reagents and vaccines for Salmonellosis
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CHAPTER 2.9.10.
TOXOPLASMOSIS
SUMMARY
Definition and description of disease: Toxoplasmosis is a zoonotic infection of animals caused
by the protozoan parasite Toxoplasma gondii. It has the capacity to infect all warm-blooded animals
and, while infection does not cause clinical illness in the majority of animal species, in some it
causes acute life-threatening disease and in others, particularly sheep and goats, it may manifest
itself as a disease of pregnancy by multiplying in the placenta and fetus. In these latter animals it
can result in the abortion or the birth of weak lambs/kids, which may be accompanied by a
mummified fetus. Characteristically, in these cases, the placental intercotyledonary membranes are
normal, but white foci of necrosis, approximately 23 mm in diameter, may be visible in the
cotyledons. Microscopically, these foci appear as areas of coagulative necrosis that are relatively
free of inflammation. Inflammation, when present, is nonsuppurative. Toxoplasma tachyzoites are
seen only rarely in association with these foci, usually at the periphery of the lesion. Examination of
the brain may reveal focal microgliosis. The lesions often have a small central focus of necrosis that
might be mineralised. Focal leukomalacia in cerebral white matter, due to anoxia arising from
placental pathology, is often present. Focal microgliosis is more specific, as leukomalacia reflects
placental damage but may occur in other pathological conditions where the placenta is
compromised, including, though rarely, ovine chlamydiosis. Infection in pigs may cause severe fetal
losses in pregnant sows, but more usually is mild and unnoticed. Acute fatal infections affect New
World monkeys, marsupials and certain other animals.
Identification of the agent: Toxoplasma gondii is an obligate intracellular parasite that has a
sexual cycle in felidae and a two-stage asexual cycle in all warm-blooded animals. It predominantly
comprises three clonal lineages (I, II and III). In the acute phase of infection, tachyzoites multiply in
cells to cause varying degrees of tissue destruction and, in fatal cases, tachyzoites may be
demonstrated in ascitic fluid or in lung impression smears. With the onset of an immune response,
tachyzoites are transformed into bradyzoites that multiply slowly in cells to produce tissue cysts. In
aborting sheep, goats and pigs, T. gondii is often difficult to find in tissue sections, but is more likely
to be seen in sections of brain and placenta. Its identity can be confirmed by
immunohistochemistry, while the polymerase chain reaction may be used to identify parasite DNA
in tissues. Isolation of T. gondii from samples is expensive and slow but, if required, is best
achieved by inoculation of mice with tissue homogenate derived from fetal brain or placenta. The
sexual life-cycle of the parasite takes place exclusively in epithelial cells of the feline intestine and
can result in the excretion of large numbers of oocysts in the faeces. Oocysts may remain viable in
the environment for many months.
Serological tests: The dye test is the longest established serological method, and in many ways
represents the gold standard, at least in humans. The dye test uses live, virulent Toxoplasma
tachyzoites, a complement-like accessory-factor and test serum. When specific antibody acts on
the tachyzoites, the latter do not stain uniformly with alkaline methylene blue. The test has proven
unreliable in some species. In addition, as live Toxoplasma is used, the test carries a potential risk
of human infection as well as being expensive to conduct. The indirect fluorescent antibody (IFA)
test is safer, gives titres comparable with the dye test and can be used to differentiate IgM and IgG
antibodies. The direct agglutination test and the latex agglutination test are both relatively rapid and
neither requires complex laboratory facilities. The enzyme-linked immunosorbent assay requires
more sophisticated laboratory equipment but can handle large numbers of samples and does not
rely on human interpretation for the result.
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Chapter 2.9.10. Toxoplasmosis
Requirements for vaccines and diagnostic biologicals: A vaccine composed of live T. gondii
tachyzoites is available commercially for use in sheep in certain European countries and New
Zealand. The vaccine is supplied as a concentrated suspension of tachyzoites with an approved
diluent and delivery system. The vaccine must be maintained and handled strictly according to the
manufacturers instructions as it has a very short shelf life.
A. INTRODUCTION
Toxoplasma gondii is a zoonotic, obligate intracellular protozoan parasite that has the capacity to infect all warmblooded animals and, while infection does not cause clinical illness in the majority of animal species, in some it
causes acute life-threatening disease and in others, particularly sheep and goats, but also pigs, it manifests itself
as a disease of pregnancy by multiplying in the placenta and fetus. Acute, potentially fatal, infections are recorded
in New World monkeys (Cunningham et al., 1992), marsupials (Canfield et al., 1990) and certain other animals
(see below). In these cases clinical signs may include lymphadenopathy, hepatomegaly, interstitial pneumonia
and nervous signs. At post-mortem examination lymph nodes, spleen and liver may be enlarged and the latter
may have pale foci. In sheep, goats and pigs a primary infection established during pregnancy may result in
apparent infertility or in stillbirths and abortion, according to the stage of pregnancy at which infection was
initiated. In a typical case of abortion, a ewe or doe infected in mid-gestation produces a stillborn lamb/kid a few
days earlier than the predicted end of pregnancy. The aborted fetus is often accompanied by either a weak sibling
or a mummified fetus (Buxton, 2000). The ewe/doe remains clinically normal. In such cases, placental
cotyledons are typically speckled with white foci around 23 mm in diameter while the intercotyledonary
membranes appear normal. Infection in early pregnancy, when the fetus has only a rudimentary immune system,
results in fetal death and resorption. In this case the mother may present as barren, which in turn can mimic a
flock/herd infertility problem. Mothers that become infected in late pregnancy would be expected to produce
infected but clinically normal offspring. Following an infection, either during or outside of pregnancy, the parasite
would not be expected to cause abortion in any subsequent pregnancy. While recent research has questioned
this conclusion (Duncanson et al., 2001; Williams et al., 2005) the majority of current thinking tends towards the
view that recrudescence of a persistent infection during pregnancy leading to repeat abortions is not normally a
significant occurrence (Rodger et al., 2006). Infection in pigs may cause severe fetal losses in pregnant sows but
under modern intensive farming conditions, when contamination of the housing and feed by T. gondii oocysts is
minimal or absent, infection would generally be expected to be at a very low incidence and cause only mild or
unnoticed signs of infection (Lind & Buxton, 2000). However when pigs are maintained outdoors under extensive
systems, they are much more likely to encounter oocysts, and so infection would be expected to be more
common (Kijlstra et al., 2004).
Toxoplasma gondii is an obligate intracellular parasite that has a two-stage asexual cycle in warm-blooded
animals and a sexual cycle in felidae. The parasite comprises three clonal lineages (I, II and III) in the main, with
type II and III being associated with disease in animals while type II is the predominant form identified in human
disease (Howe & Sibley, 1995; Khan et al., 2006). In the asexual cycle, the two developmental stages are the
rapidly multiplying tachyzoite and the slowly multiplying bradyzoite. In acute infection, tachyzoites actively
penetrate host cells where they multiply causing the cell to rupture and release organisms locally and into the
bloodstream. As the host develops immunity, the parasite retains its overall size and shape but transforms into
the bradyzoite stage and multiplies more slowly within tissue-cysts to establish a persistent infection. These
microscopic tissue cysts are present most frequently in brain and skeletal muscle and represent the quiescent
stage of the parasite within the host. Viable tissue cysts within muscle (meat) are a significant source of human
infection. In animals that succumb to acute infection tachyzoites may be demonstrated in ascitic fluid or in lung
impression smears as well as in tissue sections of the liver and other affected organs.
The sexual cycle occurs in enteroepithelial cells of the feline definitive host and results in the production of
Toxoplasma oocysts. Following a primary infection of a cat, oocysts may be shed in the faeces for several days.
The oocysts sporulate in the environment over the next 15 days (depending on aeration, humidity and
temperature), at which time they become infective. They are very resistant to environmental conditions and may
remain infective for a year or more. Sporulated oocysts are 11 13 m in diameter and each contains four
sporozoites in each of two sporocysts (Dubey & Beattie, 1988). When a susceptible animal ingests sporulated
oocysts the sporozoites are released to penetrate the intestinal lining, become tachyzoites and establish an
infection.
In sheep, goats, pigs, horses and humans, tissue cysts may remain for the rest of the life of the individual (Dubey
& Beattie, 1988). Toxoplasma does not usually cause clinical illness in cattle, camelids or deer but, as noted, can
cause fatal disease in New World monkeys, marsupials and certain other animals including hares (Lepus
europaeus; L. timidus) (Gustafsson & Uggla, 1994), the Pallas cat (Brown et al., 2005), the arctic fox (Srensen et
al., 2005), some birds (Dubey et al., 2001) and sea mammals (Fayer et al., 2004). It is suggested that these and
other similarly affected animals have had minimal exposure to T. gondii in their natural habitat through the ages,
making them particularly vulnerable to the parasite.
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Abortion in sheep and goats due to T. gondii must be differentiated from that caused by other infectious agents,
including infections with Chlamydophila abortus (see Chapter 2.7.7 Enzootic abortion of ewes), Coxiella burnetii
(see Chapter 2.1.12 Q fever), Brucella melitensis (see Chapter 2.7.2 Caprine and ovine brucellosis [excluding
Brucella ovis]), Campylobacter fetus fetus (see Chapter 2.4.5 Bovine genital campylobacteriosis), Salmonella spp.
(see Chapter 2.9.9 Salmonellosis), border disease (see Chapter 2.7.1 Border disease), and the viruses that cause
bluetongue, Wesselsbrons disease and Akabane disease. In pigs, Brucella suis (see Chapter 2.8.5 Porcine
brucellosis) may also cause fetal death, mummification and abortion.
Human health risks
Toxoplasma gondii readily infects human beings and while infection is relatively common (approximately 30% of
the population depending on age and environment), clinical illness is relatively uncommon. Those particularly at
risk of developing clinical illness include pregnant women, as the parasite can pose a serious threat to the unborn
child if the mother becomes infected for the first time while pregnant, and individuals who are immunosuppressed,
such as tissue transplant patients, AIDS patients, patients with certain types of cancer and those undergoing
certain forms of cancer therapy. These individuals are at risk of developing acute lethal infection if left untreated.
The very young and very old may also be more susceptible. On occasions, people with no apparent immune
deficiency may develop an illness characterised by general malaise, fever and lymphadenopathy. The most likely
sources of human infection are ingestion of raw or lightly cooked meat containing live T. gondii tissue cysts,
ingestion of raw or lightly cooked vegetables contaminated with oocysts or exposure to oocysts derived from cat
faeces, such as may be encountered in gardens and childrens sand pits. Toxoplasmosis is now also recognised
to be a water-borne zoonosis (Dubey, 2004). This method of transmission occurs where water treatment is
ineffective or non-existent and there is a sizeable local felid population that contaminates surface water with
oocysts (Bowie et al., 1997; Dubey, 2004). Linked to this there is now also an appreciation that sea mammals are
becoming infected by waters from contaminated land and from untreated urban sewage (Fayer et al., 2004).
1.
a)
Isolation
Isolation of T. gondii from aborted ovine and caprine fetuses and fetal membranes is best made by
inoculation of laboratory mice. The best tissues for inoculation are fetal brain and placental cotyledons, and
optimum results are obtained with fresh samples free from contamination. Samples must not be frozen at
any stage, as this kills the parasite;
i)
With aseptic precautions, remove 25 g of placental cotyledon or brain tissue from the aborted fetus.
ii)
Homogenise the tissue in an equal volume of 0.3 M sterile phosphate buffered saline (PBS), pH 7.4,
with added antibiotics (100 International Units [IU]/ml penicillin and 745 IU/ml streptomycin) in a
stomacher (Seward Laboratory, London) or other suitable macerating equipment. Brain tissue may be
effectively homogenised by passing it through a 16-gauge needle ten times by means of a syringe.
iii)
Inoculate each of three Toxoplasma-free mice intraperitoneally with 0.5 ml of the homogenate.
iv)
Kill the mice 68 weeks after inoculation and remove the brains. Blood should also be recovered from
the mice at this stage and the serum separated and stored at 20C. Brains from mice that die before
68 weeks should also be harvested.
v)
Homogenise each mouse brain with an equal volume of sterile PBS by passing through a 16-gauge
needle ten times by means of a syringe.
vi)
Spread one drop (5 l) of a given suspension on each of five slides.
vii)
Dry and stain with Giemsa, dehydrate and mount under a cover-slip.
viii) Examine slides under a microscope. Tissue cysts appear as circular structures measuring 550 m
filled with blue-staining, crescent-shaped bradyzoites.
An alternative method for examining the mouse brain is to take a small portion of forebrain (approximately
match-head size) squashed flat with a cover-slip. Tissue cysts should be easily detected under the
microscope.
If the tissues inoculated are heavily infected with T. gondii, mice may die at 12 weeks.
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Failure to demonstrate tissue cysts does not rule out a positive diagnosis. Serum from the mice may be
analysed for the presence of antibodies to Toxoplasma (e.g. using an indirect fluorescent antibody [IFA]
test); if this analysis is also negative, infection with Toxoplasma is unlikely.
b)
Tissue sections
In animals that die with acute toxoplasmosis, focal mononuclear inflammation with or without focal necrosis
may be seen in a number of tissues, including the liver, heart and lungs. The latter may be oedematous.
Lymph nodes may have undergone expansion and there may or may not be focal necrosis with or without
haemorrhage. Typically Toxoplasma tachyzoites may be demonstrable in association with necrosis and
inflammation.
In cases of abortion and stillbirth in sheep and goats, affected placental cotyledons typically contain large
foci of coagulative necrosis that may have become mineralised with time. Any associated inflammation is
characteristically slight and nonsuppurative. Well preserved samples of placental cotyledons may show
moderate oedema of the mesenchyme of the fetal villi with a diffuse hypercellularity due to the presence of
large mononuclear cells. Sometimes small numbers of intracellular and extracellular toxoplasms are visible,
usually on the periphery of a necrotic area or in a villus that is in the early stages of infection. The
Toxoplasma tachyzoites appear ovoid, 26 m long, with nuclei that are moderately basophilic and located
centrally or towards the posterior end.
In the fetal brain primary and secondary lesions may develop. Microglial foci, typically with a necrotic and
sometimes mineralised centre and often associated with a mild focal lymphoid meningitis, represent a fetal
immune response following direct damage by local parasite multiplication. Toxoplasma tissue cysts are only
rarely found, usually at the periphery of these lesions. Focal leukomalacia is also common and is considered
to be due to fetal anoxia in late gestation caused by advanced lesions in the placentome preventing
sufficient oxygen transfer from mother to fetus. Such foci occur most commonly in the cerebral white matter
cores, but sometimes also in the cerebellar white matter. Focal leukomalacia on its own suggests placental
disease or acute insufficiency but the two types of neuropathological change seen together are characteristic
of Toxoplasma infection. Confirmation of the identity of T. gondii-like structures in tissue sections from such
cases, as well as from instances of acute toxoplasmosis, may be achieved by immunohistochemistry that
labels intact T. gondii or antigenic debris. The method is both convenient and sensitive and is used with fixed
tissues (including archive tissues) that may also exhibit a degree of decomposition, where isolation would
not be appropriate or possible. The ABC indirect immunoperoxidase method and the peroxidase
antiperoxidase (PAP) technique (Uggla et al., 1987) are equally good.
c)

Several polymerase chain reaction (PCR)-based assays have been developed for the detection of DNA from
T. gondii. The main target regions are the B1 repetitive sequence (Burg et al., 1989), the P30 (SAG1) gene
(Savva et al., 1990) and 18S ribosomal RNA (rRNA) (Ellis, 1998). The sensitivity of the PCR is dependent
on the copy number of the target sequence (P30: 1 copy; B1: 35 copies; rRNA: 110 repeat units).
Customised synthetic DNA oligonucleotides are commercially available (e.g. www.sigma-genosys.co.uk).
Recently, the method for amplification of the B1 repetitive sequence has been used to analyse the lens
aspirates of congenitally infected human cataract patients (Mahalakshima et al., 2007) and was found to be
more sensitive than the conventional method used (enzyme-linked immunosorbent assay [ELISA]).
However, although the PCR is extremely sensitive, care should be taken if it is the only test available, as in
many situations a more reliable diagnosis will be gained if it is used in combination with other diagnostic
data.
Recently, a real-time PCR has been developed to allow simultaneous quantification and amplification of
DNA. It is very similar to existing PCR methods and can be carried out on 96-well microtitre plates. After
each round of amplification, fluorogenic dyes intercalate with the double-stranded DNA and the results,
shown on an amplification plot, allow quantification of the parasite DNA in the sample. Real-time PCR has
been used to amplify and quantify DNA from the T. gondii B1 gene (Costa et al., 2000; Lin et al., 2000). This
quantification of parasite DNA can be used to determine the number of parasites in tissues and fluids, such
as the amniotic fluid of patients suspected of being congenitally infected with T. gondii. (Nagy et al., 2007).
The real-time PCR is a highly sensitive and specific method, however it is expensive and requires
specialised detection systems and therefore may only be cost-effective in laboratories where analysis of
large numbers of samples is carried out.
The following method is a nested form of the PCR, amplifying the B1 repetitive sequence of DNA (Wastling
et al., 1993). Parasite DNA can be extracted and purified from several tissues, including placenta, the central
nervous system, heart and skeletal muscle.
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Contaminating red blood cells in tissues are removed by washing in 10 mM Tris/NH4Cl lysis buffer, pH 7.6,
followed by centrifugation at 2000 g for 15 minutes. DNA is then extracted from the resultant pellet and
resuspended in 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 containing proteinase K 100 g/ml and 0.5%
Tween 20.
Samples are incubated at 55C for at least 1 hour, then the proteinase K is inactivated by boiling. The PCR
procedure is performed in 50 l volumes. Amplification of the B1 gene is performed by modifying the
procedure described in ref. 1. The reaction mixture contains 10 mM Tris, pH 8.3, 2.5 mM MgCl2, 40 mM KCl,
0.01% gelatin, 0.1 mM dNTPs, 0.2 M of each primer (oligonucleotide primers are those described in Bowie
et al., 1997), two sense primers P1 and P2 and two antisense primers P3 and P4) and 2.5 units of Taq
polymerase.
Primary amplification is performed with primers 1 and 4 to give a 193 bp product over 25 cycles of 93C for
1 minute, 50C for 1.5 minutes and 72C for 3 minutes. The amplified product is then diluted 1/20 in distilled
water to reduce amplification of non-specific products.
Secondary amplification using nested primers 2 and 3 and the same reaction conditions, is carried out over
15 cycles to give a 94 bp product. The final product is then visualised on 1% agarose gels. Southern blotting,
using a labelled probe, can be used to confirm the identity of the B1 PCR products and to distinguish them
from non-specific products.
d)
Oocyst detection in drinking water

Toxoplasma gondii oocysts have been detected in drinking water using the method for the detection of
Cryptosporidium oocysts (Issac-Renton et al., 1998). The method relies on the collection of a large-volume
sample of water and passing it through a cartridge filter. Identification of Toxoplasma oocysts was by means
of inoculation of rodents.
2.
Serological tests
There are several serological tests available for the detection of T. gondii antibodies. In one type of test the
observer judges the given colour of tachyzoites under a microscope, such as with the dye test (DT) and IFA test.
Another depends on the principle of agglutination of Toxoplasma tachyzoites, red blood cells or latex particles, as
with the direct agglutination test (DAT) and indirect haemagglutination test (IHA) and latex agglutination (LA) test,
respectively. With the ELISA, the degree of colour change defines the quantity of specific antibody in a given
solution. The DT, IFA test, DAT and ELISA are outlined below and the IFA test is given in more detail.
The DT (Sabin & Feldman, 1948) is the so-called gold standard serological test for Toxoplasma antibody in
humans. Live Toxoplasma tachyzoites are incubated with a complement-like accessory factor and the test serum
at 37C for 1 hour before methylene blue is added. Specific antibody induces membrane permeability in the
parasite so that the cytoplasm is able to leak out and the tachyzoite does not incorporate the dye and so appears
colourless. Tachyzoites not exposed to specific antibody (i.e. a negative serum sample) take up the dye and
appear blue. The DT is both specific and sensitive in humans, but may be unreliable in other species. In addition,
it is potentially hazardous as live parasite is used. It is expensive and requires a high degree of technical
expertise. It should be noted that on animal welfare grounds, tachyzoites should be grown in tissue culture rather
than in mouse peritoneum whenever possible.
The IFA test (Munday & Corbould, 1971) is a simple and widely used method. Whole, killed Toxoplasma
tachyzoites are incubated with diluted test serum, the appropriate fluorescent antispecies serum is added, and the
result is then viewed with a fluorescence microscope. Fluorescent-labelled antibodies are available commercially
for a variety of animal species, the method is relatively inexpensive and kits are commercially available. However,
the method requires a fluorescence microscope and the results are read by eye, so individual variation may
occur. It may be difficult to find some species-specific conjugates and there is a risk of possible cross-reactivity
with rheumatoid factor and anti-nuclear antibodies.
The DAT (Desmonts & Remington, 1980) is both sensitive and specific. Formalinised Toxoplasma tachyzoites are
added to U-shaped well microtitre plates and dilutions of test sera are then applied. Positive samples will produce
agglutination that can be graded, while negative samples will produce a button of precipitated tachyzoites at the
bottom of the well. The test is simple and easy to perform although relatively large amounts of antigen are
required. Kits are commercially available. The method of growth and harvesting of parasites is given below. A
commercially available latex agglutination test (LAT) is also available. The DAT and LAT are not species specific
and are suitable for use in all species.
The original ELISA (Voller et al., 1976) uses a soluble antigen preparation made from Toxoplasma RH strain
tachyzoites (as described below) and layered into wells in a microtitre plate. Test sera (e.g. ovine in origin) are
1291
added, followed by an anti-species enzyme-labelled conjugate such as horseradish peroxidase-labelled antiovine-IgG. Any attached conjugate causes a colour change in the substrate that is directly related to the amount
of bound antibody, and which can be read with a spectrophotometer at the absorbance specific to the substrate
used. The assay is simple, can readily test a large number of samples and is easy to perform with the chosen
anti-species conjugate. Defined anti-species conjugates, substrates and whole kits are commercially available.
However, the assay does require a spectrophotometer. The ELISA is well suited to laboratories required to
analyse large numbers of samples.
Recently, a kinetics ELISA (KELA) has been developed (Werre et al., 2002). The KELA system measures the rate
of reaction between bound enzyme and the substrate solution that leads to development of colour. Three optical
densities (OD) are read at 45-second intervals (using the KELA data management program) and the results are
reported in terms of slopes. The correlation between the ELISA and the KELA is very high, and therefore, the two
tests are very good diagnostic tools, differing only in their convenience of application.
To improve the specificity of the conventional ELISA, assays that use recombinant antigens (Johnson & Illana,
1991) and affinity purified Toxoplasma-specific antigens (Lekutis et al., 2001) have been developed for use in
sheep (Sager et al., 2003; Tenter et al., 1992) but these tests are not yet routinely used.
With the conventional ELISA the detection of Toxoplasma-specific IgG and IgM antibodies allows a degree of
discrimination between acute and chronic toxoplasmosis. More recently avidity assays have been developed. As
the immune response matures, after infection is established, so antibodies of increasing avidity (functional affinity)
for the antigen develop. This avidity can be measured and used to indicate active or recent T. gondii infection. An
assay for the detection of avidity of IgG for the P30 antigen of T. gondii in sheep has been developed (Sager et
al., 2003). This test is a good diagnostic tool for discriminating relatively recent from more established infections.
Preparation of antisera and antigens
Antisera to T. gondii and conjugated antisera for use in the IFA test and ELISA, to allow screening of a variety of
animal species, may be obtained commercially. International standards for animal sera are not available.
Below are protocols for the preparation of tachyzoite antigen for use in the IFA test and ELISA. Tachyzoites may
be grown in mice or in tissue culture and retained as whole parasites for the IFA test, or prepared as soluble
antigen for the ELISA.
Production of Toxoplasma tachyzoites in mice

i)
Inject each of six Toxoplasma-free mice intraperitoneally using a 1-ml syringe and a 23-gauge needle,
with 0.2 ml of 1 107/ml T. gondii tachyzoites of the RH strain, collected fresh from a previous mouse
passage or from tissue culture. (For optimum recovery of tachyzoites having minimal host mononuclear
cells, mice should be more than 68 weeks of age and weigh approximately 2225 g.)
ii)
Kill the mice 3 days later by CO2 inhalation (avoid cervical dislocation as this may cause contamination
of peritoneal fluid with red blood cells).
iii)
Pin the mouse out on its back on a clean cork mat. Reflect the abdominal skin with aseptic precautions,
remove any peritoneal fluid by means of a 21-gauge needle attached to a 1 ml syringe and gently eject
the harvested exudate into an equal volume of PBS.
The optimum time to collect tachyzoites is 72 hours after initial inoculation, when there will be sufficient
organisms but before there is significant contamination by host cells. It is also important not to delay
harvesting peritoneal fluid much past 3 days as the mice will die. (If tachyzoites for mouse inoculation
are taken from a frozen stabilate, it may be necessary to harvest mice 4 or 5 days after the initial
inoculation and passage the parasite once more through mice before using it as an antigen in the
above tests.)
iv)
Centrifuge the fluid at 500 g for 5 minutes, aspirate supernatant and resuspend in Hanks balanced salt
solution (HBSS). Alternate between PBS and HBSS washes by centrifugation.
v)
Calculate the concentration of tachyzoites and contaminating host cells with an improved Neubauer
counting chamber (Count numbers of tachyzoites at 1/1000 dilution and cellular contamination at 1/10).
vi)
Carry out further washes (step iv above) as required to reduce cellular contamination to <0.5% host
mononuclear cells and <0.25% for red blood cells.
vii)
Resuspend the tachyzoites in PBS to give a final concentration of 1 107/ml.
viii) Tachyzoites may be maintained by continual passage in this manner without the addition of
penicillin/streptomycin by observing strict aseptic procedures.
1292
Preparation of aliquots of a frozen stabilate of T. gondii tachyzoites

i)
Produce tachyzoites in mice or tissue culture as described.
ii)
Centrifuge at 500 g for 5 minutes and resuspend in Iscoves modified Dulbeccos medium (IMDM)
(Gibco BRL, Paisley, UK) approximately three times.
iii)
Add the following solutions to give these concentrations: 10% dimethyl sulphoxide; 20% normal horse
serum (free from antibody to T. gondii); 70% resuspended tachyzoites to give a final concentration of
1 108 tachyzoites/ml.
iv)
Allow the preparation to stand on the bench for 1 hour.
v)
Dispense into 1-ml aliquots using screw-topped tubes appropriate for liquid nitrogen storage.
vi)
Put the tubes into a small container, wrap in thick insulating material and place in 70C freezer to
allow the tachyzoites to freeze gradually.
vii)
The next day transfer to liquid nitrogen, keeping well insulated while transferring.
viii) This stabilate may then be used for mouse inoculation or tissue culture growth of the parasite. When
removing from storage thaw the sample rapidly in warm water.
Production of Toxoplasma tachyzoites in tissue culture

i)
Toxoplasma gondii can be grown and maintained in tissue culture by twice-weekly passage in African
green monkey kidney (Vero) cells.
ii)
Cells and parasite are grown in IMDM supplemented with 50 IU/ml penicillin, 50 g/ml streptomycin
and 2% fetal bovine serum.
iii)
Tachyzoites are harvested from tissue culture flasks by scraping the cell monolayer using a sterile cell
scraper.
iv)
Using 25 cm2 vented tissue culture flasks that have each been seeded with 1 105 Vero cells, add
tachyzoites at the rate of two tachyzoites per monolayer cell and incubate at 37C in a 5% CO2
humidified chamber. Harvest after 34 days.
Preparation of whole tachyzoites for use in the IFA test

i)
Produce 4 107/ml suspension of RH strain T. gondii tachyzoites in PBS.
ii)
Add formaldehyde (40%) to give a final concentration of 0.2% (v/v).
iii)
Incubate at 4C overnight and divide into aliquots in suitable sealed tubes and store frozen until
required.
Production of soluble antigen for ELISA

i)
Produce a suspension of RH strain T. gondii tachyzoites in PBS.
ii)
Centrifuge at 2000 g for 15 minutes, retain the pellet and resuspend it in nine times its volume of
distilled water.
iii)
Rupture the tachyzoites by freezing and thawing three times.
iv)
The antigen preparation is then sonicated for 20 seconds at 4C at an amplitude of 20 microns.
v)
Remove any cellular debris by centrifugation at 10,000 g for 30 minutes at 4C.
vi)
Retain the supernatant and store at 20C until required. (Protein estimation might be expected to give
a value of between 2 and 4 g/ml.)
Protocol for the IFA test

The following is a protocol for carrying out an IFA test for anti-Toxoplasma IgG antibodies in sheep serum. It
only requires minor modifications for testing different species or for measuring IgM antibody.
i)
Clean the required number of tissue culture 15-well multitest slides (Flow laboratories) and allow to dry.
ii)
Layer 5 l of a whole tachyzoite preparation on to each well and allow to dry.
iii)
Fix in methanol for 10 minutes.
1293
iv)
Wash twice for 10 minutes each time in 0.3 M PBS, pH 7.4.
v)
Add 5 l of the given test sheep serum (diluted in PBS) to each well. (Prepare serial dilutions of the test
sera, e.g. 1/16, 1/32, etc. up to 1/1024.) Ensure that positive and negative control sera are included in
each test as well as a PBS-only sample. Incubate for 30 minutes at room temperature.
vi)
Wash twice for 10 minutes each time in PBS.
vii)
Add 5 l of an appropriate dilution of rabbit-anti-sheep IgG conjugated to fluorescein isothiocyanate,

diluted in 0.2% filtered Evans blue dye in PBS, to each well and incubate for 30 minutes at room
temperature.
viii) Wash three times for 10 minutes each time in PBS.

ix)
Mount the slides under cover-slips with buffered glycerol (nine parts PBS one part glycerol) or Citifluor
(Citifluor Ltd, London).
x)
Examine using a fluorescence microscope, fitted with 20 and 40 objective lenses.
With a negative test serum result the whole parasites will appear red due to the autofluorescence of the
Evans blue dye. They may also present with a green fluorescent cap at the parasite pole (nonspecific polar
fluorescence). With a positive test serum the parasites will fluoresce red and at least 80% of them within a
given well will be surrounded by an unbroken band of green fluorescence. In an adult sheep/goat a positive
titre could be defined as 1/64 and a negative titre as 1/32. For lamb/kid and fetal sera, respective titres
could be defined as 1/32 and 1/16.
An example slide set-up is shown below:
Sample 1
1/16
1/32
1/512
1/1256
1/1024
1/128
1/64
PBS only
1/64
|
|
|
1/128
1/1024
1/32
1/256
1/512
1/16
Sample 2

The only available vaccine is a commercially produced live preparation for sheep (Toxovax, Intervet BV, The
Netherlands; Toxovax, AgVax, Ag Research, New Zealand), currently licensed for use in the UK, Ireland, France,
Portugal and Spain and New Zealand. It consists of tissue culture grown S48 T. gondii tachyzoites attenuated by
over 3000 passages in mice. The vaccine stimulates effective protective immunity for at least 18 months following
a single subcutaneous injection, but as it is unable to produce tissue cysts, sheep are not left with a persistent
vaccinal infection. The vaccine has a short shelf life and is a potential risk to immunosuppressed and pregnant
female operatives (Buxton, 1993). The vaccine should be stored and used strictly according to the manufacturers
instruction, thus it should not be frozen at any time, should be maintained cool (around 4C) and not exposed to
direct sunlight. The diluent provided should be added to the concentrated suspension of tachyzoites immediately
prior to use.
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*
* *
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CHAPTER 2.9.11.
VEROCYTOTOXIGENIC ESCHERICHIA COLI
SUMMARY
Escherichia coli are normal inhabitants of the gastrointestinal tract of animals and humans. Some
strains have become highly adapted to cause diarrhoea and a range of extra-intestinal diseases.
Since 1977, it has been recognised that some diarrhoegenic strains of E. coli produce toxins that
have an irreversible cytopathic effect on cultured Vero cells. Such verocytotoxigenic E. coli (VTEC)
belong to over 100 different serotypes. Escherichia coli O157:H7 is the predominant and most
virulent serotype in a pathogenic subset of VTEC, designated enterohaemorrhagic E. coli (EHEC).
This designation is based on their capacity to cause haemorrhagic colitis and haemolytic uraemic
syndrome in humans, their ability to produce verocytotoxins, their ability to cause attaching and
effacing lesions on epithelial cells, and their possession of a characteristic large plasmid. In the past
two decades, VTEC O157:H7 has risen in importance world-wide as a public health problem. Other
non-O157 serogroups, including O26, O91, O103, O104, O111, O113, O117, O118, O121, O128
and O145, have been associated with occasional outbreaks of human disease, and others may be
associated with sporadic cases. Ruminants represent the main natural host of VTEC and are
generally healthy carriers of the organisms. VTEC have also been isolated from pigs, cats, dogs,
chickens and wild birds. Cattle are considered to be the main reservoir of E. coli O157:H7 infection
for humans. Despite its pathogenicity for humans, infection in animals with E. coli O157:H7 is
invariably asymptomatic. By contrast, the EHEC serogroups, O26, O111 and O103 may be
pathogenic for both humans and animals. The presence of VTEC in animal faeces provides the
potential for these organisms to enter the food chain by faecal contamination of milk products,
contamination of meat with intestinal contents during the slaughter process or contamination of fruit
and vegetables by contact with infected manure. VTEC are also transmitted through contaminated
water and by direct contact with infected people or animals.
Identification of the agent: Diagnostic procedures for VTEC have been developed, primarily for
E. coli O157:H7, and seek to overcome the problems of isolating low numbers of target organisms
from complex matrices such as animal faeces, food and clinical specimens. Identification of E. coli
O157:H7 in subclinical animal carriers depends on enrichment of faeces samples in liquid media,
usually buffered peptone water with or without the addition of vancomycin, cefsulodin and cefixime,
for 6 hours at 37C followed by immunomagnetic separation using commercially available
paramagnetic particles or beads coated with anti-O157 lipopolysaccharide antibody. Beads with
bound bacteria are plated on to selective agar, commonly 1% sorbitol MacConkey agar containing
cefixime and potassium tellurite, and incubated for 18 hours at 37C. Non-sorbitol-fermenting
colonies are confirmed biochemically as E. coli and by serum or latex agglutination as possessing
the O157 somatic antigen and/or H7 flagellar antigen. Potential virulence for humans is confirmed
by the demonstration of verocytotoxin production by Vero cell assay, enzyme-linked
immunosorbent assay (ELISA) or agglutination tests or the demonstration of genes encoding
verocytotoxin by polymerase chain reaction. Detection of non-O157 VTEC relies on direct analysis
of colonies on nonselective plates by, for example, immunoblotting or DNA probing for
verocytotoxin production. Numerous immunological and nucleic acid-based recognition tests have
been described to provide a more rapid presumptive diagnosis of VTEC and many are available
commercially. Phage typing and pulsed field gel electrophoresis are widely used by reference
laboratories for subtyping VTEC O157 for epidemiological purposes.
Serological tests: Serological tests are not used routinely in animals to diagnose VTEC infection,
but it has been shown that cattle infected with VTEC produce serum antibodies to the O157
lipopolysaccharide that can be detected by ELISA.
1297
Chapter 2.9.11. Verocytotoxigenic Escherichia coli
Requirements for vaccines and diagnostic biologicals: No vaccines are currently available for
controlling VTEC infections in animals or humans, but a variety of experimental vaccines are being
developed.
A. INTRODUCTION
Escherichia coli are normal inhabitants of the gastrointestinal tract of animals and humans of which only some
strains have become highly adapted to cause diarrhoea and a range of extra-intestinal diseases. Escherichia coli
are routinely characterised by serological identification of somatic O, flagellar H and capsular K antigens.
However, while some serotypes correlate closely with certain clinical syndromes, differentiation of pathogenic
strains from the normal flora depends on the identification of virulence characteristics. Since 1977, it has been
recognised that some diarrhoegenic strains of E. coli produce toxins that have an irreversible cytopathic effect on
cultured Vero cells (Konowalchuk et al., 1977). Such verocytotoxigenic E. coli (VTEC) have been shown to belong
to over 100 different serotypes (Johnson et al., 1996a; Strockbine et al., 1998). They are also described as Shiga
toxin-producing E. coli (STEC) due to the similarity demonstrated between verocytotoxins (VT) and Shiga toxins
(Stx) of Shigella dysenteriae (OBrien & Laveck, 1983). In the past two decades, VTEC O157:H7 has increased in
importance world-wide as a public health problem. Escherichia coli O157:H7 is the predominant and most virulent
serotype in a pathogenic subset of VTEC, designated enterohaemorrhagic E. coli (EHEC). This designation is
based on their capacity to cause haemorrhagic colitis and haemolytic uraemic syndrome in humans, their ability to
produce VT, their ability to cause attaching and effacing lesions on epithelial cells, and their possession of a
characteristic large plasmid (Nataro & Kaper, 1998). Other non-O157 serotypes, including O26:H11, O104:H21,
O111:H and O145:H, have been associated with occasional outbreaks of human disease, and others still with
sporadic cases (Johnson et al., 1996a).
Ruminants represent the main natural host of VTEC and are generally healthy carriers of the organisms. VTEC
have also been isolated from pigs, cats, dogs, chickens and wild birds; these species can be transiently colonised
by the organisms (Beutin et al., 1993; Johnson et al., 1996a). Surveys have shown that O157 strains normally
represent a minority of the VTECs that colonise the intestinal tract of animals. The presence of VTEC in animal
faeces provides the potential for these organisms to enter the food chain via faecal contamination of milk,
contamination of meat with intestinal contents during slaughter or contamination of fruit and vegetables by contact
with contaminated manure. VTEC are also transmitted through water and by direct contact with infected people,
animals or animal waste. Contaminated water, used for irrigating or for washing vegetables, can also be source of
infection for humans or animals. Cattle are considered to be the main reservoir of E. coli O157:H7 infection for
humans, although the organism has been isolated from a variety of farmed animals, horses, dogs, rabbits, birds
and flies. Despite its ability to cause severe disease in humans (Paton & Paton, 1998), infection in animals with
E. coli O157:H7 is invariably subclinical. Some non-O157 serotypes, however, are pathogenic for animals and
humans and include O26:H11; O103:H2; O111:H (Bettelheim, 2000; Johnson et al., 1996a).
VTEC are also associated with oedema disease in piglets with four serotypes responsible for the majority of
outbreaks world-wide, namely O45:K+, O138:K81, O139:K82 and O141:K. The main virulence factors are a
fimbrial adhesin, F18, involved in colonisation and the VT2e toxin, which is responsible for clinical signs. A high
degree of genetic relatedness between O101 strains harbouring stx2e genes of human and porcine origin has
been demonstrated. The role of pigs as subclinical carriers of STEC in the epidemiology of human disease needs
further research.
Because E. coli O157:H7 has become the predominant zoonotic VTEC, diagnostic methods have been developed
to detect selectively this serotype in human clinical cases (Strockbine et al., 1998) and in food sources (VernozyRozand, 1997). For the latter, a validated International Standard detection method is available (EN ISO
16654:2001). In this chapter, however, emphasis will be given to the isolation and identification of O157 and other
VTEC from carrier animals (Clifton-Hadley, 2000).
1.
Samples
In most cases, samples taken from animals for VTEC isolation will be faeces collected for surveillance purposes
or as part of an epidemiological trace-back exercise following an outbreak of disease in humans. Samples may be
taken from the rectum or from freshly voided faeces on the farm or from intestinal contents after slaughter. A
variety of VTEC are present in healthy animals and not all are thought to be pathogenic for humans. Escherichia
1298
coli O157:H7, which is the most significant VTEC in human disease, is carried subclinically in animals. Cattle are
thought to be the most important reservoir of this serotype. In an infected herd, only a proportion of the animals
will be detectably infected, the organism is usually present in carriers in low numbers and is shed intermittently in
faeces. Shedding is influenced by the age of the animals, diet, stress, population density, geographical location
and season (Meyer-Broseta et al., 2001). Some animals are thought to contribute disproportionately to
transmission of infection and have been termed super-shedders (Matthews et al., 2006). Isolation rates may be
improved by taking faeces samples in preference to rectal swabs, by increasing the sample size, by increasing
the number of individuals sampled and by repeat sampling. Use of recto-anal mucosal swabs is reported to
improve detection of colonised as distinct from transiently infected cattle (Rice et al., 2003). Precautions should
be taken to avoid cross-contamination of samples in transit and at the laboratory. Samples should be kept cool
and cultured as soon as possible after collection.
Safety
Care should be exercised when handling VTEC-positive samples as the infective dose capable of causing severe
human infection may be low (possibly 100 organisms for VTEC O157:H7) and laboratory-acquired infections have
been reported (see Chapter 1.1.3 Biosafety and biosecurity in the veterinary microbiology laboratory and animal
facilities).
Isolation
a)
Liquid enrichment media

Clinical samples are routinely plated directly on to solid media for isolation of E. coli, but the number of target
VTEC organisms in faeces from healthy carriers is usually low and enrichment in liquid media improves
recovery. Commonly used enrichment media are buffered peptone water either unsupplemented (which
gives good recovery) or supplemented with 8 mg/litre vancomycin, 10 mg/litre cefsulodin and 0.05 mg/litre
cefixime (BPW-VCC) to suppress the growth of Gram-positive organisms, Aeromonas spp. and Proteus
spp.; modified trypticasesoy broth (mTSB) supplemented with 20 mg/litre novobiocin or 10 mg/litre
acriflavin to reduce the growth of Gram-positive organisms; or modified E. coli broth with 20 mg/litre
novobiocin (mEC+n). EHEC E. coli grow poorly at 44C. The optimal incubation for bovine faeces to
minimise overgrowth by other organisms is 6 hours at 37C. For meat samples, enrichment for 6 hours at
4142C is used and for water and dairy products, 24 hours at 4142C. Nonselective pre-enrichment is
necessary for the effective recovery of low levels of stressed E. coli O157. Enrichment broths should be prewarmed to prevent cold-shocking the organisms and slowing their initial growth; 24 hours incubation may
increase recovery if the organisms are stressed.
b)
Immunomagnetic separation
Immunomagnetic separation (IMS) has been used as a selective concentration technique to improve
isolation of E. coli O157:H7 where numbers of the organism are low (Chapman et al., 1994). Commercially
available paramagnetic particles or beads coated with anti-lipopolysaccharide (LPS) antibody are mixed with
an aliquot of incubated broth. Beads with bound bacteria are separated from the supernatant by a magnetic
field and after washing are plated on to selective agar and incubated for 18 hours at 37C to isolate suspect
colonies. The technique is serogroup specific. Commercial systems are available for manual or automated
separation (Chapman & Cudjoe, 2001). Recovery may be affected by the bead-to-organism ratio (optimum
is 3:1), the enrichment broth used and the problem of nonspecific adsorption of E. coli to the magnetic beads
(which can be reduced by the use of a low ionic strength solution in the IMS procedure and careful washing).
These factors should be taken into account when trying to maximise the sensitivity of the technique for
detecting target E. coli.
c)
Selective culture for Escherichia coli O157

There are no biochemical characteristics that distinguish the majority of VTEC from other E. coli, however,
the inability of most strains of E. coli O157:H7 to ferment D-sorbitol rapidly and their lack of betaglucuronidase activity can be exploited in the isolation and identification of these organisms. However, the
less common sorbitol fermenting and beta-glucuronidase positive E. coli O157:H variants (nonmotile due to
lack of expression of the H7 antigen), will not be identified by isolation in such selective media chosen for
these biochemical characteristics (Karch & Bielaszewska, 2001). MacConkey agar containing 1% D-sorbitol
instead of lactose (SMAC) is a useful and inexpensive medium on which non-sorbitol-fermenting E. coli grow
as small, round greyish-white colonies. Selectivity is improved by the addition of 0.5% rhamnose, and
addition of 0.05 mg/litre cefixime (CR-SMAC) inhibits overgrowth by Proteus spp. While fewer presumptive
colonies require testing on this medium, rhamnose is an expensive supplement. An alternative modification
is the addition of 2.5 mg/litre potassium tellurite in addition to cefixime (CT-SMAC), which has a greater
inhibitory effect against non-O157 E. coli and other non-sorbitol fermenters, such as Aeromonas,
Plesiomonas, Morganella and Providencia, than against E. coli O157 (OBrien & Laveck, 1983). This is
currently the most commonly used medium for isolating E. coli O157.
1299
Media containing fluorogenic or chromogenic glucuronides are used to distinguish non-beta-glucuronidaseproducing E. coli O157:H7 from beta-glucuronidase-producing E. coli. Hydrolysis of 4-methylumbelliferylbeta-D-glucuronide (MUG) by beta-glucuronidase activity produces a fluorescent compound visible under
UV light. The addition of 0.1 g/litre 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide (BCIG) to SMAC
differentiates white colonies of E. coli O157:H7 from green-blue colonies of sorbitol negative, betaglucuronidase positive organisms. Commercially available chromogenic and fluorogenic media may be
found by reference to media catalogues. While advances have been made in improving the selectivity of
media for E. coli O157:H7, isolation rates, particularly of stressed organisms, may be adversely affected by
the additives used. To mitigate against these effects, addition of recovery agents such as 1% sodium
pyruvate to tryptonesoy agar or delaying exposure of stressed cells to selective agents can aid recovery of
the organism (Blackburn. & McCarthy, 2000).
Sorbitol-fermenting (SF) E. coli O157:H have been isolated from patients with diarrhoea and HUS but the
epidemiology of this infection is poorly understood and only rarely has the organism been isolated from
animals, including cattle (Lee & Choi, 2006). The majority of SF E. coli O157:H isolates are susceptible to
tellurite and cannot be identified on CT-SMAC. Microbiological analysis for this organism is laborious and
entails plating IMS-separated organisms onto SMAC and testing individual SF colonies by latex agglutination
for the O157 antigen. Alternatively, colony sweeps are tested by polymerase chain reaction (PCR) for the
presence of vt2, eae, rfbO157 and sfpA (see below). Well-spaced colonies from growth positive by PCR are
then tested by colony hybridisation with probes for vt2,, eae and sfpA or colony immunoblot using specific
antibody (Karch & Bielaszewska, 2001; Lee & Choi, 2006).
d)
Isolation of other VTEC

Non-O157 VTEC grow well on media that permit the growth of E. coli, such as blood agar or MacConkey
agar, and the majority can only be differentiated from other E. coli by their ability to produce VT. The large
number of different VTEC serotypes precludes the use of O-antisera for the routine screening and
presumptive identification of colonies on these media. IMS can be used for selective concentration of
serogroups O26, O103, O111 and O145 from a pre-enriched sample, as for the O157 strains. These
serogroups are the non-O157 VTECs most commonly associated with human disease, and commercially
produced beads are currently available.
The inability of O26 strains to ferment rhamnose has led to the recent development of media that may prove
to be useful in differentiating O26 E. coli from other enteric organisms. The first is rhamnose-MacConkey
agar (RMAC) in which the lactose in the MacConkey medium is replaced by 10 g/litre rhamnose. Addition of
2.5 mg/litre potassium tellurite and 0.05 mg/litre cefixime (CT-RMAC) is said to improve specificity. The
second is a chromogenic rhamnose agar incorporating 10 g/litre rhamnose and 0.02 g/litre phenol red in ES
coliform agar (an indicator medium for beta-galactosidase activity) to which is added 0.5 mg/litre potassium
tellurite and 0.05 mg/litre cefixime. On this medium, O26 colonies are reported to be dark blue to black, other
E. coli serotypes are green, and enterobacteria other than E. coli are green, yellow or colourless.
One potentially useful virulence marker for VTEC is enterohaemolysin production, which causes haemolysis
of washed sheep erythrocytes after overnight incubation on blood agar supplemented with calcium. This
characteristic is shared by 90% of VT-producing E. coli isolated from human infections. However, the finding
that a proportion of disease-producing VTEC can be negative for enterohaemolysin production reduces the
value of enterohaemolysin agar as a screen.
In most cases, therefore, isolation of VTEC relies on direct analysis of colonies on plates by immunoblotting
or DNA probing for VT production to identify colonies for further characterisation. Colonies are first replicated
so that positive colonies can be isolated after replicates have been tested. Colonies may be blotted on to
suitable membranes (nitrocellulose or nylon) from which replicates are made or picked off into 96-well
microtitre plates containing broth for replication before transferring aliquots to appropriate filters. Colonies
are then analysed using nucleic acid probes or antibodies to identify any VTEC (Strockbine et al., 1998). Hull
et al. (1993) developed a mitomycin immunoblot assay for detecting VTEC in faeces that was simple enough
to use in routine diagnostic laboratories. Serial dilutions of faeces in broth are inoculated on to MacConkey
agar plates and incubated overnight at 37C. Using standard replica plating techniques, growth from the
plate yielding approximately 200 colonies is transferred to two 0.45-m pore-size nitrocellulose filters laid on
Syncase agar with 25 ng mitomycin/ml. This medium induces vegetative growth of bacteriophages carrying
the genes for VT and enhances toxin expression. (Alternatively, bacterial or faecal suspensions may be
plated directly on to the filters.) The plates are incubated overnight at 37C. After overnight growth, filters are
removed from the plates, immersed in a chloroform bath for 15 minutes, then blocked for 1 hour with 5%
non-fat milk in 10 mM Tris, 150 mM NaCl, 0.05% Tween (pH 8) (TNT). The filters are incubated for 1 hour in
antisera raised against VT1 or VT2, given three 5-minute washes in TNT, then incubated for 1 hour with
alkaline phosphatase-conjugated anti-immunoglobulin G followed by three further 5-minute washes in TNT.
Any reaction is visualised by colour development with nitroblue and 5-bromo-4-chloro-3-indolyl-phosphate.
VT1, VT2 and VT-negative control E. coli are tested in parallel. The use of polyclonal antibodies results in
some false positives that are eliminated by using monoclonal antibodies. When the use of DNA probes was
1300
compared with the use of the mitomycin immunoblot colony assay, it was shown that the results were
comparable. The immunoblot assay has the advantage of being simpler to perform than DNA probing.
Mitomycin plates have a long shelf life when stored in the dark at 4C.
Colony immunoblotting or probing are labour intensive techniques and may be better applied to samples that
have been screened and shown to be positive for the presence of VT or VT genes by, for example, enzymelinked immunosorbent assay (ELISA) or PCR.
Identification and characterisation of suspect colonies
Colonies growing on solid media that are suspected to be VTEC must be confirmed biochemically or genotypically
(e.g. by GadA PCR) to be E. coli. Somatic O and flagellar H antigens are identified serologically. Not all VTEC
isolated from animals are thought to be pathogenic for people. Some isolates of E. coli O157, particularly from
pigs, are non-verocytotoxigenic and nonpathogenic for humans. Diagnosis, therefore, must include the
demonstration of known virulence factors in the isolates. These include the verocytotoxins VT1 (Stx1) and VT2
(Stx2) and their genes and an outer membrane adhesion protein associated with attaching and effacing lesions,
intimin, which is encoded by the eae gene (Law, 2000). For VTEC O157 strains, subtyping methods are available
in reference laboratories for epidemiological investigations.
a)
Biochemical tests
VTEC are biochemically similar to other E. coli. VTEC O157:H7 strains differ in failing to ferment sorbitol,
failing to produce beta-glucuronidase and fermenting raffinose and dulcitol. Escherichia coli can be
distinguished from E. hermanii by lack of growth in the presence of potassium cyanide and failure to ferment
cellobiose. Escherichia hermanii is positive for both tests. Ninety-eight per cent of E. hermanii strains have a
characteristic yellow pigment on nutrient agar that is not seen in VTEC. Escherichia coli may be confirmed
by demonstration of the use of tryptophan and beta-galactosidase activity (see below) or by commercially
available biochemical test strips.
b)
Serological tests
Commercial latex kits are available for O157, O26, O91, O103, O111, O128, O145 and H7. Tests should be
carried out according to the manufacturers instructions and should incorporate positive and negative control
organisms and control latex. Presumptive diagnosis may also be made using slide or tube agglutination tests
with anti-O LPS antiserum (antisera to 181 O antigens are available). O157 antiserum has been shown to
cross-react with other organisms including E. hermanii (frequently found in foods), Salmonella O group N,
Yersinia enterocolitica serotype O9 and Citrobacter freundii, indicating the need to confirm putative VTEC
colonies as E. coli. Isolates can be tested for the presence of flagellar antigen (antisera have been raised to
56 H antigens), but this may require passage through motility medium. Some pathogens are nonmotile.
c)
Verocytotoxin production in Vero cell assay (Johnson et al., 1996a)

The Vero cell assay remains a standard method for the confirmation of VT production (see below). Vero
cells have a high concentration of globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) toxinbinding receptors in their plasma membranes and will normally detect all variants of VT. The test can be
used on faecal suspensions, culture filtrates or live cultures. In mixed faecal cultures, the sensitivity of the
assay is increased by treating the suspension with polymyxin B or mitomycin to release cell-associated
toxin. While the test is sensitive, it is not available in most routine diagnostic laboratories. It is labour
intensive and results can take 34 days after the cell culture is inoculated. Where tissue culture facilities are
not available, other methods may be used for detecting VT production, including ELISA or agglutination and
PCR can detect the vt genes. All of these methods are now available as commercial kits.
d)
Subtyping of Escherichia coli O157 for epidemiological studies

A variety of methods is available in reference laboratories to help discriminate between strains of E. coli
O157:H7 to aid epidemiological investigations of outbreaks of human disease (Hopkins & Hilton, 2000;
Strockbine et al., 1998). These methods vary in technical complexity and more than one technique is
required to provide useful differentiation. Techniques include phage typing, biotyping and antimicrobial
sensitivity testing (resistance being uncommon in strains from most countries), plasmid profiling, restriction
fragment length polymorphism analysis, ribotyping, pulsed field gel electrophoresis (PFGE) and various
PCR-based analyses (random amplification of polymorphic DNA; repetitive DNA element PCR; amplified
fragment length polymorphism analysis). Of these, only phage typing and PFGE are widely used. Despite
some difficulties with interpretation of profiles, PFGE has emerged as the standard method used by public
health reference laboratories for subtyping VTEC O157 due to its high level of discrimination and accuracy
and reproducibility. It is used in Pulsenet, a network of public health laboratories performing a standardised
PFGE method that allows comparison of fingerprints held on an electronic database by the Centres for
Disease Control and Prevention in the USA (http://www.cdc.gov/pulsenet/). The European Unions Enter-
1301
net system for the surveillance of Salmonella and VTEC relies largely on phage typing to subtype E. coli
O157:H7 strains. Use of subtyping of genes for intimin and VT has proved valuable for epidemiological
studies and source attribution (Beutin et al., 2004; 2007). Subtyping methods for non-O157 serotypes have
been less well explored, however, similar molecular approaches to those used for VTEC O157 can be taken.
Non-culture techniques for detecting VTEC
Although definitive diagnosis of VTEC relies on the isolation and characterisation of pure cultures, cultural
methods for VTEC are time-consuming and labour intensive. This has led to the development of a range of
immunological and nucleic acid hybridisation tests for rapid identification of O and H antigens, VT or genes
associated with VT production in the sample. As the tests have a detection level above the numbers at which the
target organism is normally present in the faeces, an enrichment step (preferably nonselective for isolation of
injured or stressed bacteria) is required to increase the numbers prior to testing.
a)
Immunoassays to identify O and H antigens and VT may be used to confirm the identity of the organisms
once isolated from clinical, food or environmental samples, while others, including dipstick and membrane
technologies, microplate assays, colony immunoblotting, immunofluorescence and ELISA, are used as rapid
methods for detecting the presence of potential pathogens in samples prior to isolation, thus shortening the
time for a presumptive diagnosis. Most assays for somatic and flagellar antigens are designed to detect the
O157 LPS and H7 flagellar antigen. Toxin assays have the advantage of detecting all VTEC. Enzyme
immunoassays for O157 and VT, visual immunoassays for O157 and agglutination tests for O157, H7 and
VT are available commercially as kits (De Boer & Heuvelink, 2001; Clifton-Hadley, 2000; Nataro & Kaper,
1998; Strockbine et al., 1998). Not all have been validated for use with faeces. Specialised reagents in which
anti-O157 LPS antibodies are conjugated to fluorescein, peroxidase or phosphatase are also available. Of
the enzyme immunoassays, the most commonly used format is a sandwich assay. Antibody is bound to a
carrier surface to capture a specific VTEC antigen; following the addition of an appropriate substrate, a
second antibody with an enzyme label binds to this antigen and produces a colour reaction. The kits have
been validated with specific pre-enrichment protocols and reagents to ensure reproducible results. Some
use heat-treated samples thus improving the safety of the test, and some incorporate an automated
processing system to screen large numbers of samples. Others are blot ELISAs developed to screen
colonies for O157 antigen. The commercial kits have the advantage of being easy to perform in routine
laboratories, and tests should be carried out according to the manufacturers instructions. Kits validated for
food and carcase samples or for human clinical samples may lack sensitivity for animal faeces samples.
Immunological assays only give a presumptive result, which must be confirmed by isolation and
characterisation of the organisms producing the O157 antigen or the toxin. The availability of kits is changing
and the OIE Reference Laboratories should be able to provide the latest information on validated diagnostic
kits.
b)

i)
Colony hybridisation assays

Colony hybridisation is a useful means of detecting VTEC in mixed culture for further characterisation.
DNA probes and synthetic oligonucleotide probes are available labelled with digoxigenin or biotin and
therefore suitable for use in routine diagnostic laboratories. Assays have been described to detect VT
genes, the 60 MDa plasmid in E. coli O157 and the eae gene individually and in combination (Nataro &
Kaper, 1998; Paton & Paton, 1998; Strockbine et al., 1998). Hybridisation assays are less sensitive for
detecting VTEC in broth cultures or faecal extracts.
ii)
PCR for VT genes and other virulence markers

Many PCRs are described in the literature for detection of VT1, VT2 and VT2 variant genes (Nataro &
Kaper, 1998; Paton & Paton, 1998; Strockbine et al., 1998), and a number of these toxin-typing PCR
methods has recently been compared (Ziebell et al., 2002). Demonstration of the genes associated
with VT-production does not confirm gene expression and hence production of toxin. PCR can be used
on pure or mixed plate or broth cultures, and extracts from food or faeces. It can also be used to detect
genes in non-viable organisms. As well as its role in diagnosis, PCR has the potential to be used to
screen samples for VTEC in epidemiological studies. Amplification of target genes in bacterial DNA
extracts from faeces is less successful than from pure cultures, and careful preparation of the sample
is required to improve sensitivity. Faeces contain nonspecific PCR inhibitors and no single method of
removing these is ideal. Sensitivity is improved by nonselective enrichment prior to testing, but remains
lower than using IMS or the Vero cell cytotoxicity assay. Commercial assays are available.
DNA probes, PCR assays and microarrays have also been developed to detect other genes in VTEC
shown to be associated with virulence in humans, including eae (encoding for intimin), ehx (encoding
for enterohaemolysin production), fliC (encoding the H7 antigen), O157 rfb (encoding O157 LPS), uidA
(the mutant glucuronidase gene in beta-glucuronidase-negative E. coli O157:H7) and katP (a gene
1302
carried on the large plasmid of E. coli O157:H7 encoding a novel catalase peroxidase) (Bekal et al.,
2003; Nataro & Kaper, 1998; Paton & Paton, 1998; Strockbine et al., 1998). A variety of multiplex
assays has been developed to detect simultaneously several diagnostic genes. These assays are of
value in the characterisation of pure cultures. On mixed populations of bacteria in food or faeces
samples, they may have a use in identifying samples to which isolation procedures should be targeted.
Screening faeces for Escherichia coli O157:H7
Escherichia coli O157:H7 is the VTEC of greatest public health concern in most countries. Its carriage in the
intestinal tract of healthy animals, particularly cattle, represents a source of direct and indirect infection to
humans. Screening relies on cultural techniques designed to overcome the problems of isolating low numbers of
organisms, possibly in a stressed state, from a competing background flora followed by identification of suspect
colonies and demonstration of known virulence characteristics. These methods are still evolving and the following
is a description of the methods routinely employed in one national veterinary laboratory. Suitable precautions
should be taken to avoid human infection (see chapter 1.1.3).
a)
b)
c)
d)
Pre-enrichment
i)
Transport faeces in sterile, leak proof, closed containers at 4C and culture as soon as possible,
preferably within 2 hours of collection. Faeces intended for long-term storage should be frozen at
70C.
ii)
Mix faeces at a dilution of 1/10 in warmed buffered peptone water (BPW) in a labelled container.
iii)
Incubate at 37C2C for 6 hours.
iv)
Include positive and negative control cultures.
Immunomagnetic separation
i)
Use of Dynabeads anti-E. coli O157 product 710.04 (Dynal Biotech, ASA, Oslo, Norway) meets the
requirements of AFNOR (DYN 16/02-0696 and DIN 10167); it is cited in the USA Food and Drug
Administrations Bacteriological Analytical Manual and the Health Canada Compendium of Analytical
Methods and is the official method of the Japanese Health Ministry.
ii)
Following the instructions of the manufacturers, carry out immunomagnetic separation (IMS) on the
pre-enriched samples using the manual (MIMS) or automated (AIMS) method. Care should be taken to
mix the beads well before use and to avoid cross-contamination between prepared tubes. If using the
manual method, adherence to the instructions for careful washing of the beadbacteria complexes is
essential.
iii)
After the final wash, use a micropipette to transfer 50 l of each beadbacteria suspension to a labelled
sorbitol MacConkey agar plate containing cefixime and potassium tellurite (CT-SMAC) (Zadik et al.,
1993) taking care to avoid cross-contamination.
iv)
Using a sterile swab, spread the drop over one-third to one-half of the plate to break up the complexes.
Using a sterile 10 l loop, dilute the beadbacteria complexes further over one quadrant by streaking
out at right angles from the previously streaked area. Using a second sterile loop, streak out at right
angles from this quadrant into the final unstreaked area of the plate to obtain single colonies. Incubate
at 37C2C for 1618 hours (sorbitol-fermenting colonies lose colour after this time and may be
confused with non-sorbitol fermenting E. coli O157). An alternative method for isolating sorbitolnegative colonies is to spread the entire inoculum over the surface of a dry CT-SMAC plate with a
sterile bent rod.
Colony identification
i)
Pick off up to 10 white, sorbitol-negative colonies per plate and test by O157 latex agglutination
following the manufacturers instructions (include appropriate positive and negative control organisms
and latex control).
ii)
Subculture agglutination-positive colonies on to solid medium without antibiotics (e.g. 5% sheep blood
agar). Streak to obtain single colonies. Incubate at 37C2C overnight.
Confirmation of Escherichia coli

i)
Inoculate o-nitrophenyl beta-D-galactopyranoside (ONPG) broth. Set up positive and negative controls.
Incubate overnight, aerobically at 37C. Escherichia coli produce a positive result indicated by a
change to yellow colouration confirming beta-galactosidase activity.
ii)
Place a circle of 0.45 m cellulose nitrate membrane filter paper on to a plate of tryptone bile agar
(TBA) using sterile forceps. Use a 1 l loop to remove a loopful of growth to be tested and inoculate a
1303
pea-sized area on the surface of the Millipore filter. Set up positive and negative controls. Incubate at
44C for at least 17 hours. Transfer the membrane to filter paper soaked with indole reagent for the
detection of the use of tryptophan. Escherichia coli show a positive reaction indicated by a purple/pink
colouration.
e)
f)
iii)
A commercial reagent for detection of indole is available. The reagent is placed on to filter paper and a
portion of the colony rubbed into the reagent spot. This requires less than 5 minutes and can be
backed up by the described test if suspicious colonies appear negative. Indole medium is also
available commercially.
iv)
Alternatively, use commercially available biochemical test kits to confirm E. coli.
Somatic determination (Matthews et al., 2006)

i)
Using a sterile loop, pick off a single colony obtained by the colony-identification method described in
point c above and subculture into 4 ml of Schlecht broth (Ring & Schlecht, 1970). Incubate at
37C2C overnight.
ii)
Boil the Schlecht broth for a minimum of 1 hour at 100C.
iii)
Dispense 25 l of 0.85% saline into wells 2 to 12 of a U-well microtitre plate. Dispense 50 l of O157
antiserum into well 1. Make a doubling-dilution series of the antiserum to 1/1024, discarding 25 l after
mixing well. Add 50 l of boiled broth suspension to wells 1 to 12. Cover the plate to prevent
evaporation and incubate at 37C for 6 hours. Use a black background to identify agglutination in the
wells.
Vero cell assay

i)
point c above and subculture into 4 ml of Mundell et al. (1976) broth. Incubate at 37C2C overnight.
ii)
Set up broths with control strains of organisms producing no toxin, thermolabile enterotoxin (LT),
cytotoxic necrotising factor (CNF) and verocytotoxin (VT). Incubate at 37C2C overnight.
iii)
Dispense Vero cells (African green monkey kidney cells, reference ATCC CCL81, seeding rate
2 105/ml) into flat-well microtest plates, 200 l to each well, 24 hours before inoculation. Incubate at
37C2C in 5% CO2 for 24 hours.
iv)
Add 100 l of a 400,000 units/ml solution of polymyxin B sulphate in sterile distilled water to each
overnight broth culture. Incubate at 37C2C for 5 hours.
vi)
Centrifuge the broths at 3000 rpm for 30 minutes.
vii)
Remove supernatants into labelled sterile containers (approximately 1.5 ml required).
viii) Place the Vero cell plate on a numbered worksheet to identify each well. Inoculate 10 l of prepared
supernatant into the relevant well of Vero cells. Return Vero cells to the CO2 incubator and incubate for
3 days.
ix)
g)
Examine cells after 24 hours, 48 hours and 72 hours to observe any cytopathic effect. Compare with
positive and negative test controls. With VT-positive samples, the cell sheet becomes disintegrated
with blackened, shrivelled cells observed between 24 and 72 hours.
Multiplex PCR for VT1, VT2 and eae (Beebakhee et al., 1992; Jackson et al., 1987; Strockbine et
al., 1988)
Multiplex PCR is used to confirm the presence of virulence determinants using primers as shown below:
Target
gene
VT1
VT2
eaeA
1304
Accession
no.
M19437
X07865
X60439
Primer sequence
Nucleotide
position
Amplicon
size (bp)
F (5-CGC-TCT-GCA-ATA-GGT-ACT-CC-3)
287306
256
R (5-CGC-TGT-TGT-ACC-TGG-AAA-GG-3)
522541
F (5-TCC-ATG-ACA-ACG-GAC-AGC-AG-3)
623642
R (5-GC-TTC-TGC-TGT-GAC-AGT-GAC-3)
788807
F (5-GC-TTA-GTG-CTG-GTT-TAG-GAT-TG-3)
271293
R (5-CCA-GTG-AAC-TAC-CGT-CAA-AG-3)
871890
185
618
i)
point c above and subculture into 1 ml of Luria-Bertani broth. Set up three appropriate control broths.
Incubate at 37C2C overnight.
ii)
Boil the broths for 15 minutes at 100C. Remove from waterbath and allow to cool.
iii)
Prepare master mix for 48 l per sample containing:

1 Saiki buffer (50 mM KCl; 10 mM Tris, pH 8.5; 100 g/ml gelatin); 3 mM MgCl2; 0.5 U Taq
polymerase; 25 pmoles of each primer (forward and reverse primers for VT1, VT2 and eaeA); 0.2 mM
each of dATP, dCTP, dGTP and dTTP.
iv)
Mix by inverting tubes and dispense 48 l into each PCR reaction tube.
v)
Add 2 l of boiled culture (crude DNA extract) to the bottom of each reaction tube (include three control
extracts and a media blank).
vi)
Run the PCR using cycling parameters of initial denaturation at 94C for 2 minutes; 25 cycles of 94C
for 1 minute, 62C for 1.5 minutes and 72C for 2 minutes; with a final extension of 72C for 5 minutes.
The reaction is held at 4C until required for electrophoresis.
vii)
Electrophorese 15 l of each PCR sample on a 1.5% agarose gel in E buffer (10 strength solution
made by adding to distilled water in the following order: 109 g/litre Tris, 55.6 g/litre ortho-boric acid,
9.3 g EDTA, made up to 1 litre with distilled water and adjusted to pH 8.0 with 10 ml concentrated
hydrochloric acid diluted in distilled water before use). Run 100 bp step ladder molecular weight marker
for comparison.
ix)
Stain in ethidium bromide and view by transillumination.
x)
Inspect control lanes to identify positions of VT1, VT2 and eae amplicons. Compare with bands present
in test sample lanes. Record the results.
2. Serological tests
In humans, serodiagnosis of VTEC can be valuable, particularly later in the course of the disease when the
causative organism becomes increasingly difficult to isolate from faeces. LPS has emerged as the antigen of choice,
and production of serum antibodies to the LPS of a wide range of prevalent serotypes of VTEC has been
demonstrated. Serological tests are not used for diagnosis of animal infection with VTEC. However, it has been
shown that exposure of cattle to E. coli O157:H7 infection results in the production of antibodies against the O157
LPS, which persist for months, demonstrable by the indirect ELISA (Johnson et al., 1996b). Cross-reactions have
been demonstrated between O157-LPS and the LPS antigens of other bacteria including E. coli O55, Salmonella
spp., Yersinia enterocolitica, Brucella abortus and V. cholerae non-O1 strains. To reduce cross-reactivity, a blocking
ELISA using a monoclonal antibody specific for E. coli O157 as the competing antibody has been developed for
detection of serum antibodies to O157 antigen in cattle (Laegreid et al., 1998). Serum antibodies to VT1, but not to
VT2, have been demonstrated in cattle by toxin neutralisation tests in Vero cell assays (Johnson et al., 1996b). Other
studies have shown greater prevalence of VT1 neutralising antibodies in cattle sera than VT2 which may be
explained by the greater prevalence of VT1-producing VTEC in cattle and/or the lesser immunogenicity of VT2.

There are currently no vaccines available to control zoonotic VTEC. Various approaches to the immunological
control of EHEC infections in humans are being explored (Levine, 1998). These are aimed at preventing
colonisation, intestinal disease or the serious sequelae of haemolytic uraemic syndrome and thrombotic
thrombocytopenic purpura. They include the use of conjugate vaccines (e.g. O157 polysaccharide linked to the Bsubunit of VT1 and VT2 as carrier proteins), live-vector vaccines, toxoid vaccine or passive immunisation with
hyperimmune globulin or monoclonal antibodies against VT. However, were an effective vaccine to become
available, there is debate about the social, political and economic consequences of widespread vaccination of
people against pathogens in their food. As animals, mainly cattle, are thought to be the reservoirs of infection for
the human population, a novel strategy being explored is to vaccinate cattle in order to reduce colonisation with
pathogenic VTEC and thereby reduce contamination of food and the environment (i.e. to make food safer as
opposed to protecting people against their food). One approach is to use a live, toxin-negative colonising strain as
an oral vaccine to induce antibodies against surface components, and another is to deliver colonisation factors,
such as intimin, as an edible vaccine in transgenic plants (Gyles, 1998).
REFERENCES
BEEBAKHEE G., LOUIE M., DE AZAVEDO J. & BRUNTON J. (1992). Cloning and nucleotide sequence of the eae gene
homologue from enterohemorrhagic Escherichia coli serotype O157:H7. FEMS Microbiol. Lett., 91, 6368.
1305
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identifying Shiga toxins. Res. Microbiol., 153, 289300.
*
* *
NB: There is an OIE Reference Laboratory for Escherichia coli
diagnostic tests and reagents for E. coli
1307
CHAPTER 2.9.12.
ZOONOSES TRANSMISSIBLE
FROM NON-HUMAN PRIMATES
For standards for testing non-human primates, please consult the following document:
Health monitoring of non-human primate Colonies. Recommendations of the Federation of
European Laboratory Animal Science Associations (FELASA) Working Group on Non-Human
Primate Health accepted by the FELASA Board of Management, 21 November 1998.
http://la.rsmjournals.com/cgi/reprint/33/suppl_1/3.pdf
*
* *
1308
PART 3
GENERAL GUIDELINES
1309
GUIDELINE 3.1.
LABORATORY METHODOLOGIES FOR BACTERIAL

ANTIMICROBIAL SUSCEPTIBILITY TESTING
SUMMARY
Historically, medical practitioners and veterinarians selected antimicrobials to treat bacterial
infectious diseases based primarily on past clinical experiences. However, with the increase in
bacterial resistance to traditionally used antimicrobials, it has become more difficult for clinicians to
empirically select an appropriate antimicrobial agent. As a result, in-vitro antimicrobial susceptibility
testing (AST) of the relevant bacterial pathogens, from properly collected specimens, should use
validated methods. Thus, AST is an important component of prudent antimicrobial use guidelines in
animal husbandry worldwide and veterinarians in all countries should have these data available for
informed decision-making.
Although a variety of methods exist, the goal of in-vitro antimicrobial susceptibility testing is to
provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the
infected host. This type of information aids the clinician in selecting the appropriate antimicrobial
agent, aids in developing antimicrobial use policy, and provides data for epidemiological
surveillance. Such epidemiological surveillance data provide a base to choose the appropriate
empirical treatment (first-line therapy) and to detect the emergence and/or the dissemination of
resistant bacterial strains or resistance determinants in different bacterial species. The selection of
a particular AST method is based on many factors such as validation data, practicality, flexibility,
automation, cost, reproducibility, accuracy, and individual preference.
The use of genotypic approaches for detection of antimicrobial resistance genes has also been
promoted as a way to increase the speed and accuracy of susceptibility testing. Numerous DNAbased assays are being developed to detect bacterial antibiotic resistance at the genetic level.
These methods, when used in conjunction with phenotypic analysis, offer the promise of increased
sensitivity, specificity, and speed in the detection of specific known resistance genes and can be
used in tandem with traditional laboratory AST methods.
INTRODUCTION
The spread of multiple antimicrobial-resistant pathogenic bacteria has been recognised by the World Organisation
for Animal Health (OIE), the Food and Agriculture Organization (FAO) and the World Health Organization (WHO)
as a serious global human and animal health problem. The development of bacterial antimicrobial resistance is
neither an unexpected nor a new phenomenon. It is, however, an increasingly troublesome situation because of
the frequency with which new emerging resistance phenotypes are occurring among many bacterial pathogens
and even commensal organisms.
Historically, many infections could be treated successfully according to the clinicians past clinical experience (i.e.
empirical therapy); however, this is becoming more the exception than the rule (Walker, 2007). Resistance has
been observed to essentially all of the antimicrobial agents currently approved for use in human and veterinary
clinical medicine. This, combined with the variety of antimicrobial agents currently available, makes the selection
of an appropriate agent an increasingly more challenging task. This situation has made clinicians more dependent
on data from in-vitro antimicrobial susceptibility testing, and highlights the importance of the diagnostic laboratory
in clinical practice.
A number of antimicrobial susceptibility testing (AST) methods are available to determine bacterial susceptibility
to antimicrobials. The selection of a method is based on many factors such as practicality, flexibility, automation,
cost, reproducibility, accuracy, and individual preference. Standardisation and harmonisation of AST
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Guideline 3.1. Laboratory methodologies for bacterial antimicrobial susceptibility testing
methodologies, used in epidemiological surveillance of antimicrobial drug resistance, are critical if data are to be
compared among national or international surveillance/monitoring programmes of OIE Members. It is essential
that AST methods provide reproducible results in day-to-day laboratory use and that the data be comparable with
those results obtained by an acknowledged gold standard reference method. In the absence of standardised
methods or reference procedures, susceptibility results from different laboratories cannot be reliably compared.
The method used to select samples for inclusion in antimicrobial resistance surveillance programmes, as well as
the methods used for primary bacterial isolation, are also important factors that should be standardised or
harmonised to allow direct comparison of data between different regions; consideration of these issues is
addressed in an OIE document (Dehaumont, 2004).
As the science of AST has progressed, a greater understanding of the multiple factors that could affect the overall
outcome of susceptibility testing has become clearer. This document provides guidelines and standardisation for
AST methodologies, and interpretation of antimicrobial susceptibility test results.
1.
Test requirements
In order to achieve standardisation of AST methods and comparability of AST results, the following requirements
apply:
i)
the use of standardised AST methods and the harmonisation of AST test parameters (including choice of
antimicrobial agents and subsequent interpretive criteria) are essential,
ii)
standardised AST methods, including all critical specifications and interpretive criteria, should be clearly
defined, documented in detail and used by all participating laboratories,
iii)
all AST methods should generate accurate and reproducible data,
iv)
all data should be reported quantitatively,
v)
establishment of national or regional designated laboratories is essential for the coordination of AST
methodologies, interpretations and appropriate operational techniques used to ensure accuracy and
reproducibility (e.g. quality controls),
vi)
microbiological laboratories should implement and maintain a formal quality management programme (see
Chapter 1.1.4 Quality management in veterinary testing laboratories),
vii)
laboratories should have acquired a third party accreditation that includes the AST methodologies to be used
within the scope of that accreditation. The accreditation body should meet accepted international Laboratory
Accreditation Cooperation [ILAC]) standards and guidelines regarding the standards used for the
accreditation process. The accreditation standards used should include the requirement for participation in
proficiency testing programmes,
viii) specific bacterial reference/quality control strains are essential for determining intra- and inter-laboratory
quality control, quality assurance and proficiency testing.
2.
Selection of antimicrobials for testing and reporting
Selecting the appropriate antimicrobials for susceptibility testing can be difficult given the vast numbers of agents
available. The following guidelines should be noted:
i)
the FAO/OIE/WHO expert workshop on non-human antimicrobial usage and antimicrobial resistance
recommends creating a list of veterinary and human critically important antimicrobials for susceptibility
testing and reporting,
ii)
selection of the most appropriate antimicrobials is a decision best made by each OIE Member in consultation
with the appropriate bodies and organisations,
iii)
antimicrobials in the same class may have similar in-vitro activities against select bacterial pathogens. In
these cases, a representative antimicrobial should be selected that predicts susceptibility to other members
of the same class,
iv)
certain microorganisms can be intrinsically resistant to particular antimicrobial classes; therefore it is

unnecessary and misleading to test certain agents for activity in vitro. The type of intrinsic resistance has to
be determined for these organisms via either the scientific literature or through testing,
v)
the number of antimicrobials to be tested should be limited in order to ensure the relevance and practicality
of AST.
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Periodic review of microorganisms that are currently predictably susceptible to certain antimicrobial agents is
recommended to ensure that emergent, unexpected resistance is detected. Emerging resistance may also be
suspected following poor response to a standard antimicrobial treatment regime.
3.
Antimicrobial susceptibility testing methodologies
The following requirements should be respected:

i)
bacteria subjected to AST must be isolated in pure culture from the submitted sample,
ii)
standard reference methods should be used for identification so that the subject bacteria are consistently
and correctly identified to the genus and/or species level,
iii)
bacterial isolates considered to be the most important and a sampling of other isolates, should be stored for
future analysis (either lyophilisation or cryogenic preservation at 70C to 80C).
The following factors influencing AST methods should be determined, optimised, and documented in a detailed
standard operating procedure:
i)
once the bacterium has been isolated in pure culture, the optimum concentration of the inocula must be
determined to obtain accurate susceptibility results. Bacteria or other organisms used in AST testing should
be from a fresh culture,
ii)
the composition and preparation of the agar and broth media used (e.g. pH, cations, thymidine or thymine,
use of supplemented media). Performance and sterility testing of media lots should also be determined and
documented as well as employed procedures,
iii)
the content of antimicrobial in the carrier (antibiotics used in microtitre plates, disk, strip, tablet),
iv)
composition of solvents and diluents for preparation of antimicrobial stock solutions,
v)
growth and incubation conditions (time, temperature, atmosphere e.g. CO2),
vi)
agar depth,
vii)
number of concentrations tested per broth and agar dilution,
viii) the test controls to be used, including the reference organisms used,
ix)
the subsequent interpretive criteria (clinical breakpoints, epidemiological cut-off values).
For these reasons, special emphasis has to be placed on the use of documented procedures and validated, well
documented methods, as sufficient reproducibility can be attained only through the use of such methodology.
4.
Selection of antimicrobial susceptibility testing methodology
The selection of an AST methodology may be based on the following factors:

i)
ease of performance,
ii)
flexibility,
iii)
adaptability to automated or semi-automated systems,
iv)
cost,
v)
reproducibility,
vi)
reliability,
vii)
accuracy,
viii) the organisms and the antimicrobials of interest in that particular OIE Member,
ix)
availability of suitable validation data for the range of organisms to be susceptibility tested.
5.
Antimicrobial susceptibility testing methods
The following three methods have been shown to consistently provide reproducible and repeatable results when
followed correctly (Clinical and Laboratory Standards Institute (CLSI), 2008; Walker, 2007):
1313
i)
disk diffusion,
ii)
broth dilution,
iii)
agar dilution.
a)
Disk diffusion method

Disk diffusion refers to the diffusion of an antimicrobial agent of a specified concentration from disks, tablets
or strips, into the solid culture medium that has been seeded with the selected inoculum isolated in a pure
culture (see section 3.i). Disk diffusion is based on the determination of an inhibition zone proportional to the
bacterial susceptibility to the antimicrobial present in the disk.
The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the
antimicrobial. When the concentration of the antimicrobial becomes so diluted that it can no longer inhibit the
growth of the test bacterium, the zone of inhibition is demarcated. The diameter of this zone of inhibition
around the antimicrobial disk is related to minimum inhibitory concentration (MIC) for that particular
bacterium/antimicrobial combination; the zone of inhibition correlates inversely with the MIC of the test
bacterium. Generally, the larger the zone of inhibition, the lower the concentration of antimicrobial required
to inhibit the growth of the organisms. However, this depends on the concentration of antibiotic in the disk
and its diffusibility.
Note: Disk diffusion tests based solely on the presence or absence of a zone of inhibition without regard to
the size of the zone of inhibition are not acceptable AST methodology.
Considerations for the use of the disk diffusion methodology
Disk diffusion is straightforward to perform, reproducible, and does not require expensive equipment. Its
main advantages are:
i)
low cost,
ii)
ease in modifying test antimicrobial disks when required,
iii)
can be used as a screening test against large numbers of isolates,
iv)
can identify a subset of isolates for further testing by other methods, such as determination of MICs.
Manual measurement of zones of inhibition may be time-consuming. Automated zone-reading devices are
available that can be integrated with laboratory reporting and data-handling systems. The disks should be
distributed evenly so that the zones of inhibition around antimicrobial discs in the disc diffusion test do not
overlap to such a degree that the zone of inhibition cannot be determined. Generally this can be
accomplished if the discs are no closer than 24 mm from centre to centre, though this is dependent on disk
concentration and the ability of the antimicrobial to diffuse in agar.
b)
Broth and agar dilution methods

The aim of the broth and agar dilution methods is to determine the lowest concentration of the assayed
antimicrobial that inhibits the visible growth of the bacterium being tested (MIC, usually expressed in g/ml
or mg/litre). However, the MIC does not always represent an absolute value. The true MIC is a point
between the lowest test concentration that inhibits the growth of the bacterium and the next lower test
concentration. Therefore, MIC determinations performed using a dilution series may be considered to have
an inherent variation of one dilution.
Antimicrobial ranges should encompass both the interpretive criteria (susceptible, intermediate and
resistant) for a specific bacterium/antibiotic combination and appropriate quality control reference organisms.
Antimicrobial susceptibility dilution methods appear to be more reproducible and quantitative than agar disk
diffusion. However, antibiotics are usually tested in doubling dilutions, which can produce inexact MIC data.
Any laboratory that intends to use a dilution method and set up its own reagents and antibiotic dilutions
should have the ability to obtain, prepare and maintain appropriate stock solutions of reagent-grade
antimicrobials and to generate working dilutions on a regular basis. It is then essential that such laboratories
use quality control organisms (see below) to assure accuracy and standardisation of their procedures.
Broth dilution
Broth dilution is a technique in which a suspension of bacterium of a predetermined optimal or appropriate

concentration is tested against varying concentrations of an antimicrobial agent (usually serial twofold
1314
dilutions) in a liquid medium of predetermined, documented formulation. The broth dilution method can be
performed either in tubes containing a minimum volume of 2 ml (macrodilution) or in smaller volumes using
microtitration plates (microdilution). Numerous microtitre plates containing lyophilised prediluted antibiotics
within the wells are commercially available. The use of identical lots in microdilution plates may assist in the
minimisation of variation that may arise due to the preparation and dilution of the antimicrobials from
different laboratories. The use of these plates, with a documented test protocol, including specification of
appropriate reference organisms, will facilitate the comparability of results among laboratories.
Due to the fact that most broth microdilution antimicrobial test panels are prepared commercially, this
method is less flexible than agar dilution or disk diffusion in adjusting to the changing needs of the
surveillance/monitoring programme.
Because the purchase of antimicrobial plates and associated equipment may be costly, this methodology
may not be feasible for some laboratories.
Agar dilution
Agar dilution involves the incorporation of varying concentrations of antimicrobial agent into an agar medium,
usually using serial twofold dilutions, followed by the application of a defined bacterial inoculum to the agar
surface of the plate. These results are often considered as the most reliable for the determination of an MIC
for the test bacterium/antimicrobial combination.
The advantages of agar dilution methods include:
i)
the ability to test multiple bacteria, except bacteria that swarm, on the same set of agar plates at the
same time,
ii)
the potential to improve the identification of MIC endpoints and extend the antibiotic concentration
range,
iii)
the possibility to semi-automate the method using an inoculum-replicating apparatus. Commercially

produced inoculum replicators are available and these can transfer between 32 and 60 different
bacterial inocula to each agar plate,
Agar dilution methods also have certain disadvantages, for example:

i)
if not automated, they are very laborious and require substantial economic and technical resources,
ii)
once the plates have been prepared, they normally should be used within a week (or less, depending
on the antimicrobials tested),
iii)
the endpoints are not always easy to read nor is the purity of the inoculum easy to verify.
Agar dilution is often recommended as a standardised AST method for fastidious organisms (CLSI, 2006c),
such as anaerobes and Helicobacter species.
c)
Other bacterial AST and specific antimicrobial resistance tests

Bacterial antimicrobial MICs can also be obtained using commercially available gradient strips that diffuse a
predetermined antibiotic concentration. However, the use of gradient strips can be very expensive and MIC
discrepancies can be found when testing certain bacteria/antimicrobial combinations compared with agar
dilution results (Ge et al., 2002; Rathe et al., 2009).
Regardless of the AST method used, the procedures should be documented in detail to ensure accurate and
reproducible results, and appropriate reference organisms should always be tested every time AST is
performed in order to ensure accuracy and validity of the data.
The appropriate AST choice will ultimately depend on the growth characteristics of the bacterium in
question. In special circumstances, novel test methods and assays may be more appropriate for detection of
particular resistance phenotypes. For example, chromogenic cephalosporin-based tests (CLSI, 2008) (e.g.
nitrocefin) may provide more reliable and rapid results for beta-lactamase determination in certain bacteria,
whereas inducible clindamycin resistance in Staphylococcus spp. may be detected using a disk diffusion
method employing standard erythromycin and clindamycin disks in adjacent positions and measuring the
resultant zones of inhibition (e.g. D-zone or D-test) (Zelazny et al., 2005).
Similarly, extended-spectrum beta-lactamase (ESBL) (CLSI, 2008) activity in certain bacteria can also be
detected by using standard disk diffusion susceptibility test methods incorporating specific cephalosporins
(cefotaxime and ceftazidime) in combination with a beta-lactamase inhibitor (clavulanic acid) and measuring
1315
the resulting zones of inhibition. Also penicillin-binding protein 2a (PBP 2a) can be detected in methicillin
resistant staphylococci with a latex agglutination test (Stepanovic et al., 2006). It is essential that testing of
known positive and negative control strains occurs alongside clinical isolates to ensure accurate results.
d)
Future directions in antimicrobial susceptibility/resistance detection

The use of genotypic approaches for detection of antimicrobial resistance genes has been promoted as a
way to increase the rapidity and accuracy of susceptibility testing (Cai et al., 2003; Chen et al., 2005).
Numerous DNA-based assays are being developed to detect bacterial antibiotic resistance at the genetic
level. The newest and perhaps most state-of-the-art approach is to predict antimicrobial resistance
phenotypes via identification and characterisation of the known genes that encode specific resistance
mechanisms.
Methods that employ the use of comparative genomics, genetic probes, microarrays, nucleic acid
amplification techniques (e.g. polymerase chain reaction [PCR]), and DNA sequencing offer the promise of
increased sensitivity, specificity, and speed in the detection of specific known resistance genes (Cai et al.,
2003; Chen et al., 2005; Perreten et al., 2005). Genotypic methods have been successfully applied to
supplement traditional AST phenotypic methods for other organisms including methicillin-resistant
staphylococci, vancomycin-resistant enterococci, and detection of fluoroquinolone resistance mutations (Cai
et al., 2003; Chen et al., 2005; Perreten et al., 2005). PCR methods have also been described for betalactamases, aminoglycoside inactivating enzymes, and tetracycline efflux genes (Cai et al., 2003; Chen et
al., 2005; Frye et al., 2010; Perreten et al., 2005).
Technological innovations in DNA-based diagnostics should allow for the detection of multiple resistance
genes and/or variants during the same test. The development of rapid diagnostic identification methods and
genotypic resistance testing should help reduce the emergence of antimicrobial resistance, by enabling the
use of the most appropriate antimicrobial when therapy is initiated. However, DNA techniques have to be
demonstrated to be complementary to AST methods and results.
Additionally, new technological advances may facilitate the ability to probe bacterial species for large
numbers of antimicrobial resistance genes quickly and cheaply, thereby providing additional relevant data for
surveillance and monitoring programmes (Frye et al., 2010). However, despite the new influx of genotypic
tests, documented and agreed upon phenotypic AST methods will still be required in the near future to
detect emerging resistance mechanisms among bacterial pathogens.
6.
Antimicrobial susceptibility breakpoints and zone of inhibition criteria
The objective of in-vitro AST is to predict how a bacterial pathogen may respond to the antimicrobial agent in vivo.
The results generated by bacterial in-vitro antimicrobial susceptibility tests, regardless of whether disk diffusion or
dilution methods are used, are generally interpreted and reported as resistant, susceptible or intermediate to the
action of a particular antimicrobial. No single formula for selection of optimal breakpoints has been established.
The process involves a review of existing data and is influenced by the subjectivity of individuals tasked with
selecting the appropriate breakpoints.
Generally, antimicrobial susceptibility breakpoints are established by national standards organisations,
professional societies or regulatory agencies. The relevant documents should be consulted. However, there can
be notable differences in breakpoints for the same antimicrobial agent within and among countries due to
differences between standards setting organisations and regulatory agencies and because of regional or national
decisions on dosing regimens (Brown & MacGowan, 2010; de Jong et al., 2009; Kahlmeter et al., 2006).
As mentioned previously, antimicrobial susceptibility testing results should be recorded quantitatively:
i)
as distribution of MICs in milligrams per litre or g/ml,
ii)
or as inhibition zone diameters in millimetres.
The following two primary factors enable a bacterium to be interpreted as susceptible or resistant to an
antimicrobial agent:
i)
The development and establishment of quality control ranges (CLSI, 2006c), using diffusion when possible
and dilution testing, for quality control reference microorganisms.
Establishment of quality control ranges is essential for validating test results obtained using a specific AST
method. The allowable interpretive category ranges for the reference organisms should be established prior
to determining breakpoints for susceptibility or resistance. The use of reference organisms is a quality
control and quality assurance activity. However, it is only necessary to require the use of reference
organisms.
1316
ii)
The determination of the appropriate interpretive criteria regarding establishment of breakpoints (CLSI,
2006c).
This involves the generation of three distinct types of data:
MIC population distributions of the relevant microorganisms,
pharmacokinetic parameters and pharmacodynamic indices of the antimicrobial agent,
results of clinical trials and experience.
The interpretation of the data involves creating a scattergram from the bacterial population distribution
(representative bacterial species), by plotting the zone of inhibition against the logarithm to the base 2 of the MIC
for each bacterial pathogen. The selection of breakpoints is then based on multiple factors, including regression
line analysis that correlates MICs and zone diameters of inhibition, bacterial population distributions, error rate
bounding, pharmacokinetics, and ultimately, clinical verification.
The development of a concept known as microbiological breakpoints, or epidemiological cut-off values, which is
based on the population distributions of the specific bacterial species tested, may be more appropriate for some
antimicrobial surveillance programmes. In this case, bacterial isolates that deviate from the normal wild-type
susceptible population would be designated as resistant, and shifts in susceptibility to the specific
antimicrobial/bacterium combination could be monitored (Kahlmeter et al., 2006; Turnidge et al., 2006). There is a
great advantage in the recording of quantitative susceptibility data in that such data may be analysed according to
clinical breakpoints as well as by using epidemiological cut-off values.
7.
Antimicrobial susceptibility testing guidelines
A number of standards and guidelines are currently available for antimicrobial susceptibility testing and
subsequent interpretive criteria throughout the world (CLSI, 2008; Kahlmeter et al., 2006). Amongst others, these
include standards and guidelines published by:
British Society for Antimicrobial Chemotherapy (BSAC, UK),
Clinical Laboratory and Standards Institute (CLSI, USA),
Comit de lAntibiogramme de la Socit franaise de Microbiologie (CASFM, France),
Commissie richtlijnen gevoeligheidsbepalingen (CRG, the Netherlands),
Deutsches Institut fr Normung (DIN, Germany),
European Committee on Antimicrobial Susceptibility Testing (EUCAST),
Japanese Society for Chemotherapy (JSC, Japan),
Swedish Reference Group for Antibiotics (SRGA, Sweden).
At this time, only the CLSI (formerly NCCLS) has developed protocols for susceptibility testing of bacteria of
animal origin and determination of interpretive criteria (CLSI, 2008). However, protocols and guidelines are
available from a number of standards organisations and professional societies, including those listed above for
susceptibility testing for similar bacterial species that cause infections in humans. It is possible that such
guidelines can be adopted for susceptibility testing for bacteria of animal origin, but each country must evaluate its
own AST standards and guidelines. Additionally, efforts focusing on both standardisation and harmonisation of
susceptibility/resistance breakpoints on an international scale are progressing. These efforts have primarily
focused on the adoption of the standards and guidelines of CLSI and EUCAST, which provide laboratories with
methods and quality control values enabling comparisons of AST methods and generated data (CLSI, 2008;
Kahlmeter et al., 2006). For those OIE Members that do not have standardised AST methods in place, the
adoption of either set of standards would be an appropriate initial step towards acceptable methods and
harmonisation.
Many bacteria that cause disease in aquatic animals require growth conditions (e.g. lower temperatures,
supplemented or semisolid media) that may vary considerably as compared to terrestrial bacterial pathogens.
This necessitated the need for the development of antimicrobial testing methods for bacteria isolated from aquatic
species. Further information with regards to methods for disk diffusion or broth dilution antimicrobial susceptibility
testing for bacteria isolated from aquatic animals can be referenced in two CLSI documents (CLSI, 2006a;
2006b). Further information with regards to methods for disk diffusion or broth dilution antimicrobial susceptibility
testing for infrequently isolated or certain fastidious bacteria (e.g. Campylobacter, Pasteurella) can also be
referenced in the CLSI M45-A document (CLSI, 2006c).
As a first step towards comparability of monitoring and surveillance data, Members should be encouraged to
strive for harmonised and standardised programme design (Brown & MacGowan, 2010; Kahlmeter et al., 2006;
White et al., 2001). Data from countries using different methods and programme design may otherwise not be
directly comparable (Brown & MacGowan, 2010). Notwithstanding this, data collected over time in a given country
may at least allow the detection of emergence of antimicrobial resistance or trends in prevalence of
1317
susceptibility/resistance in that particular country (Petersen et al., 2003). However, if results achieved with
different AST methods are to be presented side by side, then comparability of results must be demonstrated and
consensus on interpretation achieved. This will be best accomplished by the use of accurate and reliable
documented AST methods used in conjunction with monitoring of AST performance while using well
characterised reference microorganisms among participating laboratories.
8.
Comparability of results
To determine the comparability of results originating from different surveillance systems, results should be
reported quantitatively including information on the performance of the methods, the reference organisms and the
antimicrobial.
AST data, consisting of cumulative and ongoing summary of susceptibility patterns (antibiograms) among
clinically important and surveillance microorganisms should be created, recorded and analysed periodically at
regular intervals (CLSI, 2009). Data must also be presented in a clear and consistent manner so that both new
patterns of resistance can be identified and atypical findings confirmed or refuted. This data should be available
on a central data bank and published yearly.
Cumulative AST data will be useful in monitoring susceptibility/resistance trends in a region over time and
assessing the effects of interventions to reduce antimicrobial resistance.
9.
Quality control (QC) and quality assurance (QA)
Quality control/quality assurance systems should be established in accordance with chapter 1.1.4 in laboratories
performing AST:
i)
quality control refers to the operational techniques that are used to ensure accuracy and reproducibility of
AST,
ii)
quality assurance includes, but is not limited to, monitoring, record keeping, evaluating, taking potential
corrective actions if necessary, calibration, and maintenance of equipment, proficiency testing, training and
QC. A QA programme helps ensure that testing materials and processes provide consistent quality results.
The following components should be determined and monitored:

i)
precision of the AST procedure,
ii)
accuracy of the AST procedure,
iii)
qualifications, competence, and proficiency of the laboratory personnel, as well as the personnel that
interpret the results and those that are involved in monitoring of antimicrobial resistance,
iv)
performance of the appropriate reagents.
The following requirements should be respected:

i)
Strict adherence to specified and documented techniques in conjunction with quality control (i.e. assurance
of performance and other critical criteria) of media and reagents.
ii)
Record keeping of:
lot numbers of all appropriate materials and reagents,
expiration dates of all appropriate materials and reagents,
equipment calibration and monitoring,
critical specifications for AST performance (reference results, time, temperature etc.).
iii)
The appropriate reference microorganism(s) should always be used regardless of the AST method
employed.
iv)
Reference microorganisms are to be obtained from a reliable source for example, from the American Type
Culture Collection (ATCC), reliable commercial sources, or institutions with demonstrated reliability to store
and use the organisms correctly.
v)
Reference microorganisms should be catalogued and well characterised, including stable defined
antimicrobial susceptibility phenotypes. Records regarding these reference organisms should include the
established resistant and susceptible ranges of the antimicrobials to be assayed, and the reference to the
method(s) by which these were determined.
1318
vi)
Laboratories involved in AST should use the appropriate reference microorganisms in all AST testing.
vii)
Reference strains should be kept as stock cultures from which working cultures are derived and should be
obtained from national or international culture collections. Reference bacterial strains should be stored at
designated centralised or regional laboratories. Working cultures should not be subcultured from day to day
as this introduces contamination and the method of producing working cultures should ensure that stock
cultures are rarely used. This may be accomplished with the production of an intermediate stock of cultures
derived from the original cultures that are used to crate day-to-day working cultures.
viii) The preferred method for analysing the overall performance of each laboratory should test the working stock
of the appropriate reference microorganisms on each day that susceptibility tests are performed.
Because this may not always be practical or economical, the frequency of such tests may be reduced if the
laboratory can demonstrate that the results of testing reference microorganisms using the selected method
are reproducible. If a laboratory can document the reproducibility of the susceptibility testing methods used,
testing may be performed on a weekly basis. If concerns regarding accuracy, reproducibility, or method
validity emerge, the laboratory has a responsibility to determine the cause(s) and repeat the tests using the
reference materials. Depending on the cause(s), daily reference material use and any other corrective action
may be re-initiated.
ix)
Reference microorganisms should be tested each time a new batch of medium or plate lot is used and on a
regular basis in parallel with the microorganisms to be assayed.
x)
Appropriate biosecurity issues should be addressed in obtaining and dispersing microorganisms to

participating laboratories.
10. External proficiency testing

Laboratories should participate in external quality assurance and/or proficiency testing programmes in
accordance with chapter 1.1.4. Laboratories are also encouraged to participate in international inter-laboratory
comparisons (e.g. WHO External Quality Assurance System) (Hendriksen et al., 2009). All bacterial species
subjected to AST should be included.
National reference laboratories should be designated with responsibility for:
i)
monitoring the quality assurance programmes of laboratories participating in surveillance and monitoring of
antimicrobial resistance,
ii)
characterising and supplying to those laboratories a set of reference microorganisms,
iii)
creating, managing, and distributing samples to be used in external proficiency testing,
iv)
creating a central database available on the internet (e.g. European Antimicrobial Resistance Surveillance
System [EARSS]) that contains the different susceptibility/resistance profiles for each bacterial species
under surveillance.
11. Conclusion
Although a variety of methods exist, the goal of in-vitro antimicrobial susceptibility testing is the same: to provide a
reliable predictor of how a microorganism is likely to respond to antimicrobial therapy in the infected host. This
type of information aids the clinician in selecting the appropriate antimicrobial agent, provides data for
surveillance, and aids in developing antimicrobial judicious use policies (World Organisation for Animal Health,
2010).
In-vitro antimicrobial susceptibility testing can be performed using a variety of formats, the most common being
disk diffusion, agar dilution, broth macrodilution, broth microdilution, and a concentration gradient test. Each of
these procedures requires the use of specific testing conditions and methods, including media, incubation
conditions and times, and the identification of appropriate quality control organisms along with their specific QC
ranges. It is essential that AST methods provide reproducible results in day-to-day laboratory use and that the
data be comparable with those results obtained by an acknowledged gold standard reference method. In the
absence of standardised methods or reference procedures, antimicrobial susceptibility/resistance results from
different laboratories cannot be reliably compared.
The use of genotypic approaches for detection of antimicrobial resistance genes has also been promoted as a
way to increase the rapidity and accuracy of susceptibility testing. Additionally, new technological advances in
molecular techniques (e.g. microarray) may facilitate the ability to probe bacterial species for large numbers of
antimicrobial resistance genes quickly and cheaply, thereby providing additional relevant data into surveillance
and monitoring programs (Ojha & Kostrzynska, 2008; Poxton, 2005). Despite the new influx of genotypic tests
1319
however, standardised phenotypic AST methods will still be required in the near future to detect emerging
resistance mechanisms among bacterial pathogens.
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CHEN S., ZHAO S., MCDERMOTT P.F., SCHROEDER C.M., WHITE D.G. & MENG J. (2005). A DNA microarray for
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Cumulative Antimicrobial Susceptibility Test Data; Approved Guideline, Third Edition. CLSI, 940 West Valley
Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA,
CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI) (2006a). Document M42-A, Methods for Antimicrobial Disk
Susceptibility Testing of Bacteria Isolated from Aquatic Animals; Approved Guideline CLSI, 940 West Valley
CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI) (2006b). Document M49-A, Methods for Broth Dilution
Susceptibility Testing of Bacteria Isolated from Aquatic Animals; Approved Guideline. CLSI, 940 West Valley
CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI) (2006c). Document M45-A. Methods for Antimicrobial
Dilution and Disk Susceptibility of Infrequently Isolated or Fastidious Bacteria; Approved Guideline. CLSI, 940
West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA,
DEHAUMONT P. (2004). OIE International Standards on Antimicrobial Resistance. J. Vet. Med. [Series B], 51, 411
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DE JONG A., BYWATER R., BUTTY P., DEROOVER E., GODINHO, K., KLEIN U., MARION
V., VALLE, M., & WHEADON A. (2009). A pan-European survey of antimicrobial
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susceptibility towards human-use
antimicrobial drugs among zoonotic and commensal enteric bacteria isolated from healthy food producing
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MEINERSMANN R.J., BERRANG M.E., DAVIS J.A., BARRETT J.B., TURPIN J.B., THITARAM S.N. & FEDORKA-CRAY P.J.
(2010). Development of a DNA microarray to detect antimicrobial resistance genes identified in the National
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GE B., BODEIS S., WALKER R.D., WHITE D.G., ZHAO S., MCDERMOTT P.F. & MENG J. (2002). Comparison of Etest and
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HENDRIKSEN R.S., SEYFARTH A.M., JENSEN A.B., WHICHARD J., KARLSMOSE S., JOYCE K., MIKOLEIT M., DELONG S.M.,
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C.J., STEINBAKK M., SORIANO F. & STETSIOUK. (2006). European committee on antimicrobial susceptibility testing
(EUCAST) technical notes on antimicrobial susceptibility testing. Clin. Microbiol. Infect., 12, 501503.
OJHA S. & KOSTRZYNSKA M. (2008). Examination of animal and zoonotic pathogens using microarrays. Vet. Res.,
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PERRETEN V., VORLET-FAWER L., SLICKERS P., EHRICHT R., KUHNERT P. & FREY J. (2005). Microarray-based detection
of 90 antibiotic resistance genes of gram-positive bacteria. J. Clin. Microbiol., 43, 22912302.
PETERSEN A., AARESTRUP F.M., HOFSHAGEN M., SIPILA H., FRANKLIN A. & GUNNARSSON E. (2003). Harmonization of
antimicrobial susceptibility testing among veterinary diagnostic laboratories in five Nordic countries. Microb. Drug
Resist., 9, 381388.
POXTON I.R. (2005). Molecular techniques in the diagnosis and management of infectious diseases: do they have
a role in bacteriology? Med. Princ. Pract., 14, 2026.
RATHE M., KRISTENSEN L., ELLERMANN-ERIKSEN S., THOMSEN M.K. & SCHUMACHER H. (2009). Vancomycin-resistant
Enterococcus spp.: validation of susceptibility testing and in vitro activity of vancomycin, linezolid, tigecycline and
daptomycin. APMIS., 118, 6673.
STEPANOVIC S., HAUSCHILD T., DAKIC I., AL-DOORI Z., SVABIC-VLAHOVIC M., RANIN L. & MORRISON D. (2006).
Evaluation of phenotypic and molecular methods for detection of oxacillin resistance in members of the
Staphylococcus sciuri group. J. Clin. Microbiol., 44, 934937.
TURNIDGE J., KAHLMETER G., & KRONVALL G. (2006). Statistical characterisation of bacterial wild-type MIC value
distributions and the determination of epidemiological cut-off values. Clin. Microbiol. Infect. 12:418-425.
WALKER R.D. (2007). Antimicrobial susceptibility testing and interpretation of results. In: Antimicrobial Therapy in
Veterinary Medicine, Giguere S., Prescott J.F., Baggot J.D., Walker R.D., Dowling P.M. eds. Ames, IA, Blackwell
Publishing.
WHITE D.G., ACAR J., ANTHONY F., FRANKLIN A., GUPTA R., NICHOLLS T., TAMURA Y., THOMPSON S., THRELFALL J.E.,
VOSE D., VAN VUUREN M., WEGENER H., & COSTARRICA L. (2001). Standardisation and harmonisation of laboratory
methodologies for the detection and quantification of antimicrobial resistance. Rev. Sci. Tech. Off. int. Epiz., 20,
849858.
WORLD ORGANIZATION FOR ANIMAL HEALTH (OIE) (2010). Chapter 6.9. Responsible and prudent use of antimicrobial
agents in veterinary medicine. OIE Terrestrial Animal Health Code, Volume 1. OIE, Paris, France.
ZELAZNY A.M., FERRARO M.J., GLENNEN A., HINDLER J.F., MANN L.M., MUNRO S., MURRAY P.R., RELLER L.B., TENOVER
F.C. & JORGENSEN J.H. (2005). Selection of strains for quality assessment of the disk induction method for
detection of inducible clindamycin resistance in staphylococci: a CLSI collaborative study. J. Clin. Microbiol., 43,
26132615.
*
* *
NB: There is an OIE Reference Laboratory for Antimicrobial resistance
Please contact the OIE Reference Laboratory for any further information on
Antimicrobial resistance
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GUIDELINE 3.2.
BIOTECHNOLOGY IN THE DIAGNOSIS

OF INFECTIOUS DISEASES
INTRODUCTION
Modern concepts of livestock breeding and increased global trade demand the development of
early warning diagnostic platforms, global capacity and diagnostic harmonisation to enhance
livestock production and health and contribute towards uniting pathogen detection technologies as
a basis of the global one-world, one-health initiative.
The global development of improved transport infrastructure has contributed to increased
international movement of people and goods. Consequently, the risk of pathogen transfer between
countries or even between continents is increased. In response to global climate changes, diseases
endemic in certain areas, such as bluetongue and African swine fever, are showing a tendency to
move or extend to other geographical areas outside their normal range. Finally, the constant
intensification of animal production requires different approaches related to the preventive
measures, diagnosis and control of animal diseases.
In order to be widely accepted, modern diagnostic methods have to be validated, sensitive, specific,
rapid, user friendly, cost effective, and eventually automated to facilitate the evaluation of large
numbers of samples.
Eradication of rinderpest is a unique example of the application of new technologies to the control
and eradication of a disease, where the virus neutralisation test was replaced with a well validated
enzyme linked immunosorbent assay that was used in the eradication programme worldwide.
The purpose of this chapter is to provide general background information for the non-specialist.
Two issues of the OIE Scientific and Technical Review are concerned with biotechnology and the
diagnosis of animal diseases and are available on the OIE website. The following is an outline of
the topics briefly reviewed in this chapter.
A.
Detection of nucleic acids
1.
2.
3
4.
5.
6.
7.
8.
Nucleic acid extraction

Polymerase chain reaction (PCR) and real-time PCR
Isothermal amplification
Detection of nucleic acids without DNA amplification
Diagnosis by restriction fragment length polymorphisms and related DNA-based approaches
Genome sequencing
Diagnosis by DNA probes and DNA microarray technology
Metagenomics
B.
Detection of proteins
B1. Detection of antigens

1.
2.
3.
4.
5
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Antigen-capture enzyme-linked immunosorbent assay (AgELISA)

Quantitative immunoassays (radioimmunoassay and quantitative enzyme immunoassay)
Fluorescence antibody test
Immunochromatography (lateral flow devices)
Guideline 3.2. Biotechnology in the diagnosis of infectious diseases
B2. Detection of antibody

1.
2.
3.
4.
5.
6.
7.
8.
9.
Agglutination
Haemaglutination inhibition test
Indirect ELISA (iELISA)
Competitive ELISA (cELISA)
Blocking ELISA (bELISA)
Immunoblotting
Fluorescent
B3. Developing technologies for protein detection

B4. Proteomics
B5. Production of antigens by recombinant DNA technology
C.
Implications of the new technologies
A. DETECTION OF NUCLEIC ACIDS

Efficient management of infectious diseases, in particular those of a transboundary nature, require rapid,
sensitive, specific and confirmatory identification of the pathogen. Conventional diagnostic techniques such as the
isolation of the pathogen (the gold standard for disease diagnosis), are not only laborious for fastidious pathogens
that are difficult to cultivate in vitro, but could also pose a risk to diagnostic technicians if they are zoonotic in
nature. As these limitations often require high level security laboratories (e.g. BSL3 level), the amplification of
nucleic acids has been shown to have advantages. The polymerase chain reaction (PCR)-based platform
technologies have, in particular, quickly revolutionised infectious disease diagnoses. The DNA amplification
platforms are sensitive, specific, rapid and robust as they use several non-infectious nucleic acid templates as
starting material. The platforms are also cost effective and prone to automatisation and therefore ideal for high
throughput.
1.
Nucleic acid extraction
An important step in the molecular diagnostic procedure is extraction of clean nucleic acid mixtures to act as a
template for the reactions. While it is relatively easy to extract DNA from bacterial cultures or blood, it is
technically more challenging to prepare suitable material from field samples, such as faeces, internal organs or
abortion material. If the target material has not been purified of contaminants in the clinical sample, the assay is
compromised and may yield false results. On the other hand, having to process large numbers of samples
increases the risk of contamination, leading to false-positive results. Manipulation steps during the extraction of
nucleic acids are critical cause of cross contamination of samples, leading to false positive results. From this
reason, nucleic acid extaction should be performed using strict working procedures, in an isolated room,
separated from other phases of the PCR procedure (such as preparation of the master mix, addition of extracted
samples into the master mix or amplification). Multiple negative controls (such as DEPC [diethylpyrocarbonate]
water) are usually inserted between individual samples of the tested sample sets in order to detect eventual cross
contamination.
Several types of samples are known to inhibit the PCR reaction, leading to false-negative results. Including an
internal control in the assay is important for the quality control of the nucleic acid extraction, to prove the absence
of PCR inhibitors. These internal controls can be either a house-keeping gene, an endogenous gene, a constant
basal cell-expressed gene, such a the gluceraldehyde-3-phosphatse (GPDH3) or the actin, or an exogenous
nucleic acid that is not present naturally in the preparation but added at the extraction step (Belk & Thorn,
2001). A number of specialised methods for particular types of samples and tissues, most of which are now
commercially available either as manual or automated systems for robotic workstations. The development and
accessibility of the robotic extraction platforms not only minimises the risk of contamination, but also enables
processing of large numbers of samples under constant reaction conditions and minimal operator manipulation.
Consequently, these platforms have contributed to the establishment of high-throughput, robust diagnostic
assays, shortening the processing time required per sample from hours to minutes (Belk et al., 2009). These are
destined to improve the reliability of nucleic acid extraction from different samples, but it still remains a
challenging area.
As an alternative to nucleic acid extraction, biotechnologists are increasingly focusing on polymerases that are
resistant to PCR inhibitors and several are now available on the market for direct amplification of nucleic acids
from pathological specimens without any extraction step. Assays increasingly use an internal control to
demonstrate that PCR inhibitors are not present.
1323
2.
Polymerase chain reaction (PCR), real-time PCR
The PCR exploits natural DNA replication mechanisms and results in the in-vitro production of large quantities of
a desired sequence of DNA from a complex mixture of heterogeneous sequences (Saiki et al., 1988). PCR can
amplify a selected region of from fifty to several thousand base pairs into billions of copies. The specificity of the
amplified region can be targeted by specific primers (short synthetic molecules of DNA complementary to both
strands and flanking the target sequences), which are annealed to the single-stranded template and extended
with the DNA polymerase.
The amplification of DNA in PCR protocols is accomplished using a succession of cyclic incubation steps at
different temperatures. The target DNA is first heat-denatured to separate the two complementary strands to
provide a single-stranded template. Specific primers are then annealed to the single-stranded template at a low
temperature and extended with DNA polymerase at an intermediate temperature. Once the polymerase has
synthesised a new strand of DNA, the product is separated from the template by heating to a higher temperature.
These steps, referred to as cycles, are repeated 2040 times, resulting in amplification of target DNA sequences.
The key to the geometric amplification of target DNA sequences by the PCR is the selection of paired primers
that, when extended, will create additional reciprocal primer-annealing sites for primer extension in subsequent
cycles. To detect RNA (e.g. RNA viruses), a cDNA copy of the RNA must first be made using reverse
transcriptase (RT). The cDNA then acts as the template for amplification by the PCR. This technique is referred to
as reverse transcription PCR (RT-PCR).
The identity of the PCR product is defined by its characteristic size, and/or confirmed using DNA probes (see
below), or restriction digests, which can be used to provide restriction fragment length polymorphisms (RFLP)
(see Section A.5). More commonly, since the advent of automated cycle sequencing techniques, identification can
be made unequivocally via direct sequencing of the PCR product. For example, sequencing is used in the
virulence typing of avian influenza A virus, in which virulence-associated structural motifs at the haemaglutinin
gene cleavage site are reliable indicators of high pathogenicity in chickens. The sensitivity of a PCR may be
enhanced by the use of a second set of primers to amplify a sub-fragment from the PCR product of the first
reaction. This technique is commonly referred to as nested PCR and has been used to detect low levels of
pathogens in the sample. However, the use of nested PCR can increase the rate of environmental contamination
during the second (subfragment) amplification and consequently increase the risk of false-positive results.
PCR is a highly sensitive procedure for detecting infectious agents in host tissues and vectors, even when only a
small number of host cells are infected. PCR can target and amplify a gene sequence that has become integrated
into the DNA of infected host cells. It can also target and amplify unintegrated viral gene sequences. It is clear
that PCR has a role in the testing of vaccines to detect contamination. However, it does not differentiate between
viable and nonviable organisms or incomplete pieces of genomic DNA, and this may complicate interpretation of
results and affect the applicability of PCR in this role.
PCR may prove to be very useful in the diagnosis of chronic, persistent infections, such as bovine viral diarrhoea
(BVD), enzootic bovine leukosis or caprine arthritis/encephalitis virus. These diseases present serious problems
in terms of diagnosis and prevention as infected animals are a constant potential source of transmission.
To expand its utility in veterinary diagnostics and pathogen identification, PCR has been extensively modified over
the years. PCR using broadly conserved primers is designed for identification of classes of pathogens. Using
PCR primers that are complementary to these conserved sequence regions, the presence in the sample of any
bacteria of a desired class can be determined. It must be noted that a positive PCR result needs to be further
characterised by hybridisation with species-specific probes, analysis by RFLP, or by sequencing. Similarly,
consensus PCR can be designed to use degenerate primers targeted at conserved sequence regions or motifs of
a group of related pathogens. The targeting of degenerate primers (i.e. a mixture of similar primers with different
bases in some positions) has led to the identification of many previously unrecognised viruses in various animal
species. On the other hand, multiplex PCR has been designed to use two or more primer pairs directed at
pathogen-specific unique sequences within a single reaction for simultaneous detection of multiple pathogens.
Multiplex PCR has the advantage of a high degree of sensitivity and specificity. However, there have been reports
that multiplexing can reduce sensitivity compared with single reactions, because of competition. If it is important to
have a very sensitive assay, this should be considered during the validation procedure (assay development and
optimisation).
Classical PCR methods for diagnosis of pathogens, both bacterial and viral, are now widely replaced with realtime PCR assays. In real-time PCR assays, intercalating dyes or a target-specific probe or primer (labelled with
fluorescent dye) are used. The measured fluorescent signal is proportional to the number of specific DNA
fragments produced. Thus, during the real-time PCR, the accumulation of PCR products can be monitored in
each consecutive cycle as a change in the degree of fluorescence. In other words, the assay can be used for
absolute or relative quantification of the DNA or RNA content in a given sample. In contrast to conventional PCR,
real-time PCR requires less manipulation, is more rapid, has a closed-tube format (decreased risk of crosscontamination), is highly sensitive and specific thus retaining qualitative efficiency, and provides quantitative
1324
information. In many cases, real-time PCR assays have proved to be more sensitive than existing reference
methods. The development of portable real-time PCR machines and assays raises the prospect of these
techniques being used for rapid (less than 2 hours) diagnosis of disease outbreaks in the field.
When PCR is used for diagnosis, a great deal of care is required to avoid contamination of the samples because
the exquisite sensitivity of the technique can easily lead to false-positive results. Multicentre studies have shown
that positive samples are detected consistently between laboratories, but that false positives are frequently
obtained with known negative samples, indicating the continuing presence of contamination problems (Schweiger
et al., 1997). A new generation of robotic workstations is now available where PCR reactions may be set up with
only a single tube open at any one time. This greatly reduces the risk of contamination. It is also important to
control for potential negative results caused by the presence of PCR inhibitors in the reaction mixture. A
template, independent of the target DNA, known to produce a PCR product (mimics) with specific primers can be
used as a control for the PCR inhibitors, thus indicating false-negative results (Belk et al., 2009). Use of these
precautions allows the PCR to become a realistic option for the diagnostician.
The generation of the signal in a real-time PCR assay has been limited, until recently, to certain chemistries, such
as intercalating dyes (SYBR Green and EvaGreen), hydrolysis probes, molecular beacons, primer probe energy
transfer (PriProET), scorpion primers, dual hybridisation probes and dye-labelled oligonucleotide ligation (Belk et
al., 2009). Alternative labelling has been developed using tags that enable high multiplexing of the assay. The
mass tag PCR assay is an improvement of the real-time PCR platform, in which the primers are tagged with tags
of known, but different, molecular weights. After amplification of the targeted DNA fragments, the tags are
released using UV light and subsequently measured using mass spectrometry. This approach enables
multiplexing of much larger panels of target DNA fragments (and hence multiple diseases), as the assay is not
limited to the number of dyes available. Application of the mass tag PCR assay has been already proven in
differential
diagnosis
of
syndromic
diseases
(respiratory,
haemorrhagic,
enteric
pathogens,
meningitis/encephalitis syndrome) and detection of new clades of pathogens (Lipkin, 2010). A modification of this
method uses matrix-assisted laser desorption-Ionisation (MALDI), which directly measures the molecular weights
of the PCR products and compares them with known databases (Lipkin, 2010).
Additional improvement in the sequencing technology has been achieved by shiftihg from photometric to chemical
detection of the PCR reaction in real time (Neidringhaus et al., 2011). The technology is called ion torrent
sequencing (pH-mediated sequencing, silicon sequencing or semiconductor sequencing) and is based on
detection of the release of hydrogen ions, when a nucleotide is incorporated into a strand of DNA by the
polymerase. Hydrogen ions will change the pH of the solution, which can be detected by an ion sensor (micro pH
meter). The whole setup uses high-density array of micro-machined wells to perform the biochemical process in a
massively parallel way. Fourth generation sequencing sequencing platforms, such as nanopore sequencing
technologies, long read extension methods and methods based on direct video recording of nucleic bases, have
already been developed as a proof of principle (Neidringhaus et al., 2011). Their impact within the scientific
community is still to be evaluated.
3.
Isothermal amplification
Isothermal amplification technologies offer the advantage of omitting thermocycling, enabling DNA amplification at
constant temperature. These technologies include nucleic acid sequence-based amplification (NASBA),
transcription-mediated amplification (TMA), signal-mediated amplification of RNA technology (SMART), strand
displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification
(LAMP), isothermal multiple displacement amplification (IMDA), Helicase-dependent amplification (HDA), circular
helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA) and strand invasion-based
amplification (SIBA) (Gill & Ghaemi, 2008). The most widely used method is the LAMP, which deploys four
primers forming a stem-loop DNA by self-primed DNA synthesis and a DNA polymerase with strand displacement
activity. The result of the amplification process is the production of loops at the ends of the complementary
strands, which are continually extended. The amplification process is indicated by either the occurrence of
turbidity in the reaction mixture or the generation of a fluorescent signal using fluorescent dyes (Gill & Ghaemi,
2008).
4.
Detection of nucleic acids without DNA amplification
A new approach to detection of target DNA has been developed using surface-enhanced Raman scattering
(SERS) (Harpster et al., 2009). The method is based on capturing a thiol-conjugated probe and a methylene bluelabelled reporter probe, which are attached to the complementary (target) DNA. A Raman label positions the
methylene blue on a gold nanoparticle, to an optimal distance for SERS enhancement. The production of a
measurable, capture-specific signal of the Raman spectrum is produced by elicitation of surface-enhanced
plasmon resonance by laser excitation. The analytical sensitivity of the method is in the nanomolar range. The
signal can also be measured by quartz crystal microbalance-dissipation (a method based on the induction of a
piezoelectric effect), which enables quantitative interpretation of the results. A modification of this method uses
paramagnetic nanoparticles as a carrier for the capture probe. Once the target DNA is attached to the capture
1325
and reporter probes, the complexes are attracted magnetically to a defined position, where the concentrated
signal is measured. Quantitative evaluation of the results is also possible using this method.
5.
Diagnosis by restriction fragment length polymorphisms (RFLP) and related DNA-based

approaches
Serological tests that are commonly used to identify microorganisms may not distinguish between isolates of
closely related pathogens, whether they are viruses, bacteria, fungi or parasites. DNA-based procedures can offer
a higher degree of discrimination that is often required. An appropriate starting point may be analyses for
restriction fragment length polymorphisms (RFLP).
The RFLP approach is based on the fact that the genomes of even closely related pathogens are defined by
variation in sequences. For example, the linear order of adjacent nucleotides comprising the recognition
sequence of a specific restriction enzyme in one genome may be absent in the genome of a closely related strain
or isolate.
In practice the RFLP procedure consists of isolating the target pathogen, extracting DNA or RNA (with
subsequent reverse transcription to cDNA), and then digesting the nucleic acid with one or a panel of restriction
enzymes. These restriction enzymes are endonucleases that recognise and cleave double-stranded DNA at
specific nucleotide sequences called restriction sites. The individual fragments within the digested DNA are then
separated by gel electrophoresis and visualised by staining with ethidium bromide. Ideally each strain will reveal a
unique pattern, or fingerprint. Many different restriction enzymes may be considered at the outset, so that
analyses of many molecular fingerprints from digestions with several individual restriction enzymes may be
undertaken; a combination of the best set of results will allow a comprehensive differentiation between strains or
isolates (Loza-Rubio et al., 1999).
Of greater utility to the study of pathogens is a modification to the basic RFLP technique whereby the PCR is
incorporated as a preliminary step. The PCR method is used to amplify a specific region of the genome (known
already by the investigator to be variable in sequence between different pathogens), which then serves as the
template DNA for the RFLP technique. This combination (PCR-RFLP) offers a much greater sensitivity and
comparability for the identification of pathogens and is especially useful when the pathogen is present in the
infected animal in low numbers or is difficult to isolate in culture. The involvement of specific strains or types in a
disease outbreak can be thus determined and the epidemiological tracing of isolates within a country or between
countries should be possible. The incorporation of pulsed-field gel electrophoresis (PFGE) facilitates the
separation of large (up to megabase size) fragments of DNA and can be a useful adjunct to the basic RFLP
analysis. These technologies are extensively used in the official programme for detection and discrimination of
food-borne pathogens (Escherichia coli O157:H7, Salmonella, Shigella, Listeria, or Campylobacter) by the Center
for Disease Control, US Department of Health and Human Services (http://www.cdc.gov/pulsenet/whatis.htm).
RFLPs have a clear value for use in epidemiological studies but more critical interpretation of RFLP data involves
the construction of databases to determine whether the RFLP profiles are clinically significant and linked to
factors such as virulence or host range. In practice, it is usual not to rely on one restriction site but to use sites
from several locations within the genome to classify an isolate. A continuing dilemma for veterinary diagnosticians
is the correct assessment of any molecular differences that might be found between different isolates of a
pathogen, as the loss or acquisition of restriction endonuclease site(s) may not be associated with differences in
the ability of the pathogen to cause disease, i.e. an RFLP difference may not be functionally significant, except as
a distinguishing feature.
Random amplified polymorphic DNA (RAPD) is performed using 812-mer random primers that will or will not
amplify a segment of DNA depending on complementarity to the primers sequence. If a mutation occurs in the
template DNA at the site that was previously complementary to the primer, a PCR product will not be produced,
resulting in a different pattern of amplified DNA segments on the gel. RAPD markers that define individual strains,
may be sequenced and then used as a sequence-confirmed amplified region (SCAR). Thus conversion of an
anonymous polymorphic marker to a SCAR means that a single PCR may be done to more simply identify a
specific genome (Lewin et al., 2002).
Although the RFLP and PCR-RFLP are much less powerful compared with the modern sequencing technologies,
they are inexpensive, easy to perform and sufficiently descriptive for epidemiological investigations of outbreaks
and identification of individual strains of pathogens. The RFLP/PCR-RFLP motif investigation is a valuable tool to
differentiate the origin of an infection (especially where animal-to-human transfer has occurred) and/or to
differentiate between field and vaccinal strains of individual pathogens.
6.
Genome sequencing
The techniques by which DNA from a pathogen may be detected and characterised continue to improve and
evolve. Presently, the ultimate discriminatory procedure is that of genome sequencing. Since 1977, the Sanger
method (Sanger et al., 1977) has been the dominant approach and gold standard for DNA sequencing.
1326
Conventional DNA sequencing is based on cycle sequencing of targeted DNA fragments with labelled di-deoxy
nucleotides, which have a property to stop the elongation at their place of binding. Each di-deoxy nucleotide is
labelled with different dye, enabling distinction between individual di-deoxy nucleotides. As each di-deoxy
nucleotide competes with the normal nucleotides for their complementary binding sites, the result of such PCR
amplification will be a mixture of DNA fragments of different length, each ending with a defined di-deoxy
nucleotide (identifiable by its colour). The PCR mixture is then analysed using capillary electrophoresis, which
separates the fragments by length and reads the colour of each fragment. Analytical software is then used to
convert the colour signals to a layout of nucleotides.
Development of microarray technologies, as well as the improvements in DNA manipulation, have contributed
significantly to the development of direct sequencing protocols capable of detecting unknown pathogens. This
technology enables sequencing of large DNA fragments, allowing sequence comparison with sequence
databases available locally or in the public domain.
Sequencing of a well characterised portion of the genome is playing an important role in pathogen
characterisation and epidemiological studies. Sequencing the products amplified by PCR using degenerate
primers targeting a gene common to the viruses in the same family has become an important diagnostic tool,
especially for identification of previously unrecognised members of the family. Sequencing the products in a
defined region of the genome is used in epidemiological studies to evaluate the genetic similarity to other
pathogens of the same species (subtype), to determine the phylogenetic properties or to determine the origin of
an outbreak/infection. Additionally, by analysing and comparison of different sequence motifs, this technology
offers the possibility of predicting the tendency of pathogens to mutate into more pathogenic strains, allowing, to a
certain level, tracing forward the spread of an outbreak or infection (Horimoto & Kawaoka, 1995).
The state of the art technologies, such as high mass tag-PCR and high-throughput sequencing (Belk et al.,
2009; Lipkin, 2010) have opened much greater diagnostic opportunities, compared with conventional targeted
sequencing. The mass tag-PCR has been successfully used for simultaneous detection and differentiation of
syndromic diseases, such as respiratory diseases, diarrhoeas, encephalitides/meningitides and haemorrhagic
fevers (Lipkin, 2010). Moreover, high-throughput sequencing applied in multiplex platforms is capable of
generating random whole genome sequencing, giving the opportunity for simultaneous pathogen detection and
comparison in different regions of the genome.
The commercial launch of the first parallel pyrosequencing, 454 DNA sequencer platform in 2005, introduced the
new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). This allows
the sequencing of a large genome in a short time, facilitating the study of genetic material recovered directly from
environmental samples, or metagenomics. These new technologies have made it possible quickly to identify an
unknown pathogen (emerging pathogens) or one difficult to cultivate in vitro, or to identify a variant that is present
in small quantities within a mixture (Kunin et al., 2008).
High-throughput sequencing is a significant challenge for the bioinformatics solutions needed to analyse the vast
quantities of data generated, in order to answer specific biological questions including possible amplifications of
high numbers of unexpected pathogens and their interactions with the host cell genome (Kunin et al., 2008). The
process of differentiation of these pathogens, when performed using conventional blasting, is still time-consuming
and inappropriate for routine use. Several approaches are currently under development to solve this problem,
such as sample preparation (removal of the host cell eukaryotic DNA during the extraction phase), reducing the
entry datasets for evaluation (submitting parts of the obtained amplicons instead of the whole genome), targeting
towards a limited (reduced) panel of pathogens (e.g. animal pathogens, only viruses/bacteria, only a group of
viruses/bacteria, etc.) and optimisation of bioinformatics (production of specialised software platforms capable of
analysing large amounts of data using built in algorithms). Each of these approaches has advantages and
disadvantages, however it seems that careful sample preparation and optimisation of bioinformatics can offer the
best solutions.
7.
Diagnosis by DNA probes and DNA microarray technology
Conventional DNA probing and microarray analysis are different but closely related processes. Fundamental to
both processes is the binding (hybridisation) of DNA, derived from a sample suspected of containing a pathogen
(the unknown), with highly characterised DNA derived in advance from a pathogen of interest (the known DNA).
In conventional DNA probing, the unknown DNA (or RNA) the target is immobilised on a solid surface e.g. a
membrane. The known DNA, made into a probe by labelling or tagging it in some way, is in the liquid phase and
is applied to the target.
Additionally, in conventional DNA probing, the target can be nucleic acids extracted from clinical material or
cultured cells and either (a) added to membranes (a dot or slot blot) or (b), less conveniently in a diagnostic
context, transferred to a membrane after gel electrophoresis. The amount of pathogen in a clinical sample might
1327
be too low for detection. Consequently the nucleic acid might be amplified by PCR or RT-PCR, the PCR product
being applied to a membrane. To visualise a probe bound to its target, the probe can be labelled with a
radioactive nuclide or, more commonly and safely, tagged non-radioactively. For example, biotin or psoralen
biotin may be incorporated into the probe, the bound probe then being detected by addition of streptavidin linked
to an enzyme for subsequent generation of colour or light (chemiluminescence).
In microarray diagnosis, it is the known DNA (large oligonucleotides or complementary DNA) that is the target,
immobilised on a glass slide, and the unknown DNA, in the liquid phase that is labelled to make a probe.
In microarray probing the probe is made from the nucleic acid of the test sample. The nucleic acid is extracted
from a sample and a PCR or RT-PCR performed using random oligonucleotide primers. In this way, part of all the
nucleic acids in the sample both of host and pathogen origin are amplified. These PCR products,
representative of every nucleic acid in the sample, are labelled with a fluorescent dye and applied to the
microarray. Under optimised conditions only the DNA derived from the pathogen will bind to the DNA on the glass
slide. If detection of a particular pathogen or group of related pathogens is the object then pathogen-specific
oligonucleotides can be used to amplify these within the sample for probe production.
A microarray is so-called because it can comprise several thousand different known DNAs, each DNA being
spotted onto glass slides to form the array. Each spot is only around 10 m in diameter. DNAs complementary to
parts of selected genes of pathogens can be used to make the arrays (Belk et al., 2009). However, if large
numbers of pathogens are to be investigated then it would be easier logistically to use large oligonucleotides.
Microarrays for detecting pathogens can be designed for several levels of differentiation. In the case of
oligonucleotide target DNAs oligonucleotides may initially be designed to be able to detect and differentiate
pathogens at the genus level. A number may be chosen, perhaps 10 or so, of oligonucleotides with a high degree
of sequence conservation (consensus oligonucleotides) within a given genus, such that a probe made from a field
sample containing a member of that genus would be likely to hybridise to at least some of the oligonucleotides,
while not hybridising (or hybridising to a lesser degree) to those corresponding to related genera, e.g. to
differentiate Apthovirus (foot and mouth disease virus [FMDV]) isolates from Enterovirus strains in the
Picornaviridae family. Other sets of oligonucleotides, placed on the same array slide, able to characterise a
pathogen more specifically, e.g. to differentiate the seven types of FMDV, and potentially for even further
refinement at the subtype level, could then be selected.
In conventional DNA probing the detection of a pathogen is limited by the number of probes used, whereas
microarray analysis is limited only by the number of target DNAs on the array. If a microarray has 1000 different
oligonucleotides, then to achieve the same resolving power by conventional probing would require 1000 probes
and 1000 separate probing reactions. The great advantage of microarray analysis in searching for pathogens is
that hundreds of pathogens can be looked for simultaneously when probing a single microarray slide. Clearly,
microarray analysis has great potential when investigating diseases of unknown aetiology or diseases where
more than one pathogen might be present and when subtyping is required. To enhance sensitivity in pathogen
detection, microarrays can be coupled with PCR amplifications. These PCRs are usually designed to amplify one
or more conserved genes, or multiple sequences, such as PCR using broadly conserved primers, consensus
PCR and multiplex PCR. When a particular pathogen needs to be identified, then the use of a microarray would
be less justifiable, as the production and hybridisation of slides is relatively expensive. Instead, for these more
simple cases, it would be more appropriate to use pathogen/subtype specific PCRs, followed by sequencing or
RFLP for confirmation.
Currently, most of the novel technologies, such as mass tag PCR, SERS, high-throughput sequencing, etc. are
compatible with certain microarray formats. This enables high multiplexing of the tests in a single run, enabling
generation of a large amount of data from a single sample. Appropriate software are used for internal quality
control/quality assurance purposes in the microarrays, as well as for data recording related to the status of an
individual sample.
If past developments in biotechnology are indicative of the future, then microarray equipment and reagents would
be expected to become less expensive, leading to greater application of this technology in animal disease
diagnosis. It will assist in the search for hitherto undiscovered viruses or the characterisation of bacterial strains in
terms of virulence, anti-microbial sensitivity or other important markers. One of the main challenges faced when
using array-based approaches is the handling and analysis of the very large data sets that are generated.
8.
Metagenomics
Metagenomics is defined as a culture-independent study of the collective set of microbial populations

(microbiome) in a sample by analysing the samples nucleotide sequence content. (Bexfield & Kellam, 2011). The
technology is based on amplification and sequencing of the whole DNA and/or RNA content of a given sample
followed by extensive filtering of obtained data using specific software solutions. Metagenomics is a powerfool
1328
tool for random detection of existing or new pathogens. There are several consideration related to this technology,
such as the ratio between the number of target and the number of total amplified sequences (0.00135% reads in
case of detection of arena virus (Palacios et al., 2008), sample selection (amount of pathogens in the targeted
sample), time consuming data acquisition and data analysis time. Two limitations are currently of major concern:
i) as the method relies on finding similarities with known pathogens, there is still no solution for definition of
unmatched sequences, and ii) software solutions that may facilitate the interpretation of the results. To minimise
these disadvantages, three solutions are currently considered, the increased pathogen load (target samples with
hign probability of pathogen multiplication), reducing the resulting data sets by limiting the number of targeted
pathogens and/or excluding host reference sequences from data analysis and optimising bioinformatics for
profession related application.
B. DETECTION OF PROTEINS
The boundaries between what might be termed classical biological tests and biotechnology-based methods
are not clear cut, and both are used to detect proteins specific to disease agents, or antibody responses to those
proteins. Most of the methods rely on immune interactions between antigens and antibodies. It is also difficult to
clearly differentiate what is an antibody and what is an antigen detection test, as most of these assays can be
used for both, antigen or antibody detection, depending on the respective unknown antibody or antigen
component. Therefore, the classification in this section on antigen and antibody detection methods is conditional,
and a qualification is given for each method. For completeness, a brief description of conventional immunoassays
such as agglutination and complement fixation, as well as more recent developments in biotechnology, such as
the different ELISA platforms, are given in this section.
B1. DETECTION OF ANTIGENS

1.
Antigen-capture enzyme-linked immunosorbent assay (AgELISA)
The antigen-capture enzyme-linked immunosorbent assay (AgELISA) facilitates detection of antigen from
pathogens present in an animal prior to or during clinical disease. The AgELISA commonly follows a sandwich
assay format using capture and detecting antibodies (either specific MAbs or polyclonal antibodies). Antigen from
the test sample is first captured by a specific MAb or polyclonal antibody bound to a solid-phase support and its
presence is detected through use of a second MAb or polyclonal antibody, which may either be radio- or more
generally, enzyme-labelled. If the detecting antibody is not labelled then an anti-species conjugate (reactive to the
detector antibody) is used. The capture antibody selects the target antigen from other competing protein in
sample suspensions and ensures that it is semi-concentrated to increase the chances of its detection. The
desired characteristics of the capture MAb are strong binding to the pathogen, recognition of a conserved epitope
highly specific for the target agent, and the ability to attach to an ELISA plate without loss of reactivity. In addition,
a second MAb recognising an epitope other than that recognised by the capture MAb that is bound to the ELISA
plate is often used as part of the indicator system. However, it may be difficult to identify MAbs of comprehensive
intra-type reactivity and polyclonal antisera may be preferred to increase the likelihood of reaction against all
antigenic variants.
AgELISAs have been developed for detection of Anaplasma (Trueblood et al., 1991), BVDV (Mignon et al., 1991),
rinderpest and PPR (Libeau et al., 1994). Related antigen-capture methods using immunomagnetic beads are
now important and well accepted methods for detecting certain bacterial infections, including Listeria, Salmonella
and Escherichia coli. The principle of this technology relies on immunomagnetic separation, i.e. using small
super-paramagnetic particles or beads coated with antibodies against surface antigens of cells. Both intact
bacteria and their soluble antigenic determinants can be detected after magnetic extraction from the test sample
using a second antibody in a sandwich format. As solid surface-free binding, the antigen-capture assays using
immunomagnetic beads can enhance the kinetics of an antigen-antibody reaction. As a result, both nonspecific
binding and the incubation time are reduced.
2.
Quantitative immunoassays (radioimmunoassay and quantitative enzyme immunoassay)
Quantitative ELISA (qELISA) is not used specifically for disease detection. However, it is widely used for
measurement of the concentration of specific proteins (thyroxin, steroids) or other proteins or haptens in the body
or body fluids. Hence, these methods are often used for diagnosis of hormonal disorders, mainly in animal
reproduction. The principle is very similar to competitive ELISA (cELISA). The plates are coated with antibodies
against the substance that is to be measured. The competitive substance is labelled with a marker (an isotope in
a radioimmunoassay and an enzyme or biotin in an enzyme immunoassay) and is titrated to a concentration that
will saturate the coating antibodies. A set of standards of known concentration is added to the wells of the ELISA
plate. As the concentration of the standard increases, the labelled competitor is increasingly displaced in
1329
appropriate wells, resulting in reduced colouration at the end of the reaction. Based on this correlation, a standard
curve is drawn, showing the discoloration (percentage of displacement) as a function of the known concentration.
Once the standard curve is drawn, the percentage of displacement caused by the unknown sample is measured
and the concentration of the targeted substance read via the standard curve.
3.
Fluorescence antibody test
Fluorescent antibody test (FAT) is used for detection of pathogens in animal tissues or fluids, using specific
antibodies against the targeted antigen. The antibodies are labelled with fluorescent dye (most commonly used is
fluorescein isothiocyanate [FITC]). Once the sample is prepared according to a predefined laboratory procedure,
labelled antibodies are added onto the sample, the sample is incubated under defined conditions, washed to
remove the unbound antibodies and examined under fluorescent microscope. Visible fluorescence appears at the
binding sites of the specific antibodies. The method is commonly used for detection of the rabies virus in the
brains of dead animals and classical swine fever virus in tissues of suspected pigs. As the method is based on
direct binding of the labelled antibody to the antigen (pathogen) present in the sample, it is commonly called direct
immunofluorescence.
4.
As an adjunct to the isolation of causative organisms from tissue, immunohistochemistry has become a standard
tool for identification of pathogens, and also for confirmation of the results obtained using high-tech diagnostic
technologies (e.g. mass tag PCR). The in situ detection of antigens in fixed tissues offers a number of advantages
over other diagnostic techniques. These advantages are: (i) convenience of sample submission; (ii) safe handling
of potential human pathogens; (iii) retrospective studies of stored specimens; (iv) rapidity; and (v) the detection of
nonviable organisms (Haines & Clark, 1991). Immunohistochemistry is also used for the detection of abnormal
prion protein (PrPSc) in brain tissue to confirm scrapie, BSE and other transmissible spongiform
encephalopathies, and has proved to be more sensitive than standard histopathological examination
(Thorgeirsdottir et al., 2002). As the number of MAbs to defined antigens increases, the use of
immunohistochemistry for the identification of organisms and other specific markers for autoimmune diseases and
neoplasia is increasing. As formalin fixation can denature the antigenic epitopes (i.e. the three dimensional
structure recognised by MAbs) the limiting step in the application of immunohistochemistry is to identify an
appropriate MAb/antigen combination that will bind in formalin-fixed tissues. This may be overcome by using
frozen sections or employing antigen retrieval techniques (e.g. proteolytic enzyme digestion, microwaving) before
immunostaining.
5.
Immunochromatography (lateral flow devices)
Immunochromatograhy provides a convenient method for detection of antigens in several minutes without special
apparatus. The sample is applied on one end of a filter in which antibody is immobilised and microbeads (such as
colloidal gold) conjugated with antibody are applied. The antigen in the sample forms an immunocomplex with the
antibody labelled with colloidal gold. This complex moves along with the liquid sample, and makes a contact with
the antibody immobilised on the membrane, where it forms an immuno-complex with the immobilised antibody,
generating a colored product that can be visualised by eye.
B2. DETECTION OF ANTIBODIES

1.
Agglutination
Agglutination is a method using the property of specific antibodies to bind many pathogenic organisms into single
clumps thereby forming large complexes, which are easily precipitated. The precipitation can be macroscopically
or microscopically visible. Different technical applications of this principle are still widely used in veterinary
laboratories, especially for detection of specific antibodies against brucellosis and leptospirosis (see disease
specific chapters in this Terrestrial Manual). The method can be used for detection of both antibodies, when
known antigen is used, or antigen, when well defined sera are used.
2.
The haemagglutination inhibition method relies on the property of some pathogens (mainly viruses) to
nonspecifically agglutinate erythrocytes. In the presence of specific antibodies, this actvitiy of the virus can be
blocked and it will be inhibited from agglutinating the erythrocytes. The test is used for qualitative and
quantitative detection of both antigens and antibodies. It has been used extensively for detection of serotypespecific antibodies against avian influenza, peste des petits ruminants (PPR) and others. The test can be also
1330
used for detection of antigen (detecting presence of an avian influenza virus from the allantoic fluid after
attempted isolation) (Mulder & Brans, 1952).
3.
The agar gel immunodifussion test is based on the property of proteins to randomly diffuse in an agar of certain
concentration. Antibodies and antigen are placed in wells at a defined distance. After incubation, at the site in the
agar gel where antibodies and the antigen meet and bind, precipitation lines are formed, visible under a beam of
intense oblique light against a black background. The test is most commonly used for detection of antibodies
against avian influenza (matrix antibodies), equine infectious anaemia (Coggins test) and enzootic bovine
leukosis. Under certain circumstances the test can be used for detection of antigen (monitoring the success of
avian influenza virus isolation in allantoic fluid).
4.
Complement is a thermolabile system of over 25 proteins present in the blood that are sequentially activated by
antigen/antibody complexes to assist in eliminating pathogens. The complement fixation test (CFT) relies on the
property of some antibodies to use complement as a mediator. The test is based on two independent reactions,
each requiring complement as a mediator: (i) the main reaction in which the test serum (heat-inactivated to
destroy the complement) is incubated with an external source (usually rabbit or guinea pig) of complement and
the antigen; and (ii) the helper (indicator) reaction, where anti-erythrocyte antibodies (haemolysin) are incubated
with washed sheep erythrocytes in predefined concentration. If the tested serum in the main reaction is positive
(specific for the targeted antigen), the complement will have been used and the whole mixture will therefore lack
active complement. If the reaction is negative, the complement will not have been spent and the mixture will
contain active complement. After an appropriate incubation period, a defined volume from the main reaction
mixture is added to the helper reaction mixture. In the case of positive reaction (no active complement in the
main mixture), the erythrocytes in the helper reaction will not be haemolysed and will form a discrete round
button on the bottom of the tube (or well in tests where a microplate format is used). The supernatant in these
tubes (wells) will be clear. In the case of a negative reaction (complement in the main mixture is active); the
erythrocyte in the helper reaction will be haemolysed. In this case there will be no formation of the discrete
button on the bottom of the tube (or well in tests where microplate format is used). The supernatant in these tubes
(wells) will be redish and turbid. The CFT can be used for qualitative and semiquantitative detection of specific
antibodies. It is still widely used for detection of antibodies against brucellosis in cattle, sheep and goats]), Q
fever, contagious bovine pleuropneumonia (CBPP) and several other diseases.
5.
Indirect ELISA (iELISA)
In an iELISA, the target antigen (whole or purified) is bound to the solid phase in an ELISA plate. Serum samples
at an appropriate dilution are added. If specific antibodies against the coated antigen are present, they will bind to
the antigen. The ELISA plates are washed to remove any unbound antibodies. Anti-immunoglobulin antisera
conjugated with an enzyme (usually a peroxidase), specific for the immunoglobulin of the animal species
examined are then added. If specific antibodies against the coated antigen have been present in the first phase,
the conjugated anti-immunoglobulins will bind to them and will not be removed during the second washing step.
The substrate buffer is then added. In positive cases (presence of specific antibodies) the colour of the substrate
buffer will change. The colour development is measured at a defined wavelength using a spectrophotometer and
is proportional to the level of antibodies present in the sample.
6.
Competitive ELISA (cELISA)
The cELISA is an immunoassay that can be used to detect or quantify antibody or antigen using a competitive
method. The cELISA for detection of specific antibodies has largely replaced the iELISA for large-scale screening
and sero-surveillance. The cELISA offers significant advantages over an iELISA, as samples from many species
may be tested without the need for species-specific enzyme-labelled conjugates for each species under test.
Many antigens are extremely difficult or time consuming to purify. When used in an indirect assay, they can
produce high background values because of nonspecific binding. However, relatively crude antigens may be used
in the cELISA provided the detecting antibody has the desired specificity. The principle of a competitive assay for
the detection of antibodies is competition between the test serum and the detecting antibody. Specific binding of
the detecting antibody is detected using an appropriate anti-species conjugate. A reduction in the expected colour
obtained is caused by binding of antibodies present in the test serum with the antigen, which therefore prevents
binding of the specific detecting antibody.
7.
Blocking ELISA (bELISA)
The basic setup in the bELISA is similar to that described for the AgELISA (paragraph B2.1.). Antibodies specific
to the targeted antigen are bound to the solid phase of the ELISA plate. The antigen, prior to adding to the
1331
antibody coated plate, is incubated with the samples. If the samples contain antibodies against that antigen, they
will bind (block) to the antigen during this incubation. When added to the wells, the blocked antigen will be unable
to bind to the coating antibodies. Consequently, when the detecting antibody is added to the wells it will not be
able to recognise any bound antigen (no colour development). In contrast, if the sample is derived from a negative
case, binding will occur and there will be colour development.
8.
Immunoblotting
Immunoblotting combines the high resolution of gel electrophoresis with the specificity of immunochemical
detection and offers a means of identifying immunodominant proteins recognised by antibodies from infected
animals or monoclonal antibodies (MAbs) directed against the target agent. The immunoblotting procedure can be
divided into six steps: (i) Preparation of the sample, (ii) Resolution of the antigen by gel electrophoresis,
(iii) Transfer of the separated polypeptides to a membrane support (nitro-cellulose membrane, polyvinylidene
difluoride [PVDF]), (iv) Blocking nonspecific binding sites on the membrane, (v) Incubation with detecting
antibody, and (vi) Detection of bound antibody.
The choice of detecting antibody is critical. Polyclonal sera are composed of a range of antibodies reflecting the
full repertoire of the immune response to a particular complex antigen. They will therefore detect a number of
distinct polypeptides giving a characteristic profile of reactivity. MAbs bind to only one epitope; therefore they are
useful in identifying highly specific polypeptides. After incubation with the detecting antibody, any antibodies
bound to specific protein bands are visualised using enzyme-labelled anti-species antisera and a suitable
substrate/chromogen.
Immunoblotting is performed chiefly in diagnostic laboratories to identify and/or characterise infectious agents
based on antigen specificity or using known antigens to detect a specific serological response. False-positive and
false-negative results in other diagnostic assays can often be resolved by immunoblotting (Molina Caballero et al.,
1993). As an example of antigen detection, immunoblotting has been used on a large scale as a major screening
method for bovine spongiform encephalopathy (BSE) and scrapie; it has been used on millions of brain stem
samples in Europe and elsewhere for the detection of prion protein (Schaller et al., 1999). It has now largely been
replaced as a screening test by AgELISA or lateral flow device based methods but is still an important
confirmatory test and integral to the differentiation of transmissible spongiform encephalopathies strains into
typical and atypical bovine spongiform encephalopathies and scrapie. Immunoblotting is also often used to
determine the specificity of individual MAbs. Individual purified polypeptides (or recombinant expressed proteins)
may also be transferred to nitrocellulose or PVDF membranes by immunoblotting to examine the reactivity of test
sera to individual proteins. This characteristic profile of reactivity may be used to help distinguish between animals
that have been vaccinated or infected, such as the enzyme-linked immunoelectrotransfer blot (EITB), a western
blot for FMD that is widely used in South America (Bergmann et al., 1993). The major factor affecting the success
of an immunoblotting technique is the nature of the epitopes recognised by the antibodies. Most high resolution
gel techniques involve some form of denaturation of the antigen. This destroys conformational determinants and
allows only the detection of linear or non-conformational epitopes. Most polyclonal antisera contain antibodies to
both linear and conformational epitopes, but MAbs are directed at single epitopes; thus if they target
conformational epitopes, they will not react with denatured protein.
9.
Fluorescent antibody
Modification of this method can be used for detection of antibodies. Essentially, the modification is in the use of
secondary antibody, specific for the antibodies of the species examined. For example, if a dog is examined for the
presence of specific antibodies against leishmania, leishmania antigen is fixed onto a slide, incubated with the
examined dog serum, washed to remove unbound antibodies and again incubated with labelled anti-dogantobodies (antibodies rised by immunisation of different animal species with immunoglobulines of a dog).
Fluorescence appears only if the examined animal has specific antibodies against the target antigen.
B3. DEVELOPING TECHNOLOGIES FOR PROTEIN DETECTION

The following protein-detection technologies are in the developmental stage:
i)
Multiplex ELISA platforms that can be deployed to create disease package assays (BVDV, Infectious
bovine rhinotracheitis [IBR], parainfluenza virus 3 [PI3] and respiratory syncytial virus [RSV]). The test uses
the same principle as the conventional ELISA, adapted into a micro-format.
ii)
Time-resolved fluorescence resonance energy transfer (TR-FRET) used as a source of signal generation.
The labelling of the conjugate and the antigen is performed using two fluorophore dyes. The principle has
been tested for competitive and sandwich format.
1332
iii)
Potentiometric ELISA based on production of an electrical, instead of a colour signal, requiring minimal
sample preparation and producing results within 215 minutes.
iv)
A SERS assay (using colloidal gold as a ligand) for detection of DNA/RNA and proteins in immunological
reactions using piezoelectric effect, as well as amplification of the signal using magnetic capture.
v)
Multiplex protein assay based on signal generation using piezoelectric effect and the oscillation modulation
caused by specific analyte binding. The assay has been developed for rapid result generation (60 seconds),
multiplex capacity, qualitative and quantitative measurements, does not need reagents and/or incubations.
The device is the size of a mobile phone, and integrated wireless transmission of the results can be used to
expedite data analysis. The principle of the work has been scientifically proven and preliminary
investigations on its applicability to avian influenza diagnosis are in progress.
vi)
Proximity ligation assays (PLA) for detection of antigens

PLA are a modification of the conventional immunohistochemistry. It is based on the application of two
antibodies targeted towards different epitopes of a single protein, detected by secondary antibodies labelled
with short strands of known DNA. Upon binding of the secondary antibodies, their DNA strands are amplified
and complementary detector probes (labelled with fluorescent dyes) in turn, then bind to their
complementary sequence on the amplified stands. This results in multiple amplification of the signal and
enables detection of minimal amounts of target proteins present in the sample. The technique is used as a
tool for rapid scanning of multiple proteiens or proteomes
vii)
MALDI-TOF analysis for determinative bacteriology

MALDI-TOF mass spectrometry uses an ultraviolet absorbing matrix mixed with a polymer in an appropriate
solvent. The sample/matrix mixture is placed onto a sample probe tip. Under vacuum conditions the solvent
is removed, leaving co-crystallised polymer molecules homogeneously dispersed within the matrix
molecules. Molar masses are determined using a pulsed laser beam with appropriate frequency. The
technique is increasingly being used in microbiological laboratories for detection and differentiation of viral,
bacterial and fungal pathogens.
B4. PROTEOMICS
The proteome is the total complement of proteins expressed within a cell, a tissue or an organism and proteomics
is the study of proteins, including their expression level, post-translational modification and interaction with other
proteins, on a large scale. As not all proteins are expressed at all times, but are dependent on physiological and
environmental factors, proteomics can provide an excellent overall view of disease processes at the protein level.
Because the application of proteomics to novel drug discovery promises huge economic returns, companies all
over the world have rapidly poured resources into this new research field.
Many methods used in proteomics, including two-dimensional gel electrophoresis (2DGE) and mass spectrometry
(MS) were established many years ago. However recent advances in MS techniques, together with whole
genome sequencing and the development of powerful bioinformatics and robotics platforms, have revolutionised
protein identification. The general principle of proteomics is that proteins are separated, usually by 2DGE on
polyacrylamide gels, then protein spots are excised, digested with trypsin, and the resultant peptides analysed by
MS. The masses of these peptides are then compared with the predicted masses of peptides derived by
computational analyses of genome databases, resulting in gene identification. MS can also be used to deduce the
amino acid sequence of peptides and to characterise post-translational modifications such as glycosylation or
phosphorylation. 2DGE has some drawbacks, particularly for the separation of hydrophobic proteins, and other
separation techniques based on liquid chromatography are now finding favour for certain applications.
Nevertheless, 2DGE is the method of choice for creating quantitative maps of protein expression and many
thousands of proteins can be analysed in a short space of time.
Alterations in the proteome of body tissues or of fluids such as serum, urine or cerebro-spinal fluid can be
measured directly so changes that occur in a disease state can be accurately pinpointed. As well as identifying
molecules that may be targets for novel therapies, this approach is a very powerful tool for early-stage diagnosis
of disease. The best-established clinical applications of proteomics so far are in the identification of markers for
the early diagnosis of cancers, such as bladder cancers, in urine. However, considerable research efforts are also
ongoing in other areas such as heart disease, Alzheimers disease and insulin-dependent diabetes.
The use of proteomics for the diagnosis of infectious disease is in its infancy but may prove to be of considerable
importance. For example, definitive diagnosis of chronic hepatitis B virus (HBV) infection still relies on liver biopsy,
but proteomic analysis of serum samples shows that the expression of at least seven serum proteins is changed
significantly in chronic HBV patients. Similarly, the ante-mortem differential diagnosis of Creutzfeldt-Jakob
disease (CJD) may be aided by proteomics as preliminary data show that seven proteins in cerebro-spinal fluid
(CSF) are differentially expressed between patients with variant or sporadic CJD (Choe et al., 2002).
1333
An extremely useful application of proteomics to the diagnosis of infectious disease is in the identification of novel
diagnostic antigens by screening serum from infected and uninfected individuals against immunoblotted, 2DGE
mapped proteomes of infectious agents.
Within the veterinary field, proteomics-based research projects are now underway and these will undoubtedly
yield novel diagnostic tools for the future. Proteome maps are being derived for a range of veterinary pathogens
including bacteria (Mujer et al., 2002), protozoa (Rout & Field, 2001) and nematodes (Yatsuda et al., 2003).
B5. PRODUCTION OF ANTIGENS BY RECOMBINANT DNA TECHNOLOGY

Advances in molecular biology and genetics in the 1970s initiated the development of recombinant DNA
technology. Since then the impact of this technology is such that it plays a vital role in scientific research as well
as in the diagnosis and treatment of disease. Recombinant DNA technology simply refers to the transfer of genes
from one organism into another: literally the recombination of DNA from different sources. The objectives of
recombinant DNA technology include identifying genes, isolating genes, modifying genes, and re-expressing
genes in other hosts or organisms. These steps permit scientists and clinicians to identify new genes and the
proteins they encode, to correct endogenous genetic defects, and to manufacture large quantities of specific gene
products such as hormones, antigens for use in vaccines, and other proteins produced by biological agents of
interest. Of particular value is the degree of specificity attainable in diagnostic tests by the use of recombinant
protein.
Native proteins are perhaps the ideal antigens, providing sequence-specific and surface structural epitopes. Many
current diagnostic tests require test antigens that need to be continuously produced from cell culture or harvested
from an infected animal. Such antigen preparations are expensive and often have a short shelf-life, with each new
batch of antigen requiring standardisation. Natural proteins are rarely available in a completely pure form, and
antibodies often develop against contaminating polypeptides that can lead to false-positive results. Recombinant
DNA technology produces antigens that offer many advantages over antigens isolated from other biological
sources. These advantages include a high purity, high specific activity and as the protein is synthesised in
genetically modified laboratory-grown cells, each preparation of the protein product is identical to the previous
preparation, ensuring batch-to-batch consistency. When recombinant antigens are used in combination with the
cELISA format, purification of the recombinant antigen from the lysate may not be necessary as the specificity of
the cELISA resides mainly in the MAb used.
An outline of the procedure for the production of an antigen by recombinant DNA technology is as follows. The
identification of an antigen of potential diagnostic or scientific significance is achieved through the study of the
antibody response of the host to the proteins of the organism in question. Immunodominant antigens, defined
proteins of the organism against which the host responds with the highest potential antibody titre, are of particular
interest as they are likely to be major stimuli of cellular and humoral immunity against the pathogen of interest.
Antigen recognition studies are widely used to identify biologically relevant, immunodominant antigens for use in
generating MAbs as well as in vaccine development. Once a protein of interest has been identified, the gene
encoding the protein is generated using messenger RNA (mRNA) from the organism as a template for making
cDNA. This method of cloning the gene encoding the protein of interest requires a prior knowledge about the
gene sequence, either directly from the organism of interest or through the use of gene sequences from closely
related species. An alternative method, when gene sequence data are not available, is the generation of
recombinant libraries from the genomic DNA of the organism or from cDNA synthesised from mRNA. Fragments
of the recombinant libraries can be cloned into a protein expression system, which may be prokaryotic or
eukaryotic, and the gene library screened for expression of the protein.
There is a wide choice of expression systems. Protein may be expressed in bacteria, usually E. coli, yeast, insect
cells using baculovirus, or in eukaryotic cells by infection with appropriate viral vectors or by permanent
transfection. Differences in glycosylation when prepared in bacterial, insect or mammalian cell cultures can modify
protein structure and its reactivity with antibody. Antigen may need to be extracted from the cell or it may be
secreted. Purification is often, but not always, necessary. An upcoming trend in the production of antigens for use
in assays is in the development of synthetic peptide antigens. This allows antigens to be tested as diagnostic
reagents based on the gene sequence, expression of the whole protein being unnecessary, thus shortening the
process.
Genome sequences of numerous pathogens have already been determined. A gene responsible for an antigen or
immunogen can easily be cloned with PCR technologies and characterised by nucleotide sequence (Rappuoli &
Covacci, 2003). In short, the amplified DNA can be cloned in a bacterial plasmid vector and expressed as a
recombinant antigen. The cloned gene can be designed in a way that the recombinant protein is fused at its N or
C-terminal with a detection tag that can be used for its purification by affinity chromatography. There are many
tags but the most popular are polyhisdine (6-8 residues), glutathione S-transferase (GST) or the Flag peptide Flag
composed of 8 amino acid residues. The antigenicity of the gene products can then be determined. Systematic
1334
screening of the antigen gene in silico, from genome sequence data, accelerates the development of diagnostic
kits and vaccine (Tortorella et al., 2000).
B4. IMPLICATIONS OF THE NEW TECHNOLOGIES

There are a number of trends in diagnostic technologies that will have an impact on the way in which disease
diagnosis will be approached in the future, affecting the laboratory environment, data analysis and disease
control:
i)
The global development of chip technologies has led to a strong trend towards miniaturisation of the test
format in both molecular and protein detection assays. The test formats range from several millimetres to
several centimetres.
ii)
Although miniaturised, multiplex platforms can be developed that are capable of detecting from several up to
thousands of pathogens in a single sample.
iii)
Miniaturisation of the test format is being accompanied by similar miniaturisation of laboratory equipment,
enabling on-site testing. This trend is resulting in the use of sophisticated equipment, previously only used in
the laboratory, in the field, facilitating the rapid and early diagnosis of infectious diseases, as well as the
modes of reaction of appropriate Competent Authorities.
iv)
The development of alternative sources of signal generation/amplification, replacing light with mass
measurement, piezoelectric effect or concentration of the ligand will lead to the development of a whole new
platform of technologies.
vi)
Although development of new technologies can often mean improved capability, consideration should
always be paid to the actual value and the role of the confirmatory test in diagnosis!
viii) As the diagnostic platforms are continuously changing in the ways described above, several components of
the disease control chain will be affected. Appropriate communication technologies/information systems will
need to be developed in order to systematically collect, store and analyse large datasets produced by the
new technologies in a relatively short time. There is likely to be an increasing trend towards real-time inputs
of results via mobile telephones or the Internet that will require relevant development of IT and data-handling
systems.
ix)
Appropriate infrastructure and preparedness have to be an integral component of technology update. This
will include distance training in testing in the field (sample collection and preparation, testing, interpretation
of the test results, sample processing for dispatch to reference laboratories and reporting to appropriate
authorities).
x)
Adaptation of national contingency plans towards simultaneous management of multiple diseases, as well
as management of new, unexpected diseases.
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*
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GUIDELINE 3.3.
THE APPLICATION OF BIOTECHNOLOGY TO THE

DEVELOPMENT OF VETERINARY VACCINES
INTRODUCTION
The practice of vaccination for the prevention of animal disease has been used for centuries and
has proven to be a powerful tool for the alleviation of animal suffering as well as the economic well
being of producers of animal products. Up until 1520 years ago, vaccines had changed little from
those originally pioneered by Jenner and Pasteur. Since that time there have been significant
changes in the types of vaccines available owing to a number of factors, including compatibility with
eradication programmes and international trade policies as well as cost-effectiveness of production.
The first recombinant vaccines were introduced in the late 1980s to control Aujeszkys disease and
rabies in wild-life (Pastoret et al., 1988) and are the forerunners of similar products that will be
available in the future.
The approaches used in the development of vaccines have expanded rapidly as the result of
increased knowledge of the mechanisms by which protective immunity is induced, and the
explosion of genomic data on both pathogens and their hosts. The associated evolution of new
technology in the field of molecular biology and immunology has furthermore had a large impact on
the development of new vaccine strategies and the quality of the products that are produced. It has
enabled the design of vaccines targeted for the control and eradication of specific pathogens within
the framework of regional, national and international requirements. Use of recombinant
technologies bring with it the need for the application of a riskbenefit assessment that takes into
account the specific aspects that need to be considered, particularly with respect to safety (see
Appendix 1.1.6.1 Risk analysis for biologicals for veterinary use, to Chapter 1.1.6 Principles of
veterinary vaccine production of this Terrestrial Manual).
This chapter describes a range of technologies that are used to produce vaccines engineered for a
specific purpose. The categorisation is aimed to assist the reader to understand the technologies
employed, but it should be recognised that the categories are not mutually exclusive (i.e. reverse
genetics may be used to produce a chimeric vaccine). In principle, the technologies can be used to
change the target pathogen itself to alter its properties by deletion, insertion, other genetic
modifications, or they can be used to modify the isolated genes or coding sequences of pathogens
to produce specific immunogens associated with protective immunity.
A. REVERSE GENETICS
The development of a reverse genetics system for a range of different RNA and DNA viruses has revolutionised
the field of virology by making it possible to introduce designed mutations, insertions and deletions into the viral
genome of live viruses. It has by now been used in a range of applications that include the attenuation of viruses,
the modification of host specificity and the generation of replication-deficient viruses. These strategies have also
been applied to the development of new vaccine strategies and are widely used in the characterisation of the
structure and function of individual viral genes and coding sequences.
The technology of reverse genetics involves the generation of a cloned copy of complementary DNA (cDNA) from
RNA by reverse transcription in vitro, manipulating DNA in vitro followed by generating the modified live virus by
transfection of permissive cells with the cloned DNA(s).The technology was first demonstrated using the
bacteriophage Q-Beta, a positive-strand RNA virus (Taniguchi, 1978). Subsequently, a large number of positivestrand RNA viruses including severe acute respiratory syndrome (SARS) coronavirus, with large genomes have
been rescued, which has helped in the study of the biology of these viruses and the development of new live
attenuated viral vaccines. For example, reverse genetics was used to develop an infectious clone of transmissible
1338
Guideline 3.3. The application of biotechnology to the development of veterinary vaccines

gastroenteritis virus (TGEV), which induced lactogenic immunity in immunised pigs (Sola et al., 2003). This novel
technique has also been used to develop a modified porcine respiratory and reproductive syndrome virus, which
can be used as a DIVA (differentiating infected and vaccinated animals) vaccine to help differentiate between
vaccinated and infected pigs (de Lima et al., 2008).
Owing to the inherent characteristics of negative-strand RNA viruses, it took years of work before this technique
could be developed and used for generating engineered viruses containing negative-strand RNA genomes.
Reverse genetics was first developed for influenza virus, a segmented negative-strand RNA virus. Since then, this
technique has been successfully used for the generation of a number of RNA viruses containing either
unsegmented or segmented negative-strand genomes. For example, the use of this technique has led to the
development of a vaccine for avian influenza virus in which the engineered virus contained a haemagglutinin (HA)
gene from an H5N1 virus and a neuraminidase (NA) gene from a H2N3 virus, using a H1N1 backbone (Meeusen
et al., 2007). The resultant inactivated H5N3 virus vaccine induced complete protection in birds against highly
pathogenic H5N1 challenge. A reverse genetics strategy has also been used in the development of Foot and
mouth disease, Classical swine fever, and Newcastle disease vaccines (see chapters 2.1.5, 2.8.3 and 2.3.14,
respectively). More recently, reverse genetics systems have been developed for segmented double-stranded
RNA viruses including bluetongue virus (BTV) introducing the possibility of new vaccine development strategies
for these viruses (Boyce et al., 2008).
Disabled infectious single-cycle (DISC) vaccine involve the deletion of an open reading frame coding for a key
protein involved in the viral replication or viral capsid formation (Widman et al., 2008). The DISC virus is isolated
in cells expressing the key protein, thus providing the missing protein in trans. Such virus, when injected in
animals, can complete only one round of replication without producing a progeny virus. Vaccines based on DISC
viruses are more stimulatory than a killed virus vaccine and are devoid of problems associated with live vaccines.
B. RECOMBINANT VECTOR TECHNOLOGY

Advances in reverse genetics, genomics, and proteomics have facilitated the identification of mechanisms of
virulence, host-pathogen interactions, and protective antigens from many pathogenic microorganisms and also
the development of suitable vehicles/vectors for delivery of these antigens to the host. The availability of bacterial
and viral genome sequences has facilitated the rapid construction of defined deletions in the genomes of a wide
variety of pathogens, which not only results in attenuation, but also creates space for the insertion of foreign
genes coding for antigens from heterologous microbes. In general, live bacterial or viral vectors share several
characteristics including ease and economy of production, non-integration into the host genome, stability and a
reasonable capacity to insert genes coding for heterologous antigens. In addition, as with any live vaccine, the
vector should be avirulent and the impact of immunity to the vector should be evaluated.
a)
Bacterial vectors
In general bacterial vectors are attenuated by deletion of genes required for key metabolic processes or
genes associated for virulence. Although they are not used routinely in animals, rapid progress is being
made in developing and evaluating different bacteria as vectors. For several years, BCG (Bacillus Calmette
Guerin) and Salmonella have been developed as vectors for delivering vaccine antigens to animals and the
latter has been used for the generation of live vaccine strains for poultry. There are currently a number of
other bacterial vectors being developed based on commensal microorganisms (Lactococcus, Streptococcus,
Lactobacillus and Staphylococcus) or attenuated pathogenic organisms (Shigella, Bacillus, Yersinia, Vibrio,
Cornebacteria, and Bordetella), all of which are being evaluated for their ability to induce protective
immunity.
b)
Viral vectors
Most viral vectors are developed using viruses that are associated with mild or no disease or using viruses
that are pathogenic but attenuated by deletion of virulence genes. Replication competent virus vectors,
which can produce progeny virus, as well as replication-defective virus vectors, which do not produce
progeny virus, have been developed and evaluated as vaccine delivery vehicles. A number of commercial
vaccines based on DNA virus vectors, including poxviruses and herpesviruses, have been successfully
licensed for use in veterinary medicine (reviewed in Gerdts et al., 2006). These include vectors based on
vaccinia virus, canarypox virus, fowlpox virus and turkey herpesvirus. A number of viral vectors have been
developed or are in the process of being developed, improved and evaluated. These include RNA viruses
such as Venezuelan equine encephalitis virus, Newcastle disease virus and feline foamy virus as well as
DNA viruses such as adenoviruses, herpesviruses and pox viruses, Fowl pox and canary pox vectors have
been used in a wide range of applications (MacLachlan et al., 2007; Swayne, 2009) whereas replicationdeficient human adenovirus vectors have been used very successfully in the development of FMDV
vaccines (Rodriguez & Grubman, 2009). Licensed canary pox vaccines include vaccines against equine
1339

influenza and feline leukaemia. Other licensed vector vaccines include the herpesvirus of turkeys vectored
with an infectious bursal disease insert.
C. GENE-DELETED VACCINES
The knowledge of specific virulence factor(s) of a pathogen and the availability of recombinant DNA technology
has facilitated the creation of specific gene-deleted pathogens for use as live vaccines. The approach of creating
and testing defined gene deletions ultimately aids in reducing the pathogenicity/virulence of the organism without
affecting the immunogenicity. Such gene-deleted organisms can be used as vaccines as they retain the
immunogenic features of the wild-type organism but cannot cause disease. However, to be effective as viable
vaccine(s), these organisms should be genetically stable, easy to grow and easy to administer. So far, genes
involved either in determining virulence or regulating key metabolic pathways of the organism(s) have been
targeted for such deletions.
This approach has been successfully used to create several live attenuated vaccine strains of bacterial pathogens
that are genetically stable, safe to use and induce better protection than killed vaccines. Gene-deleted Salmonella
enterica serovar typhimurium and serovar enteritidis vaccines have been licensed for use in poultry (Babu et al.,
2004; Meesun et al., 2007) and similarly, an aroA gene-deleted Streptococcus equi vaccine has been licensed for
use in horses (Jakobs et al., 2000; Meesun et al., 2007).
This technology has also been successfully used to create live attenuated vaccine strains of viral pathogens that
are genetically stable and can be used as marker vaccines to differentiate between vaccinated and infected
animals. A double gene (gE and TK) deleted pseudorabies virus marker vaccine has been licensed for use in pigs
(Ferrari et al., 2000; Meesun et al., 2007) and similarly, gE deleted a bovine herpesvirus-1 marker vaccine has
been licensed for use in cattle (Meesun et al., 2007; Van Oirschot et al., 1996).
D. CHIMERIC VIRUSES
Chimeric viruses are defined as recombinant viruses that may contain parts of two closely related viral genomes.
For example, a chimeric virus could be one that contains structural genes of one viral serotype and nonstructural
genes of another serotype of the same virus. Alternatively, a chimeric virus would be one that contains part of the
genome from different members belonging to the same virus family. In principle, chimeric viruses display the
biological characteristics of both the parent viruses. One of the main advantages of this approach is that a single
dose of chimeric virus delivers the complete repertoire of antigens closely resembling the pathogen(s), which can
induce protective immune response against multiple viral pathogens belonging to or different serotypes of the
same viral pathogen.
The availability of infectious full-length complementary DNA (cDNA) clones of different RNA viruses using reverse
genetics technologies has led to novel vaccine development strategies. Chimeric pestiviruses have been
constructed using an infectious cDNA clone containing the classical swine fever virus (CSFV) genome or the
bovine viral diarrhoea virus (BVDV) genome backbones. In one instance, a chimeric pestivirus was constructed
by replacing the BVDV E2 coding sequence in the infectious DNA copy of BVDV strain CP7 with the
corresponding E2 coding sequence of CSFV strain Alfort 187 (Reimann et al., 2004). Another chimeric virus was
constructed by replacing the CSFV E2 coding sequence in the infectious DNA copy of CSFV vaccine strain C with
the corresponding E2 coding sequence from BVDV (van Gennip et al., 2000). These chimeric viruses appeared to
be attenuated in pigs, induced complete protection against CSFV challenge and helped to discriminate between
vaccinated and infected pigs (Reimann et al., 2004; van Gennip et al., 2000).
In another application, chimeric porcine circoviruses (PCVs) have been isolated using infectious cDNA clones of
porcine circovirus PCV1 in which the capsid protein from pathogenic PCV2 was used to replace the
corresponding gene in the nonpathogenic PCV1 strain (PCV1-2). Likewise, the capsid gene in PCV2 has been
replaced with the gene from PCV1 (PCV2-1). The chimeric PCV1-2 virus appeared to be attenuated in pigs and
induced protective immunity against wildtype PCV2 challenge in pigs (Fenaux et al., 2004).
This platform technology has also been used to generate chimeric flaviviruses. In one example, a chimeric virus
was generated by replacing the coding sequences for structural proteins of yellow fever YF-17D virus with those
of West Nile virus (WNV). A single dose of this chimeric flavivirus vaccine induced both cell-mediated and
humoral immune responses in horses, and provided protection against WNV challenge without causing any
clinical illness (Meeusen et al., 2007). The same platform technology has also been used to develop human
vaccines for Japanese encephalitis virus, WNV and Dengue virus. Although chimeric flavivirus vaccines have
shown satisfactory safety profiles and protective efficacies, caution should be used in evaluating chimeric viruses
for the change in virulence.
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E. SUBUNIT VACCINES
Subunit vaccines composed of semi-pure or purified proteins have been commercially available since the early
1980s, with subunit components produced by recombinant DNA technology available since the 1990s (Cohen,
1993; Rhodes et al., 1994; Ulmer et al., 1993; 1995). The latter have attracted growing interest and activity since
that time. Subunit vaccines do not include live recombinant vector technologies, which provide the delivery of
recombinant proteins in vivo. The field of genomics and related areas has revolutionised the manner in which
microbial antigens are identified. Since the first bacterial genome was sequenced in 1995, there has been a huge
increase in the number of bacterial, viral, and parasite genomes for which genome sequences are available.
Indeed, virtually all pathogens of animals are represented and those pathogens that are not can readily be
obtained in less than a day. More importantly, the development of the bioinformatics resources and tools that are
required to analyse these genomes has proceeded in parallel and it is now relatively easy to identify surfaceexposed antigens, specific B- and T-cell epitopes, etc. There is no requirement to have the ability to grow the
organism in culture: for example subunit vaccines for Piscirickettsia salmonis, a salmonid pathogen, have been
developed even though the organism could not be readily grown (Kuzyk et al., 2001).
The production of subunit antigens can be achieved by both conventional biochemical or recombinant DNA
technologies. The latter involves a range of prokaryotic and eukaryotic expression systems including yeast, insect
cell and plants (Chichester & Yusibov, 2007) by means of a variety of integrated or transient expression
strategies. Biochemical techniques remain useful in some cases where recombinant expression is not
appropriate, such as antigens requiring complex assembly (e.g. fimbriae), or when post-translational modification
is necessary. For example, Campylobacter jejuni is one bacterial species that glycosylates many surface proteins
and, as such, they are best produced in C. jejuni rather than heterologous expression systems, although
Escherichia coli strains have been engineered to carry out the same function (Wacker et al., 2002). An excellent
example of a subunit vaccine composed of an authentic antigen that retained three dimensional structure was the
original E. coli K99 vaccine for calf scours, which was tested three decades ago (Acres et al., 1979). This product
was based on the K99 fimbrial antigen, which could readily be extracted from cells by heat treatment, thus
retaining the three dimensional fimbrial structure. Another example includes a baculovirus expressed vaccine
against porcine circovirus type 2 (Fachinger et al., 2008). In a number of cases the expressed subunit vaccine
protein spontaneously assembles into well defined particles that may resemble virus particles. These virus-like
particles (VLPs) are a sub-class of subunit vaccines (Roy & Noad, 2008) and their application in vaccine
development is reviewed in section F. Vaccines containing ORF 2 protein of PCV-2 expressed in baculovirus has
been commercialised.
Subunit vaccines could have some advantages over live attenuated and inactivated vaccines, including the ability
to induce strong humoral and cell-mediated immune response. The vaccines furthermore have an excellent safety
profile, and can be used in combination with other subunit vaccines. However, efficacy is dependent on the
protective immunity being induced by inoculation of a single or set of defined recombinant proteins. Experience
has shown this may be affected by the gene expression system used. In addition, subunits vaccines may be
expensive to produce for some glycoproteins and may require the use of adjuvants to enhance immune
responses.
One of the biggest advantages of subunit vaccines is that they are generally compatible with DIVA strategies as
long as the antigen is not being used as a marker. In the case of bovine herpesvirus, glycoprotein gD has been
successfully used in subunit vaccine formulations. Although immunisation with gD has proven to be protective at
an individual animal level (Harland et al., 1992; van Drunen Littel-van den Hurk et al., 1994), it has not reduced
the prevalence of the virus in the field, thus limiting its use. Subunit vaccines against a variety of other respiratory
and enteric viruses, including BVDV, BRSV, PI3, rotavirus, and coronavirus have been successfully tested,
although none of these is used on a commercial basis. Bacterial subunits have arguably proven more successful
than their viral counterparts. This is because of the cost-effectiveness of growth of both conventional and
recombinant organisms and a general requirement for a Th2-biased immune response in many cases.
Recombinant vaccines are commercially available for respiratory pathogens such as Mannheimia haemolytica
and Actinobacillus pleuropneumoniae based upon the leukotoxins produced by these organisms, as well as
transferrin-binding proteins. Actinobacillus pleuropneumoniae is an excellent example of a vaccine composed of
subunits selected on cross-serotype reactivity, thus providing broad-spectrum protection against disease.
Likewise a vaccine against atrophic rhinitis containing a non-toxic derivative of Pasteurella multocida
dermonecrotic toxin produced by a genetically modified Escherichia coli strain together with a conventional
B. bronchiseptica bacterin has been commercialised.
Vaccines against CSF demonstrate well the need to target recombinant technology to a particular purpose.
Conventional, live attenuated CSF vaccines have a rapid onset of immunity and are effective at preventing
transmission of infection (Van Oirschot, 2003) but have the disadvantage that it is not possible to differentiate
infected pigs from those that have merely been vaccinated. Commercial E2 subunit vaccines have a slower onset
of immunity and reduce but do not prevent viral shedding. However, they enable a DIVA strategy to be followed
thereby facilitating a vaccination to live strategy. Their use is therefore likely to be of particular benefit in high
1341

value breeder pigs where the vaccine can be used to limit the clinical impact of the disease whilst allowing
individual infected pigs to be identified and eliminated.
F. VIRUS-LIKE PARTICLES
Virus-like particles (VLPs) are supra-molecular structures composed of one or more recombinant proteins. The
particles form through self-assembly and typically range from 20 to 100 nm in size. Depending on the origin they
can be icosahedral or rod-like in structure (reviewed in Jennings & Bachmann, 2008). VLPs offer the advantage of
formulating the vaccine antigen in a particulate structure, thereby increasing the immunogenicity of the vaccine.
VLPs can be used as either vaccine itself or as carrier for genetically fused (chimeric), incorporated or covalently
linked antigens. VLPs have been extensively studied for the past 20 years, with human vaccines against hepatitis
B virus (Zuckerman, 2006) and human papillomavirus (Stanley, 2008) commercially available and several
vaccines for veterinary application in development. These include vaccine for bluetongue virus, rota- and
parvovirus.
VLPs offer several advantages for the use as vaccine including a high safety profile, the similarity to viral and
bacterial structures, the ability for large-scale production and the possibility of combining the VLPs with other
adjuvants. Typically, immunisation with VLP induces rapid and strong antibody responses. Similar to viruses and
bacteria multiple copies of the vaccine antigens are displayed in a highly repetitive and ordered, quasi crystallinestructure (Bachmann & Zinkernagel, 1996), which can cross-link the B cell receptor resulting in activation of the
B cell and subsequent induction of T-independent IgM responses (Thyagarajan et al., 2003).). Furthermore, this
enables interaction with the complement system resulting in increased phagocytosis. Moreover, the particulate
structure of VLPs also enhances uptake by dendritic cells and subsequent cross-presentation of the antigen. It
was demonstrated by Lenz et al. (2001) that cross-presentation of particulate antigens was more effective than
presentations of soluble antigens. However, the induction of T cell responses overall is still not as effective as
those induced by live vaccines. To overcome this, VLPs have been successfully combined with molecular
adjuvants such as CpG ODN and single-stranded RNA. Other VLPs have been demonstrated to directly stimulate
dendritic cells (DC.) For example, L1 protein-VLPs of papillomavirus have been shown to directly activate DC.
VLPs can either be used as vaccine itself or be used as carrier for recombinant antigens, either incorporated,
directly, genetically fused or covalently linked. For example, the bovine rotavirus virus protein 6 (VP6) forms VLPs
that are highly immunogenic and already confer protection against challenge infection (Redmond et al., 1993).
However, using the VP4 and VP7, other antigens can be covalently linked to the VP6 particles and used for
immunisation (Redmond et al., 1993). Other prominent examples include the hepatitis B surface antigen VLPs
(HBsAg-VLP), human immunodeficiency virus 1, dengue virus VLPs, norovirus VLPs, and influenza A VLPs.
Examples of VLPs used as carriers include the well characterised hepatitis B core antigen VLPs (HBcAg VLPs;
[Blanchet & Sureau, 2006; Pumpens & Grens, 2001]) as carrier for the influenza a M2 protein (M2-HBcAg
[Jegerlehner et al., 2002]), or malaria B- and T-cell epitopes (Nardin et al., 2004). While mostly administered
systemically, some VLPs-based vaccines already have been tested for mucosal administration.
G. DNA VACCINES
Immunisation with DNA represents a relatively new vaccination strategy that is based on a simple concept. DNA
vaccines can be defined as antigen-encoding bacterial plasmids that are capable of inducing specific immune
responses upon inoculation into a suitable host. Immunisation is accomplished by the uptake of purified plasmid
in the host cells, where it persists extrachromosomally in the nuclei. Subsequent expression of protein results in
the presentation of normally processed or modified forms of the protein to the immune system. In the host, native
forms of the proteins have access to presentation pathways by class I major histocompatibility (MHC) antigens in
addition to class II MHC presentation, which results in a balanced immune response. The use of pure plasmid
DNA offers many advantages over other vaccine delivery vehicles. One of the greatest advantages is the ability of
DNA vaccines to induce both humoral and cell-mediated immune responses, which is critical for protection from
many diseases. There is also evidence that DNA vaccines can induce long-term immunity, which is a further
requirement for vaccine efficacy. As the vector itself does not induce immune responses, DNA vaccines can be
repeatedly administered without the interference of antibodies. From a technical viewpoint, DNA vaccines are
easy to engineer, produce and purify, so new DNA vaccines can be constructed and evaluated in animal models
within months. DNA vaccines are very stable and therefore have a long shelf life and can be transported without a
cold chain. The safety of DNA vaccines has been established in various trials in several species including humans
(Bagarazzi et al., 1998; Kim et al., 2001).
As soon as the concept of DNA immunisation began to be explored, this technology was found to be very
effective in rodents, but initially did not perform as well in larger species. However, recent progress has resulted in
the development of DNA vaccines in a number of outbred target species (Carvalho et al., 2009; Redding &
Weiner, 2009). Currently, four veterinary DNA vaccines have been licensed, against growth hormone releasing
1342

hormone for swine in Australia, infectious haematopoietic necrosis virus for salmon in Canada, WNV for horses
and melanoma for dogs in the USA (Kutzler & Weiner, 2008). To achieve higher efficacy in large animal species,
optimisation at various levels has been required, including (i) vector modifications; (ii) protein engineering to
modify subcellular localisation; (iii) improvements in DNA delivery routes and methods; (iv) inclusion of adjuvants,
as a gene or co-administered agent, and (v) antigen targeting to antigen-presenting cells (APCs). It is likely that
the often unsatisfactory efficacy of DNA vaccines in large animals was caused by inefficient transfection, as well
as immunological blandness, of the administered plasmids. The use of a needle-free vaccine delivery device was
shown to reduce the effective dose of an experimental polyvalent DNA vaccine for avian influenza, and to rapidly
deliver repeated injections in poultry (Rao et al., 2009).
H. ANTIGEN DELIVERY AND MOLECULAR ADJUVANTS

Adjuvants are substances that enhance immune responses when co-administered with antigens. They are a
critical component of killed (recombinant and subunit) vaccines, which are often poorly immunogenic. Adjuvants
can be classified into two broad categories based on their presumed mechanism of action: i) delivery systems,
and ii) immunostimulatory adjuvants. Delivery systems include many conventional adjuvants and numerous
particulate adjuvants and will be discussed separately below.
Despite the importance of adjuvants in vaccines, their mechanisms of action remains poorly understood. Recent
advances in the understanding of innate immunity have provided important clues on the molecular mechanisms of
action of immunostimulatory adjuvants. In this regard, immune cells express a variety of receptors, collectively
termed pattern recognition receptors (PRR) that broadly detect conserved microbial components referred to as
pathogen-associated molecular patterns (PAMPs). A number of PRR have been described including toll-like
receptors (TLR); e.g. TLR 9 recognises bacterial CpG nucleic acid motifs, natural agonist of TLR7/8, singlestranded viral RNA (oligoribonucleotides, ORN) strongly activate innate immune responses in mice, humans and
are particularly potent in large animals; TLR4 agonist such as lipolysaccharide (LPS) that is known for its powerful
immunostimulatory and adjuvant properties, but unfortunately this molecule is highly toxic; nucleotide
oligomerisation domain (NOD)-like receptor (NLL), retinoic acid inducible gene (RIG)-like receptors (RLL), and Ctype lectin receptors (CLR), all of which detect microbial components. Engagement of these receptors by their
agonists leads to a cascade of molecular and cellular events that result in activation of innate immunity, which
directs antigen-specific adaptive immunity. Of these receptors, TLR agonists are most widely explored and have
shown great promise as adjuvants. Interestingly, the live-attenuated yellow fever vaccine 17D (YF-17D), one of
the most successful vaccines available, activates TLR2, 7, 8 and 9 (Querec et al., 2006), suggesting that the
success of at least some of the live vaccines may be the result of their ability to activate TLRs. This has
generated a great deal of interest in TLR agonist as adjuvants.
The existing paradigm in the veterinary vaccine industry of one adjuvant-one vaccine, is driven partly by costs
associated with including more than one adjuvant in a vaccine; however, it may severely limit the efficacy of
potentially safe vaccine candidates, and may explain, at least in part, why some vaccines or adjuvants have only
achieved suboptimal efficacy. Evidence is slowly accumulating to indicate that multiple adjuvants may offer more
than can be achieved with a single adjuvant.For example, although CpG ODNs are a good adjuvant, they can
have even greater adjuvant activity if formulated or coadministered with other compounds, such as particulates,
mineral salts, saponins, liposomes, cationic peptides, polysaccharides and bacterial toxins and the synthetic
polymers, polyphosphazenes (Wack et al., 2008).
The adjuvant effect of microparticles has been known for some time and has been previously reviewed (Mutwiri et
al., 2005). Particulate delivery systems are thought to promote trapping and retention of antigens in local lymph
nodes. In addition, microparticles facilitate antigen presentation by APCs via both MHC class I and MHC class II
restricted processing and presentation pathways. One of the main advantages of microparticles for targeted
antigen delivery is that they can be a flexible delivery platform that can be used to deliver both antigens and
immunostimulatory molecules.
Other potential antigen delivery systems include polyphosphazenes, a class of synthetic polymers consisting of a
backbone with alternating phosphorus and nitrogen atoms and organic side groups attached to each phosphorus
(Mutwiri et al., 2007). Immune stimulating complex (ISCOM), which is a small 40 nm nanoparticle composed of
saponin (adjuvant), lipids and antigen, and has been described as an antigen delivery system because it not only
has adjuvant activity and but also the ability to target APC (Morein et al., 2004). A commercial ISCOM-based
vaccine against equine influenza has been licensed for many years (Heldens et al., 2009).
I. VACCINE DELIVERY
Vaccine delivery comprises a diverse range of approaches with the overall goal of providing vaccines for mass
vaccination during disease outbreaks and delivery of vaccines to wildlife. Oral vaccines used in rabies vaccination
1343

of wildlife such as foxes were initially based on attenuated rabies vaccine viruses such as the ERA strain, but
concerns that these vaccines could rarely cause rabies (Fehlner-Gardiner et al., 2008) have largely lead to their
replacement (http://ec.europa.eu/food/animal/diseases/eradication/rabies_pres_19.pdf). In Canada, a live
adenovirus-vectored rabies vaccine with a good safety profile (Knowles et al., 2009) is currently being used in
rabies vaccination campaigns directed at controlling skunk and raccoon rabies (Rosatte et al., 2009). The live oral
vaccinia-rabies glycoprotein (V-RG) vaccine is widely used elsewhere, and attempts are being made to optimise
the vaccine baits for efficacy for other species including dogs (Cliquet et al., 2008). Rabies infection in stray dogs
and wildlife represents a serious problem for humans globally, and research for safer, more stable and efficacious
live oral rabies vaccines continue (Faber et al., 2009). Other possibilities for mass vaccination using edible plantmade vaccines have been actively investigated, but in spite of biotechnological advances in plant expression of
vaccine antigens, no commercial products for oral use have been identified to date (Rice et al., 2005).
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*
* *
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GUIDELINE 3.4.
THE ROLE OF OFFICIAL BODIES IN

THE INTERNATIONAL REGULATION OF
VETERINARY BIOLOGICALS
SUMMARY
The official control of veterinary biologicals is vested in various national and regional organisations
that differ in their approach to ensuring the quality, safety and efficacy of the products. International
harmonisation of regulations concerning biological products did not begin until well after those
concerning chemically defined products. The first biological products for veterinary use were not
manufactured and distributed until the end of the nineteenth century. They were often produced
under unsophisticated conditions, and distributed or sold without any control other than those of
their manufacturers. Later, each manufacturer developed its own standards. In Europe, these were
subject to State controls as early as 1895 for certain diagnostic products (e.g. mallein, tuberculin) or
vaccines. Gradually the conditions for international harmonisation of standards evolved, beginning
with the comparative testing of products being issued by different European laboratories. It was
only in the second half of the twentieth century that national laws covering veterinary biologicals
were imposed. These demanded that precisely defined techniques be followed before biological
products for veterinary use could be licensed. This was followed by considerable efforts to
harmonise these national regulations, first at the regional level (notably in Europe and the
Americas) then at the global level, notably by the Office International des pizooties (OIE) with the
publication of the first edition of the OIE Manual of Standards for Diagnostic Tests and Vaccines in
1989.
World-wide harmonisation of standards for veterinary biologicals will be of help to Chief Veterinary
Officers who must follow the instructions given in the OIE International Animal Health Code, as they
apply to all biological products for use in international trade. It will also be of assistance to vaccine
producers, who have expressed their wish for world-wide harmonisation of registration rules so as
to simplify and facilitate marketing of their products. Evidently, it will also be of interest to farmers
and to consumers, who would benefit from the fact that the safety and efficacy of the products that
they use would have been assured to a uniformly high level.
The different sections of this chapter will review and compare regulations from the regions of the
world that have made most progress in this field and will describe current attempts at harmonising
these regulations on an international scale.
Note: In this chapter the term veterinary biological will be taken to include vaccines for use in
animals, antisera for use in animals, and in-vivo diagnostic preparations.
A. REGULATION OF VETERINARY BIOLOGICALS: PRESENT SITUATION

1.
In Japan
1.1. Introduction
Medicinal products that are exclusively used for animals, including veterinary biologicals, are under the jurisdiction
of the Ministry of Agriculture, Forestry and Fisheries, and securing their quality, efficacy and safety is stipulated in
the Pharmaceutical Affairs Law (Anon, 1960). Since 1972, registration procedures have been developed with the
aim of rationalising the examination procedure and facilitating the acquisition of approval. These procedures are
stipulated in the Pharmaceutical Affairs Law and other related regulations. Consequently, a speedy and simple
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Guideline 3.4. The role of official bodies in the international regulation of veterinary biologicals
examination procedure has been achieved with emphasis on the assurance of quality, safety and efficacy. The
Food Safety Commission was established in the Cabinet Office, Government of Japan, in July 2003. In the case
of approval examination, re-examination and re-evaluation, all veterinary vaccines, except products of dogs and
cats, must comply with the Food Safety Basic Law.
1.2. Regulations governing the approval and quality assurance of veterinary biologicals
a)
Application for approval and licence

A person intending to release veterinary biologicals on the market shall obtain the license for marketing
approval holders and the marketing approval for each biological from the Minister of Agriculture, Forestry
and Fisheries. The application for the marketing approval should be submitted with designated appended
documents, such as those on clinical studies. Of the latter, the safety studies and clinical studies using the
target animal species should have been performed in compliance with GLP (Good Laboratory Practice) and
GCP (Good Clinical Practice). A marketing approval holder shall comply with the standard of GQP (Good
Quality Practice) and the GVP (Good Vigilance Practice).
A licence to manufacture veterinary biologicals is issued by the Minister of Agriculture, Forestry and
Fisheries and must be renewed every 5 years. Conformity to GMP (Good Manufacturing Practice) is
stipulated as one of the conditions for obtaining or renewing the licence to manufacture.
b)
National assay
After receiving a licence, each batch of the veterinary biological must be examined by the National
Veterinary Assay Laboratory according to the procedures of the Assay Standard for Veterinary Biological
Products (Anon, 2002b; Makie, 1998). A marketing approval holder must apply it to the national assay. Each
product for marketing must include an official identification stamp on the container or the package as a seal.
c)
Re-examination and re-evaluation

Re-examination is performed on newly approved veterinary biologicals. Usually a field assessment of the
veterinary vaccines is conducted over a period of 6 years following initial approval of the veterinary vaccines.
During this investigation, the efficacy and the safety are re-examined.
Re-evaluation is performed on availability of approved products after marketing by order of the Minister of
Agriculture, Forestry and Fisheries. This may happen when it is suspected that a veterinary biological does
not conform to the latest standards of veterinary biological products.
d)
Minimum requirement of veterinary biological products

The examinations provide information about the consistency of the manufacturing process and the quality of
the product: manufacturing methods, properties of strains used for manufacturing, methods of quality
control, methods of storage and shelf life, according to the standards given in the Minimum Requirement of
Veterinary Biological Products (Anon, 2002a). Any product that does not conform to these product
standards cannot be manufactured, imported or marketed.
e)
Cases of rejection of approval

When the quality of the veterinary biological that has been submitted for approval is found to be
unsatisfactory, or its adverse effects are marked as compared with its indications, the product is judged to be
of little value and approval is not given.
f)
Cancellation of approvals
At the time of granting approval to market, the quality, safety and efficacy of the product are carefully
examined with reference to the latest available technology. However, if scientific knowledge acquired since
the granting of approval indicates that there could be a health hazard associated with the product, reexamination and re-evaluation are performed and an order of cancellation of approval may be made.
1.3. Procedure for marketing approval

When a person intends to market veterinary biologicals, an application for approval to market the veterinary drug
must be submitted on a designated form to an official in charge of veterinary biologicals at the Department of
Animal Hygiene of each Prefecture. If the documentation is satisfactory, the application for approval to market,
together with appended documents, are sent to and reviewed by the Secretariat of the Ministry of Agriculture,
Forestry and Fisheries. At that time, a hearing may be conducted if necessary. The application is then discussed
in the Pharmaceutical Affairs Sub-council, Pharmaceutical Affairs and Food Sanitation Council, and if any
problems are not found, notice of approval to market the veterinary product is sent to the applicant.
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2.
In the European Union
2.1. Introduction
The pharmaceutical legislation of the European Union (EU), which has evolved over a 30-year period, covers both
medicinal products for human and veterinary use. Harmonisation of requirements in the area of veterinary
medicines began in 1981 with the adoption of Directives 81/851/EEC and 81/852/EEC, laying down common
requirements for manufacturing and marketing authorisations, based on the evaluation of the quality, safety, and
efficacy of the product. These Directives, and subsequent veterinary and human pharmaceutical legislation, were
consolidated into Directive 2001/82/EC and 2001/83/EC for veterinary and human products, respectively. A series
of detailed guidelines were first published in 1994 entitled Rules Governing Medicinal Products in the EU
(European Union, 1999). These have since been updated and describe in detail the legal basis for obtaining
marketing authorisations, how dossiers should be compiled and how they should be assessed. These rules serve
as extremely useful reference publications for any authority that is setting up a system for authorisation of
veterinary biologicals. The rules were formally adopted and applied specifically to veterinary biologicals from
1993. Many additional measures were taken to further harmonise the procedures and the criteria for the
evaluation of veterinary medicinal products, such as framework requirements and interpretive guidelines for their
testing, principles and guidelines of GMP, and a Community procedure for the evaluation of high-technology
products. However, granting of authorisations remained at the national level. As a consequence, although
applications were evaluated on the basis of these harmonised criteria and procedures, and in some cases
simultaneously by the authorities of the Member States, there were differences in the decisions reached by the
Member States on individual products. This was why in 1990 the Commission proposed a new system for
marketing authorisation for medicinal products, which was adopted by the Council of Ministers in 1993 and
entered into force on 1 January 1995.
One of the first consequences was the creation of the European Medicines Evaluation Agency (EMEA) in London,
United Kingdom (UK).
New legislation for veterinary products (Regulation 726/2004 and Directive 2004/28/EC) was published in May
2004. This legislation, for the main part, came into force in 2005 and resulted in a number of changes aimed at
strengthening public and animal health, and environment protection by reinforcing requirements and controls.
Directive 2001/82/EC already stated that the competent authorities cannot grant a marketing authorisation (MA)
without having conducted a benefitrisk analysis. The document in the MA dossier must demonstrate that the
benefit bound to efficacy outweighs potential risk. But the relation between benefit and risk was not defined in
that Directive. The new Directive gives the definition of the riskbenefit balance: an evaluation of the positive
therapeutic effects of the veterinary medicinal product in relation to the risk.
The risks concern:
The animal;
The user of medicinal product;
The consumer liable to ingest animal food containing medicinal products residues;
The environment.
With the revision of the Directive, if no medicinal product is available for 3 consecutive years, its MA is secluded.
Before the revision, the MA was renewed every 5 years. Now, a single renewal is required. The
pharmacovigilance is reinforced.
Title IV of regulation 726/2004 related to responsibilities and administrative structure of the European Agency has
come into force in April 2004 in order to face the consequences of the enlargement of the EU.
In December 2009, the EMEA officially launched a new organisational structure and visual identity, and became
the European Medicines Agency (EMA). This was the second major reorganisation of the Agencys services since
it was established in 1995, and resulted from the expansion of the Agencys responsibilities and tasks, giving it a
stronger role in the protection of public and animal health in Europe.
2.2. The role of the European Medicines Evaluation Agency

In 1995, a new European system for the authorisation of medicinal products came into force. After 10 years of
cooperation between national registration authorities at the EU level and 4 years of negotiations, the Council of
the EU adopted, in June 1993, three directives and one regulation, which together form the legal basis of the
system (Brunko, 1997).
The EMEA was established by Council Regulation 2309/93/EEC of 22 July 1993 (Official Journal No. L214,
24.8.1993), and London, UK, was chosen as its location by decision made by the Heads of State and
Government on 29 October 1993. This agency formulates opinions and, apart from the administrative staff and
the management board, is composed of two scientific committees, the CHMP (Committee for Human Medicinal
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Products) in charge of medicinal products for humans and the CVMP (Committee for Veterinary Medicinal
Products) in charge of medicinal products for use in animals.
The CVMP is responsible for the evaluation of applications for marketing authorisation for products derived from
biotechnology, for productivity enhancers, new chemical entities and other innovative new products. In addition,
the CVMP makes recommendations regarding MRLs (maximum residue limits) for substances used in foodproducing animals. To support its activities, the CVMP relies on a pool of experts put at the disposal of the agency
by the EU Member States. These experts may participate in any of the CVMP working parties. Among the
working parties, the Immunologicals Working Party (CVMP/IWP) advises the CVMP on general policy issues such
as the elaboration and revision of guidelines on immunological veterinary medicinal products (IVMPs). A scientific
advice working party foreseen in regulation 726/2004 has been created. The aim of this working party is to advise
applicants during the development phase of a veterinary medicinal product. The CVMP prepares scientific
guidelines in consultation with the competent authorities of the EU Member States, to help applicants prepare
marketing authorisation applications for medicinal products for veterinary use.
Guidelines are intended to provide a basis for the practical harmonisation of the manner in which the EU Member
States and the Agency interpret and apply the detailed requirements for the demonstration of quality, safety and
efficacy contained in the Community directives. They also help to ensure that applications for marketing
authorisation are prepared in a manner that will be recognised as valid by the Agency.
Immunological guidelines are provided for general items, stability, efficacy, summary of product characteristics
(SPC), and availability (minor uses/minor species), and are available on the EMA website at: www.ema.europa.eu
2.3. Present European procedures for marketing authorisation

At the time of the revision of the Community Code in 2004, the difficulties encountered in practice as well as
jurisprudentially precise details were taken into account and modifications to various Community procedures were
adopted: tightening of the conditions of examination and the safeguard clause, official recognition of the
coordination group for mutual recognition and decentralised procedures (veterinary), arbitration and creation of a
new procedure (decentralised procedure, see below). These provisions were introduced by the Directive
2004/28/EC amending Directive 2001/82/EC.
Four registration procedures for human and veterinary medicinal products have become available through this
new legislation:
1.
The centralised procedure allows a unique marketing authorisation (MA) to be obtained and made available
in all the Member States. This applies to high technology products defined in the annex to the Regulation. It
is optional for innovative medicinal products. This procedure was extended to veterinary vaccines covering
animal diseases that are subject to Community prophylactic measures.
2.
The national procedure allows an MA to be obtained for a medicinal product in a single country or in a
country that will be the origin of a mutual recognition procedure.
3.
The mutual recognition procedure: applications for authorisation of a product may still be obtained in a single
Member State (the Reference Member State) by means of a national procedure. Following approval in the
Reference Member State, applications may be made, if desired, to other Concerned Member States for
identical authorisations to be granted on the basis of mutual recognition.
4.
The new decentralised procedure is the addition of the national one and the mutual recognition procedure,
i.e. it is based upon the principle of mutual recognition of national authorisations. At the beginning of this
procedure, all Member States are associated, but assessment is conducted by one reference Member State
chosen by the applicant; this is immediately followed by a mutual recognition procedure.
The most important change is the compulsory aspect of arbitrage in the case of a disagreement between Member
States during the mutual recognition or the decentralised procedures. If a Member State considers that there is a
serious risk to public health, a pre-arbitrage procedure must be carried out. In such a situation, an MA holder
cannot remove his/her demand. The arbitrage allows a decision to be made on whether there is a serious risk
with the use of the medicinal product. Finally, the decision (to grant or refuse the MA) is harmonised throughout
the community.
It should be noted that the European Commission is preparing a legal proposal on the review of the legal
framework governing medicinal products for veterinary use.
2.4. Manufacturing authorisation and batch release control

In accordance with Directive 2001/82/EC, authorisation is also required for the manufacture of veterinary
medicinal products, including immunologicals. This directive provides for regular inspections and stipulates that
1351
manufacture must be supervised by a qualified person, who certifies that each batch is in conformity with the
approved specifications for the product. For the implementation of these requirements, the Commission has
adopted Directive 91/412/EEC relating to the principle and guidelines of GMP, and published a detailed guide on
GMP developed by a group of pharmaceutical inspectors from the Member States (Volume 4).
The new directive also establishes GMP for active starting materials for medicinal products. This provision is
reinforced through the provision of the opportunity for Member States to carry out inspections of active materials
destined for the manufacturers of veterinary medicinal products.
Manufacturers are required to have the services of a qualified person at their disposal to certify that each batch of
product has been manufactured and checked in accordance with the conditions for marketing authorisation. This
is a basic requirement of the pharmaceutical legislation. In the case of batches imported from third countries,
each batch has to undergo a full qualitative and a quantitative analysis of at least the active ingredients in the first
Member State of import into the EU, under the supervision of a qualified person. Until this control by the qualified
person has been carried out, a batch cannot circulate within the EU without further control. When the certificate is
released, no more controls are necessary. In the special case of immunological veterinary medicinal products, an
additional step may be introduced. Article 82 of Directive 2001/82/EC, as amended by Directive 2004/28/EC, of
the European Parliament and the Council allows, for reasons of human or animal health, a Member State to
request samples of each batch of a given IVMP to be submitted to a Competent Authority (CA) for official testing
by an Official Medicine Control laboratory (OMCL) before it is placed on the market and establishes the conditions
under which a restricted test list can be applied. This is referred to as Official Control Authority Batch Release.
OCABR performed by any given Member State must be mutually recognised by all other member states requiring
OCABR for that product.
Article 81 of Directive 2001/82/EC allows a Member State, where appropriate, to ask a MA holder to provide
documentation to a control authority or an OMCL proving that control tests were carried out in accordance with
the methods laid down in the MA dossier. This is referred to as an Official Batch Protocol Review. A goodwill
agreement has been adopted by the Veterinary Batch Release Network (VBRN) to mutually recognise Official
Batch Protocol Review (OBPR) certificates between Member States provided the procedure and rules codified by
the network are followed. This legislation concerns EU/EEA Member States and is also applied by any state
having signed a formal agreement, which includes recognition of OCABR, with the EU. Currently, Switzerland has
done so via a Mutual Recognition Agreement (MRA) (source EDQM).
2.5. The role of the European Pharmacopoeia
The Convention on the elaboration of a European Pharmacopoeia (or international treaty) adopted at the Council
of Europe in 1964, laid the groundwork for the European Pharmacopoeia as a guarantee of the quality of
medicines produced in Europe.
The European Pharmacopoeia Convention has now been signed by 37 Member States1 including the EU;
moreover 24 European and non-European countries2, and the World Health Organisation (WHO) have observer
status. Close relations are maintained with the licensing authorities of the European Economic Area, where
integration is developing through contact with the EMA and the implementation of common directives and
guidelines on medicines for human and veterinary use.
The European Pharmacopoeia consists of monographs describing individual quality standards (set of control
tests applicable to one ingredient) and general quality standards applicable to families of ingredients or to dosage
forms, as well as general methods of analysis. It defines the minimum acceptable standards for products to be
authorised within the European Union because compliance with monographs is a mandatory requirement within
Directive 2001/82/EC. This requires that products must comply with the relevant specific monograph where one
exists or with the general monographs where one does not.
The European Directorate for the Quality of Medicines & Health Care (EDQM) is the administrative entity in the
Council of Europe that provides the secretariat services for the European Pharmacopoeia. EDQM creates,
maintains and distributes the international standard reagents referred to in monographs of the European
Pharmacopoeia, including standards for veterinary biologicals.
Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland,
Former Yugolsav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Montenegro, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey, United Kingdom and the European Union. Member States must apply the standards of the
European Pharmacopoeia.
Albania, Algeria, Argentina, Armenia, Australia, Belarus, Brazil, Canada, China (Peoples Rep. of), Georgia, Guinea,
Israel, Kazakhstan, Madagascar, Malaysia, Moldova, Morocco, Russian Federation, Senegal, Singapore, Syria, Tunisia,
Ukraine, United States of America and the World Health Organization. Observer States do not have to apply the European
Pharmacopoeia standards. Some of them apply the standards on a voluntary basis.
1352
In 1990, the European Pharmacopoeia co-founded, with the Japanese Pharmacopoeia and the United States
(US) Pharmacopoeia, the Pharmacopoeial Discussion Group (PDG); this group is working assiduously for
harmonisation at the global level.
The European Pharmacopoeia Commission adopted several harmonised texts for veterinary vaccines at its
142nd session. The safety tests and the tests for increased virulence performed during development of the
vaccines were harmonised in the framework of harmonisation with VICH (see below) Guidelines 41 and 44, and
to ensure consistency with European regulations.
3.
In the United States of America
3.1. Introduction
In the United States of America (USA), veterinary biologics or veterinary biological products are defined as all
viruses, sera, toxins (excluding substances that are selectively toxic to microorganisms, e.g. antibiotics), or
analogous products at any stage of production, shipment, distribution, or sale, that are intended for use in the
treatment (prevention, diagnosis, management, or cure) of diseases of animals and that act primarily through the
direct stimulation, supplementation, enhancement, or modulation of the immune system or immune response. The
term biological products includes, but is not limited to, vaccines, bacterins, allergens, antibodies, antitoxins,
toxoids, immunostimulants, certain cytokines, antigenic or immunising components of live organisms, and
diagnostic components that are of natural or synthetic origin or that are derived from synthesising or altering
various substances or components of substances such as microorganisms, genes or genetic sequences,
carbohydrates, proteins, antigens, allergens, or antibodies.
3.2. Legal basis

The Virus/Serum/Toxin Act of 1913 (the VST Act), as amended, 21 U.S.C. Sections 151 to 159, provides the
legal authority for the regulation of immunologicals and biologicals for animal use in the USA. The regulatory
programme implementing the requirements of the VST Act is administered by the Center for Veterinary Biologics
(CVB), Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture
(USDA). Administrative regulations, duly promulgated and with effect of law, are published in Title 9, Code of
Federal Regulations, Parts 101 to 118 (2006). In addition, APHIS has issued programme guidance in CVB
Notices, Veterinary Services Memoranda, Veterinary Biologics General Licensing Considerations, and the
Veterinary Biologics Program Manual. These may be accessed on the CVB web site at
www.aphis.usda.gov/vs/cvb.
The VST Act requires that products governed by the Act that enter channels of commerce be not worthless,
contaminated, dangerous or harmful. The regulatory scheme implementing these standards is structured to
require manufacturers of these products to apply for licences prior to marketing, and to place certain evidentiary
responsibilities on those applicants, i.e. manufacturers are required to demonstrate through the submission of
certain information, research data, and test results that their products are pure, safe, potent and efficacious. The
APHIS programme for immunologicals and biologicals for animal use regulates the manufacture and release of
products on to the market through a system of licensing, inspection, testing and post-marketing surveillance that
ensures that the statutory and regulatory standards are met.
3.3. Licensing and initial inspection

Any person or firm seeking to manufacture in the USA an immunological or biological for animal use must obtain
from APHIS both a licence to manufacture at a specified facility (Establishment Licence), and a licence for every
particular product to be manufactured (Product Licence). These licence requirements apply whether the product is
to be released on to the US market or is to be exported to markets abroad. Typically, an applicant will request a
facility licence at the same time as the licence for the first product. Once the facility licence and one product
licence have been obtained, a firm that seeks to manufacture and market new products needs only to apply for
additional product licences. A person or firm located overseas that seeks to market its product in the USA must
also apply for marketing authorisation. In the case of an imported product, however, the authorisation is termed a
permit rather than a licence.
To obtain a facility licence, the applicant must submit for approval the blueprint (that is, the architects plan of the
buildings) and blueprint legends for the facility. APHIS reviews these blueprints and legends to ensure that the
facility will operate in a manner consistent with GMP. If the applicant subsequently makes any physical or
operational changes to the facility, revised blueprints and legends must be submitted immediately.
To obtain a product licence, the applicant must establish the purity and identity of all master seeds and master
cell stocks that will be used in the manufacture of the product, and must submit for approval a detailed outline of
production. The outline of production includes not only the details of the method of product manufacture, but also
a description of the procedures for collecting and submitting samples and for releasing batches. The applicant
1353
must also provide information regarding the professional and technical credentials of company personnel, and
must identify a qualified individual (termed under US regulations as the government liaison) who acts as the
official contact with CVB during the licensing process, and who is subsequently responsible for the submission of
the firms test reports in conjunction with the release of the product on to the market. The applicant is required to
submit test data that demonstrate that the product produced in accordance with the outline is pure, safe, potent
and efficacious. The applicant must submit to CVB laboratories samples of three consecutive batches of the
product so that the results of the applicants tests of the product can be confirmed.
Finally, before the facility or product licences are issued, the applicants premises are subject to a comprehensive
inspection by APHIS examiners. The inspection ensures that the facility is operating in a manner consistent with
GMP by confirming that the establishment is configured in the manner set out in the approved blueprints and
legends, that the production line is set up and operating in accordance with the approved outline of production,
and that records are adequately kept and maintained for each step in production. The inspection also confirms
that the applicant follows procedures consistent with GLPs, that the in-process and final product testing
programme is conducted properly and appropriately documented, that the sampling is conducted properly, and
that adequate procedures for determining and documenting the release of the product on to the market are in
place.
3.4. Post-licensing inspection

Once a firm has been issued facility and product licences, APHIS will routinely conduct thorough follow-up
inspections of the facility to ensure that the licensee continues to operate the establishment in accordance with
the programme regulations and in the manner represented at the time of licensing. Post-licensing inspections are
conducted unannounced periodically. If the licensee proposes any significant changes to the facility or to the
method of production of a licensed product, APHIS retains the right to conduct a special inspection prior to
approving the changes.
3.5. Testing
Each licensee is responsible for thoroughly testing all of its production processes and each serial (or lot) of every
product prior to release on to the market. The type and amount of testing required depends on the particular
product, but is determined and approved by the regulatory authority prior to the issuing of the product licence. A
qualified individual employed by the licensee (government liaison) is responsible for selecting the samples to be
tested, for monitoring the licensees testing programme, and for certifying the test results to the regulatory
authority.
At the same time that the firm selects its samples for its own in-house testing, it also selects samples to be
submitted to the CVB laboratories. The CVB retains the right to conduct confirmatory testing. CVB then selects a
percentage of the samples submitted for confirmatory testing to verify the accuracy and proficiency of the
manufacturers tests. The testing is conducted prior to marketing authorisation for each serial. By regulation, CVB
policy stipulates that it is required to put its selected samples on test within 14 days of the date on which the
samples are received; ordinarily, samples are put on test sooner than the 14-day limit so that the testing of
production by the firm and the CVB proficiency testing programme are effectively being conducted at the same
time.
When the firm receives the results of its own tests, the government liaison submits those results to the regulatory
authority along with a batch release form, initiating the release procedure. If the batch has not been selected as
part of the proficiency testing programme, or if it has been selected but the CVB tests confirm the companys test
results, the release form is counter-signed by the regulatory authority completing the release procedure. If either
the company tests or the proficiency tests indicate that the batch may be unsatisfactory, the batch is not eligible
for release.
If the licensee makes a proposal to modify its facility or its operation in any way that could affect the purity, safety,
potency or efficacy of the product, the regulatory authority may require the licensee to provide data demonstrating
the purity, safety, potency and efficacy of the product as well as to submit samples of the product to CVBs
laboratories for confirmatory testing.
3.6. Post-marketing surveillance

CVB operates a post-marketing surveillance programme to monitor the performance of products on the market.
Under this programme, CVB typically learns of any problems relating to product quality through consumer reports
or complaints, although the programme regulations also place an obligation on the licensee to inform CVB of any
problem that comes to its attention regarding the purity, safety or potency of the product. CVB has the legal
authority to intervene in the marketplace where there are serious concerns with respect to the purity, safety,
potency or efficacy of the product.
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B. COMPARISON OF EUROPEAN UNION AND UNITED STATES REGULATIONS

Veterinary biologicals must meet certain basic criteria, regardless of the Regulatory Agency overseeing their
production. These criteria include:
Safety: the product must be safe in the target species and, if live, in species exposed to shed organisms;
Efficacy: the product should be effective according to claims indicated on the label;
Quality: includes purity, potency and consistency;
Purity: the product must be free from contaminating agents;
Potency: each batch of product should be formulated, and tested, to ensure effectiveness and reproducibility
of activity as demonstrated in the registration data.
Although, on a global basis, agencies and regulations differ, all strive to ensure that products offered to the endconsumer conform to these basic standards.
The EU uses a complete system that is a combination of GMP, including validated processes and specifications
of materials, together with production methods that ensure the quality of the final product. In-process and batch
controls (tests) constitute additional guarantees of the quality of IVMPs. The safety tests are conducted under
GLP and the field efficacy tests under GCP. The USA defines acceptable manufacturing processes in the outline
of production and detailed facility description (blueprints and blueprint legends), and relies on inspection and
confirmatory testing to achieve the same goal. Although different, both systems are designed to allow only pure,
safe, potent, and effective biologicals to be released to the consumer.
In addition to the data provided by the applicant, expert reports have to be included in the EU marketing
authorisation application file (dossier). Each main section of the EU dossier, including analytical, safety and
efficacy, must be reviewed by an independent expert. The assessment of the expert is included in the marketing
authorisation file. No such system of third-party review exists under the USDA registration system with the
exception of certain biotechnologically derived products.
There are many procedural differences between the EU and the USA. Harmonisation between the two systems
should be established where possible, on the recognition of equivalence for tests and procedures that are
performed to assess a vaccine and that ensure quality, safety and efficacy of the product. Mutual recognition
agreements (MRAs) covering veterinary biologicals have been signed between the EU and Australia and between
the EU and New Zealand. These MRAs are now at an operational stage. Progress on MRAs between the EU and
the USA, regarding veterinary biologicals, is likely to take longer to achieve.
C. THE ROLE OF INTERNATIONAL ORGANISATIONS

Most nations have a range of official acts that regulate the sale and use of veterinary biologicals. Almost all of
these acts stipulate minimum requirements for quality, safety and efficacy of veterinary biologicals (mostly
vaccines), to be tested at independent laboratories, usually under State supervision. These acts and tests may
differ from one country to another, and they involve costs and restrictions for producers, users and testers.
Many of the vaccines described in this Terrestrial Manual are produced and/or used in countries that do not
currently apply regimens of authorisation and testing as stringent as those described in this chapter.
Nevertheless, it is useful to be aware of the regulations operating in different regions and, therefore, the testing
and inspection that is likely to have been carried out there on a veterinary biological.
The idea of harmonising this testing to simplify and reduce costs on a regional, or even world scale, is not new,
and much has been accomplished during the past 20 years. The purpose of this section is to review the current
situation by describing the role of international organisations in the regulation of veterinary vaccines.
In this section the term international organisation refers to those concerned with animal health on a world-wide
scale (OIE, the Food and Agriculture Organization of the United Nations [FAO] and the WHO).
1.
The role of the OIE (World Organisation for Animal Health)
The OIE was founded in Paris in 1924 as the world organisation for animal health, and comprised 178 Member
Countries in the year 2012. The principal aims of the OIE are: to ensure transparency in the global animal disease
and zoonosis situation, to collect, analyse and disseminate scientific veterinary information, to provide expertise
and encourage international solidarity in the control of animal diseases, within its mandate under the Agreement
1355
on Sanitary and Phytosanitary Measures (SPS Agreement) of the World Trade Organization (WTO), to safeguard
world trade by publishing health standards for international trade in animals and animal products, to improve the
legal framework and resources of national Veterinary Services and to provide a better guarantee of the safety of
food of animal origin and to promote animal welfare through a science-based approach (Truszczynski & Blancou,
1992).
Within the OIE there are four Specialist Commissions: the Terrestrial Animal Health Standards Commission,
which deals with the Terrestrial Animal Health Code, the Biological Standards Commission, the Scientific
Commission for Animal Diseases and the Aquatic Animal Health Standards Commission (including diseases of
molluscs and crustaceans). In addition, there are three Working Groups: the Working Group on Wildlife Diseases,
the Working Group on Animal Production Food Safety and the Working Group on Animal Welfare.
Among the Specialist Commissions, the one most closely connected with standardisation is the Biological
Standards Commission. This Commission establishes standards for diagnostic methods (including diagnostic
preparations) and for vaccines. Its terms of reference reflect the Commissions obligation to participate in the
standardisation of biological products, including vaccines used for prophylactic purposes. The Biological
Standards Commission is responsible for the preparation of the OIE Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals, and the organisation of Reference Laboratories for many of the diseases on the OIE List.
However, full standardisation of vaccine testing can be achieved only when the necessary standards have been
devised. It is hoped to reach the goal of standardisation and wide availability of standards through the
participation of OIE Reference Laboratories. The functions and responsibilities of experts at the over 235 OIE
Reference Laboratories include the provision of a centre of excellence in a designated activity; standardisation of
methods; preparation, storage and distribution of standard antisera, antigens and other reagents.
Among the OIE Collaborating Centres, four may be involved at some stage in veterinary vaccine control and/or
harmonisation: the Collaborating Centre for Veterinary Medicinal Products in Fougres (France), the
Collaborating Centre for ELISA (enzyme-linked immunosorbent assay) and Molecular Techniques in Animal
Disease Diagnosis in Vienna (Austria), Collaborating Centre for Diagnosis and Control of Animal Diseases and
Related Veterinary Product Assessment in Asia in Tokyo (Japan) and the Collaborating Centre for the Diagnosis
of Animal Diseases and Vaccine Evaluation in the Americas in Ames (USA).
In 1994, following discussions with the International Technical Consultation on Veterinary Drug Registration
(ITCVDR), the OIE set up an Ad hoc Group on the Harmonisation of Veterinary Medicines, which was the first
step towards the creation of the VICH (International Cooperation on Harmonisation of Technical Requirements for
Registration of Veterinary Products) (see Section C.4 below).
In May 2003, the OIE International Committee adopted a resolution entitled OIE Procedure for Validation and
Certification of Diagnostic Assays (Test Methods) for Infectious Animal Diseases. This resolution requires the OIE
Director General to make provisions to establish an OIE registry for assays with levels of validation specified.
Fitness for purpose should be used as a criterion for validation.
2.
The role of the Food and Agriculture Organization of the United Nations
FAO, established in 1945, is responsible for agricultural development and food production. The Animal Production
and Health Division (AGA) within the Agriculture Department is concerned with livestock development, and it
includes the Animal Health Service (AGAH), the main role of which is to assist Member Countries in the control
of animal diseases, with the aim of improving livestock production as an integral component of general social,
economic and agricultural development. FAOs involvement in testing veterinary biologicals is primarily through its
technical assistance system to Member Countries to set up and even execute independent quality control of
vaccines and other biologicals. One example is FAOs assistance to the AU (African Union) in setting up a system
for continental testing of veterinary vaccines, especially against rinderpest and contagious bovine
pleuropneumonia, by the Pan African Veterinary Vaccine Center (PANVAC). FAO also commissions, at the
request of Member Countries, initiatives for either quality assurance of vaccines and other biologicals or expert
consultations on this subject, or publication of manuals on the production and quality control of vaccines.
Furthermore, two auxiliary services can be asked to intervene on matters concerning these products, namely
Codex Alimentarius and the Division of Nuclear Techniques in Food and Agriculture. The latter is operated jointly
by FAO and the International Atomic Energy Agency (IAEA) based in Vienna (Austria). It has an Animal
Production and Health Section, which assists veterinary services and research institutes in developing countries
to establish radio-immunoassay (RIA) and ELISA techniques. Linked to this activity is a quality assurance scheme
under which laboratories in receipt of FAO/IAEA ELISA kits are required to routinely monitor internal quality
controls and to periodically (once or twice a year) test a batch of unknown samples, and to forward the results to
IAEA. The overall aim is to provide assurance to all concerned that the results being generated through the use of
such internationally standardised and validated kits can be relied upon as correct.
1356
3.
The role of the World Health Organization
Currently WHO is not directly involved in preparing international reference preparations (i.e. antigens or
antibodies) for purely veterinary use, but has developed and still retains in the National Institute for Biological
Standards and Control, Potters Bar (UK) some materials related to purely veterinary diseases (e.g. Newcastle
disease live vaccine, classical swine fever serum). WHO wishes to retain a role in this area in instances where the
veterinary reference preparations and guidance documents have a direct relevance to human health (Joint
FAO/WHO Expert Committee on Brucellosis, 1986; Meslin et al., 1996; WHO Expert Committee on Biological
Standardisation, 1992; WHO Expert Committee on Rabies, 1992). This involves zoonotic and potentially zoonotic
agents and other infectious agents of animal origin that are potential contaminants of biological products, whether
these are vaccines produced in cell cultures or organs for xenotransplantation. At the meeting of the Expert
Committee on Biological Standardization in October 1998, a review of currently retained international standards
and reference preparations for veterinary medicine was carried out and a list of candidates for discontinuation,
replacement and revision was suggested (Joint FAO/WHO Expert Committee on Brucellosis, 1986). The Expert
Committee however decided to defer taking action on preparation of veterinary reference materials pending an
evaluation by WHO with its partners in the veterinary field of the need for these various biological products. In
addition, the present day topicality of certain preparations, especially veterinary vaccines against known zoonoses
(e.g. anthrax, brucellosis) adopted and/or revised in the 1960s and 1970s, also needs to be evaluated.
The format of the list of Requirements for Biological Substances published as an Appendix to each report of the
Expert Committee on Biological Standardization was revised in 1998 and should facilitate the retrieval of
information and achieve the aim of improved transparency.
4.
The role of VICH
4.1. What is VICH?

a)
Short description of VICH

VICH is a trilateral (EU-Japan-USA) programme aimed at harmonising technical requirements for veterinary
product registration. Its full title is the International Cooperation on Harmonisation of Technical Requirements
for Registration of Veterinary Medicinal Products. VICH was officially launched in April 1996.
b)
Background and history

The initiative to begin the harmonisation process came in 1983 when the first International Technical
Consultation on Veterinary Drug Registration (ITCVDR) was held. Since then a series of government and
industry initiatives have been developed, culminating in the formation of the VICH.
The Codex Alimentarius formed a Committee on Residues of Veterinary Drugs in Foods in 1985. Standard
requirements for veterinary product registration were adopted in Europe in 1981.
The US Food and Drug Administration and the European Commission have held regular bilateral meetings
for the last decade to discuss common areas of interest. This has involved mutual exchange of guidelines for
consultation.
The first International Conference on Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH) was held in Brussels in November 1991. The meeting brought
together regulators and industry representatives from the USA, the EU and Japan to address quality, safety
and efficacy requirements in the three regions.
Meetings on harmonisation of veterinary biologicals were held in Ploufragan, France, in January 1992, in
Arlington, USA, in 1994 and in Singapore in 1995.
In January 1993 the GHOST (Global harmonisation of standards) discussion document was published by
FEDESA3. It set out a programme for the international harmonisation of registration requirements for
veterinary pharmaceuticals and biologicals.
Following discussions at ITCVDR and the OIE conferences, the OIE set up an ad hoc group on
harmonisation of veterinary medicinal products in 1994.
FEDESA: Fdration Europenne de la Sant Animale. In 2002, FEDESA became the International Federation for Animal
Health (IFAH).
1357
c)
The creation and scope of VICH

Preparatory work for the establishment of VICH was carried out by this OIE ad hoc group. During 1994 and
1995, two meetings were held at which the scope of veterinary harmonisation was discussed and the
membership and objectives of the VICH proposed.
On the subject of food safety standards, it was decided that the VICH should complement the work of Codex
and JECFA4. Issues related to GLP and GMP that are already the subject of mutual agreements will not
normally come within the remit of the VICH. Issues related to biologicals were considered appropriate to fall
within the scope of VICH.
Fundamental to the selection of priority topics for consideration by the VICH was the discussion document
prepared by COMISA for the Steering Committee. This report:
assesses those ICH guidelines which could be adapted to the VICH programme;
defines in detail areas of non-harmonisation between the EU, the US and Japan and provides a series
of concept papers on key topics; and
puts forward preliminary suggestions for priority topics.
With all the ground-breaking work completed, the Steering Committee of the VICH held its first meeting in
April 1996, at which the membership and the working procedures were agreed and a work programme
established.
d)
The objectives of VICH

The objectives of the VICH are along the same lines as those of the ICH.
e)
Establish and implement harmonised regulatory requirements for veterinary medicinal products in the
VICH regions, which meet high quality, safety and efficacy standards and minimise the use of test
animals and costs of product development.
Provide a basis for wider international harmonisation of registration requirements.
Monitor and maintain existing VICH guidelines, taking particular note of the International Conference on
Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH)
work programme and, where necessary, update these VICH Guidelines.
Ensure efficient processes for maintaining and monitoring consistent interpretation of data requirements
following the implementation of VICH guidelines.
By means of a constructive dialogue between regulatory authorities and industry provide technical
guidance enabling response to significant emerging global issues and science that impact on regulatory
requirements within the VICH regions.
Progress toward achieving the VICH objectives
For veterinary immunologicals there is an ongoing programme of harmonisation in a number of areas

including target animal safety studies, reversion to virulence and tests for the presence of Mycoplasma.
To date only a relatively small number of VICH guidelines have been developed for veterinary biologicals
and it is worth emphasising the difficulties in reaching agreement on veterinary biologicals between the
three regions.
The only two adopted VICH Guidelines for veterinary biologicals are testing of residual formaldehyde
adopted May 2003 and testing of residual moisture also adopted in May 2003. Several other Guidelines
that apply to veterinary biologicals and all other veterinary medicinal products have also been adopted.
Following an in-depth reflection held by all parties concerned by VICH under the auspice of OIE, the second
phase of VICH for 20062010 was publicly launched during a public conference VICH3 held in Washington
in May 2005.
More information is available at: http://vich.eudra.org.
4
JECFA: Joint FAO/WHO Expert Committee on Food Additives.
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CONCLUSION
At the moment, there is a clear intention to achieve greater international harmonisation of regulatory requirements
for veterinary biologicals (Vannier et al., 1997). Progress has already been achieved through international
organisations to allow fair competition in the marketing of veterinary products. Although past efforts by
international organisations have not resulted in a level of harmonisation sufficient to facilitate international trade,
they have laid the groundwork for current efforts. National authorities recognise the advantages of harmonisation
and are now committed to working toward this goal.
The efforts of international organisations have made the goal of harmonisation possible and have resulted in an
organisation and process for proceeding toward this goal. Success in achieving this goal will depend on the
willingness of participating national authorities to work together and accept the compromises that will be
necessary to resolve the difficult scientific and policy issues.
REFERENCES
ANON (1960). The Pharmaceutical Affairs Law. Japan Law No. 145 in 1960, as amended on 26 June 1996.
Pharmaceutical Affairs Law, Enforcement Ordinance and Enforcement Regulations, Yakugyo Jiho, Japan, 291
(in Japanese and English).
ANON (2002a). The Minimum Requirements for Biological Products for Animal Use. Ministry of Agriculture,
Forestry and Fisheries Notice No. 1567 in 2002, as amended ON 3 May 1993. Japan Association of Veterinary
Biologics, Tokyo, Japan, 1680 (in Japanese).
ANON (2002b). The Assay Standard for Biological Products for Animal Use. Ministry of agriculture, Forestry and
Fisheries Notice No. 1568 in 2002, as amended at 25 September 1997. Japan Association of Veterinary
Biologics, Tokyo, Japan, 1507 (in Japanese).
ARTIGES A. (1997). The role of the pharmacopoeias. In: Veterinary Vaccinology, Pastoret P.P., Blancou J.,
Vannier P. & Verschueren C., eds. Elsevier Science, Amsterdam, the Netherlands, 674679.
BRUNKO P. (1997). Procedures and technical requirements in the European Union. In: Veterinary Vaccinology, Pastoret
P.P., Blancou J., Vannier P. & Verschueren C., eds. Elsevier Science, Amsterdam, the Netherlands, 705717.
CODE OF FEDERAL REGULATIONS (2006). Animals and Animal Products, Title 9, Parts 1199. The Office of the
Federal Register, National Archives and Records Administration. US Government Printing Office, Washington
DC, USA.
EUROPEAN UNION (1999). The Rules Governing Medicinal Products in the European Union. Eudralex, Vols 49.
Office Publications of the European Communities, Luxembourg.
JOINT FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO)/WORLD HEALTH ORGANIZATION (WHO)
EXPERT COMMITTEE ON BRUCELLOSIS (1986). Sixth Report. WHO Technical Report Series No. 740, World Health
Organization, Geneva, Switzerland, 132 pp.
MAKIE H. (1998). The activities of veterinary vaccine control laboratories. Rev. sci. tech. Off. Int. Epiz., 17, 578584.
MESLIN F.-X., KAPLAN M.M. & KOPROWSKI H. (1996). Laboratory Techniques in Rabies. World Health Organization,
Geneva, Switzerland, 476 pp.
TRUSZCZYNSKI M. & BLANCOU J (1992). The role of the Office International des Epizooties in the standardisation of
biologicals. In: Symposium on the First Steps Towards an International Harmonization of Veterinary Biologicals
and Free Circulation of Vaccines within the EEC, Ploufragan, 1992. Dev. Biol. Stand., 79, 9598.
VANNIER P., TRUSZCYNSKI M. & ESPESETH D. (1997). Implementing International harmonisation. In: Veterinary
Vaccinology, Pastoret P.P., Blancou J., Vannier P. & Verschueren C., eds. Elsevier Science, Amsterdam, the
Netherlands, 712717.
WORLD HEALTH ORGANIZATION (WHO) EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION (1992). Forty-second
Report. WHO Technical Report Series No. 822, World Health Organization, Geneva, Switzerland, 84 pp.
WORLD HEALTH ORGANIZATION (WHO) EXPERT COMMITTEE ON RABIES (1992). Eighth Report. WHO Technical Report
Series No. 824, World Health Organization, Geneva, Switzerland, 75 pp.
*
* *
1359
PART 4
OIE REFERENCE EXPERTS1
AND DISEASE INDEX
NB: This list is current in May 2012. For the most recent list, please consult the OIE web site at: http://www.oie.int/en/ourscientific-expertise/reference-laboratories/list-of-laboratories/
1361
LIST OF OIE REFERENCE LABORATORIES IN MAY 20121
African horse sickness Peste quine Peste equina

Dr Baratang Alison Lubisi
Onderstepoort Veterinary Institute, Agricultural Research Council, Private Bag X05, Onderstepoort 0110
AFRIQUE DU SUD SOUTH AFRICA SUDFRICA
Tel: (27-12) 529.91.17 Fax: (27-12) 529.94.18
Email: ubisia@arc.agric.za
Dr Concepcin Gmez-Tejedor Ortiz(1)
Laboratorio Central de Veterinaria de Algete (LCV), Domicilio carretera de Algete, km 8. 28110 Algete (Madrid)
ESPAGNE SPAIN ESPAA
Tel: (34-91) 347.92.77 Fax: (34-91) 629.05.98
Email: cgomezte@mapya.es
Dr Jos Manuel Snchez-Vizcano(2)
Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Facultad de Veterinaria, HCV Planta stano, Universidad
Complutense de Madrid (UCM), Avda. Puerta de Hierro s/n, 28040 Madrid
ESPAGNE SPAIN ESPAA
Tel: (34-91) 394.40.82 Fax: (34-91) 394.39.08
Email: jmvizcaino@visavet.ucm.es
Dr Javier Castillo-Olivares2
Institute for Animal Health, Pirbright Laboratory
Ash Road, Pirbright, Woking, Surrey GU24 ONF
ROYAUME-UNI UNITED KINGDOM REINO UNIDO
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: javier.castillo-olivares@iah.ac.uk
African swine fever Peste porcine africaine Peste porcina africana
Onderstepoort Veterinary Institute, Agricultural Research Council, Private Bag X05, Onderstepoort 0110
Tel: (27-12) 529.91.17 Fax: (27-12) 529.94.18
Dr Jos Manuel Snchez-Vizcano
Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Facultad de Veterinaria, HCV Planta stano, Universidad
Complutense de Madrid (UCM), Avda. Puerta de Hierro s/n, 28040 Madrid
ESPAGNE SPAIN ESPAA
Tel: (34-91) 394.40.82 Fax: (34-91) 394.39.08
Email: jmvizcaino@visavet.ucm.es
Dr Linda Dixon3
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: linda.dixon@iah.ac.uk
2
3
For certain diseases, two institutions have been jointly designated as OIE Reference Laboratory or Collaborating Centre.
Where this occurs, the superscript (1) or (2) will be placed after the name of the designated Reference Expert. Pour
certaines maladies, 2 instituts ont t dsigns conjointement comme laboratoire ou centre collaborateur de rfrence de
lOIE. Dans ce cas les mentions (1) et (2) sont places aprs les noms des experts de rfrence dsigns. Para algunas
enfermedades, se designaron conjutamente dos instituciones como Laboratorios de Referencia o Centros Colaboradores.
En tales casos los Expertos de Referencia vienen sealados con los respectivos indices notescritas (1) y (2)
Subject to confirmation by the World Assembly in May 2013.
1363
List of OIE Reference Laboratories in 2012
American foulbrood of honey bees Loque amricaine des abeilles mellifres Loque americana de las abejas melferas
Dr Adriana M. Alippi
Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Unidad de Bacteriologa del
Centro de Investigaciones en Fitopatologa (UB-CIDEFI), calle 60 y 119 s/n c.c. 31, 1900 La Plata
ARGENTINE ARGENTINA ARGENTINA
Tel.: (54-221) 423.67.58 ext. 423 Fax: (54-221) 425.23.46;
E-mail: alippi@biol.unlp.edu.ar / adrianaalippi@gmail.com
Anaplasma spp. Anaplasma spp. Anaplasma spp.
Dr Fernando Parrodi Lpez
Centro Nacional de Servicios de Constatacin en Salud Animal (CENAPA), Carretera Cuernavaca Cuautla #8534
Colonia Progreso, CB 62550, Jiutepec, Morelos
MEXIQUE MEXICO.. MXICO
Tel: (52-777) 319.02.02
Email: fernando.parrodi@senasica.gob.mx / fernandoparrodi@hotmail.com
Anthrax Fivre charbonneuse Carbunco bacteridiano
Dr Elizabeth Golsteyn-Thomas
Canadian Food Inspection Agency, Lethbridge Laboratory
P.O. Box 640, Township Road 9-1, Lethbridge, Alberta T1J 3Z4
CANADA CANADA CANAD
Tel: (1-403) 382.55.51 Fax: (1-403) 381.82.83
Email: Betty.Golsteyn-Thomas@inspection.gc.ca
Dr Ginger Harvey
National Veterinary Services Laboratories
P.O. Box 844, Ames, IA 50010
TATS-UNIS DAMRIQUE UNITED STATES OF AMERICA ESTADOS UNIDOS DE AMRICA
Tel: (1-515) 337.73.42 Fax: (1-515) 337.75.69
Email: inger.r.harvey@aphis.usda.gov
Antimicrobial resistance Rsistance microbienne Resistencia microbiana
Dr Chris Teale
AHVLA Weybridge
New Haw, Addlestone, Surrey KT15 3NB
Tel: (44-1734) 46.76.12 Fax: (44-1734) 44.10.60
Email: christopher.teale@ahvla.gsi.gov.uk
Aujeszkys disease Maladie dAujeszky Enfermedad de Aujeszky
Dr Andr Jestin
Anses Ploufragan, Laboratoire dtudes et de recherches avicoles, porcines et piscicoles,
Zoople-Les Croix, BP 53, 22440 Ploufragan
FRANCE FRANCE FRANCIA
Tel: (33-2) 96.01.62.22 Fax: (33-2) 96.01.62.53
Email: andre.jestin@anses.fr
Dr Andre T.J. Bianchi
Central Veterinary Institute of Wageningen UR (CVI)
Houtribweg 39, P.O. Box 65, 8200 AB AA Lelystad
PAYS-BAS THE NETHERLANDS PASES BAJOS
Tel: (31-320) 23.88.00 Fax: (31-320) 23.86.68
Email: andre.bianchi@wur.nl
Dr Sabrina L. Swenson
National Veterinary Services Laboratories, P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.75.51 Fax: (1-515) 337.73.48
Email: sabrina.l.swenson@aphis.usda.gov
1364
Avian chlamydiosis Chlamydiose aviaire Clamidiosis aviar

Dr Konrad Sachse
Friederich-Loeffer Institute, Federal Research Institute for Animal Health
Institute of Molecular Pathogenesis, Naumburger Str. 96a, 07743 Jena
ALLEMAGNE GERMANY ALEMANIA
Tel: (49-3641) 80.43.34 Fax: (49-3641) 80.42.28
Email: konrad.sachse@fli.bund.de
Avian mycoplasmosis Mycoplasmose aviaire Micoplasmosis aviar (Mycoplasma gallisepticum, M. synoviae)
Dr Evelin Lobo Riveroi
MYCOLAB Laboratorio para diagnstico de micoplasmas, Centro Nacional de Sanidad Agropecuaria
San Jos de las Lajas, Provincia Mayabeque
CUBA CUBA CUBA
Tel: (53-47) 86.33.14 ext. 153 Fax: (53-47) 86.38.97
Email: elobo@censa.edu.cu / evelynlobo68@hotmail.com
Dr Naola Ferguson-Noel
The University of Georgia, Poultry Diagnostic and Research Center
953 College Station Rd, Athens, Georgia 30602-4875
TATS-UNIS DAMERIQUE UNITED STATES OF AMERICA ESTADOS UNIDOS DE AMRICA
Tel: (1-706) 542.30.68 Fax: (1-706) 542.56.30
Email: naolaf@uga.edu
Avian tuberculosis Tuberculose aviaire Tuberculosis aviar
Dr Ivo Pavlik
Veterinary Research Institute, Hudcova 70, 62132 Brno
TCHEQUE (Rp) CZECH (Rep.) CHECA (Rep.)
Tel: (420-5) 33.33.16.01 Fax: (420-5) 33.33.12.29
Email: pavlik@vri.cz
Babesia spp. Babesia spp. Babesia spp.
Dr Fernando Parrodi Lpez
Centro Nacional de Servicios de Constatacin en Salud Animal (CENAPA)
Carretera Cuernavaca Cuautla #8534, Colonia Progreso, CB 62550, Jiutepec, Morelos
MEXIQUE MEXICO.. MXICO
Tel: (52-777) 319.02.02
Email: fernando.parrodi@senasica.gob.mx / fernandoparrodi@hotmail.com
Bacterial kidney disease Rnibactriose Renibacteriosis (Renibacterium salmoninarum)
Dr James R. Winton
Western Fisheries Research Center, 6505 N.E. 65th Street, Seattle, Washington 98115
Tel: (1-206) 526.65.87 Fax: (1-206) 526.66.54
Email: jim_winton@usgs.gov
Bee diseases Maladies des abeilles Enfermedades de las abejas
Dre Marie-Pierre Chauzat4
Anses Sophia Antipolis, Laboratoire d'tudes et de recherches sur les ruminants et les abeilles
105 route des Chappes, BP 111, 06902 Sophia Antipolis
Tel: (33-4) 92.94.37.00 Fax: (33-4) 92.94.37.01
Email: marie-pierre.chauzat@anses.fr
Dr Wolfgang Ritter
Chemisches und Veterinruntersuchungsamt Freiburg
P.O.B. 100462, 79123 Freiburg
Tel: (49-761) 150.21.75 Fax: (49-761) 150.22.99
Email: wolfgang.ritter@cvuafr.bwl.de
4
1365
Bluetongue Fivre catarrhale du mouton Lengua azul

Dr Peter Daniels
CSIRO, Australian Animal Health Laboratory (AAHL)
5 Portarlington Road, Private Bag 24 (Ryrie Street), Geelong 3220, Victoria
AUSTRALIE AUSTRALIA AUSTRALIA
Tel: (61-3) 52.27.52.72 Fax: (61-3) 52.27.55.55
Email: peter.daniels@csiro.au
Dr Giovanni Savini
Istituto Zooprofilattico Sperimentale dellAbruzzo e del Molise G. Caporale
Via Campo Boario, 64100 Teramo
ITALIE ITALY ITALIA
Tel: (39-0861) 33.24.40 Fax: (39-0861) 33.22.51
Email: g.savini@izs.it
Onderstepoort Veterinary Institute, Agricultural Research Council
Private Bag X05, Onderstepoort 0110
Tel: (27-12) 529.91.17 Fax: (27-12) 529.94.18
Email: lubisia@arc.agric.za
Dr Peter Mertens5
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: peters.mertens@iah.ac.uk
Dr Eileen N. Ostlund
Diagnostic Virology Laboratory, National Veterinary Services Laboratories
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.75.51 Fax: (1-515) 337.73.48
Email: eileen.n.ostlund@aphis.usda.gov
Bovine babesiosis Babsiose bovine Babesiosis bovina
Prof. Ikuo Igarashi
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine
Inada-cho Nishi 2-13, Obihiro, Hokkaido 080-8555
JAPON JAPAN JAPN
Tel: (81-155) 49.56.41 Fax: (81-155) 49.56.43
Email: igarcpmi@obihiro.ac.jp
Bovine genital campylobacteriosis Campylobactriose gnitale bovine Campilobacteriosis genital bovina
Dr Jaap Wagenaar(1)
Animal Sciences Group (ASG), Division of Infectious Diseases
P.O. Box 65, 8200 AB Lelystad
Tel: (31-320) 23.81.57 Fax: (31-320) 23.89.61
Email: j.a.wagenaar@vet.uu.nl
Dr Jaap Wagenaar(2)
Faculty of Veterinary Medicine (FVM), Department of Infectious Diseases and Immunology
P.O. Box 80.165, 3508 TD Utrecht
Tel: (31-30) 253.12.42 Fax: (31-30) 253.31.99
1366
Bovine spongiform encephalopathy Encphalopathie spongiforme bovine Encefalopata espongiforme bovina

Dr Francisco Javier Blanco
Institutos de Patobiologa y Virologa, Centro de Investigaciones en Ciencias Veterinarias y Agronmicas
(CICVyA), Instituto Nacional de Tecnologa Agropecuaria (INTA)
Las Cabaas y De Los Reseros, S/N Provincia de Buenos Aires
Tel: (54-11) 46.21.17.89 Fax: (54-11) 46.21.17.43
Email: jviera@cnia.inta.gov.ar / gpinto@cnia.inta.gov.ar
Dr Stefanie Czub
Canadian Food Inspection Agency, Lethbridge Laboratory
Township Road 9-1, P.O. Box 640, Lethbridge, Alberta T1J 3Z4
CANADA CANADA CANAD
Tel: (1-403) 382.55.49 Fax: (1-403) 382.55.83
Email: stefanie.czub@inspection.gc.ca
Prof. Takashi Yokoyama
Prion Diseases Research Unit, National Institute of Animal Health,
National Agricultural Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856
JAPON JAPAN JAPN
Tel: (81-298) 38.78.40 Fax: (81-298) 38.83.32
Email: tyoko@affrc.go.jp
Prof. Andreas Zurbriggen
NeuroCentre, Department of Clinical Research and Veterinary Public Health
Division of Experimental Clinical Research, University of Bern
Bremgartenstrasse 109A, 3012 Bern
SUISSE SWITZERLAND SUIZA
Tel: (41-31) 631.25.09 Fax: (41-31) 631.25.38
Email: andreas.zurbriggen@itn.unibe.ch
Dr Marion Simmons
AHVLA Weybridge, New Haw, Addlestone, Surrey KT15 3NB
Tel: (44-1932) 35.75.64 Fax: (44-1932) 35.78.05
Email: marion.simmons@ahvla.gsi.gov.uk
Bovine tuberculosis Tuberculose bovine Tuberculosis bovina
Dr Bernardo Alonso
Gerencia de Laboratorios (GELAB) del Servicio Nacional de Sanidad y Calidad, Agroalimentaria (SENASA)
Av. Alexander Fleming 1653, 1640 Martinez - Pcia de Buenos Aires
Tel: (54-11) 48.36.19.92 Fax: (54-11) 48.36.19.92
Email: balonso@senasa.gov.ar / dilab@inea.com.ar
Dr Mara Laura Boschiroli-Cara
Anses Alfort, Unit Zoonoses Bactriennes, Laboratoire de sant animale
23 avenue du Gnral de Gaulle, 94706 Maisons-Alfort Cedex
Tel: (33-1) 49.77.13.00 Fax: (33-1) 49.77.13.44
Email: Maria-laura.boschiroli@anses.fr
Prof. Glyn Hewinson
Tel: (44-1932) 34.11.11 Fax: (44-1932)34.70.46
Email: glyn.hewinson@ahvla.gsi.gov.uk
Bovine viral diarrhoea Diarrhe virale bovine Diarrea viral bovina
Dr Peter D. Kirkland
Elizabeth Macarthur Agriculture Institute (EMAI), Virology Laboratory
Woodbridge Rd, Menangle, PMB 8, Camden NSW 2570
AUSTRALIA AUSTRALIE AUSTRALIA
Tel.: (61-2) 46.40.63.31 Fax: (61-2) 46.40.64.29
E-mail: peter.kirkland@dpi.nsw.gov.au / peter.kirkland@industry.nsw.gov.au
1367
Bovine viral diarrhoea Diarrhe virale bovine Diarrea viral bovina

Dr Oliver Lung
Canadian Food Inspection Agency, National Centre for Animal Disease (NCAD)
P.O. Box 640, Township Road 9-1, Lethbridge, Alberta T1J 3Z4
CANADA CANADA CANAD
Tel: (1-403) 382.55.00 Fax: (1-403) 381.12.02
Email: oliver.lung@inspection.gc.ca
Prof. Trevor W. Drew
AHVLA Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB
Tel: (44-1932) 35.76.37 Fax: (44-1932) 35.72.39
Email: trevor.drew@ahvla.gsi.gov.uk
Brucellosis Brucellose Brucelosis (Brucella abortus, Brucella melitensis, Brucella suis)
Dr Ana Maria Nicola
Gerencia de Laboratorios (GELAB), Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA)
Av. Alexander Fleming 1653, 1640 Martnez, Pcia. de Buenos Aires
Tel: (54-11) 48.36.19.92 Fax: (54-11) 48.36.19.92
Email: anicola@senasa.gov.ar
Dr Bruno Garin-Bastuji
Anses Alfort, Unit Zoonoses Bactriennes, Laboratoire de Sant Animale
Tel: (33-1) 49.77.13.00 Fax: (33-1) 49.77.13.44
Email: bruno.garin-bastuji@anses.fr
Dr Heinrich Neubauer
Federal Research Centre for Virus Diseases of Animals (BFAV), Friedrich-Loeffer Institute
Institute of Bacterial Infections and Zoonoses
Naumburger Str. 96a, 07743 Jena
Tel: (49-3641) 80.42.00 Fax: (49-3641) 80.42.28
Email: heinrich.neubauer@fli.bund.de
Dr Menachem Banai6
Kimron Veterinary Institute
Department of Bacteriology, P.O. Box 12, Beit Dagan 50250
ISRAL ISRAEL ISRAEL
Tel: (972-3) 968 16 98 Fax: (972-3) 968 17 53
Email: menachemba@moag.gov.il
Dr Massimo Scacchia
CESME, Istituto Zooprofilattico Sperimentale dellAbruzzo e del Molise G. Caporale
National Centre for Exotic Diseases
ITALIE ITALY ITALIA
Tel: (39-0861) 33.24.05 Fax: (39-0861) 33.22.51
Email: m.scacchia@izs.it
Dr Suk-chan Jung
Ministry of Food, Agriculture, Forestry and Fisheries (MIAFF), Brucellosis Laboratory
Bacteriology Division, National Veterinary Research & Quarantine Service (NVRQS)
480 Anyang 6-dong, Manan-gu, Anyang-si, Kyunggi-do
CORE (RP DE) KOREA (REP. OF) COREA (REP. DE)
Tel: (82-31) 467.17.65 Fax: (82-31) 467.17.78
Email: jungsc@nvrqs.go.kr / brujung@korea.kr
Dr Judy A. Stack
AHVLA Weybridge
Tel: (44-1932) 34.76.10 Fax: (44-1932) 35.72.16
Email: judy.stack@ahvla.gsi.gov.uk
Not an OIE Reference Laboratory for Brucella suis.
1368
Variole du chameau Camelpox Viruela del camello

Prof. Ulrich Wernery
Central Veterinary Research Laboratory
P.O. Box 597, Dubai
MIRATS ARABES UNIS UNITED ARAB EMIRATES EMIRATOS RABES UNIDOS
Tel: (971-4) 337.51.65 Fax: (971-4) 336.86.38
Email: cvrl@cvrl.ae
Campylobacteriosis Campylobactrioses Campilobacteriosis
Dr Jaap Wagenaar(1)
Animal Sciences Group (ASG), Division of Infectious Diseases
P.O. Box 65, 8200 AB Lelystad
Tel: (31-320) 23.81.57 Fax: (31-320) 23.89.61
Dr Jaap Wagenaar(2)
Faculty of Veterinary Medicine (FVM), Department of Infectious Diseases and Immunology
P.O. Box 80.165, 3508 TD Utrecht
Tel: (31-30) 253.12.42 Fax: (31-30) 253.31.99
Caprine arthritis/encephalitis Arthrite/encphalite caprine Artritis/encefalitis caprina
Dr Stephen Valas
Anses-Niort, Laboratoire dEtudes et de Recherches Caprines
60 rue de Pied de Fond, B.P. 3081, 79000 Niort
Tel: (33-5) 49.79.61.28 Fax: (33-5) 49 79 42 19
Email: stephen.valas@anses.fr
Dr Donald P. Knowles, Jr
Animal Diseases Research Unit (USDA/ARS), Co-located at the College of Veterinary Medicine
Washington State University, 3003 ABBF, Pullman, Washington 99164-6630
Tel: (1-509) 335.60.01 Fax: (1-509) 335.83.28
Email: dknowles@vetmed.wsu.edu
Channel catfish virus disease Herpsvirose du poisson-chat Virosis del bagre de canal
Dr Larry A. Hanson
College of Veterinary Medicine, Fish Diagnostic Laboratory, Mississippi State University
P.O. Box 6100, Spring Street, Mississippi 39762
Tel: (1-662) 325.12.02 Fax: (1-662) 325.10.31
Email: hanson@cvm.msstate.edu
Chronic wasting disease Cachexie chronique Caquexia crnica
Dr Aru Balachandran
Canadian Food Inspection Agency, Ottawa Laboratory Laboratory (Fallowfield)
Animal Disease Research Institute, 3851 Fallowfield Road, P.O. Box 11300
Station H, Nepean, Ontario K2H 8P9
CANADA CANADA CANAD
Tel: (1-613) 221.48.54 Fax: (1-613) 221.61.03
Email: aru.balachandran@inspection.gc.ca
Dr Dr Hyun-Joo Sohn
Prion Disease Research Laboratory, Division of Foreign Animal Disease
Animal, Plant and Fisheries Quarantine and Inspection Agency
335 Anyang-ro, Manan-gu, Anyang, Gyeongii 430-757
Tel: (82-31) 467.18.67 Fax: (82-31) 467.18.30
Email: shonhj@korea.kr
1369
Classical swine fever Peste porcine classique Peste porcina clsica

Dr John Pasick
Canadian Food Inspection Agency, National Centre for Foreign Animal Disease
1015 Arlington Street, Winnipeg, Manitoba R3E 3M4
CANADA CANADA CANAD
Tel: (1-204) 789.20.13 Fax: (1-204) 789.20.38
Email: john.pasick@inspection.gc.ca
Prof. Volker Moennig
University of Veterinary Medicine of Hannover, Department of Infectious Diseases
Institute of Virology Bnteweg 17, 30559 Hannover
Tel: (49-511) 953.88.40 Fax: (49-511) 953.88.98
Email: volker.moennig@tiho-hannover.de
Dr Shunji Yamada
National Institute of Animal Health, Department of Exotic Diseases
6-20-1 Josui-honcho, Kodaira, Tokyo, 187-0022
JAPON JAPAN JAPN
Tel: (81-42) 321.14.41 Fax: (81-42) 325.51.22
Email: musasabi@affrc.go.jp
Prof. Zygmunt Pejsak
National Veterinary Research Institute
Partyzantow str. 57, 24-100 Pulawy
POLOGNE POLAND POLONIA
Tel: (48-81) 889.30.30 Fax: (48-81) 886.25.95
Email: zpejsak@piwet.pulawy.pl
Prof. Trevor W. Drew
AHVLA Weybridge
Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB
Tel: (44-1932) 35.76.37 Fax: (44-1932) 35.72.39
Contagious agalactia Agalaxie contagieuse Agalaxia contagiosa
Dr Robin A.J. Nicholas
Mycoplasma Group, Department of Statutory and Exotic Bacterial Diseases, AHVLA Weybridge
Tel: (44-1932) 35.73.79 Fax: (44-1932) 35.74.23
Email: robin.nicholas@ahvla.gsi.gov.uk
Contagious bovine pleuropneumonia Pripneumonie contagieuse bovine Perineumona contagiosa bovina
Dr Chandapiwa Marobela Raborokgwe
National Veterinary Laboratory, Sebele Content Farm, P.O.Box. 0035, Gaborone
BOTSWANA BOTSWANA BOTSUANA
Tel: (267-39) 2.88.16 Fax: (267-39) 2.89.56
Email: cmarobela-raborokgwe@gov.bw
Dr Franis Poumarat(1)
Anses Lyon, Laboratoire de Pathologie Bovine et Hygine des Viandes
Rsistance aux produits phytosanitaires
31 avenue Tony Garnier, BP 7033, 69342 Lyon Cedex 07
Tel: (33-4) 78.72.65.43 Fax: (33-4) 78.61.91.45
Email: francois.poumarat@anses.fr
Dr Franois Thiaucourt(2)
CIRAD-UMR, Control of exotic and emerging animal diseases
Campus International de Baillarguet TA A-15/G, 34398 Montpellier Cedex 5
Tel: (33-4) 67.59.37.24 Fax: (33-4) 67.59.37.98
Email: thiaucourt@cirad.fr
1370
Contagious bovine pleuropneumonia Pripneumonie contagieuse bovine Perineumona contagiosa bovina

Dr Attilio Pini
ITALIE ITALY ITALIA
Tel: (39-0861) 33.22.28 Fax: (39-0861) 33.22.51
Email: a.pini@izs.it
Dr Ana Rosa Pombo Botelho
Laboratrio Nacional de Investigao Veterinria (LNIV)
Estrada de Benfica 701, 1549-011 Lisboa
PORTUGAL PORTUGAL PORTUGAL
Tel: (351-21) 711.53.33/39/40 Fax: (351-21) 711.52.36
Email: ana.botelho@lniv.min-agricultura.pt
Contagious caprine pleuropneumonia Pleuropneumonie contagieuse caprine Pleuroneumona contagiosa caprina
Dr Franois Thiaucourt
CIRAD-UMR, Control of exotic and emerging animal diseases
Campus International de Baillarguet TA A-15/G, 34398 Montpellier Cedex 5
Tel: (33-4) 67.59.37.24 Fax: (33-4) 67.59.37.98
Email: thiaucourt@cirad.fr
Contagious equine metritis Mtrite contagieuse quine Metritis contagiosa equina
Dr Hendrik-Jan Roest
Central Veterinary Institute of Wageningen UR (CVI), Department of Bacteriology
P.O. Box 2004, 8203 AA Lelystad
Tel: (31-320) 23.80.26 Fax: (31-320) 23.81.53
Email: hendrikjan.roest@wur.nl
Dr Paul Todd
AHVLA Bury St Edmunds, Rougham Hill, Bury St Edmunds, Suffolk IP33 2RY
Tel: (44-1284) 72.44.99 Fax: (44-1284) 72.45.00
Email: paul.todd@ahvla.gsi.gov.uk
Dr Matthew M. Erdman
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.75.68 Fax: (1-515) 337.75.69
Email: matthew.m.erdman@aphis.usda.gov
Crayfish plague Peste de lcrevisse Plaga del cangrejo de ro (Aphanomyces astaci)
Dr Satu Viljamaa-Dirks
Finnish Food Safety Authority, Evira Kuopio, P.O. Box 92, 70701 Kuopio
FINLANDE FINLAND FINLANDIA
Tel: (358-207) 72.49.62 Fax: (358-207) 72.49.70
Email: satu.viljamaa-dirks@evira.fi
Dr Birdit Oidtmann
The Centre for Environment, Fisheries, and Aquaculture Science (CEFAS),
Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB
Tel: (44-1305) 20.66.61 Fax: (44-1305) 20.66.01
Email: birgit.oidtmann@cefas.co.uk
CrimeaCongo haemorrhagic fever Fivre hmorragique de CrimeCongo Fiebre hemorrgica de CrimeaCongo
Dr Michle Bouloy
Unit de gntique molculaire des Bunyavirus, Dpartement de Virologie
Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15
Tel: (33-1) 40.61.31.57 Fax: (33-1) 40.61.31.51
Email: mbouloy@pasteur.fr
1371
Dourine Dourine Durina

Prof. Vyacheslav T. Zablotsky
All-Russian Research Institute for Experimental Veterinary Medicine (VIEV), Veterinary Dept
Laboratory of Equine Viral Diseases, 24-1, Ryazanskiy prosp.
109428 Moscow
RUSSIE RUSSIA RUSIA
Tel: (7-095) 785.84.27 Fax: (7-095) 970.03.69
Email: konstyurov@yandex.ru
Echinococcosis/hydatidosis Echinococcose/hydatidose Equinococosis/hidatidosis
Prof. Masao Kamiya
Laboratory of Environmental Zoology, Department of Biosphere and Environmental Sciences,
Faculty of Environmental Systems, Rakuno-Gakuen University
Midori-machi 582, Ebetsu 069-8501
JAPON JAPAN JAPN
Tel: (81-11) 388.49.09 Fax: (81-11) 388.49.09
Email: mkamiya@kyj.biglobe.ne.jp
Prof. Allal Dakkak
Dpartement de Pathologie et Sant Publique Vtrinaire,
Unit de Parasitologie et Maladies Parasitaires, Institut Agronomique et Vtrinaire Hassan II
BP 6202, Rabat-Instituts
MAROC MOROCCO MARRUECOS
Tel: (212-37) 77.64.32 Fax: (212-37) 77.64.32
Email: a.dakkak@iav.ac.ma
Dr Philip S. Craig
Cestode Zoonoses Research Group, Biosciences Research Institute, University of Salford
Manchester M5 4WT
Tel: (44-161) 295.54.88 Fax: (44-161) 295.50.15
Email: p.s.craig@salford.ac.uk
Enteric septicaemia of catfish Entrosepticmie du poisson-chat Septicemia entrica del bagre (Edwardsiella ictaluri)
Dr Larry A. Hanson
College of Veterinary Medicine, Fish Diagnostic Laboratory, Mississippi State University
P.O. Box 6100, Spring Street, Mississippi 39762
Tel: (1-662) 325.12.02 Fax: (1-662) 325.10.31
Email: hanson@cvm.msstate.edu
Enzootic abortion of ewes (ovine chlamydiosis) Avortement enzootique des brebis (chlamydiose ovine)
Aborto enzotico de las ovejas (clamidiosis ovina)
Dr Konrad Sachse
Federal Research Institute for Animal Health, Friederich-Loeffer Institute, Institute of Molecular Pathogenesis
Tel: (49-3641) 80.43.34 Fax: (49-3641) 80.42.28
Email: konrad.sachse@fli.bund.de
Dr Nicole Borel
Institute for Veterinary Pathology (IVPZ), Vetsuisse Faculty, University of Zurich
Winterhurerstrasse 268, CH-8057 Zurich
Tel: (41-44) 635.85.71 Fax: (41-44) 635.89.34
Email: apos@vetpath.unizh.ch / n.borel@access.uzh.ch
1372
Enzootic bovine leukosis Leucose bovine enzootique Leucosis bovina enzotica

Prof. Thomas W. Vahlenkamp
Centre for Infectious Diseases, Faculty of Veterinary Medicine
Leipzig University, An den Tierkliniken 29, 04103 Leipzig
Tel: (49-341) 97.38.201 Fax: (49-341) 97.38.219
Email: Thomas.vahlenkamp@uni-leipzig.de
Dr Jacek Kuzmak
National Veterinary Research Institute, Pulawy
POLAND POLOGNE POLONIA
Tel.: (48-81) 886.30.14) Fax: (48-81) 886.25.95;
E-mail: jkuzmak@piwet.pulawy.pl
Dr Bhudipa Choudhury
AHVLA Weybridge
Tel: (44-1932) 35.75.59 Fax: (44-1932) 35.72.39
Email: bhudipa.choudhury@ahvla.gsi.gov.uk
Epizootic haematopoietic necrosis Ncrose hmatopotique pizootique Necrosis hematopoytica epizotica
Dr Alex Hyatt(1)
Tel: (61-3) 52.27.50.00 Fax: (61-3) 52.27.55.55
Email: alex.hyatt@csiro.au
Dr Richard Whittington(2)
Faculty of Veterinary Science, University of Sydney
425 Werombi Road, Private Bag 3, Camden NSW 2570
Tel: (61-2) 93.51.16.19 Fax: (61-2) 93.51.16.18
Email: richardw@camden.usyd.edu.au
Epizootic haemorrhagic disease Maladie hmorragique pizootique Enfermedad hemorrgica epizotica
Dr Stephan Zientara
Anses-Alfort, Laboratoire de sant animale de Maisons-Alfort
Laboratoire national de rfrence pour la BT/EHD et l'AHS, UMR 1161
23 Avenue du Gnral de Gaulle, 94703 Maisons-Alfort
Tel: (33-1) 43.96.72.80
Email: s.zientara@vet-alfort.fr
Epizootic ulcerative syndrome Syndrome ulcratif pizootique Sndrome ulcerante epizotico
Dr Somkiat Kanchanakhan
Inland Aquatic Animal Health Research Institute (AAHRI), Inland Fishery Research and Development Bureau
Department of Fisheries, Kasetsart University Campus, Paholyothin Road, Jatuchak, Bangkok 10900
THALANDE THAILAND TAILANDIA
Tel: (66-2) 579.41.22 Fax: (66-2) 561.39.93
Email: kanchanakhan@yahoo.com
Equine encephalomyelitis (Eastern) Encphalomylite quine de lEst Encefalomielitis equina del Este
Equine encephalomyelitis (Western) Encphalomylite quine de lOuest Encefalomielitis equina del Oeste
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.75.51 Fax: (1-515) 337.73.48
1373
Equine infectious anaemia Anmie infectieuse des quids Anemia infecciosa equina
Dr Jianhua Zhou
Laboratory of Equine Infectious Anemia
Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences
427 Maduan Street, Harbin 150001
CHINE (RP. POPULAIRE DE) CHINA (PEOPLES REP. OF) CHINA (REP. POP. DE)
Tel: (86-189) 46.06.61.24 Fax: (86-451) 82.73.31.32
Email: jianhua_uc@126.com
Dr Kenji Murakami
Viral disease Section, National Institute of Animal Health
3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856
JAPON JAPAN JAPN
Tel: (81-29) 838.78.41 Fax: (81-29) 838.79.07
Email: muraken@iwate-u.ac.jp
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.75.51 Fax: (1-515) 337.73.48
Equine influenza Grippe quine Gripe equina
Prof. Kerstin Borchers
Institute of Virology, Veterinary Medicine, Free University of Berlin
Philippstrasse 13, 10115 Berlin
Tel: (49-30) 20.93.65.63
Email: borchers@zedat.fu-berlin.de
Prof. Ann Cullinane
Head of the Virology Unit, Irish Equine Centre, Johnstown, Naas, Co. Kildare
IRLANDE IRELAND IRLANDA
Tel: (353-45) 86.62.66 Fax: (353-45) 86. 62.73
Email: acullinane@equine-centre.ie
Dr Debra Elton
Animal Health Trust, Centre for Preventive Medicine
Lanwades Park, Kentford, Suffolk CB8 7UU
Tel: (44-1638) 75.10.00 Fax: (44.1638) 55.56.59
Email: debra.elton@aht.org.uk
Dr Thomas M. Chambers
Maxwell H. Gluck Equine Research Center, Dept of Veterinary Science, University of Kentucky
108 Gluck Equine Research Center, Lexington, Kentucky 40546-0099
Tel: (1-859) 257.47.57 Fax: (1-859) 257.85.42
Email: tmcham1@uky.edu
Equine piroplasmosis Piroplasmose quine Piroplasmosis equina
Prof. Ikuo Igarashi
JAPON JAPAN JAPN
Tel: (81-155) 49.56.41 Fax: (81-155) 49.56.43
Email: : igarcpmi@obihiro.ac.jp
Tel: (1-509) 335.60.01 Fax: (1-509) 335.83.28
1374
Equine rhinopneumonitis Rhinopneumonie quine Rinoneumona equina

Prof. Kerstin Borchers
Institute of Virology, Veterinary Medicine, Free University of Berlin
Philippstrasse 13, 10115 Berlin
Tel: (49-30) 20.93.65.63
Email: borchers@zedat.fu-berlin.de
Prof. Konstantin P. Yurov
All-Russian Research Institute for Experimental Veterinary Medicine (VIEV)
Laboratory of Equine Viral Diseases, Veterinary Department,
Ministry of Agriculture and Food, 24-1, Ryazanskiy prosp., 109428 Moscow
RUSSIE RUSSIA RUSIA
Tel: (7-495) 995.88.63 Fax: (7-495) 995.88.63
Email: konstyurov@yandex.ru
Dr Debra Elton
Animal Health Trust, Centre for Preventive Medicine
Lanwades Park, Kentford, Suffolk CB8 7UU
Tel: (44-1638) 75.10.00 Fax: (44.1638) 55.56.59
Email: debra.elton@aht.org.uk
Dr Peter J. Timoney
Tel: (1-859) 257.47.57 ext. 81094 Fax: (1-859) 257.85.42
Email: ptimoney@uky.edu
Equine viral arteritis Artrite virale quine Arteritis viral equina
Prof. Falko Steinbach7
AHVLA Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB
Tel: (44-1932) 35.76.37 Fax: (44-1932) 35.72.39
Dr Peter J. Timoney
Tel: (1-859) 257.47.57 ext. 81094 Fax: (1-859) 257.85.42
Email: ptimoney@uky.edu
Escherichia coli Escherichia coli Escherichia coli
Dr John Morris Fairbrother
The Echerichia coli Laboratory (EcL), Faculty of Veternary Medicine, University of Montreal
3200 Sicotte Saint-Hyacinthe, Qubec J2S 7C6
CANADA CANADA CANAD
Tel: (1-450) 773.85.21 Fax: (1-450) 778.81.08
Email: john.morris.fairbrother@umontreal.ca
Foot and mouth disease Fivre aphteuse Fiebre aftosa
Dr Eduardo D. Maradei
Laboratorio de Fiebre Aftosa de la Direccin de Laboratorios y Control Tcnico
Av. Sir. Alexander Fleming 1653, Martnez (1640), Buenos Aires
ARGENTINA ARGENTINE ARGENTINA
Tel.: (54-11) 48.36.19.95 Fax: (54-11) 48.36.19.95
E-mail: emaradei@senasa.gov.ar / emaradei@yahoo.com
1375
Foot and mouth disease Fivre aphteuse Fiebre aftosa

Dr Onkabetse George Matlho
Botswana Vaccine Institute, Department of Animal Health and Production
Broadhurst Industrial Site, Lejara Road, Private Bag 0031, Gaborone
BOTSWANA BOTSWANA BOTSUANA
Tel: (267-39) 1.27.11 Fax: (267-39) 5.67.98
Email: gmatlho@bvi.co.bw
Dr Rossana Allende
Centro Panamericano de Fiebre Aftosa OPS/OMS (PANAFTOSA)
Av. President Kennedy 7778, Sao Bento, Duque de Caxias, ZC 20054-40 Rio de Janeiro
BRESIL BRAZIL BRASIL
Tel: (55-21) 36.61.90.64 Fax: (55-21) 36.61.90.01
Email: rallende@paho.org
Dr Xiangtao Liu
Lanzhou Veterinary Research Institute, CAAS
National Foot and Mouth Disease Reference Laboratory
Xujiaping No.1, Yanchangpu
Lanzhou, Gansu province 730046
Tel: (86-931) 834.25.85 Fax: (86-931) 834.09.77
Email: hnxiangtao@hotmail.com / shiqisun21@hotmail.com
Dr Valery M. Zakharov
Federal Governmental Institute, Centre for Animal Health, FGI-ARRIAH
600900 Vladimir, Yurevets
RUSSIE RUSSIA RUSIA
Tel: (7-4922) 26.19.14 Fax: (7-4922) 26.38.77
Email: mail@arriah.ru
Dr Rahana Dwarka
Onderstepoort Veterinary Institute, Transboundary Animal Diseases Programme
SOUTH AFRICA AFRIQUE DU SUD SUDFRICA
Tel.: (27-12) 529.95.85; Fax: (27-12) 529.95.43;
Email: DwarkaR@arc.agric.za
Dr Wilai Linchongsubongkoch
National Institute of Animal Health, Department of Livestock Development
Pakchong, Nakhonratchasima 30130
THALANDE THAILAND TAILANDIA
Tel: (66-44) 27.91.12 Fax: (66-44) 31.48.89
Email: wilaifmd@loxinfo.co.th / rrl@dld.go.th
Dr Jef Hammond
Institute for Animal Health
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: jef.hammond@iah.ac.uk
Dr Consuelo Carrillo
National Veterinary Services Laboratories, USDA-APHIS-VS
Foreign Animal Disease Diagnostic Laboratory
Plum Island Animal Disease Center, P.O. Box 848, Greenport, NY 11944
Tel: (1-631) 323.32.56 Fax: (1-631) 323.33.66
Email: consuelo.carrillo@aphis.usda.gov
Glanders Morve Muermo
Dr Henrich Neubauer
Tel: (49-3641) 80.42.00 Fax: (49-3641) 80.42.28
1376
Glanders Morve Muermo

Prof. Ulrich Wernery
Central Veterinary Research Laboratory, P.O. Box 597, Dubai
MIRATS ARABES UNIS UNITED ARAB EMIRATES EMIRATOS RABES UNIDOS
Tel: (971-4) 337.51.65 Fax: (971-4) 336.86.38
Email: cvrl@cvrl.ae
Gyrodactylosis Gyrodactylose Girodactilosis (Gyrodactylus salaris)
Dr Tor-Atle Mo
National Veterinary Institute, Section for Parasitology
P.O. Box 750, Sentrum, 0106 Oslo
NORVGE NORWAY NORUEGA
Tel: (47-23) 21.61.10 Fax: (47-23) 21.61.01
Email: tor-atle.mo@vetinst.no
Heartwater Cowdriose Cowdriosis
Dr Dominique Martinez
Centre international en recherche agronomique pour le dveloppement
TA-A 15/A, Campus International de Baillarguet, Montferriez-sur-Lez, B.P. 5035, 34398 Montpellier Cedex 5
Tel: (33-4) 67.59.37.12 Fax: (33-4) 67.59.37.98
Email: dominique.martinez@cirad.fr
Hendra and Nipah virus diseases Maladies dues aux virus Hendra et Nipah Enfermedades causadas
por los virus Hendra y Nipah
Dr Peter Daniels
Tel: (61-3) 52.27.50.00 Fax: (61-3) 52.27.55.55
Highly pathogenic avian influenza and low pathogenic avian influenza (poultry) Influenza aviaire hautement
pathogne et influenza aviaire faiblement pathogne (volailles) Influenza aviar altamente patgena
e influenza aviar levemente patgena (aves de corral)
Dr Paul W. Selleck
CSIRO, Australian Animal Health Laboratory (AAHL), Division of Animal Health, Institute of Animal Production &
Processing, 5 Portarlington Road, Private Bag 24, Geelong 3220, Victoria
Tel: (61-3) 52.27.50.00 Fax: (61-3) 52.27.55.55
Email: paul.selleck@csiro.au
Dr John Pasick
Canadian Food Inspection Agency, National Centre for Foreign Animal Disease
1015 Arlington Street, Winnipeg, Manitoba R3E 3M4
CANADA CANADA CANAD
Tel: (1-204) 789.20.13 Fax: (1-204) 789.20.38
Email: john.pasick@inspection.gc.ca
Dr Hualan Chen
National Avian Influenza Reference Laboratory, Animal Influenza Laboratory of the Ministry of Agriculture
Harbin Veterinary Research Institute, 427 Maduan Street, Harbin 150001,
Tel.: (86-451) 85.93.50.79 Fax: (86-451) 82.73.31.32
Email: hlchen@hvri.ac.cn / hlchen1@yahoo.com
Dr Timm C. Harder
Federal Research Centre for Virus Diseases of Animals (BFAV), Friedrich Loeffler Institute
Institute of Diagnostic Virology, Boddenblick 5a, 17493 Greifswald - Insel Riems
Tel: (49-38351) 71.152 Fax: (49-38351) 71.174
Email: timm.harder@fli.bund.de
1377
Highly pathogenic avian influenza and low pathogenic avian influenza (poultry) Influenza aviaire hautement
pathogne et influenza aviaire faiblement pathogne (volailles) Influenza aviar altamente patgena
e influenza aviar levemente patgena (aves de corral)
Dr Chakradhar Tosh
High Security Animal Disease Laboratory, Indian Veterinary Research Institute
Indian Council of Agricultural Research, Anand Nagar, Bhopal 462021, Madhya Pradesh
INDE INDIA INDIA
Tel: (91-755) 275.92.04 Fax: (91-755) 275.88. 42
Email: ctosh@hsadl.nic.in / chakradhar.tosh@gmail.com
Dr Ilaria Capua
Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio Virologia
Via Romea 14/A, 35020 Legnaro, Padova
ITALIE ITALY ITALIA
Tel: (39-049) 808.43.79 Fax: (39-049) 808.43.60
Email: icapua@izsvenezie.it
Prof. Hiroshi Kida
Laboratory of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University
Kita-18, Nishi-9, Kita-ku, Sapporo 060-0818
JAPON JAPAN JAPN
Tel: (81-11) 706.52.07 Fax: (81-11) 706.52.73
Email: kida@vetmed.hokudai.ac.jp
Prof. Ian Brown
Tel: (44-1932) 35.73.39 Fax: (44-1932) 35.72.39
Email: ian.brown@ahvla.gsi.gov.uk
Ms Janice Pedersen
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.72.66 Fax: (1-515) 337.73.97
Email: janice.c.pedersen@aphis.usda.gov
Infection with abalone herpesvirus Infection due lherpsvirus de lormeau Herpesvirosis del abaln
Dr Mark Crane
CSIRO Livestock Industries, Australian Animal Health Laboratory (AAHL)
5 Portarlington Road, East Geelong, Victoria 3220
AUSTRALIA
Tel: (61-3) 52.27.51.18 Fax: (61-3) 52.27.55.55
Email: mark.crane@csiro.au
Prof. Pen Heng Chang
National Taiwan University, School of Veterinary Medicine
1, Sec. 4 Roosevelt Road, Taipei 106
TAIPEI CHINOIS CHINESE TAIPEI TAIPEI CHINO
Tel: (886-2) 33.66.12.96 Fax: (886-2) 23.66.14.75
Email: penheng@ntu.edu.tw
Infection with Batrachochytrium dendrobatidis Infection Batrachochytrium dendrobatidis
Infeccin por Batrachochytrium dendrobatidis
Dr Alex Hyatt
Tel: (61-3) 52.27.50.00 Fax: (61-3) 52.27.55.55
1378
Infection with Bonamia exitiosa Infection Bonamia exitiosa Infeccin por Bonamia exitiosa
Infection with Bonamia ostreae Infection Bonamia ostreae Infeccin por Bonamia ostreae
Dr Isabelle Arzul
IFREMER, Laboratoire de Gntique et Pathologie, BP 133, 17390 La Tremblade
Tel: (33-5) 46.76.26.10 Fax: (33-5) 46.76.26.11
Email: isabelle.arzul@ifremer.fr / iarzul@ifremer.fr
Infection with Haplosporidium nelsoni Infection Haplosporidium nelsoni Infeccin por Haplosporidium nelsoni
Dr Ryan Carnegie
Virginia Institute of Marine Science, College of William and Mary
Shellfish Pathology Laboratory, P.O. Box 1346, Gloucester Point, VA 23062
Tel: (1-804) 684.77.13 Fax: (1-804) 684.77.96
Email: carnegie@vims.edu
Infection with Marteilia refringens Infection Marteilia refringens Infeccin por Marteilia refringens
Infection with Marteilia sydneyi Infection Marteilia sydneyi Infeccin por Marteilia sydneyi
Dr Isabelle Arzul
IFREMER, Laboratoire de Gntique et Pathologie, BP 133, 17390 La Tremblade
Tel: (33-5) 46.76.26.10 Fax: (33-5) 46.76.26.11
Email: isabelle.arzul@ifremer.fr / iarzul@ifremer.fr
Infection with Mikrocytos mackini Infection Mikrocytos mackini Infeccin por Mikrocytos mackini
Dr Gary Meyer
Department of Fisheries and Oceans, Pacific Biological Station
3190 Hammond Bay Road, Nanaimo, British Columbia V9T 6N7
CANADA CANADA CANAD
Tel: (1-250) 756.70.34 Fax: (1-250) 756.70.53
Email: gary.meyer@dfo-mpo.gc.ca
Infection with Perkinsus marinus Infection Perkinsus marinus Infeccin por Perkinsus marinus
Infection with Perkinsus olseni Infection Perkinsus olseni Infeccin por Perkinsus olseni
Dr Ryan Carnegie
Virginia Institute of Marine Science, College of William and Mary
Shellfish Pathology Laboratory, P.O. Box 1346, Gloucester Point, VA 23062
Tel: (1-804) 684.77.13 Fax: (1-804) 684.77.96
Email: carnegie@vims.edu
Infection with ranavirus Infection ranavirus Infeccin por ranavirus
Dr Alex Hyatt(1)
Tel: (61-3) 52.27.50.00 Fax: (61-3) 52.27.55.55
Dr Richard Whittington(2)
Faculty of Veterinary Science, University of Sydney
425 Werombi Road, Private Bag 3, Camden NSW 2570
Tel: (61-2) 93.51.16.19 Fax: (61-2) 93.51.16.18
Email: richardw@camden.usyd.edu.au
1379
Infection with Xenohaliotis californiensis Infection Xenohaliotis californiensis Infeccin por Xenohaliotis californiensis
Prof. Carolyn Friedman
Friedman Shellfish Health Laboratory, School of Aquatic and Fishery Sciences, University of Washington
Box 355020, Seattle, Washington 98195
Tel: (1-206) 789.12.96 Fax: (1-206) 616.86.89
Email: carolynf@u.washington.edu
Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis Rhinotrachite infectieuse bovine/vulvovaginite pustuleuse
infectieuse Rinotraquetis infecciosa bovina/vulvovaginitis pustular infecciosa
Dr Martin Beer
National Referance Laboratory for Bovine herpesvirus type 1, Institute of Diagnostic Virology, Federal Research
Centre for Virus Diseases of Animals (BFAV), Boddenblick 5a, 17493 Greifswald - Insel Riems
Tel: (49-38351) 7.1223 Fax: (49-38351) 7.1275
Email: martin.beer@fli.bund.de
To be decided
Tel: (44-1932) 35.75.08 Fax: (44-1932) 35.72.39
Infectious bursal disease (Gumboro disease) Bursite infectieuse (Maladie de Gumboro)
Bursitis infecciosa (Enfermedad de Gumboro)
Dr Nicolas Eterradossi
Anses Ploufragan, Laboratoire de Ploufragan-Plouzan
Laboratoire dtudes et de recherches avicoles, porcines et piscicoles
BP 53, 22440 Ploufragan-Plouzan
Tel: (33-2) 96.01.62.22 Fax: (33-2) 96.01.62.63
Email: nicolas.eterradossi@anses.fr
Dr Yehia M. Saif
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Ohio State
University, 1680 Madison Avenue, Wooster, OH 44691-4096
Tel: (1-330) 263.37.43 Fax: (1-330) 263.36.77
Email: saif.1@osu.edu
Infectious haematopoietic necrosis Ncrose hmatopotique infectieuse Necrosis hematopoytica infecciosa
Dr James R. Winton
Western Fisheries Research Center
6505 N.E. 65th Street, Seattle, Washington 98115
Tel: (1-206) 526.65.87 Fax: (1-206) 526.66.54
Email: jim_winton@usgs.gov
Infectious hypodermal and haematopoietic necrosis Ncrose hypodermique et hmatopotique infectieuse
Necrosis hipodrmica u hematopoytica infecciosa
Dr Jie Huang
Molecular Pathology Laboratory, Yellow Sea Fisheries Research Institute
Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao, Shandong 266071
Tel: (86-532) 582.30.62 ext. 802 Fax: (86-532) 581.15.14
Email: huangjie@ysfri.ac.cn / aquadis@ysfri.ac.cn
Prof. Donald V. Lightner
Aquaculture Pathology Laboratory, Department of Veterinary Science and Microbiology, University of Arizona
Building 90, Room 202 Pharmacy/Microbiology, Tucson, AZ 85721
Tel: (1-520) 621.84.14 Fax: (1-520) 621.48.99
Email: dvl@u.arizona.edu
1380
Infectious myonecrosis Myoncrose infectieuse Mionecrosis infecciosa

Tel: (1-520) 621.84.14 Fax: (1-520) 621.48.99
Infectious salmon anaemia Anmie infectieuse du saumon Anemia infecciosa del salmn
Dr Frederick Kibenge
Atlantic Veterinary College, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University
of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3
CANADA CANADA CANAD
Tel: (1-902) 566.09.67 Fax: (1-902) 566.08.51
Email: kibenge@upei.ca
Dr Birgit Dannevig
National Veterinary Institute, P.O. Box 750, Sentrum, 0106 Oslo
Tel: (47-23) 21.64.04 Fax: (47-23) 21.63.01
Email: birgit.dannevig@vetinst.no
Koi herpesvirus disease Herpsvirose de la carpe ko Herpesvirosis de la carpa koi
Dr Kei Yuasa
National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura
Minami-ise, Watarai, Mie 516-0193
JAPON JAPAN JAPN
Tel: (81-599) 66.18.30 Fax: (81-599) 66.19.62
Email: khv-lab@fra.affrc.go.jp
Dr Keith Way
The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth Laboratory
Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB
Tel: (44-1305) 20.66.39 Fax: (44-1305) 20.66.01
Email: keith.way@cefas.co.uk
Leptospirosis Leptospirose Leptospirosis
Dr Jessica Petrakovsky(1)
Direccin General de Laboratorios y Control Tcnico
Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA)
Avenida Talcahuano N 1660 (1640), Martnez, Pcia de Buenos Aires
Tel: (54-11) 48.36.11.14 int 287 / 299 Fax: (54-11) 48.36.11.17 int 288
Email: jpetrako@senasa.gov.ar
Dr Luis E. Samartino(2)
Instituto de Bacteriologa, Centro de Investigaciones en Ciencias Veterinarias (CICV)
Instituto Nacional de Tecnologa Agropecuaria (INTA)
Castelar, Casilla de Correo 77, 1708 Morn, Pcia. de Buenos Aires
Tel: (54-11) 46.21.12.89 Fax: (54-11) 46.21.17.12
Email: lsanma@correo.inta.gov.ar
Dr Lee D. Smythe
WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis
Western Pacific Region, Queensland Health Scientific Services
39 Kessels Road, Coopers Plains, P.O. Box 594, Archerfield, Queensland 4108
Tel: (61-7) 32.74.90.64 Fax: (61-7) 32.74.91.75
Email: lee_smythe@health.qld.gov.au
1381
Leptospirosis Leptospirose Leptospirosis

Dr Rudy Hartskeerl
Royal Tropical Institute, N.H. Swellengrebel Laboratory of Tropical Hygiene, Division of Health
Department of Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam
Tel: (31-20) 566.54.38 Fax: (31-20) 697.18.41
Email: r.hartskeerl@kit.nl
Dr William A. Ellis
Department of Agriculture, Veterinary Sciences Division
Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland
Tel: (44-2890) 51.94.41 Fax: (44-2890) 52.57.55
Email: bill.ellis@afbini.gov.uk
Dr Mark Wilson
Tel: (1-515) 337.75.68 Fax: (1-515) 337.75.69
Email: mark.a.wilson@aphis.usda.gov
Lumpy skin disease Dermatose nodulaire contagieuse Dermatosis nodular contagiosa
Tel: (27-12) 529.91.17 Fax: (27-12) 529.94.18
Dr Eeva Tuppurainen
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: eeva.tuppurainen@bbsrc.ac.uk
Maedi-visna Maedi-visna Maedi-visna
Dr Stephen Valas
Anses-Niort, Laboratoire dEtudes et de Recherches Caprines
60 rue de Pied de Fond, B.P. 3081, 79000 Niort
Tel: (33-5) 49.79.61.28 Fax: (33-5) 49 79 42 19
Email: stephen.valas@anses.fr
Tel: (1-509) 335.60.01 Fax: (1-509) 335.83.28
Mareks disease Maladie de Marek Enfermedad de Marek
Dr Nair Venugopal
Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN
Tel: (44-1635) 57.84.11 Fax: (44-1635) 57.72.63
Email: venu.gopal@bbsrc.ac.uk
Dr Aly M. Fadly
USDA, ARS, Avian Disease and Oncology Laboratory
33606 East Mount Hope Roas, East Lansing, Michigan 48823
Tel: (1-517) 337.68.29 Fax: (1-517) 337.67.76
Email: fadly@msu.edu
1382
Myxomatosis Myxomatose Mixomatosis

Dr Antonio Lavazza
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna Bruno Ubertini
Via Antonio Bianchi 9, 25124 Brescia
ITALIE ITALY ITALIA
Tel: (39-030) 22.90.617 Fax: (39-030) 22.90.559
Email: antonio.lavazza@izsler.it
New world screwworm (Cochliomyia hominivorax) Myiase Cochliomyia hominivorax Miasis por Cochliomyia hominivorax
Dr John Welch
COPEG (Panama-US Commission for the Eradication and Prevention of NWS)
Apartado Postal 0816-07636
PANAMA PANAMA PANAM
Tel: (507) 296.06.12 Fax: (507) 296.09.69
Email: john.b.welch@aphis.usda.gov
Newcastle disease Maladie de Newcastle Enfermedad de Newcastle
Dr Paul W. Selleck
Division of Animal Health, Institute of Animal Production & Processing
5 Portarlington Road, Private Bag 24, Geelong 3220, Victoria
Tel: (61-3) 52.27.50.00 Fax: (61-3) 52.27.55.55
Email: paul.selleck@csiro.au
Dr Zhiliang Wang
National Diagnostic Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center
Ministry of Agriculture, 369 Nanjing Road, Qingdao 266032
Tel: (86-532) 87.83.91.88 Fax: (86-532) 87.83.99.22
Email: zlwang111@yahoo.com.cn
Dr Christian Grund
Federal Research Centre for Virus Diseases of Animals (BFAV), Friedrich-Loeffler-Institute
Institute of Diagnostic Virology, Boddenblick 5a, 17493 Greifswald - Insel Riems
Tel: (49-383) 51.71.96 Fax: (49-383) 51.72.75
Email: christian.grund@fli.bund.de
Dr Ilaria Capua
Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio Virologia
Via Romea 14/A, 35020 Legnaro, Padova
ITALIE ITALY ITALIA
Tel: (39-049) 808.43.79 Fax: (39-049) 808.43.60
Dr Kang-Seuk Choi
National Veterinary Research & Quarantine Service, Ministry of Food, Agriculture, Forestry and Fisheries
(MIFAFF), 335 Joongang-ro, Manan-gu, Anyang, Gyeonggi 430-757
Tel: (82-31) 467.18.21 Fax: (82-31) 467.18.14
Email: kchoi0608@korea.kr
Prof. Ian Brown
Tel: (44-1932) 35.73.39 Fax: (44-1932) 35.72.39
Email: ian.brown@ahvla.gsi.gov.uk
Ms Janice Pedersen
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 337.72.66 Fax: (1-515) 337.73.97
Email: janice.c.pedersen@aphis.usda.gov
1383
Oncorhynchus masou virus disease Herpsvirose du saumon masou Herpesvirosis del salmn masou
Dr Mamoru Yoshimizu
Laboratory of Biotechnology and Microbiology, Microbiology Department, Faculty of Fisheries
Graduate School of Fisheries Sciences, Hokkaido University
3-1-1 Minato-cho, Hakodate, Hokkaido 0418611-8611
JAPON JAPAN JAPN
Tel: (81-138) 40.88.10 Fax: (81-138) 40.88.10
Email: yosimizu@fish.hokudai.ac.jp
Ovine epididymitis Epididymite ovine Epididimitis ovina (Brucella ovis)
Dr Ana Maria Nicola
Gerencia de Laboratorios (GELAB), Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA)
Av. Alexander Fleming 1653, 1640 Martnez, Pcia. de Buenos Aires
Tel: (54-11) 48.36.19.92 Fax: (54-11) 48.36.19.92
Email: anicola@senasa.gov.ar
Dr Bruno Garin-Bastuji
Anses Alfort, Unit Zoonoses Bactriennes, Laboratoire de Sant Animale
Tel: (33-1) 49.77.13.00 Fax: (33-1) 49.77.13.44
Email: bruno.garin-bastuji@anses.fr
Dr Heinrich Neubauer
Tel: (49-3641) 80.42.00 Fax: (49-3641) 80.42.28
Dr Menachem Banai
Kimron Veterinary Institute
Department of Bacteriology, P.O. Box 12, Beit Dagan 50250
ISRAL ISRAEL ISRAEL
Tel: (972-3) 968 16 98 Fax: (972-3) 968 17 53
Email: menachemba@moag.gov.il
Dr Massimo Scacchia
National Centre for Exotic Diseases
ITALIE ITALY ITALIA
Tel: (39-0861) 33.24.05 Fax: (39-0861) 33.22.51
Email: m.scacchia@izs.it
Dr Judy A. Stack
Tel: (44-1932) 34.76.10 Fax: (44-1932) 35.72.16
Email: judy.stack@ahvla.gsi.gov.uk
Paratuberculosis Paratuberculose Paratuberculosis
Dr Bernardo Alonso
Gerencia de Laboratorios (GELAB) del Servicio Nacional de Sanidad y Calidad, Agroalimentaria (SENASA)
Av. Alexander Fleming 1653, 1640 Martinez - Pcia de Buenos Aires
Tel: (54-11) 48.36.19.92 Fax: (54-11) 48.36.19.92
Email: balonso@senasa.gov.ar / dilab@inea.com.ar
Dr Jacek Gwozdz
Johnes Disease Laboratory, Research and Development Division, Department of Primary Industries
475 Mickleham Road, Attwood, Victoria 3049
Tel: (61-3) 92.17.42.77 Fax: (61-3) 92.17.42.99
Email: jacek.gwozdz@dpi.vic.gov.au
1384
Paratuberculosis Paratuberculose Paratuberculosis

Dr Ivo Pavlik
Veterinary Research Institute
Hudcova 70, 62132 Brno
TCHEQUE (Rp) CZECH (Rep.) CHECA (Rep.)
Tel: (420.5) 33.33.16.01 Fax: (420.5) 33.33.12.29
Email: pavlik@vri.cz
Dr Pascale Mercier
Anses-Niort, 60 rue de Pied-de-Fond, B.P. 3081, 79012 Niort Cedex
Tel: (33-5) 49.79.61.28 Fax: (33-5) 49.79.42.19
Email: pascale.mercier@anses.fr
Peste des petits ruminants Peste des petits ruminants Peste de pequeos rumiantes
Dr Genevive Libeau
CIRAD-BIOS, Control of Exotic and Emerging Animal Diseases
Programme Sant Animale, Campus International de Baillarguet TA A-15/G, 34398 Montpellier Cedex 5
Tel: (33-4) 67.59.37.98 Fax: (33-4) 67.59.38.50
Email: genevieve.libeau@cirad.fr
Dr Michael Baron
Ash Road, Pirbright, Woking, Surrey GU24 0NF
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: michael.baron@iah.ac.uk
Porcine reproductive and respiratory syndrome Syndrome dysgnsique et respiratoire du porc
Sndrome disgensico y respiratorio porcino
Dr Kegong Tian
Veterinary Diagnostic Laboratory, China Animal Disease Control Center
2 Yuanmingyuan West Road, Haidian District, Beijing
Tel: (86-010) 62.89.12.57 Fax: (86-010) 62.89.35.07
Email: tiankg@263.net
Prof. Zygmunt Pejsak
National Veterinary Research Institute, Department of Swine Diseases
Partyzantow Str. 57, 24-100 Pulawy
POLOGNE POLAND POLONIA
Tel: (48-81) 889.30.30 Fax: (48-81) 886.25.95
Email: stadejek@piwet.pulawy.pl
Rabbit haemorrhagic disease Maladie hmorragique du lapin Enfermedad hemorrgica del conejo
Dr Lorenzo Capucci
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna, B. Ubertini
Via A. Bianchi n 7/9, 25124 Brescia
ITALIE ITALY ITALIA
Tel: (39-30) 229.03.08 Fax: (39-30) 229.05.59
Email: lorenzo.capucci@izsler.it
Rabies Rage Rabia
Dr Christine Fehlner-Gardiner
Centre of Expertise for Rabies (CFIA/ACIA), Animal Diseases Research Institute, Ottawa Laboratory (Fallowfield)
3851 Fallowfield Road, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9
CANADA CANADA CANAD
Tel: (1-613) 228.66.98 Fax: (1-613) 228.66.69
Email: christine.fehlner-gardiner@inspection.gc.ca
1385
Rabies Rage Rabia

Prof. Changchun Tu
Diagnostic Laboratory for Rabies and Wildlife Associated Zoonoses, Department of Virology
Changchun Veterinary Research Institute (CVRI), Chinese Academy of Agricultural Sciences (CAAS)
Liuying Xi Road 666, Jingyue Economic Development Zone, Changchun 130122
Tel: (86-431) 86.98.59.21 Fax: (86-431) 86.98.58.62
Email: changchun_tu@hotmail.com
Dr Jacques Barrat(1)
Anses, Laboratoire de la faune sauvage de Nancy, Technople agricole et vtrinaire, B.P. 40009
54220 Malzville Cedex
Tel: (33-3) 83.29.89.50 Fax: (33-3) 83.29.89.58
Email: jacques.barrat@anses.fr
Dr Florence Cliquet(2)
Anses, Laboratoire de la faune sauvage de Nancy, Technople agricole et vtrinaire, B.P. 40009
54220 Malzville Cedex
Tel: (33-3) 83.29.89.50 Fax: (33-3) 83.29.89.58
Email: florence.cliquet@anses.fr
Dr Thomas Mller
Institute of Epidemiology, Friedrich-Loeffler Institut, Federal Research Institute for Animal Health
Seest. 55, 16868 Wustherhausen/Dosse
Tel: (49-33) 97.98.01.86 Fax: (49-33) 97.98.02.00
Email: thomas.mueller@wus.bfav.de
Dr Dong-Kun Yang
Rabies Research Laboratory, Division of Viral Disease, Animal Plant and Fisheries Quarantine and Inspection
Agency, Ministry of Food, Agriculture, Forestry and Fisheries
175 Anyang-ro, Manan-gu
Anyang, Gyeongii 430-757
KOREA (REP. OF)
Tel: (82-31) 467.17.83 Fax: (82-31) 467.17.97
Email: yangdk@hanmail.net
Dr Claude Taurai Sabeta
Onderstepoort Veterinary Institute, Rabies Unit
Tel: (27-12) 529.94.39 Fax: (27-12) 529.93.90
Email: sabetac@arc.agric.za
Dr Anthony Fooks
Rabies and Wildlife Zoonoses Group, Virology Department, AHVLA Weybridge
Tel: (44-1932) 35.78.40 Fax: (44-1932) 35.72.39
Email: tony.fooks@ahvla.gsi.gov.uk
Dr Charles Rupprecht
Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases
Division of High Consequence Pathogens and Pathology, Poxvirus and Rabies Branch
1600 Clifton Rd., MS G33, Atlanta, GA, 30333
Tel: (1-404) 639.10.53 Fax: (1-404) 639.15.64
Email: cyr5@cdc.gov
Red sea bream iridoviral disease Iridovirose de la daurade japonaise Iridovirosis de la dorada japonesa
Dr Kazuhiro Nakajima
National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4, Fukuura
Kanazawa-ku, Yokohama-Shi, Kanagawa 236-8048
JAPON JAPAN JAPN
Tel: (81-45) 788.76.15 Fax: (81-45) 788.50.01
Email: rsiv-lab@fra.affrc.go.jp
1386
Rift Valley fever Fivre de la Valle du Rift Fiebre del Valle del Rift
Dr Michle Bouloy
Unit de gntique molculaire des Bunyavirus, Dpartement de Virologie
Institut Pasteur, 25 rue du Dr Roux
75724 Paris cedex 15
Tel: (33-1) 40.61.31.57 Fax: (33-1) 40.61.31.51
Email: mbouloy@pasteur.fr
Tel: (27-12) 529.91.17 Fax: (27-12) 529.94.18
Rinderpest Peste bovine Peste bovina
Dr Genevive Libeau
CIRAD-BIOS, Control of Exotic and Emerging Animal Diseases
Programme Sant Animale, Campus International de Baillarguet TA A-15/G, 34398 Montpellier Cedex 5
Tel: (33-4) 67.59.37.98 Fax: (33-4) 67.59.38.50
Email: genevieve.libeau@cirad.fr
Dr Michael Baron
Ash Road, Pirbright, Woking, Surrey GU24 0NF
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: michael.baron@iah.ac.uk
Salmonellosis Salmonelloses Salmonelosis
Dr Cornelius Poppe
Laboratory for Foodborne Zoonoses, Guelph Laboratory, Health Canada, Public Health Agency of Canada
110 Stone Road West, Guelph, Ontario, N1G 3W4
CANADA CANADA CANAD
Tel: (1-519) 822.33.00 Fax: (1-519) 822.22.80
Email: cornelis.poppe@gmail.com
Dr Matthias Hartung
Bundesinstitut fr Risikobewertung (Federal Institute for Risk Assessment)
P.O. Box 330013, 14191 Berlin
Tel: (49-30) 84.12.22.12 Fax: (49-30) 84.12.29.52
Email: matthias.hartung@bfr.bund.de
Dr Antonia Ricci
Istituto Zooprofilattico Sperimentale delle Venezie, National Reference LaboratoryL for Salmonella
Viale DellUniversit 10, 35020 Legnaro (PD)
ITALIE ITALY ITALIA
Tel: (39-049) 808.42.96 Fax: (39-049) 883.02.68
Email: aricci@izsvenezie.it
Dr Rob Davies
AHVLA Weybridge
Tel: (44-1932) 35.73.61 Fax: (44-1932) 35.75.95
Email: rob.davies@ahvla.gsi.gov.uk
1387
Scrapie Tremblante Prurigo lumbar

Dr Francisco Javier Blanco
Institutos de Patobiologa y Virologa, Centro de Investigaciones en Ciencias Veterinarias y Agronmicas
(CICVyA), Instituto Nacional de Tecnologa Agropecuaria (INTA)
Las Cabaas y De Los Reseros, S/N Provincia de Buenos Aires
Tel: (54-11) 46.21.17.89 Fax: (54-11) 46.21.17.43
Email: jviera@cnia.inta.gov.ar / gpinto@cnia.inta.gov.ar
Dr Aru Balachandran
Canadian Food Inspection Agency, Ottawa Laboratory Laboratory (Fallowfield)
Animal Disease Research Institute, 3851 Fallowfield Road, P.O. Box 11300
Station H, Nepean, Ontario K2H 8P9
CANADA CANADA CANAD
Tel: (1-613) 221.48.54 Fax: (1-613) 221.61.03
Email: aru.balachandran@inspection.gc.ca
Prof. Andreas Zurbriggen
NeuroCentre, Department of Clinical Research and Veterinary Public Health
Division of Experimental Clinical Research, University of Bern
Bremgartenstrasse 109A, 3012 Bern
Tel: (41-31) 631.25.09 Fax: (41-31) 631.25.38
Email: andreas.zurbriggen@itn.unibe.ch
Dr Marion Simmons
Tel: (44-1932) 35.75.64 Fax: (44-1932) 35.78.05
Email: marion.simmons@ahvla.gsi.gov.uk
Sheep pox and goat pox Clavele et variole caprine Viruela ovina y viruela caprina
Dr Hamid Reza Varshovi
RAZI Vaccine and Serum Research Institute
P.O. Box 19171/148, Hessarak Karadj, Teheran
IRAN IRAN IRN
Tel: (98-21) 311.79.08 Fax: (98-261) 455.21.94
Email: int@rvsri.com or hr_varshovi@yahoo.com
Tel: (27-12) 529.91.17 Fax: (27-12) 529.94.18
Dr Eeva Tuppurainen
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 ONF
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: eeva.tuppurainen@bbsrc.ac.uk
Spherical baculovirosis (Penaeus monodon-type baculovirus) Baculovirose sphrique (Baculovirus spcifique de Penaeus
monodon) Baculovirosis esfrica (baculovirus de tipo Penaeus monodon)
Dr Grace Lo
College of Life Science, Institute of Zoology, National Taiwan University
1, Sec. 4, Roosevelt Road, Taipei
Tel: (886-2) 23.63.35.62 Fax: (886-2) 23.63.81.79
Email: gracelow@ccms.ntu.edu.tw
Tel: (1-520) 621.84.14 Fax: (1-520) 621.48.99
1388
Spring viraemia of carp Virmie printanire de la carpe Viremia primaveral de la carpa

Dr Hong Liu
Shenzhen Exit & Entry Inspection and Quarantine Bureau, AQSIQ
2049 Heping Road, Shenzhen, 518001
Tel: (86-755) 25.58.84.10 Fax: (86-755) 25.58.86.30
Email: liuhong@szciq.gov.cn
Dr David Stone
The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth Laboratory
Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB
Tel: (44-1305) 20.66.42 Fax: (44-1305) 20.66.01
Email: david.stone@cefas.co.uk
Surra Surra Surra (Trypanosoma evansi)
Dr Philippe Bscher8
Institute of Tropical Medicine Antwerp, Department of Parasitology
Nationalestraat 155, B-2000 Antwerpen
BELGIQUE BELGIUM BELGICA
Tel: (32-3) 247.65.34 Fax: (32-3) 247.63.73
Email: PBuscher@itg.be
Prof. Noboru Inoue
JAPON JAPAN JAPN
Tel: (81-155) 49.56.47 Fax: (81-155) 49.56.43
Email: ircpmi@obihiro.ac.jp
Swine influenza Grippe porcine Gripe porcina
Dr Emanuela Foni
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna B. Ubertini
Via A. Bianchi n 9, 25124 Brescia
ITALIE ITALY ITALIA
Tel: (39-0521) 29.37.33 Fax: (39-0521) 29.35.38
Email: emanuela.foni@izler.it
Dr Takehiko Saito
Viral Disease and Epidemiology Research Division, National Institute of Animal Health
National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, 305-0856
JAPON JAPAN JAPN
Tel: (81-29) 838.79.14 Fax: (81-29) 838.79.14
Email: taksaito@affrc.go.jp
Tel: (1-515) 337.75.51 Fax: (1-515) 337.73.48
Swine vesicular disease Maladie vsiculeuse du porc Enfermedad vesicular porcina
Dr Emiliana Brocchi
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna B. Ubertini
Via A. Bianchi n 9, 25124 Brescia
ITALIE ITALY ITALIA
Tel: (39-030) 229.03.10 Fax: (39-030) 229.03.69
Email: emiliana.brocchi@bs.izs.it
Subject to confirmation by the World Assembly of Delegates to the OIE in May 2013.
1389
Swine vesicular disease Maladie vsiculeuse du porc Enfermedad vesicular porcina

Dr Jef Hammond
Institute for Animal Health
Tel: (44-1483) 23.24.41 Fax: (44-1483) 23.24.48
Email: jef.hammond@iah.ac.uk
Taura syndrome Syndrome de Taura Sndrome de Taura
Tel: (1-520) 621.84.14 Fax: (1-520) 621.48.99
Tetrahedral baculovirosis Baculovirose ttradrique Baculovirosis tetradrica (Baculovirus penaei)
Tel: (1-520) 621.84.14 Fax: (1-520) 621.48.99
Theileriosis Theilriose Teileriosis
Dr Dirk Geysen
Institute of Tropical Medicine, Department of Animal Health, Nationalestraat 155, 2000 Antwerpen
BELGIQUE BELGIUM BELGICA
Tel: (32-3) 247.62.64 Fax: (32-3) 247.62.68
Email: dgeysen@itg.be
Transmissible gastroenteritis Gastro-entrite transmissible Gastroenteritis transmisible
Dr Linda J. Saif
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State
University
1680 Madison Avenue, Wooster, OH 44691-4096
Tel: (1-330) 263.37.44 Fax: (1-330) 263.36.77
Email: saif.2@osu.edu
Trichinellosis Trichinellose Triquinelosis
Dr Alvin Gajadhar
Canadian Food Inspection Agency, Centre for Animal Parasitology
116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3
CANADA CANADA CANAD
Tel: (1-306) 975.53.44 Fax: (1-306) 975.57.11
Email: alvin.gajadhar@inspection.gc.ca
Dr Edoardo Pozio
Istituto Superiore di Sanita, Laboratorio di Parasitoligia, Viale Regina Elena 299, 00161 Roma
ITALIE ITALY ITALIA
Tel: (39.06) 49.90.23.04 Fax: (39.06) 49.90.35.61
Email: edoardo.pozio@iss.it
Trypanosomosis (tsetse-transmitted) Trypanosomose (transmise par ts-ts) Tripanosomosis (transmitida por tsets)
Dr Marc Desquesnes
UMR177-Intertryp (CIRAD-IRD), CIRAD-bios, Campus international de Baillarguet, TA A-17 / G
34398 Montpellier Cedex 5
Tel: (33-4) 67.59.37.24 Fax: (33-4) 67.59.37.98
Email: marc.desquesnes@cirad.fr
1390
Turkey rhinotracheitis Rhinotrachite de la dinde Rinotraquetis del pavo

Dr Nicolas Eterradossi
Anses Ploufragan, Laboratoire de Ploufragan-Plouzan
Laboratoire dtudes et de recherches avicoles, porcines et piscicoles
BP 53, 22440 Ploufragan-Plouzan
Tel: (33-2) 96.01.62.22 Fax: (33-2) 96.01.62.63
Email: nicolas.eterradossi@anses.fr
Venezuelan equine encephalomyelitis Encphalomylite quine vnzulienne Encefalomielitis equina venezolana
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 663.75.51 Fax: (1-515) 663.73.48
Vesicular stomatitis Stomatite vsiculeuse Estomatitis vesicular
Dr Rossana Allende
Centro Panamericano de Fiebre Aftosa OPS/OMS (PANAFTOSA)
Av. President Kennedy 7778, Sao Bento, Duque de Caxias, ZC 20054-40 Rio de Janeiro
BRESIL BRAZIL BRASIL
Tel: (55-21) 36.61.90.64 Fax: (55-21) 36.61.90.01
Email: rallende@paho.org
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 663.75.51 Fax: (1-515) 663.73.48
Viral encephalopathy and retinopathy Encphalopathie et rtinopathie virales Encefalopata y retinopata virales
Dr Giovanni Cattoli
Istituto Zooprofilattico Sperimentale delle Venezie, Dipartimento di Ittioviroligia,
Via Romea 14/A, 35020 Legnaro PD
ITALIE ITALY ITALIA
Tel: (39-049) 808.42.48 Fax: (39-049) 808.43.92
Email: gcattoli@izsvenezie.it
Dr Toshihiro Nakai
Hiroshima University, Graduate School of Biosphere Science, Laboratory of Fish Pathology
Higashi Hiroshima 739-8528
JAPON JAPAN JAPN
Tel: (81-82) 424.79.47 Fax: (81-82) 424.43.80
Email: nakaitt@hiroshima-u.ac.jp
Viral haemorrhagic septicaemia Septicmie hmorragique virale Septicemia hemorrgica viral
Dr Niels Jrgen Olesen
National Veterinary Institute, Technical University of Denmark
Hangvej 2, 8200 Aarhus N
DANEMARK DENMARK DINAMARCA
Tel: (45-35) 88.68.31 Fax: (45-35) 88.69.01
Email: njol@vet.dtu.dk
West Nile fever Fivre de West Nile Fiebre del Nilo Occidental
Dr Rossella Lelli
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale, Via Campo Boario, 64100 Teramo
ITALIE ITALY ITALIA
Tel: (39-0861) 33.22.05 Fax: (39-0861) 33.22.51
Email: r.lelli@izs.it
1391
West Nile fever Fivre de West Nile Fiebre del Nilo Occidental
P.O. Box 844, Ames, IA 50010
Tel: (1-515) 663.75.51 Fax: (1-515) 663.73.48
White spot disease Maladie des points blancs Enfermedad de las manchas blancas
Dr Jie Huang
Molecular Pathology Laboratory, Yellow Sea Fisheries Research Institute
Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao, Shandong 266071
Tel: (86-532) 582.30.62 ext. 802 Fax: (86-532) 581.15.14
Email: huangjie@ysfri.ac.cn / aquadis@ysfri.ac.cn
Dr Grace Lo
College of Life Science, Institute of Zoology, National Taiwan University
1, Sec. 4, Roosevelt Road, Taipei
Tel: (886-2) 23.63.35.62 Fax: (886-2) 23.63.81.79
Email: gracelow@ccms.ntu.edu.tw
Tel: (1-520) 621.84.14 Fax: (1-520) 621.48.99
West tail disease Maladie des queues blanches Enfermedad de la cola blanca
Dr A. Sait Sahul Hameed
Aquaculture Biotechnology Division, Department of Zoology, C. Abdul Hakeem College (Affliated to Thiruvalluvar
University, Tamil Nadu), Melvisharam-632 509, Vellore Dt. Tamil Nadu
INDIA INDE INDIA
Tel: (91-4172) 26.94.87 Fax: (91-4172) 26.94.87
E-mail: cah_sahul@hotmail.com
Yellowhead disease Maladie de la tte jaune Enfermedad de la cabeza amarilla
Dr Peter Walker
CSIRO, Aquaculture and Aquatic Animal Health (AAHL)
5 Portarlington Road, Private Bag 24, Geelong 3220, Victoria
Tel: (61-3) 52.27.54.65 Fax: (61-3) 52.27.55.55
Email: peter.walker@csiro.au
_______________
1392
LIST OF OIE COLLABORATING CENTRES IN 2012
Animal Disease Surveillance Systems, Risk Analysis and Epidemiological Modelling Systmes de surveillance des maladies
animales, analyse des risques et modlisation pidmiologique Sistemas de vigilancia de enfermedades animales, anlisis de
riesgos y modelos epidemiolgicos
Centers for Epidemiology and Animal Health

USDA-APHIS-VS-CEAH
2150 Centre Ave, Building B
Fort Collins, CO 80526-8117
UNITED STATES OF AMERICA ETATS-UNIS DAMRIQUE ESTADOS UNIDOS DE AMERICA
Tel: (1-970) 494.72.00
Fax: (1-970) 494.72.29
Email: cristobal.zepeda@aphis.usda.gov Email 2: ceah@aphis.usda.gov
Web: http://www.aphis.usda.gov/about_aphis
Animal Feed Safety and Analysis Scurit sanitaire et analyses de l'alimentation animale Seguridad y anlisis de los
alimentos para animales
Food and Agricultural Materials Inspection Center

Shintoshin 2-1
Cyuo-ku, Saitama
Saitama 330-9731
JAPON JAPAN JAPN
Tel: (81-50) 37.97.18.63
Fax: (81-48) 600.23.72
Email: toshirou_takehara@nm.famic.go.jp
Animal Welfare Research Recherche en matire de bien-tre animal Investigacin en bienestar animal
Instituto de Ciencia Animal

Universidad Austral de Chile
Facultad de Ciencias Veterinarias
Independencia 641
Casilla 567
Valdivia
CHILI CHILE
Tel: (56-63) 221.690
Fax: (56-63) 221.212
Email: cgallo@uach.cl
Web: http://www.uach.cl
This bi-national OIE Collaborating Centre will include participation from the following institutions:
Ce centre binational incluera la participation des institutions suivantes :
Este Centro Colaborador bo-nacional incluye la participacin de las siguientes instituciones:
Instituto de Biociencias
Facultad de Veterinaria
Universidad de la Repblica O del Uruguay
Lasplaces 1550 CP 11300
Montevideo
URUGUAY
Tel: (598-2) 628.35.05
Fax: (598-2) 628.01.30
Email: stellamaris32@hotmail.com
Web: http://www.bienestaranimal.org.uy
1393
List of OIE Collaborating Centres 2012
Animal Welfare Science and Bioethical Analysis Science du bien-tre animal et lanalyse biothique Ciencia del bienestar
animal y el anlisis biotico
Department of Agriculture, Fisheries and Forestry

Australian Government
GPO Box 858
Canberra, ACT 2601
AUSTRALIE AUSTRALIA
Tel: (61-2) 62.72.39.25
Fax: (61 2) 62.72.37.78
Email: Peter.thornber@daff.gov.au
1394
Animal Welfare Science and Bioethics Centre

Massey University
Private Bag 11 222
Palmerston North 4442
NOUVELLE ZLANDE NEW ZEALAND NUEVA ZELANDA
Tel: (64-6) 356.6099 - Tel2: (64-6) 356.81437
Fax: (64-6) 350.56.57
Email: d.j.mellor@massey.ac.nz
Animal Behaviour and Welfare Research Centre

AgResearch Ltd
Ruakura Research Centre
Private Bag 3123
Hamiliton
NOUVELLE ZLANDE NEW ZEALAND NUEVA ZELANDA
Tel: (64-7) 838.55.69
Email: jim.webster@agresearch.co.nz
Animal Welfare Science Centre

Department of Agriculture and Food Systems
Melbourne Graduate School of Land and Environment
University of Melbourne
Parkville Bld 142
Victoria 3010
AUSTRALIE AUSTRALIA
Tel: (61-3) 83.44.89.33
Email: phh@unimelb.edu.au
Centre for Animal Welfare and Ethics

Natural Resources Agriculture and Veterinary Services
University of Queensland
St Lucia, Queensland 4072
AUSTRALIE AUSTRALIA
Tel: (61-7) 5460.331
Email: c.phillips@uq.edu.au
CSIRO Livestock Industries

Locked Bag 1
Armidale NSW 2350
AUSTRALIE AUSTRALIA
Tel: (61-2) 67.76.13.54
Email: drewe.ferguson@csiro.au
Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine Diagnostic des maladies infectieuses en
mdecine vtrinaire bas sur la biotechnologie Diagnstico de las enfermedades infecciosas de la medicina veterinaria
basado en la biotecnologa
National Veterinary Institute

Travvgen 22, 75189 Uppsala
SUDE SWEDEN SUECIA
Tel: (46-18) 67.40.00
Fax: (46-18) 67.44.67
Email: sandor.belak@sva.se
Swedish University of Agricultural Sciences

Department of Biomedical Sciences and Veterinary Public Health
P.O. Box 7036, 750 07 Uppsala
SUDE SWEDEN SUECIA
Tel: (46-18) 67.41.35
Fax: (46-18) 30.91.62
Email: sandor.belak@slu.se
Development and Production of Vaccines, Pharmaceutical Products and Veterinary Diagnostic Systems using Biotechnology
Dveloppement et production de vaccins, produits pharmaceutiques et dispositifs de diagnostic vtrinaire utilisant la
biotechnologie Desarrollo y produccin de vacunas, productos farmacuticos y dispositivos de diagnstico veterinario
mediante la biotecnologa
Centro de Ingeniera Gentica y Biotecnologa

P.O. Box 6162
Av. 31 e/ 158 y 190
Rpto. Cubanacn, Playa
Marianao, La Habana 10600
CUBA
Tel: (53-7) 271.33.13
Email: carlos.borroto@cigb.edu.cu
Diagnosis and Control of Animal Diseases and Related Veterinary Product Assessment in Asia Diagnostic et contrle des
maladies animales et valuation des produits vtrinaires en Asie Diagnstico y control de enfermedades animales y
evaluacin de productos veterinarios en Asia
National Institute of Animal Health

6-20-1 Josui-honcho
Kodaira, Tokyo 187-0022
JAPON JAPAN JAPN
Tel: (81-42) 321.14.41
Fax: (81-42) 325.51.22
Email: skenichi@affrc.go.jp
National Veterinary Assay Laboratory

1-15-1 Tokura, Kokubunji
Tokyo 185-8511
JAPON JAPAN JAPN
Tel: (81-42) 321.14.41
Fax: (81-42) 321.51.22
Email: skenichi@affrc.go.jp - Email2: nval@nval.maff.go.jp
Diagnosis and Control of Animal Diseases in Eastern Europe, Central Asia and Transcaucasia Diagnostic et contrle des
maladies animales en Europe de lEst, en Asie centrale et en Transcaucasie Diagnstico y control de las enfermedades
animales en Europa oriental, Asia central y Transcaucasia
Federal Centre for Animal Health (FGI-ARRIAH)

Ministry of Agriculture of the Russian Federation
600901 Yurevets
Vladimir
RUSSIA RUSSIE RUSIA
Tel: (7-0922) 26.38.77 - Tel 2: (7-0922) 26.06.14
Fax: (7-0922) 26.38.77
Email: mail@arriah.ru
Web: http://www.arriah.ru/portal/en
1395
Diagnosis of Animal Diseases and Vaccine Evaluation in the Americas Diagnostic des maladies animales et valuation des
vaccins dans les Amriques Diagnstico de las enfermedades animales y evaluacin de las vacunas en las Amricas

USDA, APHIS, Veterinary Services
P.O. Box 844
Ames, Iowa 50010
Tel: (1-515) 337.72.66 Tel 2: (1-515) 337.61.00
Fax: (1-515) 337.73.97
Email: elizabeth.a.lautner@aphis.usda.gov Email 2: nsvl_concerns@aphis.usda.gov
Institute for International Cooperation in Animal Biologics

College of Veterinary Medicine
Iowa State University
Ames, Iowa 50011
Tel: (1-515) 294.84.59
Fax: (1-515) 294.82.59
Email: iicab@iastate.edu
Web: http://www.cfsph.iastate.edu/iicab/
Center for Veterinary Biologics

USDA, APHIS, Veterinary Services
P.O. Box 844
Ames, Iowa 50010
Tel: (1-515) 337.72.66
Fax: (1-515) 337.73.97
Email: rick.e.hill@aphis.usda.gov Email 2: cvb@aphis.usda.gov
Web: http://www.aphis.usda.gov/animal_health/vet_biologics
Diagnosis, Epidemiology and Control of Animal Diseases in Tropical Regions Diagnostic, pidmiologie et contrle des
maladies animales en rgions tropicales Diagnstico, epidemiologa y control de las enfermedades animales en las regiones
tropicales
Centre international en recherche agronomique pour le dveloppement

TA-A 15/A
Campus International de Baillarguet
Montferriez-sur-Lez
B.P. 5035
34398 Montpellier Cedex 5
FRANCE FRANCIA
Tel: (33-4) 67.59.37.12
Fax: (33-4) 67.59.37.98
Email: dominique.martinez@cirad.fr
Web: http://umr-cmaee.cirad.fr
Diseases at the Animal/Human Interface Maladies l'interface entre l'homme et les animaux Enfermedades en la interfaz
animal-humano
1396
Istituto Zooprofilattico Sperimentale delle Venezie

Laboratorio Virologia
Via Romea 14/A
35020 Legnaro, Padova
ITALY ITALIE ITALIA
Tel: (39-049) 808 43 79
Fax: (39-049) 808 43 60
Web: http://www.izsvenezie.it
ELISA and Molecular Techniques in Animal Disease Diagnosis Diagnostic des maladies animales par lELISA et les techniques
molculaires Diagnstico de enfermedades animales por el mtodo ELISA y las tcnicas moleculares
FAO/IAEA Animal Production and Health Laboratory

Agriculture and Biotechnology Laboratory, IAEA Laboratories
Wagramerstrasse 5, P.O. Box 100
1400 Vienna
AUTRICHE AUSTRIA
Tel: (43-1) 2600.28.355
Fax: (43-1) 2600.280221
Email: adama.diallo@iaea.org
Emerging and Re-Emerging Zoonotic Diseases Zoonoses mergentes et r-mergentes Enfermedades zoonticas
emergentes y re-emergentes
National Center for Emerging and Infectious Diseases

One Health Office, Centers for Disease Control and Prevention
1600 Clifton Road, MS D-76
Atlanta, Georgia 30333
Tel: (1-404) 639.73.78
Fax: (1-404) 639.70.90
Email: chr1@cdc.gov
Web: http://www.cdc.gov/ncezid/
Epidemiology and Diagnosis of Emerging, Re-Emerging and Transboundary Diseases of Animals in the Caribbean and Central
America Epidmiologie et diagnostic des maladies animales mergentes, r-mergentes et transfrontalires dans les Carabes
et lAmrique central Epidemiologa y Diagnstico de Enfermedades Emergentes, Reemergentes y Transfronterizas de los
Animales en el Caribe y Centroamrica
Centro Nacional de Sanidad Agropecuaria

Apdo 10, San Jos de las Lajas
CP 32700, Provincia Mayabeque
HABANA
CUBA
Tel: (53-47) 86.32.06
Email: lydia@censa.edu.cu
Web: http://www.censa.edu.cu
Epidemiology and Risk Assessment of Aquatic Animal Diseases Epidmiologie et valuation du risque des maladies des
animaux aquatiques Epidemiologa y evaluacin del riesgo de enfermedades de los animales acuticos

Department for Epidemiology
P.O. Box 750 Sentrum
0106 Oslo
Tel: (47-23) 21.63.65
Fax: (47-23) 21.60.01
Email: edgar.brun@vetinst.no Email 2: ebrun25@gmail.com
Centre for Aquatic Health Science

Atlantic Veterinary College (AVC)
University of Prince Edward Island
Department of Health Management
550 University Avenue
Charlottetown, PE C1A 4P3
CANADA CANAD
Tel: (1-902) 566.07.28
Fax: (1-902) 620.50.53
Email: lhammell@upei.ca
Web: http://www.eraaad.org
1397
Epidemiology, Training and Control of Emerging Avian Diseases Epidmiologie, formation et contrle des maladies aviaires
mergentes Epidemiologa, formacin y lucha contra las enfermedades aviares emergentes
Istituto Zooprofilattico Sperimentale delle Venezie

Vialle dellUniversita 10
35020 Legnaro, Padova
ITALY ITALIE ITALIA
Tel: (39-049) 808.43.91
Fax: (39-049) 883.05.39
Email: smarangon@izsvenezie.it Email 2: dirsan@izsvenezie.it
Web: http://www.izsvenezie.it
Food Safety Scurit sanitaire des aliments Seguridad sanitaria de los alimentos
University of Tokyo
Research Center for Food Safety
Graduate School of Agricultural and Life Sciences
1-1-1 Yayoi, Bunkyo-ku
Tokyo 113-8657
JAPON JAPAN JAPN
Tel: (81-50) 37.97.18.60
Fax: (81-48) 600.23.72
Email: aksugiur@mail.ecc.u-tokyo.ac.jp
Food Safety, Diagnosis and Control of Animal Diseases in Eastern Europe, Central Asia and Transcaucasia Scurit sanitaire
des aliments, diagnostic et contrle des maladies animales en Europe de l'Est, en Asie centrale et en Transcaucasie Seguridad
sanitaria de los alimentos y el diagnstico y control de las enfermedades animales en Europa oriental, Asia central y
Transcaucasia
FGU The All-Russian State Centre for Quality and Standardisation of Veterinary Drugs and
Feed (VGNKI)
Ministry of Agriculture and Food
5 Zvenigorodskoye Shosse
123022 Moscow
RUSSIA RUSSIE RUSIA
Tel: (7-095) 253.14.91
Fax: (7-095) 253.14.91
Email: vgnki@vgnki.ru
Web: http://www.vgnki.ru
Food-Borne Zoonotic Parasites Zoonoses parasitaires dorigine alimentaire Zoonosis parasitarias de origen alimentario
Centre for Food-borne and Animal Parasitology

Canadian Food Inspection Agency
116 Veterinary Road
Saskatoon
Saskatchewan S7N 2R3
CANADA CANAD
Tel: (1-306) 975.53.44
Fax: (1-306) 975.57.11
Email: alvin.gajadhar@inspection.gc.ca
Information on Aquatic Animal Diseases Information sur les maladies des animaux aquatiques Informacin sobre las
enfermedades de los animales acuticos
1398
Centre for Environment, Fisheries and Aquaculture Sciences

Weymouth Laboratory
Barrack Road, The Nothe
Weymouth
Dorset DT4 8UB
ROYAUME UNI UNITED KINGDOM REINO UNIDO
Tel: (44-1305) 20.66.39
Fax: (44-1305) 20.66.01
Email: stephen.feist@cefas.co.uk
Web: http://www.cefas.defra.gov.uk/our-science/animal-health-and-food-safety/aquatic-animaldisease/oie-collaborating-centre-for-information-on-aquatic-animal-diseases.aspx
Laboratory Capacity Building Renforcement des capacits des laboratoires Desarrollo de capacidad de los laboratorios
Australian Animal Health Laboratory

5 Portarlington Road
Private Bag 24 (Ryrie Street)
Geelong 3220, Victoria
AUSTRALIE AUSTRALIA
Tel: (61-3) 52.27.52.72 Tel 2: (61-3) 52.27.50.00
Fax: (61-3) 52.27.55 55
Web: http://www.csiro.au
New and Emerging Diseases Maladies nouvelles et emergentes Nuevas enfermedades y enfermedades emergentes
Australian Animal Health Laboratory

5 Portarlington Road
Private Bag 24 (Ryrie Street)
Geelong 3220, Victoria
AUSTRALIE AUSTRALIA
Tel: (61-3) 52.27.50.00
Fax: (61-3) 52.27.55.55
Email: martyn.jeggo@csiro.au
Web: http://www.csiro.au
Research and Training in Population Animal Health Diagnosis and Surveillance Systems Recherche et formation en matire de
diagnostic et de systmes de surveillance des maladies animals Investigacin y formacin en el mbito del diagnstico
zoosanitario y de los sistemas de vigilancia de poblaciones
International Epilab
Technical University of Denmark
Blowsvej 27
DK-1790 Copenhagen V
DANEMARK DENMARK DINAMARCA
Tel: (45-35) 88.60.00
Fax: (45-35) 88.60.01
Email: vet@vet.dtu.dk
Web: http://www.vet.dtu.dk
Research on Emerging Avian Diseases Recherche sur les maladies aviaires mergentes Investigacin sobre las
enfermedades aviares emergentes
Southeast Poultry Research Laboratory

US Department of Agriculture, Agricultural Research Service (USDA-ARS)
934 College Station Road
Athens, Georgia 30605
Tel: (1-706) 546.34.33
Fax: (1-706) 546.31.61
Email: David.Swayne@ars.usda.gov
Research, Diagnosis and Surveillance of Wildlife Pathogens Recherche, diagnostic et surveillance des agents pathognes de
la faune sauvage Investigacin, diagnstico y vigilancia de los patgenos de la fauna silvestre
Canadian Cooperative Wildlife Health Centre

Department of Veterinary Pathology
Western College of Veterinary Medicine
University of Saskatchewan
52 Campus Drive
Saskatoon S7N 5B4
CANADA CANAD
Tel: (1-306) 966.50.99
Fax: (1-306) 966.73.87
Email: ted.leighton@usask.ca Email 2: ccwhc@usaks.ca
Web: http://www.ccwhc.ca
1399
National Wildlife Health Center

US Geological Survey
US Department of Interior
6006 Schroeder Road
Madison, Wisconsin, WI 53711
UNITED STATES OF AMERICA ETATS-UNIS DAMRIQUE ESTADOS UNIDOS DE
AMERICA
Tel: (1-608) 270.24.01
Email: jsleeman@usgs.gov
Web: http://www.nwhc.usgs.gov/
Surveillance and Control of Animal Diseases in Africa Surveillance et contrle des maladies animales en Afrique Vigilancia y
control de enfermedades animales en frica
Onderstepoort Veterinary Institute

Agricultural Research Council
Private Bag X05
Onderstepoort 0110
SOUTH AFRICA AFRIQUE DE SUD SUDFRICA
Tel: (27-12) 529.91.06
Fax: (27-12) 565.46.64
Email: musoket@arc.agric.za
Surveillance and Control of Animal Protozoan Diseases Surveillance et contrle des maladies animales protozoaires
Vigilancia y control de las enfermedades protozoicas animales
National Research Center for Protozoan Disease

Obihiro University of Agriculture and Veterinary Medicine
Nishi 2-13, Inada-cho
Obihiro, Hokkaido 080-8555
JAPON JAPAN JAPN
Tel: (81-155) 49.56.41
Fax: (81-155) 49.56.43
Email: igarcpmi@obihiro.ac.jp
Training in Integrated Livestock and Wildlife Health and Management Formation en gestion sanitaire de linterface des animaux
domestiques et animaux sauvages Formacin en gestin sanitaria de la interfase de los animales de cra y de los animales
salvajes
Department of Veterinary Tropical Diseases

Faculty of Veterinary Science
University of Pretoria
Private Bag X04
Onderstepoort 0110
SOUTH AFRICA AFRIQUE DE SUD SUDFRICA
Tel: (27-12) 529.82.69
Fax: (27-12) 529.83.12
Email: koos.coetzer@up.ac.za
Training of Official Veterinarians Formation des vtrinaires officiels Formacin de Veterinarios Oficiales
1400
Ecole interne de VetAgro Sup

Ecole nationale des Services vtrinaires
1 avenue Bourgelat
69280 Marcy-lEtoile
FRANCE FRANCIA
Tel: (33-4) 78.87.25.45
Fax: (33-4) 78.87.25.48
Email: olivier.faugere@ensv.vetagro-sup.fr
Training of Veterinary Services Formation des services vtrinaires Capacitacin de los servicios veterinarios
Centro Buenos Aires para la Capacitacin de los Servicios Veterinarios

CEBASEV
Libertad N 1240, Piso 1, Oficina 30
CP 1012, Ciudad Autnoma de Buenos Aires
ARGENTINE ARGENTINA
Tel: (54-11) 48.16.64.22
Fax: (54-11) 48.16.64.22
Email: jdillon@senasa.gov.ar Email 2: eleon@cnia.inta.gov.ar
Web: http://prosaia@prosaia.org
Training Veterinary Officials and Diagnosing Infectious Animal Diseases and Zoonoses in Tropical Africa Formation des agents
des Services vtrinaires officiels et diagnostic des maladies animales infectieuses et des zoonoses en Afrique tropicale
Formacin de veterinarios oficiales y diagnstico de epizootias infecciosas y zoonosis en frica Tropical
Ecole Inter-Etats des Sciences et Mdecine Vtrinaires

BP 5077
Dakar
SNGAL SENEGAL
Tel: (221-33) 865.10.08
Fax: (221-33) 824.42.83
Email: directioneismv@hotmail.com Email 2: ljpangui@yahoo.fr
Validation, Quality Assessment and Quality Control of Diagnostic Assays and Vaccine for Vesicular Diseases in Europe
Validation, assurance qualit et contrle qualit des preuves diagnostiques et des vaccins pour les maladies vsiculeuses en
Europe Validacin, aseguramiento y control de la calidad de los ensayos de diagnstico y de las vacunas para enfermedades
vesiculares en Europa
Centre dEtudes et de Recherches Vtrinaires et Agrochimiques de Ukkel

CODA/CERVA/VAR
Department of Virology
Section Epizootic Diseases
Groeselenberg 99
B-1180 Ukkel
BELGIQUE BELGIUM BLGICA
Tel: (32-2) 379.04.00 Tel 2: (32-2) 37.90.512
Fax: (32-2) 379.06.66
Email: krdec@coda-cerva.be
Veterinary Drug Regulatory Programmes Programmes de rglementation des mdicaments vtrinaires Programas
Reguladores de Medicamentos Veterinarios
Center for Veterinary Medicine

Food and Drug Administration (FDA)
Department of Health and Human Services
7519 Standish Place, HFV-1, Room 177
Rockville, Maryland 20855
Tel: (1-240) 276.90.25
Email: merton.smith@fda.hhs.gov
Web: http://www.fda.gov/AnimalVeterinary/default.htm
Veterinary Medicinal Products Mdicaments vtrinaires Medicamentos veterinarios
Agence Nationale du Mdicament Vtrinaire

Anses Fougres
B.P. 203
La Haute Marche, Javen
35302 Fougres Cedex
FRANCE FRANCIA
Tel: (33-2) 99.94.78.78
Fax: (33-2) 99.94.78.99
Email: Jean-Pierre.orand@anses.fr
Web: http://www.anmv.anses.fr
1401
Veterinary Services Capacity Building Renforcement des capacits des services vtrinaires Desarrollo de capacidad de los
servicios veterinarios
Center for Animal Health and Food Safety

University of Minnesota
136 Andrew Boss Laboratory
1354 Eckles Avenue
St Paul, Minnesota 55108
Tel: (1-612) 625.87.09
Fax: (1-612) 624.49.06
Email: huest001@umn.edu Email2: cahfs@umn.edu
Web: http://www.cahfs.umn.edu
Veterinary Training, Epidemiology, Food Safety and Animal Welfare Formation vtrinaire, pidmiologie, scurit sanitaire
des aliments et bientraitance animale Entrenamiento veterinario, epidemiologa, seguridad sanitaria de los alimentos y
bienestar animal
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale"

Via Campo Boario
64100 Teramo
ITALIE ITALY ITALIA
Tel: (39-0861) 33.22.05
Fax: (39-0861) 33.22.51
Email: r.lelli@izs.it
Web: http://www.izs.it
Zoonoses in Europe Zoonoses en Europe Zoonosis in Europa
Federal Research Institute for Animal Health

Friedrich-Loeffler-Institute
Sdufer 10
17493 Greifswald
Insel Riems
GERMANY ALLEMAGNE ALEMANIA
Tel: (49-38351) 71.102
Fax: (49-38351) 71.151
Email: thomas.mettenleiter@fli.bund.de Email 2: poststelle@fli.bund.de
Web: http://www.fli.bund.de
Zoonoses of Asia-Pacific zoonoses de lAsie-Pacifique Zoonosis de Asia-Pacfico
Harbin Veterinary Research Institute

Chinese Academy of Agricultural Sciences
No 427 Maduan Street,
Nangang District
Harbin, Heilongjiang, 150001
Tel: (86) 189.46.06.62.20;
EmailZj@hvri.ac.cnwww.hvri.ac.cn
*
* *
1402
ALPHABETICAL LIST OF DISEASES COVERED IN

THIS TERRESTRIAL MANUAL
Disease
Page
Acarapisosis of honey bees........................... 358

Aethina tumida (see Small hive beetle
infestation [Aethina tumida])........................... 385
African horse sickness................................... 819
African swine fever......................................... 1067
Akabane virus (see Bunyaviral diseases
of animals [excluding Rift Valley fever])
American foulbrood of honey bees................ 365
Anaplasmosis (see Bovine anaplasmosis)
Anthrax...........................................................
87
Atrophic rhinitis of swine................................ 1080
Aujeszkys disease.........................................
97
Avian chlamydiosis......................................... 401
Avian infectious bronchitis.............................
414
Avian infectious laryngotracheitis................... 427
Avian influenza..............................................
436
Avian metapneumovirus (see Turkey
rhinopneumonitis [avian metapneumovirus
infections])
Avian mycoplasmosis (Mycoplasma
gallisepticum, M. synoviae).............. 455
Avian pasteurellosis (see Fowl cholera)
Avian tuberculosis.......................................... 470
Disease
Campylobacter fetus (see Bovine genital
campylobacteriosis)
Campylobacter jejuni.................................
Caprine and ovine brucellosis
(excluding Brucella ovis)................................
Caprine arthritis/encephalitis
& Maedi-visna.................................................
Chrysomya bezziana (see Screwworm)
Classical swine fever (hog cholera)...............
Cochliomyia hominivorax (see Screwworm)
Contagious agalactia.....................................
Contagious bovine pleuropneumonia............
Contagious caprine pleuropneumonia...........
Contagious equine metritis............................
Cowdriosis (see Heartwater)
Cryptosporidiosis..........................................
Cysticercosis................................................
Dermatophilosis............................................
Dourine..........................................................
Duck virus enteritis........................................
Duck virus hepatitis.......................................
Page
1183
968
978
1089
987
704
995
831
1190
1214
717
838
480
489
Babesiosis (see Bovine babesiosis)

Bluetongue..................................................... 112
Border disease............................................... 957
Bovine anaplasmosis..................................... 589
Bovine babesiosis.......................................... 601
Bovine brucellosis.......................................... 616
Bovine cysticercosis....................................... 651
Bovine genital campylobacteriosis................. 652
Bovine spongiform encephalopathy............... 662
Bovine tuberculosis........................................ 674
Bovine viral diarrhoea..................................... 690
Brucella ovis (see Ovine epididymitis)
Brucellosis (see Bovine brucellosis, Caprine and
ovine brucellosis [excluding Brucella ovis], Ovine
epididymitis [Brucella ovis], or Porcine brucellosis)
Bunyaviral diseases of animals
(excluding Rift Valley fever)................... 1163
Echinococcosis/Hydatidosis........................... 130
Enterovirus encephalomyelitis (see Teschovirus
encephalomyelitis [previously enterovirus
encephalomyelitis or Teschen/Talfan disease])
Enzootic abortion of ewes
(ovine chlamydiosis)...................................... 1008
Enzootic bovine leukosis...............................
721
Epizootic haemorrhagic disease.................... 112
Epizootic lymphangitis...................................
846
Equine encephalomyelitis
(Eastern and Western)..................................
852
Equine infectious anaemia............................
860
Equine influenza............................................
865
Equine piroplasmosis..................................... 879
Equine rhinopneumonitis...............................
889
Equine viral arteritis.......................................
899
Escherichia coli (see Verocytotoxogenic
Escherichia coli)
European foulbrood of honey bees...............
375
Cache virus (see Bunyaviral diseases of animals

[excluding Rift Valley fever])
Camelpox...................................................... 1175
Campylobacter coli....................................... 1183
Foot and mouth disease................................

Fowl cholera (avian pasteurellosis)................
Fowl plague (see Avian influenza)
145
500
1403
Alphabetical list of diseases covered in this Terrestrial Manual
Disease
Fowl pox........................................................
Fowl typhoid and Pullorum disease..............
Page
507
514
Glanders.......................................................
913
Goat pox (see Sheep pox and goat pox)
Gumboro disease (see Infectious bursal disease)
Haemorrhagic septicaemia...........................
732
Heartwater...................................................... 174
Hendra and Nipah virus diseases................. 1225
Highly pathogenic avian influenza
(see Avian influenza)
Hog cholera (see Classical swine fever)
Horse mange.................................................
923
Hydatidosis (see Echinococcosis/Hydatidosis)
Infectious bovine rhinotracheitis/
infectious pustular vulvovaginitis....................
Infectious bursal disease
(Gumboro disease)........................................
Japanese encephalitis..................................
Johnes disease (see Paratuberculosis)
745
528
188
Leishmaniosis...............................................
198
Leptospirosis.................................................
209
Listeria monocytogenes................................. 1239
Lumpy skin disease.......................................
762
Maedi-visna.................................................... 978
Malignant catarrhal fever...............................
775
Mange............................................................ 1256
Mareks disease............................................. 545
Mycoplasma gallisepticum (see Avian
mycoplasmosis)
Mycoplasma synoviae (see Avian mycoplasmosis)
Myxomatosis.................................................. 931
Nairobi sheep disease.................................... 1017
Newcastle disease......................................... 555
New World screwworm (see Screwworm)
Nipah virus encephalitis................................ 1105
Nosemosis of honey bees.............................
380
Old World screwworm (see Screwworm)
Ovine chlamydiosis (see Enzootic abortion of ewes)
Ovine epididymitis (Brucella ovis)................. 1018
Ovine pulmonary adenocarcinoma
(adenomatosis)............................................. 1027
Paratuberculosis (Johnes disease)..............
234
Peste des petits ruminants............................ 1032
Porcine brucellosis........................................ 1106
Porcine cysticercosis...................................... 1113
1404
Disease
Page
Porcine reproductive and

respiratory syndrome..................................... 1114
Pseudorabies (see Aujeskys disease)
Pullorum disease (see Fowl typhoid and Pullorum
disease)
Pulmonary adenomatosis (see Ovine pulmonary
adenocarcinoma [adenomatosis])
Q fever...........................................................
250
Rabbit haemorrhagic disease........................

Rabies............................................................
Rift Valley fever..............................................
Rinderpest.....................................................
941
263
283
294
Salmonella abortusovis................................. 1044

Salmonellosis................................................ 1268
Scrapie.......................................................... 1045
Screwworm...................................................
223
Sheep pox and goat pox............................... 1055
Small hive beetle infestation
(Aethina tumida).....................................
385
Surra (see Trypanosoma evansi infections
[including surra])
Swine influenza............................................. 1127
Swine vesicular disease................................ 1138
Teschovirus encephalomyelitis (previously
enterovirus encephalomyelitis or
Teschen/Talfan disease)............................... 1145
Theilerosis.....................................................
785
Toxoplasmosis............................................... 1287
Transmissible gastroenteritis......................... 1152
Trichinellosis..................................................
305
Trichomonosis................................................ 801
Tropilaelaps clareae (see Tropilaelaps infestation of
honey bees)
Tropilaelaps infestation of honey bees
(Tropilaelaps spp.).......................................... 389
Tropilaelaps koenigerum (see Tropilaelaps
infestation of honey bees)
Trypanosoma evansi infections
(including surra).............................................. 314
Trypanosomosis (Tsetse-transmitted)............ 809
Tuberculosis (see Avian tuberculosis or
Bovine tuberculosis)
Tularemia......................................................
329
Turkey rhinopneumonitis (avian
metapneumovirus infections)............................ 574
Varroosis of honey bees............................
394
Venezuelan equine encephalomyelitis..........
924
Verocytotoxigenic Escherichia coli................ 1297
Vesicular stomatitis........................................ 335
West Nile fever....................................
346

OIE Manual of Diagnostic Tests and Vaccines For Terestrial Animals Vol 2 - Ecvine, Leporide, Suine, Caprine

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WORLD ORGANISATION FOR ANIMAL HEALTH

MANUAL OF DIAGNOSTIC TESTS AND VACCINES

This Terrestrial Manual has been edited by the

OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals

Manual of Recommended Diagnostic Techniques and Requirements for Biological Products:

Seventh Edition: ISBN 978-92-9044-878-5

To ensure transparency in the global animal disease and zoonosis situation.

To improve the legal framework and resources of national Veterinary Services.

OIE Terrestrial Manual 2012

Doctor Bernard Vallat

Professor Vincenzo Caporale

OIE Terrestrial Manual 2012

OIE Terrestrial Manual 2012

OIE LISTED DISEASES AND OTHER DISEASES OF IMPORTANCE TO

African horse sickness .............................................................................................

Border disease .........................................................................................................

OIE Terrestrial Manual 2012

African swine fever ...................................................................................................

Bunyaviral diseases of animals (excluding Rift Valley fever)* ..................................

Laboratory methodologies for bacterial antimicrobial susceptibility testing ..............

OIE REFERENCE EXPERTS AND DISEASE INDEX

List of OIE Reference Laboratories (as of May 2012) ...............................................................................

OIE Terrestrial Manual 2012

Arrangement of the Terrestrial Manual

Explanation of the tests described and of the table on pages xixiv

OIE Terrestrial Manual 2012

Introduction (How to use this Terrestrial Manual)

List of OIE Reference Laboratories

OIE Terrestrial Manual 2012

LIST OF TESTS FOR INTERNATIONAL TRADE

Foot and mouth disease

New World screwworm (Cochliomyia

Paratuberculosis (Johnes disease)

Rift Valley fever

Trypanosoma evansi infections

West Nile fever

Acarapisosis of honey bees

American foulbrood of honey bees

Please refer to Terrestrial Manual chapters to verify which method is prescribed.

OIE Terrestrial Manual 2012

List of tests for international trade

European foulbrood of honey bees

Small hive beetle infestation

Tropilaelaps infestation of honey bees

Varroosis of honey bees

Avian infectious bronchitis

Avian infectious laryngotracheitis

Duck virus enteritis

Duck virus hepatitis

Fowl typhoid and Pullorum disease

Agent id., Agg.

Infectious bursal disease

Turkey rhinotracheitis (avian

CF, ELISA, IFA

Bovine genital campylobacteriosis

Bovine spongiform encephalopathy

Gamma interferon test

Bovine viral diarrhoea

Contagious bovine pleuropneumonia

Enzootic bovine leukosis

Infectious bovine rhinotracheitis/

OIE Terrestrial Manual 2012

List of tests for international trade