Manual of Diagnostic Tests For Aquatic Animals: Fourth Edition, 2003

Office international des pizooties 12, rue de Prony 75017 Paris France
Tel.: 33 (0)1 44 15 18 88 Fax: 33 (0)1 42 67 09 87 www.oie.int oie@oie.int

Manual of
Diagnostic
Tests for
Aquatic Animals

Fourth Edition, 2003

This Aquatic Manual has been edited by the OIE Aquatic Animal Health Standards Commission
on the basis of comments received from Member Countries, and approved by the
International Committee of the OIE

First Edition, 1995
Second Edition, 1997
Third Edition, 2000

OIE Manual of Diagnostic Tests for Aquatic Animals
Fourth Edition 2003

ISBN 92-9044-563-7

Copyright
OFFICE INTERNATIONAL DES EPIZOOTIES, 2003
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
WWW: http://www.oie.int

Reproduction or translation is permitted for non-commercial purposes only, provided reference is made
to the source and a cutting of the reprinted material is sent to the OIE.
Manual of Diagnostic Tests for Aquatic Animals 2003 iii
F ORE WORD

The World Organisation for Animal Health (OIE) is an intergovernmental organisation that was established in 1924
in order to promote world animal health. Its main activities are as follows:
1 To collect and disseminate to its Member Countries, information (including emergency information) on the
occurrence, course and treatment of animal diseases.
2 To provide guidelines and standards for health regulations applicable to international trade in animals.
3 To promote and co-ordinate research on the pathology, diagnosis, treatment and prevention of animal diseases when
international collaboration in such research is desirable.
Aquatic animals are included in the concept of animals above. Diagnostic procedures for some aquatic animal diseases
used to be included in the OIE International Animal Health Code (1986 edition), but it became clear that separate
publications specific to aquatic animal health were needed. The reasons are that the conditions, problems and
requirements in this field are different to those encountered in other animals, and that international trade in aquatic
animals and their products is intensifying and increasing in importance.
The purpose of this Manual of Diagnostic Tests for Aquatic Animals (referred to hereafter as the Aquatic
Manual) is to provide a uniform approach to the diagnosis of the diseases listed in the OIE Aquatic Animal Health
Code (referred to hereafter as the Aquatic Code), so that the requirements for health certification in connection with
trade in aquatic animals and aquatic animal products, can be met.
Although many publications exist on the diagnosis and control of aquatic animal diseases, the Aquatic Manual is a
key document describing the methods that can be applied to the OIE-listed diseases in aquatic animal health
laboratories all over the world, thus increasing efficiency and promoting improvements in aquatic animal health world-
wide.
The task of compiling the Aquatic Manual was assigned to the OIE Aquatic Animal Health Standards Commission
(Aquatic Animals Commission), and all the chapters were circulated to OIE Member Countries for comments and
revision. The Aquatic Manual will be continually revised and updated as new information on aquatic animal diseases
in general, and new emerging diseases in particular, becomes available. It is intended to publish a new edition every two
years; intermittent changes will be available on the OIE Web site.
Dr Bernard Vallat Dr Eva-Maria Bernoth
Director General, OIE President, Aquatic Animals Commission
2003
Manual of Diagnostic Tests for Aquatic Animals 2003 v
CONT E NT S

Introduction vii
Acknowledgements ix
Abbreviations xiii
Definitions xv
PART 1 GENERAL PROVISIONS
SECTION 1.1. INTRODUCTORY CHAPTERS
1.1.1 Quality management in veterinary testing laboratories 3
1.1.2. Principles of validation of diagnostic assays for infectious diseases 10
1.1.3. Validation and quality control of polymerase chain reaction methods used
for diagnosis of infectious diseases
20
1.1.4. Requirements for surveillance for international recognition of freedom
from infection
27
1.1.5. Methods for disinfection of aquaculture establishments 46
PART 2 DISEASES OF FISH
Chapter I.1. General information 63
SECTION 2.1. DISEASES LISTED BY THE OIE
Chapter 2.1.1. Epizootic haematopoietic necrosis 77
Chapter 2.1.2. Infectious haematopoietic necrosis 86
Chapter 2.1.3. Oncorhynchus masou virus disease 100
Chapter 2.1.4. Spring viraemia of carp 108
Chapter 2.1.5. Viral haemorrhagic septicaemia 115
Chapter 2.1.6. Channel catfish virus disease 125
Chapter 2.1.7. Viral encephalopathy and retinopathy 135
Chapter 2.1.8. Infectious pancreatic necrosis 142
Chapter 2.1.9. Infectious salmon anaemia 152
Chapter 2.1.10. Epizootic ulcerative syndrome 162
Chapter 2.1.11. Bacterial kidney disease (Renibacterium salmoninarum) 167
Chapter 2.1.12. Enteric septicaemia of catfish (Edwardsiella ictaluri) 185
Chapter 2.1.13. Piscirickettsiosis (Piscirickettsia salmonis) 193
Chapter 2.1.14. Gyrodactylosis (Gyrodactylus salaris) 200
Chapter 2.1.15. Red sea bream iridoviral disease 207
Chapter 2.1.16. White sturgeon iridoviral disease 212
PART 3 DISEASES OF MOLLUSCS
Chapter 3.1.1. Bonamiosis (Bonamia exitiosus, B. ostreae and Mikrocytos roughleyi ) 230
Chapter 3.1.2. MSX diseases (Haplosporidium nelsoni) 235
Chapter 3.1.3. Marteiliosis (Marteilia refringens and M. sydneyi) 240
Chapter 3.1.4. Mikrocytosis (Mikrocytos mackini) 246
Chapter 3.1.5. Perkinsosis (Perkinsus marinus and P. olseni/atlanticus) 249
Contents
vi Manual of Diagnostic Tests for Aquatic Animals 2003

Chapter 3.1.6. SSO disease (Haplosporidium costale) 255
Chapter 3.1.7. Withering syndrome of abalone (Candidatus Xenohaliotis californiensis) 260
PART 4 DISEASES OF CRUSTACEANS
Chapter 4.1.1. Taura syndrome 275
Chapter 4.1.2. White spot disease 285
Chapter 4.1.3. Yellowhead disease 298
Chapter 4.1.4. Tetrahedral baculovirosis (Baculovirus penaei) 310
Chapter 4.1.5. Spherical baculovirosis (Penaeus monodon-type baculovirus) 319
Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis 330
Chapter 4.1.7. Crayfish plague (Aphanomyces astaci) 345
Chapter 4.1.8. Spawner-islolated mortality virus disease 350
List of OIE Reference Laboratories and Collaborating Centre for diseases of fish, molluscs
and crustaceans
355
Manual of Diagnostic Tests for Aquatic Animals 2003 vii
I NT RODUCT I ON

The clinical signs in fish with the diseases listed in the OIE Aquatic Animal Health Code (referred to
hereafter as the Aquatic Code) are not pathognomonic. Moreover, these infections may occur as subclinical
infections of asymptomatic pathogen carriers.
The only dependable approach for diagnosis of fish diseases therefore lies in the specific identification of
the pathogens using laboratory methods. These methods, which are suitable for the diagnosis of isolated
cases of disease as part of national aquatic animal health surveillance/control programmes, form the main
contents of this the Manual of Diagnostic Tests for Aquatic Animals (referred to hereafter as the Aquatic
Manual).
Such health surveillance programmes aim to determine, from the results provided by standardised
laboratory procedures performed with samples collected according to defined rules, the health status of
aquatic animal stocks from a particular production site and even a geographical zone or entire country.
The satisfactory implementation of such aquatic animal health surveillance/control programmes, requires
the existence of both adequate legislation and resources in each country interested in aquatic animal health.
The diagnostic methods presented in this Aquatic Manual are all direct diagnostic methods. Due to the
insufficient development of serological methodology, the detection of antibodies to pathogens in fish has
not thus far been accepted as a routine method for assessing the health status of fish populations.
However, the validation of some serological techniques for diagnosis of certain infections could arise in
the near future, rendering the use of serology more widely acceptable for diagnostic purposes. In earlier
editions of the Aquatic Manual, the only diagnostic methods described for screening or diagnosis of fish
diseases have been based either on isolation of the pathogen followed by its specific identification, or on
the demonstration of pathogen-specific antigens using an immunological detection method. However, in
recent years, molecular techniques such as the polymerase chain reaction (PCR), DNA probes and in-situ
hybridisation have been increasingly developed for these purposes. The experiences of the last decade
indicate that the PCR techniques will eventually supersede many of the classical direct methods of
infectious agent detection. It is clear that in many laboratories, the PCR is replacing virus isolation or
bacteria cultivation for the detection of agents that are difficult or impossible to culture. There are several
reasons for this trend, including that virus isolation requires: i) the presence of replicating viruses;
ii) expensive cell culture and maintenance facilities; iii) as long as several weeks to complete the diagnosis;
and iv) special expertise, which is missing or diminishing today in many laboratories. Although PCR assays
were initially expensive and cumbersome to use, they have now become relatively inexpensive, safe and
user-friendly tools in diagnostic laboratories. Where a PCR method has been standardised sufficiently for
its use for a listed fish disease to become widely and reliably available, it has been added to the more
traditional methods in the Aquatic Manual. For the most part, molecular methods for fish diseases are
recommended for either direct detection of the pathogen in clinically diseased fish or for the confirmatory
identification of a disease agent isolated using the traditional method. With one or two exceptions,
molecular techniques are currently not acceptable as screening methods to demonstrate the absence of a
specific disease agent in a fish population for the purpose of health certification in connection with
international trade of live fish and/or their products. There is a need for more validation of molecular
methods for this purpose before they can be recommended in the Aquatic Manual. The principles and
methods of validation and quality control of PCR methods for diagnosis of aquatic animal diseases are
described in Chapter 1.1.3.
General information on diagnostic techniques for fish diseases is given in Chapter I.1. Mollusc and
crustacean diseases differ in some ways from fish diseases. For example, diagnostic methods must be
direct because these animals do not produce antibodies to pathogens. With the unavailability of the
traditional pathogen isolation methods used for fish diseases, molecular techniques, particularly PCR, have
Introduction
viii Manual of Diagnostic Tests for Aquatic Animals 2003
increasingly supplemented the more traditional histological and tissue smear methods described in the
Aquatic Manual for the listed mollusc and crustacean diseases, not only for diagnosis of clinical cases but
also for screening programmes to demonstrate the absence of the specific disease agent for health
certification purposes. General information on diagnostic techniques for mollusc diseases is given in
Chapter I.2. and for crustacean diseases in Chapter I.3.

Manual of Diagnostic Tests for Aquatic Animals 2003 ix
ACKNOWL E DGE ME NT S

AUTHORS AND PROFESSIONAL ADDRESS AT THE TIME OF WRITING
Dr D. Alderman, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth
Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom
Dr I. Anderson, Oonoomba Veterinary Laboratory, Queensland Department of Primary Industries, P.O.
Box 1085, Oonoomba QLD 4810, Australia
Dr T. Atle Mo, National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norway
Dr S. Belak, Department of Virology, Research and Development Section, The National Veterinary
Institute, Biomedical Center, Box 585, SE-751 23 Uppsala, Sweden
Dr F. Berthe, IFREMER, Laboratoire de pathologie des invertbrs, BP 133, 17390 La Tremblade, France
Dr S. Bower, Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo,
British Columbia V9R 5K6, Canada
Dr A. Cameron, AusVet Animal Health Services, 140 Falls Road, Wentworth Falls, NSW 2782, Australia
Dr C. Cunningham, FRS Marine Laboratory, FRS Marine Laboratory, P.O. Boc 101, Victoria Road,
Aberdeen AB11 9BD, Scotland, United Kingdom
Prof. Shiu-Nan Chen, National Taiwan University, Department of Zoology and Fishery Research, No. 1
Roosevelt Road, Section 4, Taipei, Taiwan 10764, Taipei China.
Dr B. Dannevig, National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norway
Dr I. East, Aquatic Animal Health Unit, Office of the Chief Veterinary Officer, Agriculture, Fisheries and
Forestry Australia, GPO Box 858, Canberra ACT 2601, Australia
Dr B. Edgerton, Animal Biosecurity Policy, Agriculture, Fisheries and Forestry Australia,
GPO Box 858, Canberra ACT 2601, Australia
Dr K. Falk, National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norway
Dr T.W. Flegel, Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road,
Bangkok 10400, Thailand
Dr C. Friedman, Assistant Professor, Aquatic and Fishery Sciences, University of Washington, P.O. Box
355020, Seattle, WA 98195-5020, United States of America
Prof. J. Fryer, Oregon State University, Corvallis, Oregon 97331-3804, United States of America
Dr H. Grizel, IFREMER, 1 rue Jean Vilar, 34200 Ste, France
Acknowledgements
x Manual of Diagnostic Tests for Aquatic Animals 2003
Dr L.A. Hanson, Fish Diagnostic Laboratory, College of Veterinary Medicine, Mississippi State University,
Box 9825, Spring Street, Mississippi 39762, United States of America
Prof. T. Hstein, National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norway
Prof. R. Hedrick, Department of Medicine & Epidemiology, School of Veterinary Medicine, University of
California, One Shields Ave, Davis, California 95616, United States of America
Prof. B.J. Hill, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth
Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, United Kingdom
Dr P.M. Hine, National Institute of Water, and Atmospheric Research, P.O. Box 14-901, Kilbirnie,
Wellington, New Zealand
Dr M. House, Oregon State University, Corvallis, Oregon 97331-3804, United States of America
Dr A. Hyatt, Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia
Dr J. Jarp, National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156 Dep., 0033 Oslo, Norway
Dr R. Jacobson, Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY
14850-5786, United States of America
Dr N. Jrgen-Olesen, Danish Veterinary Laboratory, Hangovej 2, 8200 Aarhus N, Denmark
Dr M. Kent, Center for Fish Disease Research, Department of Microbiology, 220 Nash Hall, Oregon State
University, Corvallis, OR 97311-3804, United States of America
Dr P. de Kinkelin, Unit de Virologie et Immunologie molculaires, Centre de Recherche de Jouy-en-
Josas, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas Cedex, France
Prof. D.V. Lightner, Aquaculture Pathology Laboratory, Department of Veterinary Science, University of
Arizona, Building 90, Room 202, Pharmacy/Microbiology, Tucson, AZ 85721, Tucson, AZ 85721, United
States of America
Dr C. Michel, Unit de Virologie et Immunologie molculaires, Centre de Recherche de Jouy-en-Josas,
Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas Cedex, France
Dr B. Munday (deceased), University of Tasmania, School of Science and Technology, P.O. Box 1214,
Launceston, Tasmania 7250, Australia
Dr T. Nakai, Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima 724, Japan
Dr K. Nakajima, National Research Institute of Agriculture, 422-1 Nakatsuhamaura, Nansei-cho,
Watarai-gun, Mie 516-01, Japan
Dr L. Owens, Department of Microbiology and Immunology, James Cook University,
Townsville, QLD 4811, Australia
Dr E. Peeler, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth
Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, United Kingdom
Dr R. Subasinghe, Food and Agriculture Organization of the United Nations, Fisheries Department,
Room NF 514, Viale della Terme di Caracalla, 00100 Rome, Italy
Dr P. Thorn, Department of Virology, Research and Development Section, The National Veterinary
Institute, Biomedical Center, Box 585, SE-751 23 Uppsala, Sweden
Acknowledgements
Manual of Diagnostic Tests for Aquatic Animals 2003 xi
Dr K. Tonguthai, Aquatic Animal Health Research Institute (AAHRI), Department of Fisheries, Kaesarts
University Campus, Jatujak, Bangkok 10900, Thailand
Dr P.J. Walker, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road,
St Lucia, QLD 4067, Australia
Dr R. Whittington, Elizabeth Macarthur Agricultural Institute, PMB 8, Camden NSW 2570, Australia
Dr A. Wiegers, USDA, APHIS, National Veterinary Services Laboratories, P.O. Box 844, Ames Iowa
50010, United States of America
Dr J. Winton, Western Fisheries Research Center, 6505 N.E. 65th St., Seattle, Washington 98115,
United States of America
Dr M. Yoshimizu, Microbiology Department, Faculty of Fisheries, Hokkaido University, 3-1-1
Minato-cho, Hakodate, Hokkaido 041-0821, Japan
*
* *
Manual of Diagnostic Tests for Aquatic Animals 2003 xiii
ABBRE VI AT I ONS

Ab antibody
ABTS 2,2-Azino-di-(3-ethyl-benzthiazoline)-
6-sulphonic acid
AEC aminoethyl carbazole
Ag antigen
AO acridine orange
AS Atlantic salmon (cell line)
ASK Atlantic salmon kidney (cell line)
BCIP 5-bromo-4-chloro-3-indoyl phosphate
BF-2 bluegill fry (cell line)
BKD bacterial kidney disease
BP Baculovirus penaei
BSA bovine serum albumin
BSS balanced salt solution
CCO channel catfish ovary (cell line)
CCV(D) channel catfish virus (disease)
CFA complete Freunds adjuvant
CHSE-214 chinook salmon embryo (cell line)
CIA Cowdry type A inclusion bodies
CMS cardiomyopathic syndrome
CPE cytopathic effect
CSHV coho salmon herpesvirus
CSTV coho salmon tumour virus
DEPC diethyl pyrocarbonate
DIG digoxigenin
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
dNTP deoxynucleotide triphosphate
DTT dithiotreitol
ECV European catfish virus
EDTA ethylene diamine tetra-acetic acid
EHN(V) epizootic haematopoietic necrosis
(virus)
ELISA enzyme-linked immunosorbent assay
EPC Epithelioma papulosum cyprini (cell line)
ERA EUS-related Aphanomyces
ESC enteric septicaemia of catfish
ESV European sheatfish virus
EUS epizootic ulcerative syndrome
FAT fluorescent antibody test
FBS fetal bovine serum
FCS fetal calf serum
FEV fish encephalitis virus
FITC fluorescein isothiocyanate
GAV gill-associated virus
GP glucose peptone (broth)
GPY glucose peptone yeast (broth)
H&E hematoxylin and eosin
HBSS Hanks balanced salt solution
HEPES N-2-hydroxyethyl-piperazine-N-2-
ethanesulfonic acid
HP hepatopancreas
HRPO horseradish peroxidase
IF immunofluorescence
IFAT indirect fluorescent antibody test
Ig immunoglobulin
IHHNV infectious hypodermal and
haematopoietic necrosis virus
IHN(V) infectious haematopoietic necrosis
(virus)
IPN(V) infectious pancreatic necrosis (virus)
ISA infectious salmon anaemia
ITS internal transcribed spacer
KDM-2 kidney disease medium
LOS lymphoid organ spheroids
LOV lymphoid organ virus
LPS lipopolysaccharide
MAb monoclonal antibody
MBV Penaeus monodon-type baculovirus
MCMS mid-crop mortality syndrome
MEM minimal essential medium
m.o.i. multiplicity of infection
M-MLV Moloney murine leukaemia virus
MSX multinucleate sphere X
NAb neutralising antibody
NBT nitroblue tetrazolium
OCT embedding medium for frozen tissue
specimens
OKV Oncorhynchus kisutch virus
OMV(D) Oncorhynchus masou virus (disease)
OPD o-phenylenediamine
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PBST phosphate buffered saline containing
Tween
PCR polymerase chain reaction
PFU plaque forming units
PIB polyhedral inclusion body
POB polyhedral occlusion body
RDS runt deformity syndrome
RFLP restriction fragment length
polymorphism
RHV rainbow trout herpesvirus
RKV rainbow trout kidney virus
Abbreviations
xiv Diagnostic Manual for Aquatic Animal Diseases 2003
RNA ribonucleic acid
RSD red spot disease
RSIV(D) red sea bream iridoviral (disease)
RTG-2 rainbow trout gonad (cell line)
RT-PCR reverse-transcription polymerase
chain reaction
RVC ribonucleoside vanadyl complex
SBL seabass larva (cell line)
SDS sodium dodecyl sulfate
SHK-1 salmon head kidney (cell line)
SJNNV striped jack nervous necrosis virus
SKDM selective kidney disease medium
SM(V) spawner-isolated mortality (virus)
SPF specific pathogen free
SSC standard saline citrate
SSO seaside organism
SSS sonicated salmon sperm
SVC(V) spring viraemia of carp (virus)
TEM transmission electron microscopy
TMB tetramethylbenzidine
TRITC tetramethylrhodamine-5-(and-6-)
isothiocyanate
Tris Tris (hydroxymethyl) aminomethane
TS(V) Taura syndrome (virus)
VER viral encephalopathy and retinopathy
VHS(V) viral haemorrhagic septicemia (virus)
VN virus neutralisation
VNN(V) viral nervous necrosis (virus)
WSBV white spot disease baculovirus
WSD white spot disease
WSIV(D) White sturgeon iridoviral (disease)
WSSV white spot syndrome virus
WSV white spot virus
YHD yellowhead disease
YHV yellowhead virus
YTV yamame tumour virus

Manual of Diagnostic Tests for Aquatic Animals 2003 xv
DE F I NI T I ONS 1
2
The Aquatic Animal Health Code (companion volume to this Aquatic Manual) contains a list of definitions 3
that may be consulted for the meaning of terms used in this Aquatic Manual. Some terms that are not used 4
in the Aquatic Code but that appear in the Aquatic Manual, are defined below: 5
Confidence In the context of demonstrating freedom from infection (in which the null 6
hypothesis is that infection is present), the confidence is the probability that a 7
surveillance system or combination of surveillance systems would detect the presence 8
of infection if the population were infected. The confidence depends on the 9
design prevalence, or the assumed level of infection in an infected population. 10
Confidence therefore refers to our confidence in the ability of a surveillance 11
system to detect disease, and is equal to the sensitivity of the system. This is 12
distinct from (but may be used to calculate) the probability that a given 13
population is free from infection, based on the results of one or more surveillance 14
systems. 15
Early detection system A system for the timely detection and identification of the incursion or 16
emergence of infection or disease in a country, zone or aquaculture 17
establishment. An early detection system must be under the control of the 18
Competent Authorities and must include the following characteristics: 19
a) representative coverage of target animal populations by field services; 20
b) ability to undertake effective disease investigation and reporting; 21
c) access to laboratories capable of diagnosing and differentiating relevant 22
diseases; 23
d) a training programme for veterinarians or fish health specialists for 24
detecting and reporting unusual disease occurrence. 25
Epidemiological unit A group of animals that share approximately the same risk of exposure to a 26
disease agent with a defined location. This may be because they share a 27
common aquatic environment (e.g. fish in a pond, caged fish in a lake), or 28
because management practices make it likely that a disease agent in one group 29
of animals would quickly spread to other animals (e.g. all the ponds on a farm, 30
all the ponds in a village system). 31
Fry Newly hatched fish larvae. 32
Population A group of units sharing a common defined characteristic. 33
Prescribed biosecurity A set of conditions applying to a particular disease or infection, and a particular 34
conditions zone or country, required to ensure adequate biosecurity, namely: 35
a) the disease is legally notifiable to the Competent Authority; 36
b) an early detection system is in place within the zone or country; 37
c) no vaccination against the disease is carried out; 38
Definitions
xvi Manual of Diagnostic Tests for Aquatic Animals 2003
d) infection is not known to be established in wild populations; 39
e) import requirements to prevent the introduction of disease or infection 40
into the country or zone, as outlined in the Aquatic Code, are in place. 41
Probability sampling A sampling strategy in which every unit has a known non-zero probability of 42
inclusion in the sample. 43
Sensitivity the proportion of true positive tests given in a diagnostic test, i.e. the number of 44
true positive results divided by the number of true positive and false negative 45
results. 46
Specificity the probability that absence of infection will be correctly identified by a 47
diagnostic test, i.e. the number of true negative results divided by the number of 48
true negative and false positive results. 49
Study population The population from which evidence of freedom from infection is derived. This 50
may be the same as the target population or a subset of it. 51
Surveillance system A method of surveillance that generates a source of information on the animal 52
health status of populations. 53
Target population For the purposes of demonstrating freedom from infection, the population of 54
interest, usually made up of all aquatic animals of species susceptible to a 55
specified disease agent in a defined country, zone or aquaculture establishment 56
Targeted surveillance Surveillance targeted at a specific disease or infection. 57
Test A procedure used to classify a unit as either positive or negative with respect to 58
an infection or disease. Tests may be classified as: 59
a) diagnostic, when applied to clinically diseased individuals; 60
b) screening, when applied to apparently healthy individuals; or 61
c) confirmatory, when applied to confirm the result of a previous test. 62
Test system A combination of multiple tests and rules of interpretation that are used for the 63
same purpose as a test. 64
Units Individually identifiable elements. This is a generic concept used to describe, for 65
example, the members of a population, or the elements selected when sampling. 66
In these contexts, examples of units include individual animals, to ponds, nets, 67
cages, farms, villages, districts, etc. 68
69
Manual of Diagnostic Tests for Aquatic Animals 2003 9
70
C HA P T E R 1 . 1 . 1 .

QUAL I T Y MANAGE ME NT I N VE T E RI NARY
T E S T I NG L ABORAT ORI E S

SUMMARY
Valid laboratory results are essential for diagnosis, surveillance, and trade. Such results may be achieved by
the use of good management practices, valid test and calibration methods, proper technique, quality control,
and quality assurance, all working together within a quality system. These subjects comprise one complex
area of critical importance in the conduct of testing and in the interpretation of test results. This subject may
be called laboratory quality management, and includes managerial, operational, and technical elements. A
quality management programme should enable the laboratory to demonstrate that it operates a viable quality
system, is technically competent, and is able to generate technically valid results. Additionally, a laboratory
should implement a quality management programme that is appropriate for its mandate, clients, needs, and
goals, and that can be shown to be effective in meeting quality objectives. The need for the mutual recognition
of test results for international trade and the acceptance of international standards such as the ISO/IEC
1

International Standard 17025 (3) for laboratory accreditation also affect the need and requirements for
laboratory quality management programmes. The OIE has published detailed standard on this subject (5).
This chapter is not intended to reiterate the requirements of these ISO or OIE documents. Rather, it
outlines the important issues and considerations a laboratory should address in the design and maintenance
of its quality management programme.
KEY CONSIDERATIONS FOR THE DESIGN AND MAINTENANCE OF A LABORATORY QUALITY
MANAGEMENT PROGRAMME
In order to ensure that the quality management programme is appropriate and effective, the design must
be carefully thought out. The major categories of consideration and the key issues and activities within
each of these categories are outlined in the following seven sections of this chapter.
1. THE WORK, RESPONSIBILITIES, AND GOALS OF THE LABORATORY
Many factors affect the necessary elements and requirements of a quality management programme.
These factors include:
i) The type of testing done;
ii) The use of the test results;
iii) The impact of a questionable or erroneous result;
iv) The tolerance level of risk and liability;
v) Client needs (e.g. sensitivity and specificity of the test method, costs, turnaround time);
vi) The role of the laboratory in legal work or in regulatory programmes;
vii) The role of the laboratory in assisting with, confirming, and/or overseeing the work of other
laboratories; and

1 International Organization for Standardization/International Electrochemical Commission.
Chapter 1.1.1. - Quality management in veterinary testing laboratories
4 Manual of Diagnostic Tests for Aquatic Animals 2003
viii) The business goals of the laboratory, including the need for any third party recognition and/or
accreditation.
2. STANDARDS, GUIDES AND REFERENCES
It is recommended that the laboratory choose reputable and accepted standards and guides to assist in
designing the quality management programme. The OIE standard on this subject is a useful guideline
(5). For laboratories seeking accreditation, the use of ISO/IEC 17025 (3) or the OIE standard (5) will
be essential. Further information on standards may be obtained from the national standards body of
each country, from the International Laboratory Accreditation Cooperation (ILAC), and from
accreditation bodies (e.g. the National Association of Testing Authorities, Australia; the American
Association of Laboratory Accreditation, United States of America; and the Standards Council of
Canada, Canada). Technical and international organisations such as the AOAC International (formerly
the Association of Official Analytical Chemists) and the ISO publish useful references, guides, and/or
standards that supplement the general requirements of ISO/IEC 17025. ISO International Standard
9001 (4), a general standard for quality management systems and one of the many standards in the
group commonly termed the ISO 9000 series, is not usable for accreditation, as conformity with its
requirements does not necessarily ensure or imply technical competence (see Section 3. below). While a
laboratory may implement a quality management system meeting the requirements of ISO 9001,
registration or certification is used to indicate conformity with this standard. ISO/IEC 17025 contains
a tabular comparison by clause numbers of itself, ISO 9001 (1994), and ISO 9002 (1994). Both of these
latter documents have been replaced by the 2000 version of 9001 (4).
3. ACCREDITATION
If the laboratory has determined that it needs formal recognition of its quality management
programme, then third party verification of its conformity with the selected standard(s) will be
necessary. ILAC has published specific requirements and guides for laboratories and accreditation
bodies. The terms accreditation, a procedure by which an authoritative body gives formal recognition
that a body or person is competent to carry out specific tasks (1), and laboratory accreditation, the
formal recognition of the competence of a laboratory to carry out specific tests or specific types of
tests (1), are specific to ILAC requirements and to the use of ISO/IEC 17025 or the OIE standard as
the basis for the accreditation. The use of competence is significant: it means much more than having and
following documented procedures. Having competence also means that the laboratory:
i) Has technically valid and validated test methods, procedures, and specifications that are
documented in accordance with the requirements of the selected standard(s) and/or guidelines;
ii) Has adequate qualified personnel who understand the science behind the procedures;
iii) Has correct and adequate equipment;
iv) Has adequate facilities and environmental control;
v) Has procedures and specifications that ensure accurate and reliable results;
vi) Can foresee technical needs and problems;
vii) Can cope with and prevent technical problems that may arise;
viii) Can accurately calculate and control the uncertainty in testing; and
ix) Can demonstrate proficiency to conduct the test methods used.
4. SELECTION OF AN ACCREDITATION BODY
In order for accreditation to facilitate the acceptance of the laboratorys test results for trade, the
accreditation must be recognised by the international community. Therefore, the accreditation body
should be recognised as competent to accredit laboratories. Programmes for the recognition of
accreditation bodies are, in the ILAC scheme, based on the requirements of ISO/IEC Guide 58 (2).
One may obtain information on recognised accreditation bodies from the organisations that recognise
them, such as the National Cooperation for Laboratory Accreditation (NACLA), the Asia-Pacific
Laboratory Accreditation Cooperation (APLAC), the Interamerican Accreditation Cooperation
(IAAC), and the European Co-operation for Accreditation (EA).
5. DETERMINATION OF THE SCOPE OF THE QUALITY MANAGEMENT PROGRAMME AND/OR OF THE LABORATORYS
ACCREDITATION
The quality management programme should ideally cover all areas of activity affecting all testing that is
done at the laboratory. However, for the purpose of accreditation, the laboratory should determine the
scope of testing to be included in the accreditation. Factors that might affect the laboratorys choice of
scope of accreditation include:
i) The availability and cost of necessary personnel, facilities and equipment;
ii) The cost of environmental monitoring against the possibility of cross contamination;
iii) The deadline for accreditation;
iv) The impact of the test results;
v) The number of tests done;
vi) Whether the testing done is routine or non-routine;
vii) Whether any part of testing is subcontracted out;
viii) The quality assurance necessary for materials, reagents and media;
ix) The availability of reference standards (e.g. standardised reagents, internal quality control
samples, reference cultures);
x) The availability of proficiency testing;
xi) The availability, from reputable sources, of standard and/or fully validated test methods;
xii) The evaluation and validation of test methods to be done; and
xiii) The technical complexity of the method (s).
Accreditation bodies also accredit the providers and operators of proficiency testing programmes, and
may require the use of an accredited provider in order to issue the laboratory a certificate of
accreditation.
6. TEST METHODS
ISO/IEC 17025 requires the use of appropriate test methods and has requirements for selection,
development, and validation. The OIE document (5) also provides requirements for selection and
validation.
In the veterinary profession, other standard (methods published in international, regional, or national
standards) or fully validated methods (methods having undergone a full collaborative study and that are
published or issued by an authoritative technical body such as the AOAC International), while
preferable to use, may not be available. Many veterinary laboratories develop or modify methods, and
most of these laboratories have test programmes that use non-standard methods, or a combination of
standard and non-standard methods. In veterinary laboratories, even with the use of standard methods,
some in-house evaluation, optimisation, and/or validation generally must be done to ensure valid
results.
Clients and laboratory staff must have a clear understanding of what performance can be expected
from a test. Many veterinary testing laboratories will therefore need to demonstrate competence in the
development, adaptation, and validation of test methods.
This Aquatic Manual provides more detailed and specific guidance on test selection, optimisation,
standardisation, and validation in Chapter 1.1.2.
The following items discuss test method issues that are of most interest to those involved in the quality
management of the laboratory.
a) Selection of the test method
Valid results begin with the selection of an appropriate test method for the diagnostic issues at
hand. Considerations for the selection of a test method include:
i) International acceptance;
ii) Scientific acceptance;
iii) Method not outdated;
iv) Performance characteristics (e.g. analytical and diagnostic sensitivity and specificity, isolation
rate, precision [repeatability, reproducibility, replicability], accuracy, and uncertainty);
v) Behaviour in species and population of interest;
vi) Money and time available for development, adaptation, and/or evaluation;
vii) Performance time and turnaround time;
viii) Type of sample (e.g. serum, tissue) and its expected quality or state on arrival at the
laboratory;
ix) Analyte (e.g. antibody, antigen);
x) Resources and technology of the laboratory;
xi) Nature of the intended use (e.g. export, import, surveillance, screening, confirmatory,
individual animal use);
xii) Client expectations;
xiii) Safety factors;
xiv) Number of tests to be done;
xv) Cost of test, per sample;
xvi) Existence of reference standards, including reference materials; and
xvi) Availability of proficiency testing schemes.
b) Optimisation and standardisation of the test method
Once the method has been selected, it must be set up at the laboratory. Whether the method was
developed in-house or imported from an outside source, generally some additional optimisation is
necessary. Optimisation is a series of experiments and subsequent data analysis. Optimisation
establishes critical specifications and performance standards for the test process and for use in
monitoring the correct performance of the test. Optimisation should ensure that a method is
brought under statistical control. Optimisation should also determine:
i) Critical specifications for equipment and instruments;
ii) Critical specifications for reagents (e.g. chemicals, biologicals);
iii) Critical specification for reference standards, reference materials, and internal controls;
iv) Ruggedness (if applicable);
v) Critical control points and acceptable ranges, attributes or behaviour at critical control points,
using statistically acceptable procedures;
vi) The quality control activities necessary to monitor critical control points;
vii) The type, number, range, frequency, and/or arrangement of test run controls needed;
viii) The requirements for control behaviour for the non-subjective acceptance or rejection of test
results;
ix) The elements of a fixed, documented test method for use by laboratory staff; and
x) The level of technical competence required of those who carry out and/or interpret the test.
c) Validation of the test method
Validation further evaluates the test for its fitness for a given use. Validation establishes
performance characteristics for the test method, such as sensitivity, specificity, and isolation rate;
and diagnostic parameters such as positive/negative cut-off, and titre of interest or significance.
Validation should be done using an optimised, documented, and fixed procedure. Depending on
logistical and risk factors, validation may involve any number of activities and amount of data,
with subsequent data analysis using appropriate statistics. Validation activities might include:
i) Field and/or epidemiological studies;
ii) Comparison with other methods, preferably standard methods;
iii) Comparison with reference standards (if available);
iv) Collaborative studies with other laboratories using the same documented method, and
including the exchange of samples, preferably of undisclosed composition or titre. It is
preferable that these be issued by a qualified conducting laboratory that organises the study
and evaluates the results provided by the participants;
v) Reproduction of data from an accepted standard method, or from a reputable publication;
vi) Experimental challenge studies; and
vii) Analysis of internal quality control data.
ISO/IEC 17025 recognises that Validation is always a balance between costs, risks, and technical
possibilities. It also recognises that there are many cases in which quantities such as accuracy and
precision can only be given in a simplified way.
d) Uncertainty
Laboratories should be able to estimate the uncertainty of the test methods as performed in the
laboratory. This includes methods used by the laboratory to calibrate equipment (3). ISO/IEC
17025 requirements concerning this topic are stated in sections 5.1.2, 5.4.4k, 5.4.6.2, 5.4.6.3 and
5.10.3.1c of that document.
The determination of measurement uncertainty (MU) is not new to metrology. However, the
application of the principles of MU to the life sciences is relatively new, much of the work has
been theoretical, and valid approaches are being developed. It is important to note that MU does
not imply doubt about the validity of a test result or other measurement, nor is it equivalent to
error, as it may be applied to all test results derived from a particular procedure. It may be viewed
as a quantitative expression of reliability, and is commonly expressed as a number after a +/ sign
(i.e. the true value lies within the stated range, as MU is expressed as a range). Standard deviation
and confidence interval are examples of the expression of MU. An example of the use of standard
deviation to express uncertainty is the allowed limits on the test run controls for an enzyme-linked
immunosorbent assay, commonly expressed as +/ n SD.
Although the determination and expression of MU has not been standardised for veterinary
testing laboratories, some sound guidance exists.
Many chemical and microbiological test methods are published with stated values for MU.
Government agencies may also set MU values for official methods (e.g. Health Canada).
Reputable technical organisations (e.g. the AOAC International) and accreditation bodies teach
courses on MU for laboratories seeking accreditation. Many international organisations produce
standards and guidance on this subject (e.g. ISO, Co-Operation on International Traceability in
Analytical Chemistry [CITAC], and Eurachem). Codex Alimentarius, which specifies standards for
food testing, has taken the approach that it is not necessary for a laboratory to take a further
estimate of MU if the laboratory complies with Codex principles regarding quality: i.e. that the
laboratory is accredited to ISO/IEC 17025, uses validated methods (e.g. knows applicable
parameters such as sensitivity and specificity, as well as the confidence interval around such
parameters), participates in proficiency testing programmes and collaborative studies, and uses
appropriate internal quality control procedures.
The use of appropriate internal quality control procedures implies that the laboratory must
identify all sources of uncertainty, establish acceptable specifications, criteria, and/or ranges at
critical control points; and implement appropriate quality control at the critical control points
associated with each source. Sources of uncertainty include sampling, storage conditions, sample
effects, extraction and recovery, reagent quality, volumetric manipulations, environmental
conditions, contamination, equipment effects, analyst or operator bias, and random effects. The
laboratory would also be expected to correct for any known systematic error (see also Section 6.b.
steps ivii).
e) Implementation and use of the test method
Analysts should be able to demonstrate proficiency in using the test method prior to producing
reported results, and on an ongoing basis.
The laboratory should ensure, using appropriate and documented project management, records
keeping, data management, and archiving procedures, that it can recreate at need all events relating
to test selection, development, optimisation, standardisation, validation, implementation, and use.
This includes quality control and quality assurance activities.
7. STRATEGIC PLANNING
Continuous improvement is essential. It is recommended that the laboratory be knowledgeable of and
stay current with the standards and methods used to demonstrate laboratory competence and to
establish and maintain technical validity. The methods by which this may be accomplished include:
i) Attendance at conferences;
ii) Participation in local and international organisations;
iii) Participation in writing national and international standards (e.g. participation on ILAC and ISO
committees);
iv) Consulting publications;
v) Visits to other laboratories;
vi) Participation in cooperative programmes;
vii) Exchange of procedures, methods, reagents, samples, personnel, and ideas; and
viii) Wherever possible, accreditation by a third party that is itself recognised as competent to issue the
accreditation.
REFERENCES
1. ISO/IEC GUIDE 2 (1996). Standardisation and Related Activities General Vocabulary.
International Organisation for Standardisation (ISO), ISO Central Secretariat, 1 rue de Varemb,
Case Postale 56, CH - 1211, Geneva 20, Switzerland.
2. ISO/IEC GUIDE 58 (1993). Calibration and Testing Laboratory Accreditation Systems General
Requirements for Operation and Recognition, op. cit.
3. ISO/IEC INTERNATIONAL STANDARD 17025 (1999). General Requirements for the Competence of
Testing and Calibration Laboratories. International Organisation for Standardisation (ISO), ISO
Central Secretariat, 1 rue de Varemb, Case Postale 56, CH - 1211, Geneva 20, Switzerland.
4. ISO INTERNATIONAL STANDARD 9001 (2000). Quality management systems Requirements.
International Organization for Standardization (ISO), ISO Central Secretariat, 1 rue de Varemb,
Case Postale 56, CH - 1211, Geneva 20, Switzerland.
5. OFFICE INTERNATIONAL DES EPIZOOTIES (2000). Standard for Management and Technical
Requirements for Laboratories Conducting Tests for Infectious Animal Diseases. Office
International des Epizooties, 12 rue de Prony, 75017 Paris, France.
*
* *
C HA P T E R 1 . 1 . 2 .

P RI NCI P L E S OF VAL I DAT I ON OF DI AGNOS T I C
AS S AYS F OR I NF E CT I OUS DI S E AS E S

SUMMARY
Validation is the evaluation of a process to determine its fitness for a particular use. A validated assay
yields test results that identify the presence of a particular analyte (e.g. an antibody) and allows predictions
to be made about the status of the test subjects. Assays applied to individuals or populations have many
purposes, such as aiding in: documenting freedom from disease in a country or region, preventing spread of
disease through trade, eradicating an infection from a region or country, confirming diagnosis of clinical
cases, estimating infection prevalence to facilitate risk analysis, identifying infected animals toward
implementation of control measures, and classifying animals for herd health or immune status post-
vaccination. A single assay may be validated for one or several intended purposes by optimising its
performance characteristics for each purpose (e.g. setting diagnostic sensitivity [D-SN] high [such as
99.99%] with associated lower diagnostic specificity [D-SP] for a screening assay, or conversely, setting D-
SP high with associated lower D-SN for a confirmatory assay).
By considering the variables that affect an assays performance, the criteria that must be addressed in assay
validation become clearer. The variables can be grouped into three categories: (a) the sample
host/organism interactions affecting the analyte composition and concentration in the serum sample; (b) the
assay system physical, chemical, biological and technician-related factors affecting the capacity of the assay
to detect a specific analyte in the sample; and (c) the test result the capacity of a test result, derived from
the assay system, to predict accurately the status of the individual or population relative to the analyte in
question.
Factors that affect the concentration and composition of analyte in the serum sample are mainly attributable
to the host and are either inherent (e.g. age, sex, breed, nutritional status, pregnancy, immunological
responsiveness) or acquired (e.g. passively acquired antibody, active immunity elicited by vaccination or
infection). Nonhost factors, such as contamination or deterioration of the sample, may also affect the analyte
in the sample.
The principles of validation discussed in this chapter will focus primarily on methods to detect antibody in
sera. However, these same principles could be applied to validation of tests for other analytes in sera or
tissues. Chapter 1.1.3. Validation and quality control of polymerase chain reaction methods used for the
diagnosis of infectious diseases extends the principles outlined here to a direct method of infectious agent
detection, the molecular diagnostic assays.
Factors that interfere with the analytical accuracy of the assay system include instrumentation, technician
error, reagent choice (both chemical and biological) and calibration, accuracy and acceptance limits of
controls, reaction vessels, water quality, pH and ionicity of buffers and diluents, incubation temperatures
and durations, and error introduced by detection of closely related analytes, such as antibody to cross-reactive
organisms, rheumatoid factor, or heterophile antibody.
Factors that influence the capacity of the test result to predict accurately the infection or analyte status of the
host
1
are D-SN, D-SP, and prevalence of the disease in the population targeted by the assay. D-SN and
D-SP are derived from test results on samples obtained from selected reference animals. The methods used to

1 In this chapter, the terms positive and negative have been reserved for test results and never refer to infection or antibody/antigen status
of the host. Whenever reference is made to infection or analyte, any method of exposure to an infectious agent that could be detected
directly (e.g. antigen) or indirectly (e.g. antibody) by an assay, should be inferred.
Chapter 1.1.2. - Principles of validation of diagnostic assays for infectious diseases
select the reference animals are critical to the accuracy of the estimates (4). The degree to which the reference
animals represent all of the host and environmental variables in the population targeted by the assay has a
major impact on the accuracy of test-result interpretation. For example, experienced diagnosticians are
aware that an assay, validated by using samples from northern European cattle, may not give valid results
for the distinctive populations of cattle in Africa.
The capacity of a positive or negative test result to predict accurately the infection status of the animal or
population of animals is the most important consideration of assay validation. This capacity is not only
dependent on a highly precise and accurate assay and carefully derived estimates of D-SN and D-SP, but is
heavily influenced by prevalence of the infection in the targeted population or the likelihood that an animal
is infected based on clinical criteria. Without a current estimate of the disease prevalence in that population
or likelihood of infection in an individual animal, the interpretation of a positive or negative test result may
be compromised.
Many variables obviously must be addressed before an assay can be considered to be validated (4, 11).
However, there is no consensus whether the concept of assay validation is a time-limited process during which
only those factors intrinsic to the assay are optimised and standardised, or whether the concept includes an
ongoing validation of assay performance for as long as the assay is used. Accordingly, the term validated
assay elicits various interpretations among laboratory diagnosticians and veterinary clinicians. Therefore, a
working definition of assay validation is offered as a context for the guidelines outlined below. Ideally, all
diagnostic assays would be fully validated for one or more purposes, but in practice there are sometimes
limitations to the completeness of validation.
A. DEFINITION OF ASSAY VALIDATION
A validated assay consistently provides test results that identify animals as positive or negative for an
analyte or process (e.g. antibody, antigen, or induration at skin test site) and, by inference, accurately
predicts the infection status of animals with a predetermined degree of statistical certainty. This chapter
will focus on the principles underlying development and maintenance of a validated assay. Guidelines for
the initial stages in assay development are included because they constitute part of the validation process.
How this early process is carried out heavily influences the capacity of the eventual test result to provide
diagnostic accuracy.
It must be emphasised that an assay, when applied to target populations, will minimise misclassifications
of animals as false positive or false negative only to the extent that validity is assured for all stages of the
assay validation process. This assumes that a well designed and documented test method and proper
reagents, in combination with well trained technicians, will give a stable assay within the laboratory. It also
assumes a thorough use of the most rigorous experimental design and epidemiological and statistical tools.
These are required to reduce bias, random error, and false assumptions about the reference population of
animals upon which the assay performance estimates are made (4). Furthermore, it assumes that the assay
is fitted to the purpose for which it is intended (e.g. a confirmatory assay will likely yield many false-
positive results if used as a screening assay).
The working definition of assay validation used in this paper generalises a process that takes on many
different forms depending on the intended use of the assay being developed. No matter what the form of
the assay, without scientific rigour in the validation process, the assay will not perform to expectations.
However, there are many traditional techniques in widespread use that have not been subjected to the full
validation process.
B. STAGES OF ASSAY VALIDATION
Development and validation of an assay is an incremental process consisting of at least five stages:
1) Determination of the feasibility of the method for a particular use; 2) Choice, optimisation, and
standardisation of reagents, techniques and methods; 3) Determination of the assays performance
characteristics; 4) Continuous monitoring of assay performance; and 5) Maintenance and enhancement of
validation criteria during routine use of the assay (Figure 1). Although some scientists may question the
relevance of the fourth and fifth stages as validation criteria, they are included here because an assay can be
considered to be valid only to the extent that test results are valid, i.e. whether they fall within statistically
defined limits and provide accurate inferences. In practice, there are many traditional techniques in
widespread use that have not been through the formal validation process given above. Efforts should be
made to formally validate the assays performance characteristics, either through use of historical data, or
in a prospective study. This will lend credence to the belief that the assay is accomplishing what it purports
to do. In such cases the process may appropriately be entered at stages 4 and 5, thus building up a data set
of validation data for assays that lack the necessary information for stages 13.
It is of utmost importance not to stop after the first two stages of assay validation that does not
constitute a validated assay for diagnostic use. Although reagent and protocol refinement are important,
the selection of the reference populations is probably the most critical factor. It is no surprise when
reviewing the literature to find a wide range of estimates for diagnostic sensitivity (D-SN) and diagnostic
specificity (D-SP) for the same basic assay. Although part of the variation may be attributed to the
reagents chosen, it is likely that the variation in estimates of D-SN and D-SP is due to biased selection of
sera on which the test was validated. This stage in assay validation needs more attention than it has been
given previously. This is particularly true in the current atmosphere of international trade agreements and
all their implications with regard to movement of animals and animal products.
Select method
Select serum controls Select serum standards
Feasibility
studies
Optimise and
standardise
reagents and
protocols
Test
samples
from
reference
animals
Sera from known
infected and uninfected
reference animals
Serum standards
of known status
Normalise
data
Calculate
D-SP and D-SN
Normalised
test results
from routine
assay use
Categorical
(Pos/Neg)
test data
Calculate PV+and PV- for valid
interpretation of test results
Monitor precision
and accuracy
Establish
cut-off
Estimate prevalence
in target population
Analytical sensitivity
Analytical specificity
Preliminary repeatability estimate
Extend validation to other
populations by testing sera of
reference animals representing
those populations
STAGE 2
Development
and
standardisation
STAGE 1
Feasibility
STAGE 3
Characterisation
of assay
performance
STAGE 4
Monitoring assay
performance for
validity
STAGE 5
Maintenance and
extension of validation
criteria
Replacement reagents
standardised by comparison
with reagents currently in use
Calculate
precision and
accuracy
Problems requiring
reassessment

Fig. 1. Stages in the incremental process of assay validation.
An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibody will be used in this
chapter to illustrate the principles of assay validation. It is a test format that can be difficult to validate
because of signal amplification of both specific and nonspecific components (2). This methodology serves
to highlight the problems that need to be addressed in any assay validation process. The same basic
principles are used in validation of other complex or simple assay formats. Chapter 1.1.3. Validation and
quality control of polymerase chain reaction methods used for the diagnosis of infectious diseases
describes the principles for validating gene-amplification techniques.
Because of space limitations, this introductory chapter provides only basic guidelines for the principles
concerned with assay validation. It is derived from a more detailed treatment of the subject (6).
STAGE 1. FEASIBILITY STUDIES
In the ELISA example, feasibility studies are the first stage in validating a new assay. They are carried out
in order to determine if the selected reagents and protocol have the capacity to distinguish between a range
of antibody concentrations to an infectious agent while providing minimal background activity. Feasibility
studies also give initial estimates of repeatability, and of analytical sensitivity and specificity.
1. CONTROL SAMPLES
It is useful to select four or five samples (serum in our example) that range from high to low levels of
antibodies against the infectious agent in question. In addition, a sample containing no antibody is
required. These samples will be used to optimise the assay reagents and protocol during feasibility
studies, and later as control samples. The samples ideally should represent known infected and
uninfected animals from the population that eventually will become the target of the validated assay.
The samples should have given expected results in one or more serological assay(s) other than the one
being validated. The samples are preferably derived from individual animals, but they may represent
pools of samples from several animals. A good practice is to prepare a large volume (e.g. 10 ml) of each
sample and divide it into 0.1 ml aliquots for storage at or below 20C. One aliquot of each sample is
thawed, used for experiments, and ideally then discarded. If it is impractical to discard the aliquot, it
may be held at 4C between experiments for up to about 2 weeks; however, there is a possibility of
sample deterioration under these circumstances. Then, another aliquot is thawed for further
experimentation. This method provides the same source of serum with the same number of freeze
thaw cycles for all experiments (repeated freezing and thawing of serum can denature antibodies so
should be avoided). Also, variation is reduced when the experimenter uses the same source of serum
for all experiments rather than switching among various sera between experiments. This approach has
the added advantage of generating a data trail for the repeatedly run samples. After the initial stages of
assay validation are completed, one or more of the samples can become the serum control(s) that are
the basis for data expression and repeatability assessments both within and between runs of the assay.
They may also serve as standards if their activity has been predetermined; such standards provide
assurance that runs of the assay are producing accurate data (11).
It is highly desirable to include OIE International Standard Sera or other international standard sera if
they are available. This may lead to harmonisation between the assay under development and a
standard test method in which international standard sera are normally used (10).
2. SELECTION OF METHOD TO ACHIEVE NORMALISED RESULTS
Normalisation adjusts raw test results of all samples relative to values of controls included in each run
of the assay (not to be confused with transformation of data to achieve a normal [Gausian]
distribution). The method of normalisation and expression of data should be determined, preferably no
later than at the end of the feasibility studies. Comparisons of results from day to day and between
laboratories are most accurate when normalised data are used. For example, in ELISA systems, raw
optical density (absorbance) values are absolute measurements that are influenced by ambient
temperatures, test parameters, and photometric instrumentation. To account for this variability, results
are expressed as a function of the reactivity of one or more serum control samples that are included in
each run of the assay. Data normalisation is accomplished in the indirect ELISA by expressing
absorbance values in one of several ways (11). A simple and useful method is to express all absorbance
values as a percentage of a single high-positive serum control that is included on each plate. This
method is adequate for most applications. More rigour can be brought to the normalisation procedure
by calculating results from a standard curve generated by several serum controls. It requires a more
sophisticated algorithm, such as linear regression or log-logit analysis. This approach is more precise
because it does not rely on only one high-positive control sample for data normalisation, but rather
uses several serum controls, adjusted to expected values, to plot a standard curve from which the
sample value is extrapolated. This method also allows for exclusion of a control value that may fall
outside expected confidence limits.
For assays that are end-pointed by sample titration, such as serum (viral) neutralisation, each run of the
assay is accepted or rejected based on whether control values fall within predetermined limits. Because
sample values usually are not adjusted to a control value, the data are not normalised by the strict
definition of the term.
Whatever method is used for normalisation of the data, it is essential to include additional controls for
any reagent that may introduce variability and thus undermine attempts to achieve a validated assay.
The normalised values for those controls need to fall within predetermined limits (e.g. within an
appropriate multiple of the standard deviation of the mean of many runs of each control). The chosen
limits should reflect a reasonable and tolerable assay run rejection rate and an acceptable risk that some
test samples may be misclassified.
STAGE 2. ASSAY DEVELOPMENT AND STANDARDISATION
When the feasibility of the method has been established, the next step is to proceed with development of
the assay and standardisation of the selected reagents and protocols.
1. SELECTION OF OPTIMAL REAGENT CONCENTRATIONS AND PROTOCOL PARAMETERS
Optimal concentrations/dilutions of the antigen adsorbed to the plate, serum, enzymeantibody
conjugate, and substrate solution are determined through checkerboard titrations of each reagent
against all other reagents, following confirmation of the best choice of reaction vessels (usually
evaluation of two or three types of microtitre plates, each with its different binding characteristics, to
minimise background activity while achieving the maximum spread in activity between negative and
high-positive samples). Additional experiments determine the optimal temporal, chemical, and physical
variables in the protocol, including incubation temperatures and durations; the type, pH, and molarity
of diluent, washing and blocking buffers; and equipment used in each step of the assay (for instance
pipettes and washers that give the best reproducibility).
Optimisation of the reagents and protocol includes an assessment of accuracy by inclusion, in each run
of the assay, of one or more serum standards of a known level of activity for the analyte in question.
An optimised assay that repeatedly achieves the same results for a serum standard and the serum
controls may be designated as a standardised assay.
2. REPEATABILITY PRELIMINARY ESTIMATES
Preliminary evidence of repeatability (agreement between replicates within and between runs of the
assay) is necessary to warrant further development of the assay. This is accomplished by evaluating
results from replicates of all samples in each plate (intraplate variation), and interplate variation using
the same samples run in different plates within a run and between runs of the assay. For ELISA, raw
absorbance values are usually used at this stage of validation because it is uncertain whether the results
of the high-positive control serum, which could be used for calculating normalised values, are
reproducible in early runs of the assay format. Also, expected values for the controls have not yet been
established. Three-to-four replicates of each sample run in at least five plates on five separate occasions
are sufficient to provide preliminary estimates of repeatability. Coefficients of variation (standard
deviation of replicates/mean of replicates), generally less than 20% for raw absorbance values, indicates
adequate repeatability at this stage of assay development. However, if evidence of excessive variation
(>30%) is apparent for most samples within and/or between runs of the assay, more preliminary
studies should be done to determine whether stabilisation of the assay is possible, or whether the test
format should be abandoned. This is important because an assay that is inherently variable has a high
probability of not withstanding the rigours of day-to-day testing on samples from the targeted
population of animals.
3. DETERMINATION OF ANALYTICAL SENSITIVITY AND SPECIFICITY
The analytical sensitivity of the assay is the smallest detectable amount of the analyte in question, and
analytical specificity is the degree to which the assay does not cross-react with other analytes. These
parameters are distinguished from diagnostic sensitivity and specificity as defined below. Analytical
sensitivity can be assessed by end-point dilution analysis, which indicates the dilution of serum in
which antibody is no longer detectable. Analytical specificity is assessed by use of a panel of sera
derived from animals that have experienced related infections that may stimulate cross-reactive
antibodies. If, for instance, the assay does not detect antibody in limiting dilutions of serum with the
same efficiency as other assays, or cross-reactivity is common when sera from animals with closely
related infections are tested, the reagents need to be recalibrated or replaced, or the assay should be
abandoned. However, there is a place for a test with low specificity, providing the sensitivity is high, for
use as a screening test in situations where a large number of samples have to be screened and where
there is a confirmatory test that has high specificity.
STAGE 3. DETERMINING ASSAY PERFORMANCE CHARACTERISTICS
If the feasibility and initial development and standardisation studies indicate that the assay has potential for
field application, the next step is to establish the assays performance characteristics.
1. DIAGNOSTIC SENSITIVITY AND SPECIFICITY
a) Principles and definitions
Estimates of D-SN and D-SP are the primary parameters established during validation of an assay.
They are the basis for calculation of other parameters from which inferences are made about test
results. Therefore, it is imperative that estimates of D-SN and D-SP are as accurate as possible.
Ideally, they are derived from testing a series of samples from reference animals of known history
and infection status relative to the disease/infection in question and relevant to the country or
region in which the test is to be used. Diagnostic sensitivity is the proportion of known infected
reference animals that test positive in the assay; infected animals that test negative are considered
to have false-negative results. Diagnostic specificity is the proportion of uninfected reference
animals that test negative in the assay; uninfected reference animals that test positive are
considered to have false-positive results.
The number and source of reference samples used to derive D-SN and D-SP are of paramount
importance if the assay is ever to be properly validated for use in the population of animals
targeted by the assay. It is possible to calculate the number of reference samples, from animals of
known infection/exposure status, required for determinations of D-SN and D-SP that will have
statistically defined limits. Formulae and tables for determining the number of samples required
are provided elsewhere (4, 6). However, the number of reference samples derived from these
formulae/tables is likely to be inadequate for a fully validated assay because of the very large
number of variables in target populations that must be accounted for in the reference sera. These
limitations and a discussion of the selection criteria for standard sera are detailed elsewhere (4, 6).
Because of the many variables that must be accounted for, it is desirable to include at least 300
reference samples from known-infected animals, and about 1000 samples from known-uninfected
animals to determine initial estimates of D-SN and D-SP, respectively. However, such a large
number of samples from reference animals may be difficult and may even be impossible to
achieve. Starting with fewer animals, followed by updating the D-SN and D-SP estimates as more
samples from reference animals become available may be the only practical way to get initial
estimates of D-SN and D-SP (See Stage 5 below).
b) Standards of comparison for the new assay
In serology, the standard of comparison is the results of a method or combination of methods
with which the new assay is compared. Although the term gold standard is commonly used to
describe any standard of comparison, it should be limited to methods that unequivocally classify
animals as infected or uninfected. Some isolation methods themselves have problems of
repeatability and sensitivity. Gold standard methods include unequivocal isolation of the agent or
pathognomonic histopathological criteria. Because a true gold standard is impossible to achieve,
relative standards of comparison are often necessary; these include results from other serological
assays and from experimentally infected or vaccinated animals. Calculations of D-SN and D-SP
are most reliable when the gold standard of comparison is available. When only relative standards
of comparison are available, estimates of D-SN and D-SP for the new assay may be compromised
because the error in the estimates of D-SN and D-SP for the relative standard is carried over into
those estimates for the new assay.
c) Precision, repeatability, reproducibility, and accuracy
Repeatability and reproducibility are estimates of precision in the assay. Precision is a measure of
dispersion of results for a repeatedly tested sample; a precise assay has only a small amount of
dispersion. Repeatability in a diagnostic assay has two elements: the amount of agreement between
replicates (usually two or three) of each sample within a run of the assay, and the amount of
between-run agreement for the normalised values of each control sample. Reproducibility is the
amount of agreement between results of the same samples tested in different laboratories.
Accuracy is the amount of agreement between a test value and the expected value for an analyte in
a standard sample of known activity (e.g. titre or concentration). An assay system may be precise,
but not accurate, if the test results do not agree with the expected value for the standard.
Reliable estimates of repeatability and accuracy, both within and between runs of the assay, can be
obtained by use of normalised results from the many runs of the new assay that were required to
assess the sera of reference animals (less reliable estimates were obtained from preliminary data
using raw absorbance values). At least 10, and preferably 20 runs of the assay will give reasonable
initial estimates of these parameters. Methods for evaluating these parameters have been described
in detail (6).
Accuracy can be assessed by inclusion of one or more standards (samples of known titre,
concentration, etc.) in each run of the assay. The standards may be control sera provided that the
amount of analyte (e.g. titre, concentration) in each one has been previously determined by
comparison with primary or secondary reference standards (11), and the control sera are not used
in the data normalisation process.
Reproducibility of the assay is determined in several laboratories using the identical assay
(protocol, reagents, and controls) on a group of at least 10 samples, preferably duplicated to a total
of 20 samples. These samples need to represent the full range of expected analyte concentrations
in samples from the target population. The extent to which the collective results for each sample
deviate from expected values is a measure of assay reproducibility. The degree of concordance of
between-laboratory data is one more basis for determining whether the assays performance
characteristics are adequate to constitute a validated assay.
2. SELECTION OF THE CUT-OFF (POSITIVE/NEGATIVE THRESHOLD)
To achieve estimates of D-SN and D-SP of the new assay, the test results first must be reduced to
positive or negative categories. This is accomplished by insertion of a cut-off point (threshold or
decision limit) on the continuous scale of test results. Although many methods have been described for
this purpose, three examples will illustrate different approaches, together with their advantages and
disadvantages. The first is a cut-off based on the frequency distributions (6) of test results from
uninfected and infected reference animals. This cut-off can be established by visual inspection of the
frequency distributions, by receiver-operator characteristics (ROC) analysis (5, 12), or by selection that
favours either D-SN or D-SP, depending on the intended use for a given assay (9). A second approach
is establishing a cut-off based only on uninfected reference animals; this provides an estimate of D-SP
but not D-SN. The third method provides an intrinsic cut-off based on test results from sera drawn
randomly from within the target population with no prior knowledge of the animals infection status
(3). Although no estimates of D-SN and D-SP are obtained by this method, they can be determined as
confirmatory data are accumulated.
If considerable overlap occurs in the distributions of test values from known infected and uninfected
animals, it is difficult to select a cut-off that will accurately classify these animals according to their
infection status. Rather than a single cut-off, two cut-offs can be selected that define a high D-SN (e.g.
inclusion of 99% of the values from infected animals), and a high D-SP (e.g. 99% of the values from
uninfected animals). The values that fall between these percentiles would then be classified as
suspicious or equivocal, and would require testing by a confirmatory assay or retesting for detection of
seroconversion.
3. CALCULATION OF DIAGNOSTIC SENSITIVITY AND SPECIFICITY
The selection of a cut-off allows classification of test results into positive or negative categories.
Calculations of D-SN and D-SP are aided by associating the positive/negative categorical data with the
known infection status for each animal using a two-way (2 2) table (Table 1). After the cut-off is
established, results of tests on standard sera can be classified as true positive (TP) or true negative (TN)
if they are in agreement with those of the gold standard (or other standard of comparison).
Alternatively, they are classified as false positive (FP) or false negative (FN) if they disagree with the
standard. Diagnostic sensitivity is calculated as TP/(TP FN) whereas diagnostic specificity is
TN/(TN FP); the results of both calculations are usually expressed as percentages (Table 1).
Table 1. Calculations of D-SN and D-SP aided by a 2 x 2 table that associates infection status with
test results from 2000 reference animals

Reference animals of known
infection status

Infected (n = 600) Uninfected (n = 1400)

Positive 570 46

Test

TP FP

Result

FN TN

Negative 30 1354

Diagnostic sensitivity Diagnostic specificity

TP
TP FN
570
600
95.0%
+
= =

TN
TN FP
1354
1400
96.7%
+
= =

4. HARMONISATION OF ASSAYS
When an international standard method (10) is available for detection of an analyte, it is possible to
compare the performance of that method with the one under development. This process requires use
of the same serum controls and/or standards in both assays. If OIE Standard Sera or other
international standard sera are available, preferably at least three (negative, low positive, and high
positive), they should be included in the assay-comparison study. This could lead to a new assay that is
indexed to an international standard method and international standard sera (10). Harmonisation of the
two assays may then be realised.
STAGE 4. MONITORING VALIDITY OF ASSAY PERFORMANCE
1. INTERPRETATION OF TEST RESULTS FACTORS AFFECTING ASSAY VALIDITY
An assays test results are useful only if the inferences made from them are accurate. A common error
is to assume that an assay with 99% D-SN and 99% D-SP will generate one false-positive and one
false-negative result for approximately every 100 tests on animals from the target population. Such an
assay may be precise and accurate, but produce test results that do not accurately predict infection
status. For example, if the prevalence of disease in a population targeted by the assay is only 1 per 1000
animals, and the false-positive test rate is 1 per 100 animals (99% D-SP), for every 1000 tests on that
population, ten will be false positive and one will be true positive. Hence, only approximately 9% of
positive test results will accurately predict the infection status of the animal; the positive test results will
misclassify the animal 91% of the time. This illustrates that the capacity of a positive or negative test
result to predict infection status is dependent on the prevalence of the infection in the target
population (7). Of course, the prevalence will probably have been determined by use of a serological
test.
An estimate of prevalence in the target population is necessary for calculation of the predictive values
of positive (PV) or negative (PV) test results. When test values are reported without providing
estimates of the assays D-SP and D-SN, it is not possible to make informed predictions of infection
status from test results (6). It is, therefore, highly desirable to provide an interpretation statement with
test results accompanied by a small table indicating PV and PV for a range of expected prevalences
of infection in the target population. Without provision of such information, test results from the assay
may have failed to accurately classify the infection status of animals, and thus do not reflect a fully
validated assay.
STAGE 5. MAINTENANCE AND ENHANCEMENT OF VALIDATION CRITERIA
A validated assay needs constant monitoring and maintenance to retain that designation. Once the assay is
put into routine use, internal quality control is accomplished by consistently monitoring the assay for
assessment of repeatability and accuracy (1).
Reproducibility between laboratories should be assessed at least twice each year. It is highly desirable to
become part of a consortium of laboratories that are interested in evaluating their output. In the near
future, good laboratory practice, including implementation of a total quality assurance programme, will
become essential for laboratories seeking to meet national and international certification requirements (see
Chapter 1.1.1.).
Proficiency testing is a form of external quality control for an assay. It is usually administered by a
reference laboratory that distributes panels of samples, receives the results from the laboratories, analyses
the data, and reports the results back to the laboratories. If results from an assay at a given laboratory
remain within acceptable limits and show evidence of accuracy and reproducibility, the laboratory may be
certified by government agencies or reference laboratories as an official laboratory for that assay (8). Panels
of sera for proficiency testing should contain a full representation of an analytes concentration in animals
of the target population. If the panels only have high-positive and low-positive sera (with none near the
assays cut-off), the exercise will only give evidence of reproducibility at the extremes of analyte
concentration, and will not clarify whether routine test results on the target population properly classify
infection status of animals.
Because of the extraordinary set of variables that impact on the performance of serodiagnostic assays, it is
highly desirability to expand the number of standard sera from animals of known infection status because
of the principle that error in the estimates of D-SN and D-SP is reduced with increasing sample size.
Furthermore, when the assay is to be transferred to a completely different geographical region, it is
essential to re-validate the assay for this new intended use by subjecting it to sera from populations of
animals that reside under local conditions.
When a serum control sample is nearing depletion, it is essential to prepare and repeatedly test a
replacement before the serum control is depleted. The prospective control sample is included in 1020
runs of the assay before depletion of the original control to establish its proportional relationship to the
nearly depleted control. If the depleted sample was a positive control in ELISAs where the normalised
value is expressed as a per cent of that positive control, the proportional difference in ELISA activity
between the original and replacement sera must be factored into the normalisation algorithm to retain the
same cut-off, and thus the same D-SN and D-SP in the assay. When other reagents, such as antigen for
capture of antibody, must be replaced, they should be produced using the same criteria as for the original
reagents, and tested in at least five runs of the assay using a panel of sera that has been designed for this
purpose. Whenever possible, it is important to change only one reagent at a time to avoid the compound
problem of evaluating more than one variable at a time.
REFERENCES
1. CEMBROWSKI G.S. & SULLIVAN A.M. (1992). Quality control and statistics. In: An Introduction to
Clinical Chemistry, Bishop M.L., Duben-Engelkirk J.L. & Fody E.P., eds. Lippincott, Philadelphia,
USA, 63101.
2. CROWTHER J.R. (1995). ELISA theory and practice. In: Methods in Molecular Biology. Humana
Press, Totowa, NJ, USA, 1256.
3. GREINER M., FRANAKE C.R., BOHNING D. & SCHLATTMANN P. (1994). Construction of an intrinsic
cut-off value for the sero-epidemiological study of Trypanosoma evansi infections in a canine
population in Brazil: a new approach towards unbiased estimation of prevalence. Acta Trop., 56, 97
109.
4. GREINER M. & GARDNER I. (2000). Epidemiologic issues in the validation of veterinary diagnostic
tests. Vet. Prev. Med., 45, 322.
5. GREINER M., PFEIFFER D. & SMITH R.D. (2000). Principles and practical application of the receiver
operating characteristic (ROC) analysis for diagnostic tests. Vet. Prev. Med., 45, 2341.
6. JACOBSON R.H. (1998). Validation of serological assays for diagnosis of infectious diseases. Rev. sci.
tech. Off. int. Epiz., 17, 469486.
7. JACOBSON R.H. (1991). How well do serodiagnostic tests predict the infection of disease status of
cats. J. Am. Vet. Med. Assoc., 199, 13431347.
8. OFFICE INTERNATIONAL DES EPIZOOTIES (2002). OIE Guide 3: Laboratory Proficiency Testing. In:
OIE Quality Standard and Guidelines for Veterinary Laboratories: Infectious Diseases. OIE, Paris,
France, 5363.
9. SMITH R.D. (1991). Clinical Veterinary Epidemiology. Butterworth-Heinemann, Stoneham, MA,
USA, 1223.
10. WRIGHT P.F. (1998). International standards for test methods and reference sera for diagnostic tests
for antibody detection. Rev. sci. tech. Off. int. Epiz., 17, 527533.
11. WRIGHT P.F., NILSSON E., VAN ROOIJ E.M.A., LELENTA M. & JEGGO M.H. (1993). Standardization
and validation of enzyme-linked immunosorbent assay techniques for the detection of antibody in
infectious disease diagnosis. Rev. sci. tech. Off. int. Epiz., 12, 435450.
12. ZWEIG M.H. & CAMPBELL G. (1993). Receiver-operating characteristic (ROC) plots: a fundamental
evaluation tool in clinical medicine. Clin. Chem., 39, 561577.
*
* *
C HA P T E R 1 . 1 . 3 .

VAL I DAT I ON AND QUAL I T Y CONT ROL OF
P OL YME RAS E CHAI N RE ACT I ON ME T HODS US E D
F OR T HE DI AGNOS I S OF I NF E CT I OUS DI S E AS E S

SUMMARY
The diagnosis of infectious diseases is performed by direct and/or indirect detection of infectious agents. By
direct methods, the particles of the agents and/or their components, such as nucleic acids, structural or
nonstructural proteins, enzymes, etc., are detected. The indirect methods demonstrate the antibodies induced
by the infections.
The most common direct detection methods are virus isolation, bacteria cultivation (the gold standards),
electron microscopy, immunofluorescence, immunohistochemistry, antigen enzyme-linked immunosorbent
assay (ELISA), nucleic-acid hybridisation (NAH) and nucleic acid amplification, such as the polymerase
chain reaction (PCR). As NAH and PCR assays have nucleic acid molecules as targets, they are also
termed methods of molecular diagnosis.
The most common indirect methods of infectious agent detection are virus neutralisation, antibody detection
by ELISA and haemagglutination inhibition tests. In general, diagnostic laboratories simultaneously apply
both the direct and the indirect methods, in order to assure the certainty of a diagnosis.
To date, OIE principles of validation have been developed for indirect detection methods, i.e. for antibody
ELISA (see Chapter 1.1.2.). The purpose of this chapter is to extend the rules to a direct method of
infectious agent detection, i.e. to adapt the principles of validation to the PCR assays.
The experiences of the last decade indicate that the PCR techniques will eventually supersede many of the
classical direct methods of infectious agent detection. It is clear that in many laboratories, the PCR is
replacing virus isolation or bacteria cultivation for the detection of agents that are difficult or impossible to
culture. There are several reasons for this trend, including that virus isolation requires: i) the presence of
replicating viruses; ii) expensive cell culture and maintenance facilities; iii) as long as several weeks to
complete the diagnosis; and iv) special expertise, which is missing or diminishing today in many laboratories.
Although PCR assays were initially expensive and cumbersome to use, they have now become relatively
inexpensive, safe and user-friendly tools in diagnostic laboratories (2, 7).
A. PCR METHODS USED IN ROUTINE MOLECULAR DIAGNOSTICS
1. THE PRINCIPLES OF THE PCR
Polymerase chain reaction (PCR) implies that there is an amplification reaction in the assay. The term
chain reaction refers to several cycles of copying a specified stretch of DNA from a target nucleic
acid, in this case from the genome of an infectious agent. The amplified region is defined by two (or
more) short oligonucleotides, and two primers that are complementary to DNA regions flanking the
target sequence. Using a heat-stable DNA polymerase, which is not denatured during heat cycling, it is
possible to copy the DNA sequence between the primers. By repeating 2040 times a heat-cycling
regime, the amount of copied target DNA gained is enough for further operations, such as detection,
cloning or sequencing. The diagnostic sensitivity of the PCR is very high because several million copies
of the selected target are produced. The specificity of the reaction may also be very high, as determined
Chapter 1.1.3. - Validation and quality control of PCR methods used for diagnosis of infectious diseases
by the specific nucleotide sequences of the oligonucleotides (primers). The primers are designed to
detect specific nucleotide sequences in the genomes of the selected target infectious agents.
a) DNA amplification
If the genome of the infectious agent is DNA, the amplification is performed directly, with or
without previous purification of the target DNA.
b) RNA amplification (reverse-transcription PCR)
The genomes of many infectious agents contain ribonucleic acid (RNA) that cannot be amplified
directly by the PCR. For PCR amplification, a double-stranded DNA target is necessary, and this
is not available in the case of RNA viruses. This problem can be solved by the addition of a step
before the PCR is begun. Using reverse transcriptase it is possible to transcribe the RNA into
complimentary DNA (cDNA), which is double-stranded DNA and hence can be used in a PCR
assay (the procedure is termed reverse transcriptase PCR: RT-PCR). Traditionally, the reverse
transcription reaction is performed in a separate reaction vessel and the cDNA produced is then
transferred to a new tube for the PCR. However, heat-stable DNA polymerases with reverse
transcriptase activity are now readily available. When used in specific buffers, these enzymes
enable the RT-PCR reaction to take place in the same tube and in direct sequence without any
further handling.
Some examples of PCR methods currently used are given below.
2. SINGLE PCR
Single PCR (or simply PCR) uses one pair of oligonucleotide primers to amplify a small part of the
genome of the infectious agent. Analytical sensitivity is relatively high with a minimum number of 100
to 1000 copies of the genome detectable. Analytical specificity is also rather high. Both analytical
sensitivity and specificity can be further improved by applying nested PCR (see point 3 below). Using
Southern blotting and checking the PCR products with a labelled probe can further improve specificity,
but this is time-consuming and is not a common practice in diagnostic laboratories today.
3. NESTED PCR
Nested PCR assays use two amplification cycles with four primers, termed external and internal
primers. In general, nested PCR assays provide higher analytical sensitivity and specificity compared
with single PCR. Analytical sensitivity is typically <10 genomic copies of the infectious agent, and
analytical specificity is also enhanced because in the nested PCR, four oligonucleotides have to bind
specifically to the selected targets in order to yield a positive reaction (2).
4. REAL-TIME PCR
Real-time PCR is an amplification where the PCR products are detected directly during the
amplification cycles using fluorescence-labelled probes. Various real-time methods, such as TaqMan or
Molecular Beacon assays, have become popular tools for detection of infectious agents. Real-time PCR
has been used for the detection of bacteria, viruses or parasites from a range of animal species (2, 6, 8).
These new assays have several advantages over the classical single or nested PCR methods. Only one
primer pair is used, providing sensitivity often close or equal to traditional nested PCR but with a much
lower risk of contamination. Fluorescence, indicating the presence of the amplified product, is
measured through the lid or side of the reaction vessel thus there is no need for post-PCR handling of
the products. These procedures are considerably less time-consuming compared with traditional PCR
product detection in agarose gels followed by ethidium bromide staining and again, the risk of
contamination is reduced. The use of a 96-well microtitre plate format, without the need for nested
PCR, allows the procedure to be automated (5). Diagnosis can be further automated by using robots
for DNA/RNA extractions and pipetting. Compared with classical amplification methods, a further
advantage of the real-time PCR is that it is possible to perform quantitative assays (6).
5. MULTIPLEX PCR
Probes for real-time PCR can be labelled with a large number of different fluorophores, which function
as reporter dyes. The use of fluorescent probes emitting different colours enables multiplexing of the
assays. In multiplex PCR, various infectious agents can be detected and differentiated in a single
reaction vessel at the same time. The classical PCR technique was also found to be suitable for the
development of multiplex systems. However, the use of classical nested PCR methods for the
construction of a multiplex assay is complicated because of the large number of primers that may
compete with each other in the same reaction mix. In contrast, the concept of real-time PCR (single
primer pairs) provides excellent possibilities for the construction of highly sensitive multiplex systems
(2, 4).
B. VALIDATION OF MOLECULAR DIAGNOSTIC ASSAYS
When performing diagnostic analyses of clinical material it is important to produce data of good quality.
For this, some key criteria have to be fulfilled. The establishment of quality assurance (QA) and quality
control (QC) systems is required, i.e. a set of quality protocols, including the use of control samples, that
ensure that the system is working properly and confirms data quality. QA and QC systems, together with
trained and competent personnel, have already been established in many laboratories world-wide. Assay
validation is another essential factor for assuring that test results reflect the true status of the samples (3).
To predict the performance of a diagnostic assay, it is necessary to use a validation methodology to
validate the assay in question. Validation is the evaluation of a diagnostic assay for the purpose of
determining how fit the assay is for a particular use. The general principles of assay validation can be
found in Chapter 1.1.2. Principles of Validation of Diagnostic Assays for Infectious Disease. This chapter
extends these validation principles to molecular diagnostic assays. For explanations of terms and
definitions please consult Chapter 1.1.2.
C. MEASURES OF VALIDITY
Performance characteristics (or assay parameters) give information about how a method functions under
specified conditions. Some typical performance characteristics are given in Chapter 1.1.2. and some others,
important to PCR methods, are given here.
D. STAGES OF ASSAY VALIDATION
In Chapter 1.1.2., the five stages of assay validation are described in detail. In this chapter, these stages are
presented briefly with special emphasis on molecular diagnostic assays.
STAGE 1. FEASIBILITY STUDIES
A feasibility study is a preliminary step in validating a new assay. The goal is to determine whether or not a
new assay can detect a range of target concentrations without background activity. At least ten samples
(for example, infectious agents produced in the laboratory in cell or bacterial culture) are chosen, ranging
from low to high levels of the infectious agent. It is also necessary to include at least ten samples
containing no target. Usually it is difficult to separate this stage from stage 2, as preliminary optimisation is
necessary before further studies can be conducted. Assays that look promising are subjected to further
development in stage 2. Note that it is sometimes possible to substantially improve an assay by proper
optimisation schemes and thus exclusion of non-optimal assays should be done with caution.
Primer selection is critical, and account should be taken of the nature of the infectious agent, its genome
structure and the diversity of genetic sequences among different strains.
The result obtained by PCR may be influenced by the performance of the thermocycler, which should
therefore be monitored on a continual basis. Regular temperature calibration is crucial and, for real-time
PCR instruments, the optical systems must be controlled regularly. Assays developed and validated using a
specific brand of thermocycler should be revalidated or otherwise controlled when new equipment is used.
STAGE 2. ASSAY DEVELOPMENT AND STANDARDISATION
1. SELECTION OF OPTIMAL REAGENT CONCENTRATIONS, PROTOCOL PARAMETERS AND EQUIPMENT
Sample collection, preparation and transport (see Chapter I.1.) and nucleic acid extraction methods (see
Chapter I.1.) are all critical parameters in test performance and should be optimised for disease
diagnosis. Suitable methods vary depending on sample and organism type. In general, blood and serum
are suitable samples for easy extraction of target nucleic acids, while faeces and semen samples are
more difficult to handle. Extraction of RNA targets differs from extraction of DNA targets, and RNA
is more prone to degradation. Both commercial (robotic, spin columns, etc.) and in-house methods are
used for DNA or RNA extraction. It is crucial to determine the most suitable method before further
validation of the assay is performed. If the method of extraction is changed, the entire validation
procedure should be repeated.
All equipment used during the process must be properly maintained. Apparatus (heating blocks,
refrigerators, freezers, thermocyclers, pipettes, etc.) that require calibration must be calibrated
according to the laboratorys quality protocols.
When developing classical or real-time PCR assays, all parameters, protocols and reagents need to be
optimised. A standardised assay is a method that consistently gives the same result for a given sample
when repeated several times.
2. REPEATABILITY PRELIMINARY ESTIMATES
Agreement between replicates within and between runs of the assay should be accessed at this stage.
This gives important information about the assay before further validation is carried out. If excessive
variability is encountered, it should be corrected before continuing the validation process.
3. DETERMINATION OF CRITICAL CONTROL PARAMETERS
During the optimisation of the PCR assay, it is also possible to estimate the capacity of the method to
remain unaffected by small changes in the main parameters. Introduction of intentional variations in
the validation process will characterise critical parameters in the assay. Examples of such parameters
include: incubation times and temperatures, concentrations of buffers, primers, MgCl
2
, etc., pH,
amounts of other components added (e.g. dNTP, bovine serum albumin, etc.). The characterisation of
critical control parameters is crucial for identifying critical points that must be properly controlled in
the assay.
4. ANALYTICAL SENSITIVITY AND SPECIFICITY
Analytical sensitivity (or limit of detection) is defined as the smallest number of genome copies of the
infectious agent that can be detected and distinguished from a zero result. To determine analytical
sensitivity, an end-point dilution is used until the assay can no longer detect the target in question in
more than 5% of the replicates (2 standard deviations). Cloned fragments of the PCR products in
question can be used as standard samples, either as DNA or for RNA targets, the RNA being
transcribed in vitro into DNA.
Analytical specificity is defined as the ability of an assay to distinguish the target agent from other
infectious agents. This ability is determined by analysing closely related pathogens using the assay in
question.
5. RANGE
Analytical techniques can rarely be scaled up or down arbitrarily; the assay should be optimised in the
linear phase of the doseresponse curve. The range of an assay is defined as the interval between the
upper and lower concentration of an infectious agent in a sample in which the agent can be reliably
detected.
STAGE 3. DETERMINING ASSAY PERFORMANCE CHARACTERISTICS
1. DIAGNOSTIC SENSITIVITY AND SPECIFICITY
Diagnostic sensitivity (D-SN; proportion of known infected reference animals that are tested positive
in the assay) and specificity (D-SP; proportion of known uninfected reference animals that are tested
negative in the assay) are the most important parameters obtained during the validation of an assay.
They form the basis for calculating other parameters and hence they are critical to the whole validation
process. The number of reference samples required to determine estimates and allowable error of both
D-SN and D-SP can be calculated. To do this, a reasonable prediction of both D-SN and D-SP must
be used. Generally, confidence in the estimate is set at 95%. However, no formula can account for the
numerous host/organism factors that can affect the outcome of the test. The number of samples to
determine estimates of D-SN and D-SP is outlined in Chapter 1.1.2. It is recognised that achieving
these numbers for molecular assays might be difficult and costly. Testing smaller numbers will result in
a reduction in confidence of the estimate. The status of known infected and uninfected animals should
be established using comparisons with other assays. The use of spiked samples in PCR is not
appropriate as these might not be representative of naturally infected samples and thus the whole
validation process could potentially be jeopardised.
2. REPEATABILITY AND REPRODUCIBILITY
Repeatability and reproducibility are both important parameters in assay precision. Repeatability is
measured as both the amount of agreement between replicates within the same run or between
replicates tested in different runs. Reproducibility is determined in several laboratories using the
identical assay (protocol, reagents and controls).
Currently, OIE stage 3 is rarely performed to its full extent in veterinary diagnostic laboratories
carrying out PCR assays. Traditionally, many laboratories have used tests developed in-house, probably
for practical reasons. Where there are published standardised and validated methods, these should be
followed. Inter-laboratory validation processes have to be carried out even if they are costly and labour
intensive. This work will lead to standardised assays, allowing harmonised diagnostic activity in various
countries.
STAGE 4. MONITORING VALIDITY OF ASSAY PERFORMANCE
The estimation of the prevalence of a virus in the population is necessary for calculating the predictive
value of positive (PV+) or negative (PV) test results. This applies equally to molecular test methods as it
does to other methods such as the enzyme-linked immunosorbent assay.
Reference Laboratories are encouraged to determine values for D-SN and D-SP as accurately as possible,
as these are extremely important for judging the real performance of an assay when used in the field. It is
also important to estimate the predictive values (PV+ or PV) in the local situation.
STAGE 5. MAINTENANCE AND ENHANCEMENT OF VALIDATION CRITERIA
When the assay is used as a routine test it is important to maintain the internal QC. The assay needs to be
consistently monitored for repeatability and accuracy. Reproducibility between laboratories (ring tests) is
recommended by the OIE to be estimated at least twice a year (9).
If the assay is to be applied in another geographical region and/or population, it might be necessary to
revalidate it under the new conditions. This is especially true for PCR assays as it is very common for
point mutations to occur in many infectious agents (i.e. RNA viruses). Mutations, which may occur within
the primer sites, can affect the performance of the assay and by doing so the established validation is no
longer valid. It is also advisable to regularly sequence the selected genomic regions in the national isolates
of the infectious agents. This is especially true for the primer sites, to ensure that they remain stable so that
the validation of the assay cannot be questioned.
1. PRECAUTIONS AND CONTROLS
Considering the uncertainty about the safety and reliability of the PCR in routine diagnosis, special
precautions should be applied in any laboratory using PCR for detecting infectious agents, in order to
avoid false-positive or false-negative results. These, together with internal controls (mimics) assure the
safe evaluation of the results.
a) Precautions to avoid false-positive results
False-positive results (negative samples showing a positive reaction), may arise from either
product carryover from positive samples or, more commonly, from cross-contamination by PCR
products from earlier experiments. Various practices and tools have been applied to prevent false-
positive results. Samples and mixes should be handled in laminar air-flow hoods, which are
regularly decontaminated using UV light and bleach. Constructing and using special tube-holders
and openers help prevent false-positive PCR. In addition, safe laboratory practices should be
applied, i.e. to perform the basic steps of nested PCR (mix and primer preparation, sample
preparation, etc.) in separated laboratory areas (1, 2). It is also very important to include negative
controls, i.e. samples that are as similar to the test samples as possible but without having the
target. At least one negative control per five diagnostic samples should be used.
b) Internal controls (mimics) to avoid false-negative results
False-negative results (infected samples tested as negative) are mostly due to inhibitory effects
and/or pipetting errors. Therefore, internal controls (termed mimics) are used as indicators of
amplification efficiency. The mimics have the same primer-binding sequences as the template of
the agent, but flank a heterologous DNA fragment of a different size. The identical primer-
binding nucleotide sequences allow co-amplification of template and mimic in the same tube with
minimal competition. The size differences provide easy discrimination. The internal controls
increase the reliability of the diagnostic PCR (1, 2). Caution must be used when designing and
validating mimics. Extensive testing is necessary to ensure that the added mimic and its
amplification is not competing with the diagnostic PCR and thus lowering the analytical
sensitivity. The mimic is used in a concentration slightly higher than the detection limit of the
diagnostic PCR to ensure the tests performance.
In real-time PCR assays, it is also possible to apply internal controls by detecting a selected
fragment of the host animals genome. By including such an intrinsic control with a specifically
coloured reporter fluorophore, it is possible to check the sample quality and to control pipetting
errors simultaneously as the target agent is detected (8).
2. PREPARATION OF STANDARDS
Reference laboratories should provide standard samples representative of a given infectious agent. Such
samples can be cultivated infectious agents, clinical specimens, etc., which are distributed in such a
manner that the infectious agent is well preserved. Thus, the samples are distributed frozen, in organic
solvents (e.g. Trizol) or other suitable ways. The samples can also be sent as nucleic acids (frozen,
freeze-dried or in ethanol). For specific details, see the individual disease chapters.
REFERENCES
1. BALLAGI-PORDANY A. & BELAK S. (1996). The use of mimics as internal standards to avoid false
negatives in diagnostic PCR. Mol. Cell. Probes, 10, 159164.
2. BELAK S. & THOREN P. (2001). Molecular diagnosis of animal diseases. Expert Rev. Mol. Diagn., 1,
434444.
3. BURKHARDT H.J. (2000). Standardization and quality control of PCR analyses. Clin. Chem. Lab. Med.,
38, 8791.
4. ELNIFRO E.M., ASHSHI A.M., COOPER R.J., & KLAPPER P.E. (2000). Multiplex PCR: optimization
and application in diagnostic virology. Clin. Microbiol. Rev., 13, 559570.
5. JUNGKIND D. (2001). Automation of laboratory testing for infectious diseases using the polymerase
chain reaction our past, our present, our future. J. Clin. Virol., 20, 16.
6. HEID C.A., STEVENS J., LIVAK K.J. & WILLIAMS P.M. (1996). Real-time quantitative PCR. Genome
Res., 6, 986994.
7. LOUIE M., LOUIE L. & SIMOR A.E. (2000). The role of DNA amplification technology in the
diagnosis of infectious diseases. CMAJ., 163, 301309.
8. MACKAY I.M., AARDE K.E. & NITSCHE A. (2002). Real-time PCR in virology. Nucleic Acids Res., 30,
12921305.
9. OFFICE INTERNATIONAL DES EPIZOOTIES (2002). OIE Guide 3: Laboratory Proficiency Testing. In:
OIE Quality Standard and Guidelines for Veterinary Laboratories: Infectious Diseases. OIE, Paris,
France, 5363.
*
* *
C HA P T E R 1 . 1 . 4 .

RE QUI RE ME NT S F OR S URVE I L L ANCE
F OR I NT E RNAT I ONAL RE COGNI T I ON OF
F RE E DOM F ROM I NF E CT I ON

PART 1
A. INTERNATIONAL RECOGNITION OF FREEDOM FROM INFECTION
1. GENERAL PRINCIPLES
General principles are provided below for declaring a country, zone or aquaculture establishment free
from infection in relation to the time of last occurrence, and in particular for the recognition of
historical freedom.
An essential prerequisite to provide the guarantees required for the recognition of freedom from
infection is that the particular Member Country complies with the requirements of Chapter 1.4.3 of the
Aquatic Code for the evaluation of the Competent Authorities.
The general principles are:
in the absence of infection or vaccination, the animal population would be] susceptible to clinical
disease, or infection, over a period of time;
the disease agents to which these provisions apply are likely to produce identifiable clinical or
pathological signs in susceptible animals;
an animal population may be free from some specified pathogens but not from others
there are competent and effective personnel of the Competent Authority able to investigate,
diagnose and report disease or infection, if present;
the absence of infection over a long period of time in susceptible populations can be substantiated
by effective disease investigation and reporting by the Competent Authority of the Member
Country.
2. REQUIREMENTS TO DECLARE A COUNTRY, ZONE OR AQUACULTURE ESTABLISHMENT FREE FROM INFECTION
WITH A SPECIFIED PATHOGEN
The requirements to declare a country, zone or aquaculture establishment free from infection differ
depending on the previous infection status of the country, zone or aquaculture establishment, namely:
Absence of susceptible species;
Historically free;
Last known occurrence within the previous 25 years;
Previously unknown infection status.
Chapter 1.1.4. - Requirements for surveillance for international recognition of freedom from infection
2.1. Absence of susceptible species
Unless otherwise specified in the relevant disease chapter, a country, zone or aquaculture
establishment may be recognised as being free from infection without applying targeted surveillance
if there are no susceptible species (as listed in the relevant chapter of the Aquatic Code, or in the
scientific literature) present in that country, zone or aquaculture establishment, provided that the
prescribed biosecurity conditions have been in place continuously in the country, zone or aquaculture
establishment for at least the previous 10 years.
2.2. Historically free
Unless otherwise specified in the relevant disease chapter, a country, zone or aquaculture
establishment may be recognised as being free from infection without formally applying targeted
surveillance when:
there has never been any observed occurrence of disease;
or
eradication has been achieved or the disease has ceased to occur for at least 25 years,
provided that the prescribed biosecurity conditions have been in place continuously in the country,
zone or aquaculture establishment for at least the previous 10 years.
2.3. Last known occurrence within the previous 25 years
For countries or zones that have achieved eradication (or in which the disease has ceased to
occur) within the previous 25 years, in addition to the prescribed biosecurity conditions, appropriate
targeted surveillance must have been applied to demonstrate the absence of the infection, consistent
with the provisions of Section B of this chapter.
2.4. Previously unknown infection status
For countries or zones with previously unknown infection status, or which have not previously
met the requirements of the Sections A.2.1, A.2.2 or A.2.3 above, the prescribed biosecurity conditions
must be introduced in addition to targeted surveillance consistent with the provisions of Section B of
this chapter.
3. GUIDELINES FOR THE MAINTENANCE OF CONTINUED RECOGNITION OF FREEDOM FROM INFECTION
A country, zone or aquaculture establishment that has been recognised free from infection following
the provisions of Sections A.2.1 or A.2.2, may maintain its official status as infection-free provided that
the prescribed biosecurity conditions are continuously maintained.
A country, zone or aquaculture establishment that has been recognised free from infection following
the provisions of Sections A.2.3 or A.2.4, may discontinue targeted surveillance and maintain its official
status as infection-free provided that the prescribed biosecurity conditions are continuously maintained.
The different paths to recognition of freedom from infection are summarised in the diagram below.

Historically free
Last occurrence wtihin
the previous 25 years
Previously unknown
disease status
Meetprescribed
biosecurity conditions
and
Absence of
susceptible species
Implement targeted
surveillance
(Section B)
Discontinue
targeted surveillance
Freedom from Infection
Maintain prescribed
Meetprescribed

B. TARGETED SURVEILLANCE FOR DEMONSTRATION OF FREEDOM FROM INFECTION
1. INTRODUCTION
This section provides standards to be applied when demonstrating country, zone or aquaculture
establishment freedom from infection, in accordance with the principles of Section A. Standards
described in this section may be applied to all diseases, their agents and susceptible species as listed in
the Aquatic Code, and are designed to assist with the development of surveillance methodologies. More
detailed information in each disease chapter (where it exists) of this Aquatic Manual may be used to
further refine the general approaches described in this chapter. Where detailed disease/infection-
specific information is not available, suitable values should be chosen based on the guidelines in this
chapter.
Demonstrating freedom from infection involves providing sufficient evidence to demonstrate that
infection with a specified agent is not present in a specified population. In practice, it is not possible to
definitively prove that a population is free from infection (unless every member of the population is
examined simultaneously with a perfect test with both sensitivity and specificity equal to 100%). Instead,
the aim is to provide adequate evidence (to an acceptable level of confidence), that infection, if present, is
present in less than a specified proportion of the population.
Methodologies to demonstrate freedom from infection should be flexible to deal with the complexity
of real life situations. No single method is applicable in all cases. Methodologies must be able to
accommodate the variety of aquatic animal species, the multiple diseases of relevance, varying
production and surveillance systems, and types and amounts of data and information available.
The methodology used should be based on the best available information that is in accord with current
scientific thinking. The methodology should be well documented and supported with references to the
scientific literature and other sources, including expert opinion.
Consistency in methodologies should be encouraged and transparency is essential in order to ensure
fairness and rationality, consistency in decision making and ease of understanding by all the interested
parties. Applications for recognition of infection-free status should document the uncertainties, the
assumptions made, and the effect of these on the final estimate.
3. GENERAL REQUIREMENTS FOR DEMONSTRATION OF FREEDOM FROM INFECTION
3.1. Population
The target population to which the demonstration of freedom from infection applies is all
individuals of all species susceptible to the infection in a country, zone or aquaculture
establishment.
The study population may be the same as the target population or a subset of it. The study population
should be (in order of preference):
The appropriate study population as defined in the relevant disease chapter of the Aquatic Code
(if such a definition exists),
A subset of the target population that defines a group of animals which, if infection were
present, would be most likely to have a higher prevalence of infection than the target
population. This subset should be defined in terms of:
species;
time (e.g. season or month of year);
stage of life-cycle or growth period;
production system and/or management characteristics;
location;
readily identifiable physical or behavioural characteristics.
The same as the target population,
A subset of the target population with the same or lower probability of infection. The nature
and impact of any biases on the results of the analysis must be considered, documented and
taken into account in the analysis.
3.2. Sources of evidence
Evidence of freedom from infection may be based on a number of different sources, including:
structured, population-based surveys using one or more tests for the presence of the agent;
other surveillance, including structured non-random surveillance, such as:
sentinel sites;
disease notifications and laboratory investigation records;
academic and other scientific studies;
a knowledge of the biology of the agent, including environmental, host population
distribution, known geographical distribution, vector distribution and climatic information;
history of imports of potentially infected material;
biosecurity measures in place;
evaluation of the official services; or
any other sources that provide contributory evidence that infection is not present in the
country, zone or aquaculture establishment.
The sources of evidence used to demonstrate freedom from infection must be fully described. In
the case of a structured survey, this must include a description of the sampling strategy used for
the selection of units for testing. For complex surveillance systems, a full description of the system is
required including consideration of any biases that may be inherent in the system.
3.3. Statistical methodology
Analysis of data for evidence of freedom from infection involves estimating the probability ()
that the evidence observed (the results of surveillance) could have been produced under the null
hypothesis that infection is present in the population at a specified prevalence(s) (the design
prevalence[s]). The confidence in (or, equivalently, the sensitivity of) the surveillance system that
produced the evidence is equal to 1. If the confidence level exceeds a pre-set threshold, the
evidence is deemed adequate to demonstrate freedom from infection.
The required level of confidence in the surveillance system (probability that the system would detect
infection if infection were present at the specified level) must be greater than or equal to 95%.
The power (probability that the system would report that no infection is present if infection is
truly not present) may be set to any value. By convention, this is often set to 80%, but may be
adjusted according to the countrys or zones requirements.
Different statistical methodologies for the calculation of the probability , including both
quantitative and qualitative approaches, are acceptable as long as they are based on accepted
scientific principles.
The methodology used to calculate the confidence in the surveillance system must be scientifically
based and clearly documented, including references to published work describing the
methodology.
3.4. Clustering of infection
Infection in a country, zone or aquaculture establishment usually clusters rather than being
uniformly distributed through a population. Clustering may occur at a number of different levels
(e.g. a cluster of moribund fish in a pond, a cluster of ponds in a farm, or a cluster of farms in a
zone). Except when dealing with demonstrably homogenous populations, approaches to
demonstrating freedom must take this clustering into account in the design and the statistical
analysis of the data, at least at what is judged to be the most significant level of clustering for the
particular animal population and infection.
3.5. Design prevalence
Calculation of the confidence of a surveillance system is based on the null hypothesis that infection is
present in the population. The level of infection is specified by the design prevalence. In the
simplest case, this is the prevalence of infection in a homogenous population. More commonly, in
the presence of disease clustering, two design prevalence values are required, for instance, the
animal-level prevalence (proportion of fish infected in an infected farm) and the group-level
prevalence (proportion of infected farms in the country, zone or aquaculture establishment).
Further levels of clustering may be considered, requiring further design prevalence values.
The values for design prevalence used in calculations must be those specified in the relevant
disease chapter (if present) of this Aquatic Manual. If not specified for the particular disease,
justification for the selection of design prevalence values must be provided, and should be based
on the following guidelines:
At the individual animal level, the design prevalence is based on the biology of the infection
in the population. It is equal to the minimum expected prevalence of infection in the study
population, if the infection had become established in that population. It is dependent on the
dynamics of infection in the population and the definition of the study population (which may
be defined to maximise the expected prevalence in the presence of infection).
A suitable design prevalence value at the animal level (e.g. prevalence of infected animals in a
cage) may be:
between 1% and 5% for infections that are transmitted slowly; and
over 5% for more contagious infections.
At higher levels (e.g. cage, pond, farm, village, etc.) the design prevalence usually reflects the
prevalence of infection that is practically and reasonably able to be detected by a surveillance
system. Detection of infection at the lowest limit (a single infected unit in the population) is
rarely feasible in large populations. The expected behaviour of the infection may also play a
role. Infections that have the ability to spread rapidly between farms may have a higher farm-
level design prevalence than slow-moving infections.
A suitable design prevalence value for the first level of clustering, (e.g. proportion of infected
farms in a zone) may be up to 2%.
3.6. Test characteristics
All surveillance involves performing one or more tests for evidence of the presence of current or
past infection, ranging from detailed laboratory examinations to farmer observations. The
performance level of a test at the population level is described in terms of its sensitivity and specificity.
Imperfect sensitivity and/or specificity impact on the interpretation of surveillance results and
must be taken into account in the analysis of surveillance data.
All calculations must take the performance level (sensitivity and specificity) of any tests used into
account. The values of sensitivity and specificity used for calculations must be specified, and the
method used to determine or estimate these values must be documented. Where values for
sensitivity and/or specificity for a particular test are specified in this Aquatic Manual, these values
may be used without justification.
Where more than one test is used in a surveillance system (sometimes called using tests in series or
parallel), the overall test system sensitivity and specificity must be calculated using a scientifically valid
method.
Pooled testing involves the pooling of specimens from multiple individuals and performing a
single test on the pool. Pooled testing is an acceptable approach. Where pooled testing is used, the
results of testing must be interpreted using sensitivity and specificity values that have been
determined or estimated for that particular pooled testing procedure and for the applicable pool
sizes being used. Analysis of the results of pooled testing must, where possible, be performed
using accepted, statistically based methodologies, which must be fully documented, including
published references.
3.7. Multiple sources of evidence
Where multiple different data sources providing evidence of freedom from infection exist or are
generated, each of these data sources may be analysed according to the provisions of Sections B.3,
B.4 (for structured surveys) and B.5 (for complex data sources). The resulting estimates of the
confidence in each data source may be combined to provide an overall level of confidence for the
combined data sources.
The methodology used to combine the estimates from multiple data sources:
must be scientifically valid, and fully documented, including references to published material;
and
should, where possible, take into account any lack of statistical independence between
different data sources.
Surveillance information gathered from the same country, zone or aquaculture establishment at
different times may provide cumulative evidence of freedom from infection. Such evidence
gathered over time may be combined into an overall level of confidence. For instance, repeated
annual surveys may be analysed to provide a cumulative level of confidence. However, a single
(larger) survey may be able to achieve the same level of confidence in just 1 year.
Analysis of surveillance information gathered intermittently or continuously over time should,
where possible, incorporate the time of collection of the information to take the decreased value
of older information into account.
4. SPECIFIC REQUIREMENTS FOR STRUCTURED SURVEY DESIGN AND ANALYSIS
One method of generating evidence for freedom from infection is the use of structured, population-
based, targeted surveys. In addition to the requirements specified in Section B.3, the following
guidelines should be used when implementing and analysing surveys to demonstrate freedom from
infection.
4.1. Survey design
The most important unit of diagnosis is the epidemiological unit. The population of epidemiological units
must be clearly defined.
The design of the survey will depend on the size and structure of the population being studied. If
the population is relatively small and can be considered to be homogenous with regards to risk of
infection, a single-stage survey can be used.
In larger populations where a sampling frame is not available, or when there is a likelihood of
clustering of disease, multi-stage sampling is required. In two-stage sampling, at the first stage of
sampling, groups of animals (e.g. ponds, farms or villages) are selected. At the second stage,
animals are selected for testing from each of the selected groups.
Stratification may be used in survey design.
4.2. Sampling
The objective of sampling from a population is to select a subset of units from the population that is
representative of the population with respect to the characteristic of interest (in this case, the
presence or absence of infection). Sampling should be carried out in such a way as to provide the
best likelihood that the sample will be representative of the population, within the practical
constraints imposed by different environments and production systems.
Biased or targeted sampling in this context involves sampling from a defined study population that
has a different probability of infection than the target population of which it is a subpopulation.
Once the study population has been identified, the objective is still to select a representative sample
from this subpopulation.
4.3. Sampling methods
The survey design may involve sampling at several levels.
For sampling at the level of the epidemiological units or higher units, a formal probability sampling (e.g.
simple random sampling) method must be used.
When sampling below the level of the epidemiological unit (e.g. individual animal), the sampling
method used should provide the best practical chance of generating a sample that is representative
of the population of the chosen epidemiological unit. Collecting a truly representative sample of
individual animals (whether from a pond, cage or fishery) is often very difficult. To maximise the
chance of finding infection, the aim should be to bias the sampling towards infected animals, e.g.
selecting moribund animals, life stages with a greater chance of active infection, etc.
The sampling method used at all levels must be fully documented and justified.
4.4. Sample size
The number of units to be sampled from a population should be calculated using a statistically valid
technique that takes at least the following factors into account:
The sensitivity and specificity of the diagnostic test, or test system;
The design prevalence (or prevalences where a multi-stage design is used);
The level of confidence that is desired of the survey results.
Additionally, other factors may be considered in sample size calculations, including (but not
limited to):
The size of the population (but it is acceptable to assume that the population is infinitely large);
The desired power of the survey;
Uncertainty or variability in estimates of sensitivity and specificity.
The specific sampling requirements will need to be tailor-made for each individual disease, taking
into account its characteristics and the specificity and sensitivity of the accepted testing methods
for detecting the disease agent in host populations.
Detailed guidelines are to be provided in the next (fifth) edition of the Aquatic Manual. In the
meantime, the sampling procedures given in Chapters I.1, I.2 and I.3 may be applied.
4.5. Data analysis
Analysis of test results from a survey shall be in accordance with the provisions of Section B.3 and
take at least the following considerations into account:
The survey design;
The sensitivity and specificity of the test, or test system;
The design prevalence (or prevalences where a multi-stage design is used);
The results of the survey.
4.6. Quality assurance
Surveys should include a documented quality assurance system, to ensure that field and other
procedures conform to the specified survey design. Acceptable systems may be quite simple, as
long as they provide verifiable documentation of procedures and basic checks to detect significant
deviations of procedures from those documented in the survey design.
5. SPECIFIC REQUIREMENTS FOR COMPLEX NON-SURVEY DATA SOURCES
Data sources that provide evidence of freedom from infection, but are not based on structured
population-based surveys may also be used to demonstrate freedom, either alone or in combination
with other data sources. Different methodologies may be used for the analysis of such data sources, but
the methodology must comply with the provisions of Section B.3. The approach used should, where
possible, also take into account any lack of statistical independence between observations.
Analytical methodologies based on the use of step-wise probability estimates to describe the surveillance
system may determine the probability of each step either by:
the analysis of available data, using a scientifically valid methodology; or where no data are
available,
the use of estimates based on expert opinion, gathered and combined using a formal, documented
and scientifically valid methodology.
Where there is significant uncertainty and/or variability in estimates used in the analysis, stochastic
modelling or other equivalent techniques should be used to assess the impact of this uncertainty
and/or variability on the final estimate of confidence.
PART 2
EXAMPLE SURVEILLANCE SYSTEMS
The following examples describe surveillance systems and approaches to the analysis of evidence that are
able to meet the requirements of Part 1 of this chapter. The purpose of these examples is:
to illustrate the range of approaches that may be acceptable;
to provide practical guidance and models that may be used for the design of specific surveillance
systems; and
to provide references to available resources that are useful in the development and analysis of
surveillance systems.
While these examples demonstrate ways in which freedom from infection may be successfully
demonstrated, they are not intended to be prescriptive. Countries are free to use different approaches, as
long as they meet the requirements of Part 1 of this chapter.
The examples deal with the use of structured surveys and are designed to illustrate different survey
designs, sampling schemes, the calculation of sample size, and analysis of results. It is important to note
that alternative approaches to demonstrating freedom using complex non-survey-based data sources are
also currently being developed and may soon be published
1
.

1 International EpiLab, Denmark, Research Theme 1: Freedom from disease.
http://www.vetinst.dk/high_uk.asp?page_id=196
EXAMPLE 1 ONE-STAGE STRUCTURED SURVEY (FARM ACCREDITATION)
Context
A freshwater aquaculture industry raising fish in tanks has established a farm accreditation scheme. This
involves demonstrating farm-level freedom from a particular (hypothetical) disease (Disease X). The
disease does not spread very quickly, and is most common during the winter months, with adult fish at the
end of the production cycle being most severely affected. Farms consist of a number of grow-out tanks,
ranging from 2 to 20, and each tank holds between 1000 and 5000 fish.
Objective
The objective is to implement surveillance that is capable of providing evidence that an individual farm is
free from Disease X. (The issue of national or zone freedom, as opposed to farm freedom, is considered
in the next example.)
Approach
The accreditation scheme establishes a set of standard operating procedures and requirements for
recognition of freedom, based on the guidelines given in this chapter. These require farms to undertake a
structured survey capable of producing 95% confidence that the disease would be detected if it were
present. Once farms have been surveyed without detecting disease, they are recognised as free, as long as
they maintain a set of minimum biosecurity standards. These standards are designed to prevent the
introduction of Disease X into the farm (through the implementation of controls specific to the method of
spread of that disease) and to ensure that the disease would be detected rapidly if it were to enter the farm
(based on evidence of adequate health record keeping and the prompt investigation of unusual disease
events). The effective implementation of these biosecurity measures is evaluated with annual on-farm
audits conducted by independent auditors.
Survey standards
Based on the guidelines given in this chapter, a set of standards are established for the conduct of surveys
to demonstrate freedom from infection with causative agent of Disease X. These standards include:
The level of confidence required of the survey is 95% (i.e. Type I error = 5%).
The power of the survey is arbitrarily set at 95% (i.e. Type II error = 5%, which means that there is a
5% chance of concluding that a non-diseased farm is infected).
The target population is all the fish on the farm. Due to the patterns of disease in this production
system, in which only fish in the final stages of grow-out, and only in winter are affected, the study
population is defined as grow-out fish during the winter months.
The issue of clustering is considered. As fish are grouped into tanks, this is the logical level at which
to consider clustering. However, when a farm is infected, the disease often occurs in multiple tanks,
so there is little evidence of strong clustering. Also, the small number of tanks on a single farm means
that it is difficult to define a design prevalence at the tank level (i.e. the proportion of infected tanks
that the survey should be able to detect on the farm). For these reasons, it is decided to treat the
entire grow-out population of each farm as a single homogenous population.
Stratification is also considered. In order to ensure full representation, it is decided to stratify the
sample size by tank, proportional to the population of each tank.
The design prevalence at the animal level is determined based on the epidemiology of the disease.
The disease does not spread quickly, however, in the defined target population, it has been reported
to affect at least 10% of fish, if the population is infected. In order to take the most conservative
approach, an arbitrarily low design prevalence of 2% is used. A prevalence of 10% may have been
used (and would result in a much smaller sample size), but the authorities were not convinced by the
thought that the population could still be infected at a level of say 5%, and disease still not be
detected.
The test used involves destructive sampling of the fish, and is based on an antigen-detection enzyme-
linked immunosorbent assay (ELISA). Disease X is present in some parts of the country (hence the
need for a farm-level accreditation programme). This has provided the opportunity for the sensitivity
and the specificity of the ELISA to be evaluated in similar populations to those on farms. A recent
study (using a combination of histology and culture as a gold standard) estimated the sensitivity of the
ELISA to be 98% (95% confidence interval 96.799.2%), and the specificity to be 99.4% (99.2
99.6%). Due to the relatively narrow confidence intervals, it was decided to use the point estimates of
the sensitivity and specificity rather than complicate calculations by taking the uncertainty in those
estimates into account.
Sample size
The sample size required to meet the objectives of the survey is calculated to take the population size, the
test performance, the confidence required and the design prevalence into account. As the population of
each farm is relatively large, differences in the total population of each farm have little effect on the
calculated sample size. The other parameters for sample size calculation are fixed across all farms.
Therefore, a standard sample size (based on the use of this particular ELISA, in this population) is
calculated. The sample size calculations are performed using the FreeCalc software
2
. Based on the
parameters listed above, the sample size required is calculated to be 410 fish per farm. In addition, the
program calculates that, given the imperfect specificity, it is still possible for the test to produce up to five
false-positive reactors from an uninfected population using this sample size. The authorities are not
comfortable with dealing with false-positive reactors, so it is decided to change the test system to include a
confirmatory test for any positive reactors. Culture is selected as the most appropriate test, as it has a
specificity that is considered to be 100%. However, its sensitivity is only 90% due to the difficulty of
growing the organism.
As two tests are now being used, the performance of the test system must be calculated, and the sample
size recalculated based on the test system performance.
Using this combination of tests (in which a sample is considered positive only if it tests positive to both
tests), the specificity of the combined two tests can be calculated by the formula:
) (
2 1 2 1
Sp Sp Sp Sp Sp
Combined
+ =
which produces a combined specificity of 1 + 0.994 (1 0.994) = 100%
The sensitivity may be calculated by the formula:
Se Se Se
Combined
=
1

which produces a combined sensitivity of 0.9 0.98 = 88.2%
These new values are used to calculate the survey sample size yielding a result of 169 fish. It is worth
noting that attempts to improve the performance of a test (in this case increase specificity) generally result
in a decrease in the performance of the other aspect of the test performance (sensitivity in this example).
However, in this case, the loss of sensitivity is more than compensated for by the decreased sample size
due to the improved specificity.

2 FreeCalc Cameron, AR. Software for the calculation of sample size and analysis of surveys to demonstrate freedom from
disease. Available for free download from http://www.ausvet.com.au.
It is also worth noting that, when using a test system with 100% specificity, the effective power of the
survey will always be 100%, regardless of the figure used in the design. This is because it is not possible to
make a Type II error, and conclude that the farm is infected when it is not.
A check of the impact of population size on the calculated sample size is worthwhile. The calculated
sample size is based on an infinitely large population. If the population size is smaller, the impact on
sample size is shown in the following table:
Population size Sample size
1000 157
2000 163
5000 166
10,000 169
Based on these calculations, it is clear that, for the population sizes under consideration, there is little
effect on the sample size. For the sake of simplicity, a standard sample size of 169 is used, regardless of
the number of grow-out fish on the farm.
Sampling
The selection of individual fish to include in the sample should be done in such a manner as to give the
best chance of the sample being representative of the study population. A fuller description of how this
may be achieved under different circumstances is provided in Survey Toolbox
3
. An example of a single farm
will be used to illustrate some of the issues.
One farm has a total of eight tanks, four of which are used for grow-out. At the time of the survey (during
winter), the four grow-out tanks have 1850, 4250, 4270 and 4880 fish, respectively, giving a total
population of 15,250 grow-out fish.
Simple random sampling from this entire population is likely to produce sample sizes from each tank
roughly in proportion to the number of fish in each tank. However, proportional stratified sampling will
guarantee that each tank is represented in proportion. This simply involves dividing the sample size
between tanks in proportion to their population. The first tank has 1850 fish out of a total of 15,250,
representing 12.13%. Therefore 12.13% of the sample (21 fish) should be taken from the first tank. Using
a similar approach the sample size for the other three tanks is 47, 47 and 54 fish, respectively.
Once the sample for each tank is determined, the problem remains as to how to select 21 fish from a tank
of 1850 so that they are representative of the population. Several options exist.
If the fish can be handled individually, random systematic sampling may be used. This is likely to be
the case if, for example:
fish are harvested during winter and samples can be collected at harvest; or
routine management activities involving handling the fish (such as grading or vaccination) are
conducted during the winter.
If fish are handled, systematic sampling simply involves selecting a fish at regular intervals. For
instance, to select 21 from 1850, the sampling interval should be 1850/21 = 88. This means that
every 88
th
fish from the tank should be sampled. To ensure randomness, it is good practice to use a

3 Survey Toolbox for Aquatic Animal Diseases A Practical Manual and Software Package. Cameron A.R. (2002). Australian
Centre for International Agricultural Research (ACIAR), Monograph No. 94, 375 pp. ISBN 1 86320 350 8. Printed version
available from ACIAR (http://www.aciar.gov.au) Electronic version available for free download from
http://www.ausvet.com.au.
random number between 1 and 88 (in this case) to select the first fish (e.g. using a random number
table), and then select every 88
th
fish after that.
If fish cannot be handled individually (by far the most common, and more difficult, circumstance)
then the fish to be sampled must be captured from the tanks. Fish should be captured in the most
efficient and practical way possible, however every effort should be made to try to ensure that the
sample is representative. In this example, a dip net is the normal method used for capturing fish.
Using a dip net, convenience sampling would involve capturing 21 fish by repeatedly dipping at one
spot and capturing the easiest fish (perhaps the smaller ones). This approach is strongly discouraged.
One method of increasing the representativeness is to sample at different locations in the tank
some at one end, some at either side, some at the other end, some in the middle, some close to the
edge. Additionally, if there are differences among the fish, an attempt should be made to capture fish
in such a way as to give different groups of fish a chance of being caught (i.e. do not just try to catch
the small ones, but include big ones as well).
This method of collecting a sample is far from the ideal of random sampling, but due to the practical
difficulties of implementing random sampling of individual fish, this approach is acceptable, as long
as the efforts made to increase the representativeness of the sample are both genuine and fully
documented.
Testing
Specimens are collected, processed and tested according to standardised procedures developed under the
accreditation programme and designed to meet the requirements of this Aquatic Manual. The testing
protocol dictates that any specimens that test positive to ELISA be submitted for culture, and that any
positive culture results indicate a true positive specimen (i.e. that the farm is not free from disease). It is
important that this protocol be adhered to exactly. If a positive culture is found, then it is not acceptable
to retest it, unless further testing is specified in the original testing protocol, and the impact of such testing
accounted for in the test system sensitivity and specificity estimates (and therefore the sample size).
Analysis
If the calculated sample size of 169 is used, and no positive reactors are found, then the survey will have a
confidence of 95%. This can be confirmed by analysing the results using the FreeCalc software mentioned
above (which reports a confidence level of 95.06%).
It may happen in some cases that the survey is not conducted exactly as planned, and the actual sample
size is less than the target sample size. However, the size of the farm may also be smaller. In these cases, it
is advisable to analyse the farm data on a farm-by-farm basis. For example, if only 165 specimens were
collected from a farm with only 2520 fish, the resulting confidence would still be 95%. If only 160 fish
were collected, the confidence is only 94.5%. If a rigid target of 95% confidence is used, then this survey
would fail to meet that target and more evidence would be required.
EXAMPLE 2 TWO-STAGE STRUCTURED SURVEY (NATIONAL FREEDOM)
Context
A country aims to declare freedom from Disease Y of crustaceans. The industry in this country is based
largely on small-holder ponds, grouped closely together in and around villages. The disease is reasonably
highly contagious, and causes mass mortality mid to late in the production cycle, with affected animals
becoming moribund and dying in a matter of days. Affected animals show few characteristic signs, but an
infected pond will almost invariably break down with mass mortality unless harvested beforehand. It is
more common in late summer, but can occur at any time of year. It also occurs occasionally early in the
production cycle. In this country, there are some limitations to the availability of laboratory facilities and
the transport infrastructure. However, there is a relatively large government structure, and a
comprehensive network of fisheries officers.
Objective
The objective is to establish national freedom from Disease Y. The surveillance system must meet the
requirements of Part 1 of this chapter, but must also be able to be practically implemented in this small-
holder production system.
Approach
The aquaculture authorities decide to use a survey to gather evidence of freedom, using a two-stage survey
design (sampling villages at the first level, and ponds at the second). Laboratory testing of specimens from
a large number of farms is not considered feasible, so a combined test system is developed to minimise the
need for expensive laboratory tests.
The unit of observation and analysis is, in this case, the pond, rather than the individual animal. This
means that the diagnosis is being made at the pond level (an infected pond or a non-infected pond) rather
than at the animal level.
The survey is therefore a survey to demonstrate that no villages are infected (using a random sample of
villages and making a village-level diagnosis). The test used to make a village-level diagnosis is, in fact,
another survey, this time to demonstrate that no ponds in the village are affected. A test is then performed
at the pond level (farmer observation followed, if necessary, by further laboratory testing).
Survey standards
The confidence to be achieved by the survey is 95%. The power is set at 95% (but is likely to be
virtually 100% if the test system used achieves nearly 100% specificity, as demonstrated in the
previous example).
The target population is all ponds stocked with shrimp in the country during the study period. The
study population is the same, except that those remote areas to which access is not possible are
excluded. As outbreaks can occur at any time of year, and at any stage of the production cycle, it is
decided not to further refine the definition of the population to target a particular time or age.
Three tests are used. The first is farmer observation, to determine if mass mortality is occurring in a
particular pond. If a pond is positive to the first test (i.e. mass mortality is detected), a second test is
applied. The second test used is polymerase chain reaction (PCR). Cases positive to PCR are further
tested using transmission experiments.
Farmer observation can be treated as a test just like any other. In this case, the observation of
mass mortality is being used as a test for the presence of Disease Y. As there are a variety of
other diseases that are capable of causing mass mortality, the test is not very specific. On the
other hand, it is quite unusual for Disease Y to be present, and not result in mass mortality, so
the test is quite sensitive. A standard case definition is established for mass mortality (for
instance, greater than 20% of the ponds population of shrimp observed dead in the space of
less than 1 week). Based on this definition, farmers are able to diagnose each pond as having
mass mortality. Some farmers may be over-sensitive and decide that mass mortality is occurring
when only a small proportion of shrimp are found dead (false positives, leading to a decrease in
specificity) while a small number of others fail to recognise the mortalities, decreasing sensitivity.
In order to quantify the sensitivity and specificity of farmer observation of mass mortalities, as a
test for Disease Y, a separate study is carried out. This involves both a retrospective study of the
number of mass mortality events in a population that is thought to be free from disease, as well
as a study of farmers presented with a series of mortality scenarios, to assess their ability to
accurately identify a pond with mass mortality. By combining these results, it is estimated that
the sensitivity of farmer-reported mass mortalities as a test for Disease Y is 87% while the
specificity is 68%.
When a farmer detects a pond with mass mortality, specimens are collected from moribund
shrimp following a prescribed protocol. Tissue samples from 20 shrimp are collected, and
pooled for PCR testing. In the laboratory, the ability of pooled PCR to identify a single infected
animal in a pool of 20 has been studied, and the sensitivity of the procedure is 98.6%. A similar
study of negative specimens has shown that positive results have occasionally occurred, probably
due to laboratory contamination, but maybe also because of the presence of non-viable genetic
material from another source (shrimp-based feed stuffs are suspected). The specificity is
therefore estimated at 99%.
Published studies in other countries have shown that the sensitivity of transmission tests, the
third type of test to be used, is 95%, partly due to variability in the load of the agent in
inoculated material. The specificity is agreed to be 100%.
Based on these figures, the combined test system sensitivity and specificity are calculated using
the formulae presented in Example 1, first with the first two tests, and then with the combined
effect of the first two tests and the third test. The result is a sensitivity of 81.5% and a specificity
of 100%.
The design prevalence must be calculated at two levels. First, the pond-level design prevalence (the
proportion of ponds in a village that would be infected if disease were present) is determined. In
neighbouring infected countries, experience has shown that ponds in close contact with each other
are quickly infected. It is unusual to observe an infected village with fewer than 20% of ponds
infected. Conservatively, a design prevalence of 5% is used. The second value for design prevalence
applies at the village level, or the proportion of infected villages that could be identified by the survey.
As it is conceivable that the infection may persist in a local area without rapid spread to other parts of
the country, a value of 1% is used. This is considered to be the lowest design prevalence value for
which a survey can be practically designed.
The population of villages in the country is 65,302, according to official government records. Those
with shrimp ponds number 12,890, based on records maintained by the aquaculture authorities.
These are generated through a five-yearly agricultural census, and updated annually based on reports
of fisheries officers. There are no records available of the number of ponds in each of these villages.
Sample size
Sample size is calculated for the two levels of sampling, first the number of villages to be sampled and
then the number of ponds to be sampled. The number of villages to be sampled depends on the sensitivity
and the specificity of the test used to classify villages as infected or not infected. As the test used in each
village is really just another survey, the sensitivity is equal to the confidence and the specificity is equal to
the power of the village-level survey. It is possible to adjust both confidence and power by changing the
sample size in the village survey (number of ponds examined), which means that we can determine, within
certain limits, what sensitivity and specificity we achieve.
This allows a flexible approach to sample size calculation. If a smaller first-stage sample size is desired (a
small number of villages), a high sensitivity and specificity are needed, which means that the number of
ponds in each village that need to be examined is larger. A smaller number of ponds will result in lower
sensitivity and specificity, requiring a larger number of villages. The approach to determining the optimal
(least cost) combination of first- and second-stage sample sizes is described in Survey Toolbox.
A further complication is presented by the fact that each village has a different number of ponds. In order
to achieve the same (or similar) confidence and power (sensitivity and specificity) for each village, a
different sample size may be required. The authorities choose to produce a table of sample sizes for the
number of ponds to sample in each village, based on the total ponds in each village.
An example of one possible approach to determining the sample size follows:
The target sensitivity (confidence) achieved by each village-level survey is 95%. The target specificity is
100%. Using the FreeCalc software, with a design prevalence of 1% (the survey is able to detect disease if
1% or more villages are infected), the first-stage sample size is calculated as 314 villages. Within each
village, the test used is the combined test system described above with a sensitivity of 81.5% and a
specificity of 100%. Based on these figures the following table is developed, listing the number of ponds
that need to be sampled in order to achieve 95% sensitivity.
Population Sample size
30 29
40 39
60 47
80 52
100 55
120 57
140 59
160 61
180 62
200 63
220 64
240 64
260 65
280 65
300 66
320 66
340 67
360 67
380 67
400 67
420 68
440 68
460 68
480 68
500 68
1000 70
Sampling
First-stage sampling (selection of villages) is done using random numbers and a sampling frame based on
the fisheries authorities list of villages with shrimp ponds. The villages are listed on a spreadsheet with
each village numbered from 1 to 12,890. A random number table (such as that included in Survey Toolbox)
or software designed for the generation of random numbers (such as EpiCalc
4
) is used.

4 http://www.myatt.demon.co.uk/epicalc.htm
The second stage of sampling involves random selection of ponds within each village. This requires a
sampling frame, or list of each pond in the village. The fisheries authorities use trained local fisheries
officers to coordinate the survey. For each selected village, the officer visits the village and convenes a
meeting of all shrimp farmers. At the meeting, they are asked how many ponds they have and a list of
farmers names and the number of ponds is compiled. A simple random sample of the appropriate
number of ponds (between 29 and 70, from the table above, depending on the number of ponds in the
village) is selected from this list. This is done either using software (such as Survey Toolboxs
RandomAnimal program), or manually with a random number table or decimal dice for random number
selection. Details of this process are described in Survey Toolbox. This selection process identifies a
particular pond in terms of the name of the owner, and the sequence number amongst the ponds owned
(e.g. Mr Smiths 3
rd
pond). Identification of the actual pond is based on the owners own numbering system
for the ponds.
Testing
Once ponds have been identified, the actual survey consists of testing those ponds. In practice, this
involves the farmers observing the ponds during one complete production cycle. The local fisheries officer
makes weekly visits to each farmer to check if any of the selected ponds have suffered mass mortality. If
any are observed (i.e. the first test is positive), 20 moribund shrimp are collected for laboratory
examination (first PCR, and then, if positive, transmission experiments).
Analysis
Analysis is performed in two stages. First, the results from each village are analysed to ensure that they
meet the required level of confidence. If the target sample size is achieved (and only negative results
obtained), the confidence should be 95% or greater in each village. At the second stage, the results from
each village are analysed to provide a country level of confidence. Again, if the target sample size (number
of villages) is achieved, this should exceed 95%.
EXAMPLE 3 SPATIAL SAMPLING AND THE USE OF TESTS WITH IMPERFECT SPECIFICITY
Context
A country has an oyster culture industry, based primarily on rack culture of oysters in 23 estuaries
distributed along the coastline. In similar regions in other countries, Disease Z causes mortalities in late
summer/early autumn. During an outbreak a high proportion of oysters are affected, however, it is
suspected that the agent may be present at relatively low prevalence in the absence of disease outbreaks.
Objective
The national authorities wish to demonstrate national freedom from Disease Z. If the disease should be
detected, a secondary objective of the survey is to collect adequate evidence to support zoning at the
estuary level.
Approach
The authorities conclude that clinical surveillance for disease outbreaks is inadequate because of the
possibility of low level subclinical infections. It is therefore decided to base surveillance on a structured
two-stage survey, in which sampled oysters are subjected to laboratory testing. The first stage of the survey
is the selection of estuaries. However, due to the objective of providing evidence for zoning (should
disease be found in any of the estuaries), it is decided to use a census approach and sample every estuary.
In essence this means that there will be 23 separate surveys, one for each estuary. A range of options for
sampling oysters are considered, including sampling at harvest or marketing, or using farms (oyster leases)
as a level of sampling or stratification. However the peak time of activity of the agent does not correspond
to the harvest period, and the use of farms would exclude the significant numbers of wild oysters present
in the estuaries. It is therefore decided to attempt to simulate simple random sampling from the entire
oyster population in the estuary, using a spatial sampling approach.
Survey standards
The target population is all of the oysters in each of the estuaries. The study population is the oysters
present during the peak disease-risk period in late summer early autumn. Wild and cultured oysters
are both susceptible to disease, and may have associated with them different (but unknown) risks of
infection. They are therefore both included in the study population. As will be described below,
sampling is based on mapping. Therefore the study population can more accurately be described as
that population falling within those mapped areas identified as oyster habitats.
A design prevalence value is only required at the oyster level (as a census is being used at the estuary
level). While the disease is often recognised with very high prevalence during outbreaks, a low value is
used to account for the possibility of persistence of the agent in the absence of clinical signs. A value
of 2% is selected.
The test used is histopathology with immuno-staining techniques. This test is known to produce
occasional false-positive results due to nonspecific staining, but is very sensitive. Published studies
indicate values of 99.1% for sensitivity and 98.2% for specificity. No other practical tests are
available. This means that it is not possible to definitively differentiate false positives from true
positives, and that in a survey of any size, a few false positives are expected (i.e. 1.8%).
The confidence is set at 95% and the power at 80%. In the previous examples, due to the assumed
100% specificity achieved by use of multiple tests, the effective power was 100%. In this case, with
imperfect specificity, there will be a risk of falsely concluding that a healthy estuary is infected, so the
power is not 100%. The choice of a relatively low figure (80%) means that there is a 1 in 5 chance of
falsely calling an estuary infected when it is not infected, but it also dramatically decreases the survey
costs, through a lower sample size.
Sample size
Based on the assumption that the sampling procedure will mimic simple random sampling, the sample size
(number of oysters to sample per estuary) can be calculated with FreeCalc. The population size (number of
oysters per estuary) is assumed to be very large. The calculated sample size, using the sensitivity, specificity
and design prevalence figures given above, is 450. FreeCalc also reports that, based on this sample size and
the specificity of the test, it is possible to get 10 or fewer false-positive test results, and still conclude that
the population is free from disease. This is because, if the population were infected at 2% or greater, the
anticipated number of positive reactors from a sample of 450 would be greater than 10. In fact, we would
expect 9 true positives (450 2% 99.1%) and 8 false positives (450 98% 1.8%) or a total of 17
positives if the population were infected at a prevalence of 2%.
This illustrates how probability theory and adequate sample size can help differentiate between true- and
false-positive results when there is no alternative but to use a test with imperfect specificity.
Sampling
The aim is to collect a sample of 450 oysters that represent an entire estuary. Simple random sampling
depends on creating a sampling frame listing every oyster (not possible) and systematic sampling depends
on being able to (at least conceptually) line up all the oysters (again, not possible). The authorities decide to
use spatial sampling to approximate simple random sampling. Spatial sampling involves selecting random
points (defined by coordinates), and then selecting oysters near the selected points. In order to avoid
selecting many points with no oysters nearby, the estuary is first mapped (the fisheries authorities already
have digital maps defining oyster leases available). To these maps areas with significant concentrations of
wild oysters are also added, based on local expertise. Pairs of random numbers are generated such that the
defined point falls within the defined oyster areas. Other schemes are considered (including using a rope
marked at regular intervals, laid out on a lease to define a transect, and collecting an oyster adjacent to each
mark on the rope) but the random coordinate approach is adopted.
Survey teams then visit each point by boat (using a GPS [Global Positioning System] unit to pinpoint the
location). A range of approaches is available for selecting which oyster to select from a densely populated
area, but it should involve some effort at randomness. Survey staff opt for a simple approach: when the
GPS receiver indicates that the site has been reached, a pebble is tossed in the air and the oyster closest to
the point where it lands is selected. Where oysters are arranged vertically (e.g. wild oysters growing up a
post), a systematic approach is used to determine the depth of the oyster to select. First, an oyster at the
surface, next, an oyster halfway down, and thirdly, an oyster as deep as can be reached from the boat.
This approach runs the risk of bias towards lightly populated areas, so an estimate of the relative density of
oysters at each sampling point is used to weight the results (see Survey Toolbox for more details).
Testing
Specimens are collected, processed, and analysed following a standardised procedure. The results are
classified as definitively positive (showing strong staining in a highly characteristic pattern, possibly with
associated signs of tissue damage), probably positive (on the balance of probabilities, but less characteristic
staining), and negative.
Analysis
The interpretation of the results when using a test with imperfect specificity is based on the assumption
that, in order to conclude that the population is free from infection, any positive result identified is really a
false positive. With a sample size of 450, up to 10 false positives may be expected while still concluding
that the population is free from disease. However, if there is reasonable evidence that there is even a single
true positive, then the population cannot be considered free. This is the reason for the classification of
positive results into definitive and probable positives. If there are any definitive positives at all, the
population in that estuary must be considered infected. The probable positives are consistent with false
positives, and therefore up to 10 may be accepted. Using FreeCalc the actual confidence achieved based on
the number of (presumed) false positives detected can be calculated. For instance, if 8 probably positive
results were detected from an estuary, the confidence level for the survey would be 98.76%. On the other
hand, if 15 probably positive results were detected, the confidence is only 61.9%, indicating that the
estuary is likely to be infected.
Discussion
Normally, it may be safely assumed that a surveillance system aimed at demonstrating freedom from
disease is 100% specific. This is because any suspected occurrence of disease is investigated until a
definitive decision can be made. If the conclusion is that the case is truly a case of disease, then there is no
issue of declaring freedom the disease is known to be present. This example presents a different
situation where, due to lack of suitable tests, it is not possible for the surveillance system to be 100%
specific. This may represent an unusual situation in practice, but illustrates that methods exist for dealing
with this sort of problem. In practice, a conclusion that a country (or estuary) is free from infection, in the
face of a small (but statistically acceptable) number of positive results, will usually be backed up by further
evidence (such as the absence of clinical disease).
*
* *
C HA P T E R 1 . 1 . 5 .

ME T HODS F OR DI S I NF E CT I ON OF
AQUACUL T URE E S T ABL I S HME NT S

A. METHODS FOR DISINFECTION OF FISH FARMS
The choice of disinfection procedures depends on the size, type and nature of the materials and sites to
be disinfected. With the exception of the skin of personnel and eggs, which must be disinfected with
non-corrosive products, the surfaces to be disinfected consist of fabric or woven material (clothes,
nets), hard surfaces (plastic, cement) or permeable materials (earth, gravel). Disinfection is more
difficult for permeable surfaces and requires more time. Table 1 indicates the most common
ingredients and the methods to be used on the basis of these criteria.
The use of chemical products entails the implementation of measures to protect personnel and the
cultured animals and to mitigate environmental effects. It is first necessary to protect the skin and eyes
from contact with dangerous substances by using impermeable clothing, boots, eye protection and a
hat. The respiratory tract must be protected by a mask and the operator must not touch any food
without having thoroughly washed his/her hands. Finally, the products must be stored in such a way as
not to present direct or indirect danger to animal/fish or human life or the environment.
The material must be thoroughly cleaned before being disinfected. Organic material generated/
removed during the cleaning process, such as pond sludge, etc., should also be disposed of in an
appropriate manner that prevents spread of disease by such material and is environmentally safe.
Ideally, approved procedures for the use of disinfectants in aquaculture should be established. An
approval scheme should consider disinfection effect against target pathogens, toxicological and
ecotoxicological properties of the products.
Following disinfection or stamping-out, the farm should be restocked from a disease-free source.
Table 1. Disinfection and method of use
Processes Indications Method of use* Comments
Physical
Desiccation, sunlight Fish pathogens on earthen
bottoms
Dry for 3 months at an
average temperature
of 18C
Drying period can be
reduced by the use of a
chemical disinfectant
Dry heat Fish pathogens on concrete,
stone, iron, ceramic surfaces
Flame-blower,
blow-lamp

Damp heat Fish pathogens in
transportation vehicle tanks
Steam at 100C or more
for 5 minutes

Ultra-violet rays
UV-C (254 nm)
Viruses and bacteria 10 mJ/cm
2
Minimum lethal dose
Chapter 1.1.5. Methods for disinfection of aquaculture establishments
Table 1 (continued). Disinfection and method of use
Physical
Ultra-violet rays
UV-C (254 nm)
Infectious pancreatic
necrosis (IPN) and
nodavirus (VNN/VER
1
) in
water
125200 mJ/cm
2

Chemical
Acetic acid Infectious salmon anaemia
(ISA)
0.040.13%
Quartenary
ammonia
Virus, bacteria, hands, plastic
surfaces
0.11 g/litre for
115 minutes
IPN virus resistant
Calcium oxide
a
Fish pathogens on dried
earth-base
0.5 kg/m
2
for 4 weeks Replace in water and
empty disinfected pools
keeping the effluents at
pH <8.5
Calcium
hypochlorite
a

Bacteria and viruses on all
clean surfaces and in water
30 mg available
chlorine/litre. Leave to
inactivate for several
days or neutralise with
Na thiosulfate after
3 hours
Can be neutralised with
sodium thiosulfate.
See special
recommendations
Calcium cyanamide
a
Spores on earthen bottoms 3000 kg/ha on dry
surfaces; leave in contact
for 1 month

Chloramine T Destroys ISA 1% for 5 minutes
Chloramine T Destroys IPN 1% for 30 minutes
Chlorine dioxide ISA 100 ppm for 5 minutes In water of low organic
loading
Formic acid Ensile fish waste pH <4 after at least
24 hours
Destroys bacterial fish
pathogens and ISA
butnot IPN
Formalin Fish pathogens in sealed
premises
Released from
formogenic substances,
generally
trioxymethylene. Comply
with instructions
Nodavirus resistant
Hydrogen peroxide ISA virus 0.020.06%
Iodine (iodophors) Bacteria, viruses on nets,
boots and clothing
200 mg iodine/litre for a
few seconds
See special
recommendations
Iodine (iodophors) Hands, smooth surfaces >200 mg iodine/litre a
few seconds

1 Viral nervous necrosis /Viral encephalopathy and retinopathy
Table 1 (continued). Disinfection and method of use
Chemical
Ozone Sterilisation of water, fish
pathogens
0.21 mg/litre for
3 minutes
Costly and very toxic for
fish and humans
Ozone in seawater Surfaces, equipment 0.51 mg/litre TRO
2
for
3060 minutes

Poroxy compounds,
e.g. Virkon
IPN virus 1% for 1 minute
Peracetic acid ISA virus 0.080.25%
Sodium
hydroxide
a

Fish pathogens on resistant
surfaces with cracks
Mixture:
Sodium hydroxide, 100 g
Teepol, 10 g
Calcium hydroxide, 500
g
Water, 10 litres
Spray, 1 litre/10 m
2

Leave for 48 hours
The most active
disinfectant Ca(OH)
2

stains the surfaces
treated; Teepol is a
tensio-active agent.
Sodium
hypochlorite
a

Bacteria and viruses on all
clean surfaces and in water
30 mg available
chlorine/litre. Leave to
inactivate for a few days
or neutralise with Na
thiosulfate after 3 hours

Sodium
hypochlorite
a

Nets, boots and clothing 200 mg to 1 g available
chlorine/litre for several
minutes. Leave to
inactivate for a few days
or neutralise with Na
thiosulfate after 3 hours

Sodium
hypochlorite
a

Hands Rinse with clean water or
neutralise with
thiosulfate

a Dangerous See precautions indicated in general recommendations
* The concentrations indicated are those for the active substance. NB: The chemicals must be approved for the
prescribed use and used according to the manufacturers specifications.
2. NEUTRALISATION OF HALOGENS
Chlorine and iodine are highly toxic for aquatic animals and, in order to prevent serious accidents that
could result from a manipulation error, it is recommended to neutralise these products with sodium
thiosulfate five moles of thiosulfate neutralise four moles of chlorine. The molecular proportions are
the same for iodine.
Accordingly, in order to inactivate chlorine, the amount of thiosulfate should be 2.85 times the amount
of chlorine (in grams):
Number of grams of thiosulfate = 2.85 number of grams of chlorine.
For iodine, the amount of thiosulfate should be 0.78 times the amount of iodine in grams:
Number of grams of thiosulfate = 0.78 number of grams of iodine.
It is also possible to prepare a thiosulfate solution at 1% by weight, in which case the neutralising
volumes will be as follows (in ml):
1. for chlorine:
28.5 [number of litres of the disinfecting solution concentration mg/litre] / 100
2. for iodine:
it is necessary to multiply by 7.8 instead of by 28.5.
B. METHODS FOR DISINFECTION OF MOLLUSCS FARMS
The general principles pertaining to disinfection of mollusc farms (hatcheries, holding facilities) involve
the application of chemical treatments in sufficient concentrations, and for sufficient periods, to kill all
pathogenic organisms that would otherwise gain access to surrounding water systems. As the inherent
toxicity of disinfectants prohibits safe use in open water, or open water systems, disinfection can only
reasonably be applied to hatcheries and tank holding facilities, and, as a rule, all disinfectants must be
neutralised before release into the surrounding environment. In addition, as mollusc farms are generally
seawater based, compounds produced during seawater disinfection (residual oxidants) must also be
disposed of carefully.
Disinfection of eggs and larval stages is not considered practical for most molluscan systems. In
addition, there is little information on specific disinfection procedures for pathogens of molluscs (i.e.
Marteilia spp., Haplosporidium spp., Bonamia spp., Perkinsus spp., iridovirus and pathogenic levels of
marine microbes) or seawater. Therefore, disinfectants and concentrations are based on related
pathogens or seawater sterilisation. Three stages of disinfection can be applied to hatcheries:
a) pretreatment of influent water, e.g. filters (1.0 and 0.22 m) or chemical disinfection (see Section
B.2.) = protection of stocks of molluscs;
b) treatment within the facilities (especially recycling systems) = protection of stocks of molluscs;
c) treatment of effluent water = protection of the environment.
2. DISINFECTANTS* PIPELINES AND TANKS
Routine disinfection of pipelines and tanks is highly recommended; the frequency of disinfection will
vary according to the turnover of stocks of molluscs. High concentrations of molluscs should be
rotated between disinfected tanks as often as practical and/or kept in seawater that has been
disinfected with ozone (see Section B.3.a) or chlorine (see Section B.3.b) and subsequently neutralised.
Each new batch of molluscs introduced into a facility should be placed in predisinfected tanks.
As the presence of organic matter will reduce the disinfection capacity of most disinfectants, filtering
influent water (see Section B.1.a) is recommended. In addition, all surfaces must be thoroughly cleaned
prior to disinfection. The detergent used must be compatible with the disinfectant and both must be

2 TRO: Total residual oxidant
* The products specified have proven satisfactory for the purposes indicated; this does not mean that other products may not
be equally satisfactory.
compatible with the surface being treated (e.g. iodophor solutions are generally acidic so cannot be
used on concrete, which is alkaline). Ensure that the waste produced from washing is disinfected
before disposal. Complete coverage of the surfaces is required, e.g. using a high pressure spray or soak.
Wear appropriate protective clothing when working with any disinfectant (see Section A.1).
Regular air- or heat-drying of pipelines (daily), tanks and other equipment (e.g. algal culture carboys), in
addition to disinfection of their surfaces, is also recommended (especially for disease outbreaks of
unidentified aetiology).
a) Chlorine is usually applied as sodium hypochlorite (Chlorox, household bleach, etc.). Fill all
pipelines with 50 mg chlorine/litre (= 50 parts per million [ppm]). Allow an exposure time of at
least 30 minutes before flushing with clean seawater. This solution is effective against most
microbial agents as well as labyrinthulid protozoans. Chlorinated seawater must be neutralised
prior to release from the holding facility. Optimal neutralisation is achieved by passage through
activated charcoal (removes excess chlorine and chloramines). Reducing agents such as sodium
thiosulfate or aeration (which do not remove toxic chloramines) may also be used.
b) Iodophors are generally applied as alkaline solutions (Wescodyne, Betadine) at 200250 mg
iodine/litre (ppm) with a contact time of at least 10 minutes.
NOTE: Iodophors are not effective against certain protozoans in suspension, e.g. over 1000 mg
iodine/litre is tolerated by Labyrinthuloides haliotidis of abalone. Iodophors may be effective against
protozoan parasites following air or heat drying of tank surfaces and pipelines.
3. DISINFECTANTS EFFLUENT WATER
a) Ozone has been used successfully in controlling the microbial content of effluent water from
quarantine facilities. Residual compounds, formed as a result of the interaction of ozone with
seawater (residual oxidants), at levels of 0.081.0 mg/litre are considered sufficient to significantly
reduce live microbes (principally bacteria).
NOTE: The measurement of residual ozone in seawater is problematic due to the rapid and
continuous formation of oxidant products in seawater. Residuals formed between ozone and
seawater (hypobromite, bromine or hypobromous acid) are toxic to oyster larvae (and possibly
other mollusc larvae) and should be removed using a charcoal filter before passing through/out of
the mollusc facility. UV treatment of seawater post-ozonation may be required for complete
sterilisation, e.g. for quarantine.
b) Chlorine administered as sodium hypochlorite at a concentration of 25 mg chlorine/litre is
effective against certain protozoans (L. haliotidis); however, 50 mg chlorine/litre is recommended
for complete microbial sterilisation (as for pipelines and tanks see Section B.2.a). Higher
concentrations may be used under certain conditions (e.g. quarantine); however, these require
proportionately greater neutralisation treatments and exhaust systems to deal with the toxic fumes
produced.
c) Iodophors are not as effective as the above two treatments for killing protozoans.
4. DISINFECTANTS CLOTHING AND EQUIPMENT
Clean surfaces with detergent and disinfectants prior to proper disinfection.
a) Iodophors (e.g. Wescodyne, Betadine) at 200250 mg iodine/litre can be used as a footbath.
NOTE: Iodophors will stain clothing.
b) Chlorine (household bleach solution at 50 mg chlorine/litre) is also an effective footbath or
equipment wash.
c) Sodium hydroxide (1% NaOH + 0.1% Teepol or other detergent) makes an effective footbath
for rubber boots. NOTE: Do NOT use for dress shoes/boots.
5. SPECIAL RECOMMENDATIONS
a) Both chlorine and ozone produce long-lived residual oxidant compounds in seawater. Seawater at
35 parts per thousand (ppt) salinity contains 60 ppm bromide ion, which produces hypobromite in
the presence of ozone. Disinfected artificial seawater, at the same salinity, produces bromine and
hypobromous acid. As these, along with other residual compounds, are toxic to larval oysters (and
possibly other molluscs), treated seawater must be passed through an activated charcoal filter
before being used for live mollusc larvae.
Alternative protocols for halogen neutralisation involve treatment with sodium or potassium
thiosulfate (see Section A.2).
b) Monitoring of residual oxidants must be carried out regularly, especially where temperature
fluctuations occur. As residual ozone cannot be measured accurately in seawater, alternative
monitoring protocols must be installed, such as a feedback loop.
Exhaust systems should also be in place to remove toxic fumes (produced during disinfection)
from enclosed work areas. Ensure compliance with local atmospheric regulations when
discharging toxic fumes.
c) The following management practices can be used to reduce opportunistic pathogen proliferation
within a mollusc hatchery or holding facility:
i) maintain pathogen-free algal stocks and cultures;
ii) use appropriate water filtration, regular disinfection of tanks, pipes and equipment, and
footbaths, and water changes;
iii) isolate infected stocks and associated equipment at the first sign of disease;
iv) discard infected stock and sterilise equipment;
v) identify the source of infection within the holding facility to prevent further infection (algal
stocks, seawater influent system, broodstock, larval stock).
C. METHODS FOR DISINFECTION OF CRUSTACEAN FARMS
The choice of a disinfection method for use in a crustacean farm depends on many factors, which may
include: the reason(s) for disinfection, whether the farm is a broodstock facility, hatchery, or growout
farms; and the type of growout farm. Because the penaeid shrimp are the hosts for all but one of the
crustacean diseases currently listed in the Aquatic Code, this Section will focus on penaeid shrimp.
2. REASON(S) FOR DISINFECTION
Disinfection is employed as a common disease management tool in shrimp farming. It may be used as
a routine practice in biosecurity programmes designed to exclude specific diseases, as well as a routine
sanitary measure employed to reduce disease incidence within farms, or it may be used in disease
eradication (stamping out) efforts. The specific reason for disinfection, will determine the disinfection
strategy used and how it is applied.
3. OCCURRENCE OF LISTED DISEASES
When an OIE listed disease, or an important but unlisted emerging disease occurs for the first time at a
particular farm, at a particular site (i.e. at a quarantine facility), or within a region or country previously
believed to be free of that disease, it may be advisable, if not required, to eradicate the disease by
depopulating the facility and performing a thorough disinfection of all or part of the facility. Fallowing
of the affected facility for a defined period of time may be warranted in some situations (see Chapter
1.7.1, Guidelines for fallowing in aquaculture in the Aquatic Code).
4. PREVENTION OF DISEASE SPREAD TO WILD POPULATIONS
The direct disposal of diseased populations of live shrimp (any life stage; i.e. fertilised or unfertilised
eggs, larvae, postlarvae, juveniles, or adults) or waste products derived from them (i.e. processing plant
wastes such as shells, broken shrimp pieces, etc.) into receiving waters (i.e. creeks, rivers, estuaries,
bays, littoral areas) is a dangerous practice that facilitates the spread of disease from farmed populations
to wild crustacean stocks or to neighbouring farms that use the same water supply, and it should not be
permitted to occur. With cultured stocks, when the decision is made to discard a population (i.e. that is
being cultivated in a hatchery tank or growout pond) due to the presence of disease (or poor culture
performance which may be due to an undiagnosed disease), the stock in the tank or pond should be
harvested and/or humanely killed in the tank or pond. The water in the tank or pond should be
disinfected (see specific subsections on disinfection of tanks and growout ponds in the Section 5) prior
to discharge. The emptied tank or pond should be disinfected prior to restocking.
5. ROUTINE SANITATION AND BIOSECURITY
Many crustacean farms, especially those cultivating penaeid shrimp, employ measures that use a
number of disinfection methods for disease prevention and control. These measures may be part of a
farms routine biosecurity programme that may be designed for exclusion of specific diseases as well as
serving as general pest and disease exclusion measures.
5.1. Disinfectants
The following list comprises the disinfectants recommended for use in shrimp farms (the
appropriate disinfectant regimes for each specific application are discussed in the appropriate
subsection):
chlorine (as calcium hypochlorite, HTH or a bleach solution containing a sufficient
concentration of hypochlorite);
formaldehyde gas (from sublimated paraformaldehyde or concentrated formalin/potassium
permanganate reaction);
iodine (as contained in iodophors);
lime (as calcium oxide or calcium hydroxide);
UV light (from natural sunlight);
ozone;
steam;
hot water (60C);
concentrated acids;
desiccation;
detergents (for general cleaning, with some degree of disinfection capability for many
products).
5.2. Hatcheries and broodstock rearing/holding facilities
Virtually all penaeid shrimp hatcheries and broodstock holding/rearing facilities use seawater that
has been disinfected to remove potential pathogens, pests, and disease-carrying agents via
mechanical filtration, UV irradiation, and/or chemical disinfection. This may be by passive source
water filtration (i.e. by the use of seawater wells or well points) or by mechanical filtration using
high pressure pumps and a variety of water filtration devices and pore sizes. Some facilities use
filtration coupled with UV light disinfection of source water, while others use chemical
disinfection methods, using either chlorination and de-chlorination or high doses of ozone and
subsequent removal of residual oxidants. Chemical disinfection of source water typically requires
the use of one or more water storage reservoirs in which the water is treated and detoxified before
use in the shrimp hatchery or broodstock facility. Numerous manuals are available that provide
specifics on hatchery and broodstock facility design and operation for shrimp culture, and in
which details on source water disinfection are provided.
a) Disinfection of eggs and larvae in penaeid shrimp hatcheries
Certain penaeid shrimp viral diseases (i.e. Spherical baculovirosis, Tetrahedral baculovirosis,
Hepatopancreatic parvovirus infections) are transmitted by faecal contamination of spawned
eggs. These diseases, as well as infections due to certain other shrimp viruses such as White
spot disease virus, and certain bacterial and fungal disease agents, can be eliminated or have
their incidence reduced through the routine use of disinfection protocols when used to surface
disinfect eggs and/or recently hatched nauplii. A widely used method is given below:
For fertilised eggs
3

Collect fertilised eggs. Rinse with running seawater for 12 minutes. Fully immerse eggs in
100 ppm (parts per million) formalin for 1 minute. Fully immerse eggs in iodophor (0.1 ppm
iodine) for 1 minute. Rinse in running seawater 35 minutes. Transfer to disinfected larval
rearing tanks.
For nauplii
4

Using phototaxic response to light, collect nauplii with netting or screen. Rinse with running
seawater for 12 minutes. Fully immerse nauplii in 400 ppm formalin for 3060 seconds. Fully
immerse nauplii in iodophor (0.1 ppm iodine) for 1 minute. Rinse in running seawater 3
5 minutes. Transfer to disinfected larval rearing tanks.
b) Disinfection of tanks, equipment, pipes, air stones, etc.
For routine sanitation, hatchery and broodstock tanks (i.e. tanks for broodstock maturation,
matting, spawning, larval rearing and indoor nursery) should be cleaned, disinfected and dried
between use. Tanks used for the above-named purposes in crustacean (especially shrimp)
hatcheries are typically precast fiberglass tanks or they are constructed of concrete or wood
and either coated or painted with resin-based coatings (e.g. epoxy or fiberglass resin) or lined
with plastic liners manufactured for that purpose. After harvest of the stock from the tank, all
loose objects and large-sized organic debris such as algae, faeces and left-over feed should be
removed. With relatively small tanks, it is advisable after harvest of the stock to fill the tank to
capacity, immerse all nonporous corrosion resistant equipment (i.e. airlines, air stones, stand
pipes, screens, sampling containers, etc.) in the tank, and then add calcium hypochlorite to
provide a minimum of 200 ppm of free chlorine. This should be allowed to set overnight.
After the proper chlorinated soak-time, the tank can be drained and freshwater rinsed. Before
draining the system, the treated water should be dechlorinated (see specific subsections on
chlorination described in this Section), unless appropriate effluent collection and treatment
systems are in place. After the tank has been rinsed it should be allowed to completely dry. In

3 Fertilised eggs are more sensitive than nauplii to formalin.
4 Nauplii are much easier to collect than are fertilised eggs in hatcheries.
the case of large tanks, an initial cleaning to remove loose debris should be followed by
disinfection with a concentrated (~1600 ppm as chlorine) solution of calcium hypochlorite. All
inside and outside surfaces should then be sprayed with this chlorine solution. The tank should
then be allowed to set for several hours and then rinsed, filled and flushed. Surfaces should
then be scrubbed free of all remaining material. After disinfection with chlorine, small or large
tanks should be rinsed with clean water, then filled and flushed to ensure that no chlorine
residues remain before the tank is restocked for another crop.
5.3. Disinfection of growout ponds
Following the routine harvest of a crop from a growout pond (or from a large tank or raceway
used for growout of a crop), the pond (tank) bottom should be inspected. Large deposits of
organic debris should be treated or removed. This is easily accomplished in lined tanks, raceways,
or ponds (i.e. by flushing with a high pressure hose), but poses more of a challenge in large earth
bottom ponds. Many methods of pond bottom disinfection and treatment between crops are
practiced. These methods are given in detail in a number of shrimp farming manuals, and some
will be listed here only with minimal details:
a) Chlorination
This disinfectant may be used for routine treatment of ponds between crops or when disease
eradication is the goal. After draining the pond, remove (and dispose of [see section on carcass
disposal in Section C.6]) as many animals from the system as is possible (this may be difficult
in pond systems where the removal of large numbers of dead shrimp would not be practical).
Partially refill the pond (or fill to capacity if required), discontinue the addition of new water,
stop the discharge of effluent water, and remove any internal or external sources of aeration or
aeration devices, which might be subject to corrosion. Then evenly distribute sufficient
granulated calcium hypochlorite (such as Olin HTH) to provide a minimum residual free
chlorine concentration of 10 ppm within the entire system s water. (NB: The person(s)
applying the chlorine should wear waterproof outer ware to protect their skin, an approved
chlorine mask, and goggles or a face shield for eye protection.) Redistribute additional calcium
hypochlorite as often as required to maintain the residual concentration at near or 10 ppm.
Allow the system to set for a minimum of 2448 hours (especially if applied to large systems)
at this minimal chlorine concentration. The chlorine will kill all shrimp and most, if not all, of
the other organisms occupying the water column or resident in the pond. After the pond has
been treated with chlorine for the required minimum time and before any water is discharged,
neutralise the chlorine either passively by exposure to sunlight and air for approximately an
additional 48 hours (without the addition of new chlorine) or by the addition of sodium
thiosulphate at a rate of five (5) molecules of sodium thiosulphate for each four (4) molecules
of chlorine (or the weight of sodium thiosulphate being 2.85 times the weight of chlorine in
the water, see example table below).
Pond size Average
depth
Volume Chlorine
dose
Chlorine
required
HTH
(65% active Cl)
Thiosulphate
required
1 hectare 1 m 10,000 m
3
10 ppm 100 kg 154 kg 285 kg
Periodic testing should be done for residual chlorine; water should not be discharged until it
has reached 0 ppm. Once the chlorine levels have been ascertained to be at 0 ppm, the system
water can be safely dumped into the farms effluent system. In some culture systems, in
particular raceways, tanks and small lined ponds (i.e. those systems in which the majority of the
shrimp were not removed prior to disinfection), the dead shrimp should be collected for
proper disposal (see section on carcass disposal in Section C.6).
b) Liming
The lime, as calcium oxide or calcium hydroxide, should be applied to a very moist bottom at a
rate of 5000 kg/ha or 1500 kg/ha, respectively. Great care should be taken to assure that the
lime is spread evenly over the soil surface. The pond should then be allowed to set for at least
a week, or at least until the soil has dried to the point of cracking to a depth of approximately
1020 cm. Additional lime may be applied after ploughing (see below) at a rate of 50% of that
normally prescribed. The pond should again be dried for at least a week, depending on the
weather.
c) Drying and ploughing
Whether or not a pond is treated by chlorination or liming or left to dry untreated, ploughing
is a commonly used method of treating a pond bottom to reduce its organic content, improve
nutrient recycling, buffer pH, eliminate pests, and achieve disinfection through a combination
of microbial degradation, exposure to sunlight, aeration, and desiccation. In some regions,
drying and ploughing of dry pond bottoms may only be possible during the dry season. When
pond drying is an option, the pond bottom should be allowed to dry until the surface has
cracked to a depth of approximately 10 cm. Once this level of drying has been reached, the soil
should be broken up to a depth of approximately 20 cm with a plough, tiller, disk harrow, tine
harrow or other similar farm implement. Ponds treated in this manner should be left for at
least a week before being refilled and restocked.
5.4. Disinfection of source water
Because several of the listed diseases of shrimp listed in the Aquatic Code, as well as a number of
other important diseases, can be introduced into shrimp farms with source water when it contains
vectors or carriers (i.e. wild infected crab or shrimp larvae), most farms operate with biosecurity
plans that include provisions for the disinfection of source water. This may be accomplished by a
variety of means which may include one or some combination of the following strategies:
a) Filtration of source water source water is pumped into a supply/settling canal where it first
passes through coarse bar screens to remove large aquatic animals and debris. Then the water
is passed through a series of progressively finer screens, and final filtration is accomplished
by passing source water through a fine mesh (150250 m mesh size) bag screen before
being introduced into a culture pond or storage reservoir.
b) Instead of using mesh nets, some farms place filtration structures in the supply canal system.
A series of compartments within these structures are filled with filter matrixes, beginning
with coarse gravel for initial removal of large aquatic animals and debris, an intermediate
section which contains a finer matrix of sand and gravel, and the end section which contains
fine sand.
c) Chlorination and de-chlorination source water is pumped to a supply canal or directly into
culture ponds or reservoirs (with or without filtration) and treated with sufficient chlorine to
kill any potential vectors or carriers in the source water.
d) Zero or reduced water exchange: Some farms use supplemental aeration and re-circulation
of water in culture ponds and within the supply and discharge systems of the shrimp farm to
reduce source water requirements. This reduces the volume of source water that must be
disinfected before use, as well as reducing nutrient loss from farms with effluent.
6. DISEASE ERADICATION AND TOTAL FACILITY CLEAN-UP
This action may be necessary for disease control when significant, untreatable diseases occur at sites
where eradication is an option. The confirmed diagnosis of an listed diseases, or of an important but
unlisted emerging disease occurring for the first time at a particular farm, at a particular site (i.e. at a
quarantine facility), or within a region or country previously believed to be free of that disease, are
events wherein it may be advisable, if not required, to eradicate the disease by depopulating the
affected facility and performing a thorough disinfection of all or part of the facility.
Fallowing of the affected facility for a defined period of time may be warranted in some situations (see
Chapter 1.7.1, Guidelines for fallowing in aquaculture in the Aquatic Code).
The following steps/actions may be used to achieve eradication of a disease through a total facility
clean-up (TCU):
6.1. Depopulate all living shrimp stocks from the affected facility
a) Discontinue stocking of the facility.
b) Harvest and sell (if permitted) marketable stocks through normal market channels. In some
circumstances cooking the product before marketing may be advisable. Cooking, either by
steam or boiling water, will kill or deactivate all known disease agents of shrimp.
c) For unmarketable stocks the following are options for disposable after harvest:
i) Incineration: burn collected shrimp in a government approved (if required) incinerator.
The limitations to this procedure are inherent to the disposal of shrimp. That is, shrimp
contain large amounts of water and therefore this procedure may only be feasible for
small quantities of shrimp or to larger quantities if the shrimp have been dried prior to
incineration.
ii) Burial: although this technique should be applicable to a greater number of instances, it
still has its limitations. The shrimp should be placed in a pit of sufficient depth to
accommodate all of the shrimp and still provide for at least 50 cm of fill covering the
shrimp. The pit should be located some distance from the facility undergoing TCU and
a comparable distance from any other facility culturing shrimp. Drainage from the pit
area should not be into the aquifer from which the TCU site (or any shrimp culture site)
may pump its source water or into the area (estuary or beach) from which source water
is drawn. Once a proper site has been selected, then the actual burial can take place. The
bottom of the pit should be covered with calcium oxide (quicklime) at a rate of
approximately 500 g/m
2
(5000 kg/ha) or with calcium hydroxide (slaked or hydrated
lime) at a rate of approximately 150 g/m
2
(1500 kg/ha). The shrimp should be placed in
the pit in layers of approximately 10 cm depth, each covered by a quantity of slaked lime
or quicklime sufficient to completely cover the layer (equivalent to approximately 33
100% of the weight of the shrimp). The entire pit, including the top layer of shrimp
carcasses, should then be overlaid with a minimum of 50 cm of fill dirt. In some
locations, local environmental, public health and zoning officials should be consulted
before the shrimp burial pit(s) is (are) dug.
6.2. Disinfection of culture tanks and ponds
For methods see appropriate subsections in Section C.5.
6.3. Clean-up procedures for facility components other than culture areas
In order for a TCU to be effective, it may be necessary to disinfect the entire facility after all the
shrimp have either been harvested or disposed of in some other manner. After depopulation of
the facility, every possible animate and inanimate carrier of the disease agent must be identified
and either removed from the facility or thoroughly disinfected. The movement of disease agents
between live shrimp or dead numerous shrimp can be easily understood, while the same can not
be said for their movement via inanimate components. Hence, all areas, units, subunits or
components which are contaminated or potentially contaminated must go through a cleaning and
disinfection process. See Table 1 in Section A and Section C.5.1 for a list of disinfectants and their
methods of application.
a) Buildings
The disinfection regime used should be building-specific and dependent upon the use-pattern
of that particular building.
i) Office buildings: these buildings would most often be subject only to foot traffic from
people who have been in contaminated buildings or culture areas. Because of this, the
greatest focus of attention should be the floors and cold storage units in the building.
Floors should be thoroughly cleaned (if they are non-porous) with standard detergents
and cleaning solutions, followed by a thorough drying. If the floors are carpeted, they
should be vacuumed and cleaned with a detergent appropriate for carpets, or steam
cleaned. All other areas within these buildings, such as walls, bathrooms, desks,
refrigerators, freezers, etc. should be examined for possibly contaminated materials (i.e.
frozen shrimp in freezers) and any such item found and its container should be cleaned
and disinfected or disposed of in a sanitary manner.
ii) Culture buildings: it must be assumed that these buildings have had direct contact with
the disease agents and will therefore be handled in a different manner from that of the
office buildings. The disinfection regime for these buildings will consist of two steps.
First, the building should be thoroughly swept and/or vacuumed (where appropriate) to
remove as much large-sized organic and inorganic debris as possible. This should be
followed with the second step, treatment with chlorine. Chlorine solution (~1600 ppm)
should be applied (by spraying) to all surfaces which are not prone to the corrosive
actions of chlorine. Those surfaces which should not be chlorinated, can first be sponged
with a iodophor solution minimally providing 200 ppm of free iodine. These can then be
covered with plastic or any other protective material. Floor surfaces can be soak-
chlorinated to a depth of 5 cm with a 200 ppm chlorine solution. This should be allowed
to set for a minimum of 48 hours. If many of the sprayed surfaces are somewhat
susceptible to corrosion by chlorine, those surfaces can be freshwater-rinsed after the 48-
hour treatment.
In buildings where disinfection with chlorine is not practical, fumigation with
formaldehyde gas should be considered. After a general cleaning, fumigation of a sealable
building can be initiated. The entire process, from the time the building is first gassed
until it can be occupied again, should take a minimum of 3660 hours. The entire
building must be totally sealed off during the actual fumigation; there should be no means
by which the gas can escape once it is placed in the building. If possible, the electrical
service for the building should be turned off. The required environment for formaldehyde
gas disinfection is a minimum temperature of 18C with a high relative humidity (at
saturation is best, i.e. floors should be wet, etc.). Generation of formaldehyde gas is
accomplished by the addition of 17.5 g potassium permanganate to each 35 ml of 100%
formalin (a 3739% aqueous solution of formaldehyde gas) for each 2.83 m
3
(100 ft
3
) of
space. Ideally, each room in the structure should have its own source of formaldehyde gas
to assure that all areas of the building are uniformly treated. The correct amount of each
compound (potassium permanganate and formalin) should be weighed out into separate
containers, the formalin should be placed in a non-plastic container that is at least
10 times the combined volume of both the formalin and the potassium permanganate.
(The person applying a formaldehyde gas fumigation should wear waterproof outer ware
to protect their skin, an approved formaldehyde gas mask, and goggles or a face shield for
eye protection.) The containers with the proper amounts of the two reagents should then
be placed on the floor in the centre of the room, on a large disposable protective (plastic)
mat. The formalin and potassium permanganate should not be mixed at this time. Once
all rooms have the correct amounts of the two compounds, the building has been
completely sealed and the environment modified as necessary, the actual fumigation can
begin. The mixing of the two compounds must be done very rapidly and carefully as the
reaction is immediate and somewhat violent as formaldehyde gas is emitted. Starting with
the room farthest from the exterior door, add the permanganate to the formalin and
proceed to the next room. After all rooms have been completed, lock the exterior door
and seal it from the outside with tape. The building should be allowed to set for a
minimum of 12 hours. After this disinfection period the building should be flushed with
clean air for 2448 hours. There should be no detectable odour of formaldehyde when
people are allowed to reoccupy the building.
An alternate method for the generation of formaldehyde gas is the sublimation of
powdered paraformaldehyde. For each 2.83 m
3
(100 ft
3
) of space, approximately 28 g
paraformaldehyde should be used. It can be sublimated by being placed in an electric fry
pan, which has been set on high. This procedure is somewhat more dangerous, because
formaldehyde is flammable and a spark from such a heating device could theoretically
ignite the gas. The same procedures noted above for the formalin/permanganate mixture
in regards to venting, etc. should also be followed for the use of paraformaldehyde.
iii) Processing buildings: these buildings are typically constructed to permit routine
disinfection. For the most part, the procedures followed in the routine operation of such
buildings are appropriate for a TCU, provided that the building, its cold rooms, and its
freezers are also disinfected and thoroughly dried. If considered necessary, fumigation
with formaldehyde gas may be done to insure destruction of the disease agent(s) of
concern.
iv) Other buildings: buildings (feed storage, maintenance, tool rooms, etc.) should be treated
somewhat like the office building. Care should be taken to remove all the large-sized
debris, which would normally be found in relative abundance within these types of
buildings. Potentially contaminated surfaces within such buildings should next be spray-
chlorinated and allowed to set for 2448 hours. This should be followed by a freshwater
rinse. All equipment, which should not be exposed to the corrosive action of chlorine,
should be removed before the spraying, and they should be disinfected by surface
disinfection with 200 ppm of iodophor. Once the equipment has been disinfected, it can
be brought back into the building. Fumigation with formaldehyde gas is another option
for this type of building.
b) Culture support equipment and systems
These are operational units of the shrimp culture facility which may be housed in a building.
i) Artemia systems: all Artemia decapsulation and cyst hatching units and tanks should be
treated in the same manner as other tanks. They should be cleaned of all large debris, then
filled to the top with clean water and calcium hypochlorite added to achieve a final
concentration of 200 ppm (free Cl
2
). Chlorination should be allowed to continue for 24
48 hours. The outside of such tanks may be spray-chlorinated (1600 ppm chlorine).
Treated tanks can then be dechlorinated with sodium thiosulphate, drained, freshwater
rinsed, and allowed to dry for a minimum of one week. Unopened containers of Artemia
cysts at the facility can be retained. These should, however, be surface disinfected with
chlorine (200 ppm) or iodophor (200 ppm).
ii) Algae systems: containers, tanks, incubators and rooms used to produce algae for feeding
the larval stages of shrimp may be handled and disinfected in nearly the same way as
other tanks systems. The only major difference being that special care must be taken to
assure that all chlorine residues have been rinsed from the units before they are used
again. In the case of the culture tubes, flasks, carboys, and flasks used to culture algae, a
combination of acid (10% HCl) rinse or steam sterilisation can be used in lieu of
disinfection with chlorine or idophor.
Disinfection of stock cultures of living algae is not possible. The use of disinfection is
clearly out of the question; any compound which would kill the disease agent would
likewise kill the algae. Hence, there are two basic methods of minimising the chance of a
disease agent being present in the stock cultures.
Dilution: all stock cultures can be cloned from the existing stocks. Each culture
should be diluted either by means of serial dilutions (for broth cultures) or streaked
for single colonies (agar cultures). All dilutions must be performed using strict
aseptic techniques with all media being properly autoclaved. Passages from the stock
cultures should not occur until the algae culture room has itself been disinfected as
per the above building procedures. Once a culture has been diluted and cloned by
either of these methods, to the point where there remains only one cell of the
original culture, the risk is negligible that a (shrimp) disease agent may be present.
New Stock Cultures: If existing stock culture are discarded in a TCU, new stocks
should be purchased from algae supply laboratories, or obtained from other sources
where contamination with (shrimp) disease agents is unlikely, such as isolating
desired species from wild populations of algae. New stock cultures should not be
obtained from any facility that also cultures shrimp and may be contaminated with
(shrimp) disease agents of concern.
iii) Farm equipment: nets, seines, porous air-line tubing, etc. which are relatively inexpensive
and easily obtainable should be discarded and removed from the facility during a TCU
rather than being disinfected as they are not readily disinfected and chlorine is likely to
damage them and shorten their useful life.
Non-expendable equipment such as large size flexible plastic tubing, pumps and pipes,
transfer tanks, cages, harvest cages, harvest tables, Secchi disks, laboratory glassware, etc.
should be soak-chlorinated in 200 ppm solutions for 2448 hours. This is most easily
accomplished by placing these objects in the tanks that are filled with 200 ppm solutions
of chlorine. Care should be taken to have all items completely submerged (use heavy
items to weigh-down more buoyant objects). A good guide is to place everything (except
those that are to be thrown away) that is loose or can be unsecured from its point of
attachment, into the 200 ppm chlorine solution in their respective tanks.
In the case of those similar type items which are associated with ponds, they should be
placed in a special series of tanks set up near their respective ponds. These tanks should
be filled with 200 ppm chlorine solutions. Following soak-chlorination, these items
should be allowed to dry and be exposed to natural UV (sunlight) sterilisation. They
should be turned at least once to expose all areas of the items to direct sunlight.
Tools and machinery, such as tractors, trucks, portable and stationary power tools, etc.,
should be thoroughly cleaned with standard cleaning solutions. All traces of mud, shrimp
feed, etc. must be removed from these items. Following this, disinfection of surfaces
likely to have been contaminated in normal use should be rinsed off with an iodophor
solution (at a concentration of 200 ppm) or cleaned with steam.
Small tools and instruments such as, scales and balances, test instruments, small power
tools, etc., should be gently sponged off with 200 ppm of chlorine solutions if they are
inert plastic or 200 ppm of iodophor if they are otherwise. These should then be placed
back in their respective buildings during the formaldehyde fumigation. High precision
electronic test equipment should not be subjected to the fumigation, especially if there has
been little chance that it was ever contaminated.
iv) New-Water Plumbing: all new-water plumbing which is contained within buildings,
especially those which have blind ends or terminate in manifolds, should be filled with a
minimum 200 ppm chlorine solution. The chlorine solution should be held in the lines for
2448 hours minimum, followed by clean water rinsing. Pipes may also be disinfected by
recirculating hot water (>60C) through them for several hours.
v) Uniforms, boots, etc.: all items worn or used by employees should be either disposed of
or thoroughly washed and disinfected. In the case of clothing, such as coveralls, normal
washing which incorporates a chlorine bleach is sufficient, especially if accompanied by
sun drying. Other items, such as boots, gloves and other non-cloth items can be safely
soak-chlorinated in a 200 ppm chlorine solution. This should be followed by a freshwater
rinse. These items should also be contained within their respective buildings during
formaldehyde fumigation.
vi) Feed items: all feed items, such as prepared feeds, fresh feeds (i.e. squid, bloodworms,
frozen Artemia, bivalve molluscs, etc.) should be removed from the facility and replaced
with new feeds from sources known to be free of contamination by shrimp disease-
causing agents.
7. RE-STOCKING OF DISINFECTED FARMS
Following a TCU, restocking of the disinfected facilities or farms should be accomplished only with
stocks known to be free of the diseases listed in the Aquatic Code or other emerging or significant
diseases of concern.
*
* *
C HA P T E R I . 1 .

GE NE RAL I NF ORMAT I ON

A. GENERAL BASIS FOR FISH HEALTH SURVEILLANCE/CONTROL PROGRAMMES
1. TARGET PATHOGENS AND DISEASES
Target pathogens and fish diseases are included in the Aquatic Animal Health Code (the Aquatic Code)
according to the following basic considerations: they resist or respond poorly to therapy, have a
restricted geographical range, are of high socio-economic importance, and occur in species involved in
international trade. The list of fish diseases considered for notification and certification is currently
restricted to the following diseases:
Epizootic haematopoietic necrosis (EHN)
Infectious haematopoietic necrosis (IHN)
Oncorhynchus masou virus disease (OMVD)
Spring viraemia of carp (SVC)
Viral haemorrhagic septicaemia (VHS)
Channel catfish virus disease
Viral encephalopathy and retinopathy
Infectious pancreatic necrosis
Infectious salmon anaemia
Epizootic ulcerative syndrome
Bacterial kidney disease (Renibacterium salmoninarum)
Enteric septicaemia of catfish (Edwardsiella ictaluri)
Piscirickettsiosis (Piscirickettsia salmonis)
Gyrodactylosis (Gyrodactylus salaris)
Red sea bream iridoviral disease
White sturgeon iridoviral disease
2. OVERALL APPROACH FOR ANIMAL HEALTH CONTROL IN FISH CULTURE
A comprehensive approach for animal health control in fish culture requires:
Assessment of the health status of animals using methods based on the provision in Chapter 1.1.4.
The constraint of restocking open waters and farming facilities only with aquatic animals having a
health status higher than or equal to that of animals already living in the considered areas.
Eradication of disease when possible, by slaughtering infected stocks, disinfecting facilities and
restocking with fish from approved disease-free sources.
Notification by every Member Country of its particular requirements, besides those provided by
the Aquatic Code, for importation of aquatic animals and aquatic animal products.
If the above procedures are followed, it becomes possible to give adequate assurance of the health
status of aquaculture products for specified diseases, according to their country, zone or site of origin.
The issue of a health certificate by the appropriate official, based on a health status report and
examinations of aquatic animals, provides assurance that the aquaculture products in a defined
consignment originate from a whole country, a zone or a farm/harvesting site free of one or more of
Chapter I.1. - Diseases of fish: General information
the specified diseases listed in the Aquatic Code and possibly of other specified diseases (see model of
international certificate in the Aquatic Code).
The assessment of the health status of fish stocks is based on inspection of fish production sites and
further laboratory examination of samples originating from fish specimens taken among the stock of a
defined fish population. This endeavour requires the fish sample to be collected according to defined
sampling size charts and the samples to be processed according to accepted methods.
Several techniques are applicable for aquatic animal pathogens. For screening and diagnostic purposes,
the Aquatic Manual has established two types of examination procedures that will be acceptable for
such work; 1) Screening methods, and 2) Diagnostic methods. The accepted methods are listed under
each disease chapter.
B. SAMPLING PROCEDURES
1. COLLECTION OF FISH SPECIMENS
1.1. Diagnosis in a disease situation
A minimum number of ten moribund fish or ten fish exhibiting clinical signs of the diseases in
question must be collected: fish should be alive when collected, and should be sent to the
laboratory alive or killed and packed separately in sealed aseptic refrigerated containers or on ice.
The freezing of collected fish must be strictly avoided. However, it is highly preferable and
recommended to collect organ samples from the fish immediately after they have been selected at
the fish production site and to store and process the samples as described in Sections 2 and 3. An
identification label that includes information on the place and time of sampling must be attached
to the sample.
1.2. Fish appear to be clinically normal
Fish collection must encompass a statistically significant number of specimens, but it is obvious
that failure to detect certain pathogens from the sample does not guarantee the absence of these
agents in the specimen examined or in the stock. This is particularly true of free-ranging or feral
stocks from which it is difficult to collect a representative and random sample. However, the risk
of a pathogen escaping the surveillance system is reduced in fish farms whose fish stocks have
been inspected and checked for pathogens for several years (at least 2), insofar as they are not
exposed to possible recontamination by feral fish.
When a given fish production site harbours a broodstock, it is essential for one of the sample
collections made each year to be focused on the sexual products (sperm and ovarian fluid)
released by broodfish at the time of spawning (see below). If an adult broodstock includes fish of
different ages, the older fish should be selected for sampling:
Samples must comprise all susceptible species on the site (see relevant chapters of this
Aquatic Manual for the list of species susceptible to each disease), with each lot of a species
being represented in the sample group. A lot is defined as a group of the same fish species
that shares a common water supply and that originates from the same broodfish or spawning
population.
The geographical origin of samples should be defined by the name of the sampling site
associated with either its geographical co-ordinates or its location along a river course or
body of water.
If any moribund fish are present in the fish population to be sampled, they should be
selected first for sample collection and the remainder of the sample should comprise
randomly selected live fish from all rearing units that represent the lot being examined.
A general approach to surveillance and sampling is given in Chapter 1.1.4. of this Aquatic
Manual. The sampling should be designed in order to enable detection, at a 95% confidence
level, of infected animals. The following section gives information relevant to sampling
finfish. Until disease-specific details are included in the individual disease chapters in this
Aquatic Manual, Table 1 can be used to calculate sample size.
As in the case of clinically infected fish, organ and fluid samples must be taken and processed
as soon as possible after fish specimen collection. Sample freezing must be avoided.
Table 1. Sample size based on assumed pathogen prevalence in lot
Lot size At 2% prevalence,
size of sample
At 5% prevalence,
size of sample
At 10% prevalence,
size of sample
50 50 35 20
100 75 45 23
250 110 50 25
500 130 55 26
1000 140 55 27
1500 140 55 27
2000 145 60 27
4000 145 60 27
10,000 145 60 27
100,000 or more 150 60 30
After Ossiander & Wedemeyer, 1973.
1.3. Sampling specifications according to the objectives of a given fish surveillance
programme
A general approach to surveillance and sampling is given in Chapter 1.1.4. of this Aquatic Manual.
The sampling should be designed in order to enable detection, at a 95% confidence level, of
infected animals. The following section gives information relevant to sampling finfish. Until
disease-specific details are included in the individual disease chapters in this Aquatic Manual, Table
1 can be used to calculate sample size.
a) Achievement of the health status of a fish stock/population at a given inspection site
A fish culture unit must be inspected twice a year for 2 years at the appropriate life stage
of the fish and at times of the year when temperature and season offer the best
opportunity for observing clinical signs and isolating pathogens. On each occasion, fish of
the susceptible species listed in the Aquatic Code for the disease in question must be
collected in order to detect a prevalence of infection equal to or higher than 2% at 95%
confidence level. Most often, 150 fish will thus be collected on each occasion. If
broodfish are present during one of the two inspections, up to 150 ovarian fluid samples
will be taken from broodfish in the given fish culture unit.
If fish health surveillance is focused on wild fish populations at a given site of inspection
or on rearing ponds without holding facilities in which different fish crops may be
pooled, 150 fish specimens must be collected once a year for 2 years. Insofar as it is
possible, specimens of the oldest fish and/or ovarian fluid must be collected as a priority.
During this 2-year period, the fish production unit may only receive fish from a unit
whose health status has already been approved and is equal to or higher than the health
status sought for the facility being inspected.
b) Maintenance of the health status
Once a fish production unit, including pond fish production units equipped with holding
facilities, has been recognised to be free from all or certain diseases listed in the Aquatic
Code after 2 years of surveillance with laboratory tests and in the absence of any suspect
clinical signs, twice-yearly inspections must continue. However, collection of fish
specimens may be reduced to 30 fish, including broodfish when available. Moribund fish
observed during inspection visits must, however, be collected for further laboratory
examination.
Maintenance of health status of wild fish populations relevant to diseases listed in the
Aquatic Code at a given site of inspection, can only be ascertained by annual collection of
150 individuals including as many broodfish as possible.
The fish production unit may only receive fish having a health status higher than or equal
to that of those already present.
If, during viral testing of samples, a cytopathic effect (CPE) appears in cell cultures
inoculated with dilutions of the samples being tested, virus identification procedures have
to be undertaken immediately (see the relevant chapters). Provisions must be taken to
suspend the approved health status of the production unit and/or the zone (if it was
approved previously) from which the virus-positive sample originated. The suspension of
approved status will be maintained until it is demonstrated that the virus in question is
not the one referred to in the granting of disease-free status.
The above sampling specifications for the achievement and maintenance of the health
status of fish at given fish production sites imply that all provisions given in Section A.2.
(Overall approach for animal health control in fish culture) are in force.
2. SAMPLE MATERIAL TO BE USED IN VIRAL AND BACTERIOLOGICAL TESTS
Sample material depends both on the size of animals and the objective of testing, i.e. diagnosis of overt
disease or detection of fish that are asymptomatic pathogen carriers.
2.1. Specifications according to fish size
Alevin and sac fry: sample the entire fish but remove the yolk sac if present.
Fish 46 cm: take the entire viscera including the kidney. A piece of encephalon can be obtained
after severing the head at the level of the rear edge of the operculum and pressing it laterally.
Fish over 6 cm: take the kidney, spleen and encephalon.
Broodfish: take the ovarian fluid and/or tissues as described in Chapters 2.1.1.2.1.5.
2.2. Specifications according to clinical status
In the case of clinical infection, besides whole alevin or entire viscera, organs to be sampled are
anterior kidney, spleen and encephalon for virus screening, and kidney and spleen for bacterial
screening. Samples from ten diseased fish will thus be taken and combined to form pools of a
maximum of five fish each. The amount of material should be approximately 1.5 g/pool of
material from five fish.
For detecting asymptomatic carriers, samples may be combined as pools of no more than five
fish/pool, for a total weight of about 1.5 g. Pools of ovarian fluid from five broodfish should not
exceed a total volume of 5 ml, i.e. 1 ml/broodfish. These ovarian fluid samples are to be taken
individually from every sampled female and not collected following the pooling of ova.
Once aseptically removed from fish, the organs and/or ovarian fluid sampled are each split into
two parts if both bacteriological and virological examinations are to be done.
3. GENERAL PROCESSING OF ORGANS/FLUID SAMPLES FOR VIROLOGICAL EXAMINATION
3.1. Transportation and antibiotic treatment of samples
Pools of organs or of ovarian fluids are placed in sterile vials and stored at 4C until virus
extraction is performed at the laboratory. Virus extraction should optimally be carried out within
24 hours after fish sampling, but is still acceptable for up to 48 hours.
Organ samples may also be transported to the laboratory by placing them in vials containing cell
culture medium or Hanks balanced salt solution (HBSS) with added antibiotics to suppress the
growth of bacterial contaminants (one volume of organ in at least five volumes of transportation
fluid). Suitable antibiotic concentrations are: gentamycin (1000 g/ml) or penicillin
(800 International Units [IU]/ml) and streptomycin (800 g/ml). The antifungal compounds
Mycostatin or Fungizone may also be incorporated into the transport medium at a final
concentration of 400 IU/ml. Serum or albumen (510%) may be added to stabilise the virus if the
transport time will exceed 12 hours.
3.2. Virus extraction
This procedure is conducted below 15C and preferably at between 0 and 4C.
Decant antibiotic-supplemented medium from organ sample.
Homogenise organ pools in transport medium at a final dilution of 1/10 using a mortar and
pestle or electric homogeniser until a paste is obtained.
If organ samples have not been treated with antibiotics prior to homogenisation, organ
homogenates are to be resuspended in antibiotic-supplemented medium and incubated in
this medium for 24 hours at 15C or overnight at 4C. Likewise, ovarian fluid samples may
be treated with antibiotics to control microbial contamination. In neither case can
homogenates or ovarian fluid samples be diluted more than twofold.
Clarify the diluted homogenates by centrifugation at 2000 g for 15 minutes and collect the
supernatants.
Ovarian fluid samples should be centrifuged in the same way as organ homogenates, and
their supernatants used directly in subsequent steps.
3.3. Treatment to neutralise enzootic viruses
In some countries, fish are often asymptomatic carriers of enzootic viruses, such as infectious
pancreatic necrosis virus (IPNV), which induce a CPE in susceptible cell cultures and thus
complicate isolation and identification of target pathogens. In such situations, the infectivity of the
enzootic viruses must be neutralised before testing for the viruses listed in the Aquatic Code.
However, when it is important to determine whether one of the enzootic viruses is present,
samples must be tested with and without the presence of neutralising antibodies (NAbs).
To neutralise birnaviruses, mix equal volumes (200 l) of a solution of NAbs against the
indigenous birnavirus serotypes with the supernatant to be tested. Allow the mixture to react for 1
hour at 15C or overnight at 4C prior to inoculation on to susceptible cell monolayers. The titre
of the NAb solution used (it may be a multivalent serum) should be at least 2000 in a 50% plaque
reduction test versus the viral serotypes present in the given geographical area.
When samples are from a country, region, fish population or production unit considered to be
free from enzootic viral infections, this treatment of the organ homogenate should be omitted.
This approach can also be used to neutralise other viruses enzootic to the area being tested.
4. GENERAL PROCESSING OF SAMPLES INTENDED FOR BACTERIOLOGICAL EXAMINATION
As in viral infections, internal organs may be used as a source of isolation whenever systemic infection
is suspected. However, active proliferation of saprophytic microorganisms is such a disadvantage that
live fish are preferred for bacteriological examination. The fact that no antibiotic substances may be
added to the transport medium in which the samples are collected reinforces this preference.
C. MATERIALS AND BIOLOGICAL PRODUCTS REQUIRED FOR THE ISOLATION AND
IDENTIFICATION OF FISH PATHOGENS
1. FISH VIRUSES
1.1. Fish cell lines
The following five fish cell lines will be required to test for the fish pathogens mentioned in the
Aquatic Code:
Bluegill fry (BF-2)
Channel catfish ovary (CCO)
Chinook salmon embryo (CHSE-214)
Epithelioma papulosum cyprini (EPC)
Rainbow trout gonad (RTG-2)
Technical information on the use of these cells for the isolation of the fish pathogens listed in the
Aquatic Code is given in Table 2.
1.2. Culture media
Traditional Eagles minimal essential medium (MEM) with Earles salt supplemented with 10%
fetal calf serum (FCS), antibiotics and 2 mM L-glutamine is the most widely used medium for fish
cell culture.
Stokers medium, however, which is a modified form of the above medium comprising a double-
strength concentration of certain amino acids and vitamins, is particularly recommended to
enhance cell growth, using the same supplementations as above 10% tryptose phosphate.
These media are buffered with either sodium bicarbonate, 0.16 M tris-hydroxymethyl
aminomethane (Tris) HCl, or, preferably, 0.02 M N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic
acid (HEPES). The use of sodium bicarbonate alone is restricted to those cell cultures made in
tightly closed cell culture vessels.
Table 2. Technical information on the most suitable fish cell lines for detection of the
viral agents listed in the Aquatic Code
Cell line nomenclature
Cell line nomenclature
Properties Culture characteristics
BF-2 CCO CHSE-214 RTG-2 EPC
Cell morphology Fibroblastic Fibroblastic/
epithelioid
Epithelioid Fibroblastic Epithelioid
Temperature range (C) 1528 1535 425 425 1033
Optimum growth temperature (C) 20 30 20 20 30
Inoculum (number of cells 10
4
/cm
2
)
1

20 35 50 40 30
Saturation density (number of cells 10
4
/cm
2
)

150 300 300 200 300
For cell growth, the fetal bovine serum content of the medium is usually 10%, whereas for virus
isolation or virus production it may be reduced to 2%. Similarly, the pH of the culture medium for
cell growth is 7.37.4 and is adjusted to 7.6 for virus production or virus assay.
The composition of the most frequently used antibiotic mixture is penicillin (100 IU/ml) and
dihydrostreptomycin (100 g/ml). Mycostatin (50 IU/ml) may be used if fungal contamination is
expected. Other antibiotics or antibiotic concentrations may be used as convenient for the
operator depending on the antibiotic sensitivity of the bacterial strains encountered.
1.3. Virus positive controls and antigen preparation
a) Virus nomenclature
Epizootic haematopoietic necrosis virus (EHNV)
European catfish virus (ECV)
European sheatfish virus (ESV)
Infectious haematopoietic necrosis virus (IHNV)
Oncorhynchus masou virus (OMV) (snonym Salmonid herpesvirus type 2)
Spring viraemia of carp virus (SVCV)
Viral haemorrhagic septicaemia virus (VHSV) (snonym Egtved virus)
b) Virus production
For the production of most of these viruses, the susceptible cell cultures (see relevant sections
in this Aquatic Manual) must be inoculated with fairly low multiplicities of infection (m.o.i.), i.e.
10
2
to 10
3
plaque-forming units (PFU) per cell. However, the m.o.i. for OMV must be
increased up to 0.11 PFU/cell. Furthermore, the best yields for OMV are obtained using
inocula containing cell debris from a monolayer previously infected with the virus.
c) Virus preservation and storage
Dilute virus-containing cell culture fluids in order to obtain virus titres averaging 12
10
6
PFU/ml.
Dispense the resulting viral suspensions into sterile vials at volumes of 0.30.5 ml each.
Freeze and store each series of standard virus stocks at 80C or liquid nitrogen, and
check the titre of each virus stock at regular intervals if it has not been used during that

1 To achieve loose confluency within 24 hours.
time period. This is especially important for OMV, which is relatively labile, even when
stored at 80C.
Lyophilisation: long-term storage (decades) of the seeds of standard virus strains is achievable
by lyophilisation. For this purpose, viral suspensions in cell culture medium supplemented with
10% FCS are mixed (v/v) with a 50% sodium glutamate solution in distilled water before
processing. Seal or plug under vacuum and store at 4C, in the dark.
2. FISH BACTERIA
2.1. Culture media
Few species of fish pathogenic bacteria require special media for cultivation, and most of the
commonly used isolation peptones (trypsin-soya, brainheart, etc.) can conveniently be employed.
However, the low optimal temperatures of some species result in slow growth, and small colonies
are frequently obtained during isolation. In these cases, it is usual to add enrichment factors, such
as serum or blood at 510%, in order to improve cultivation. Conversely Renibacterium
salmoninarum is a very fastidious organism and requires special media enriched with cysteine
(No. A11B).
Bacteriology of fish is generally conducted at temperatures between 20 and 26C. It is sometimes
necessary or useful to have access to several incubators. Renibacterium salmoninarum, Flavobacterium
psychrophilus and others need 15C for optimal growth and many of the bacteria isolated from
warm water fish may be incubated at 30 or 37C to accelerate the diagnostic steps.
2.2. Storage of cultures
Bacterial strains can be stored for a short term on ordinary media, placing the slants or broths at
4C. For most strains, the use of commercial media or agar slants with mineral oil overlay will
extend viability to 12 years under the same conditions without further special requirements.
Freezing is probably the best way to preserve bacterial suspensions of high titre. However, it does
not always prevent some phenotype characteristics from changing. When stability of
characteristics such as virulence is important, it may be better to use lyophilisation, although the
number of viable bacteria may be decreased dramatically.
Different kinds of supports have been proposed to improve the efficacy of freezing and
lyophilisation, namely glycerol (515%) in the former case, and skim milk, lactose, and dextran (5
10%) in the latter. There is no general rule, and convenient conditions have to be determined in
prior trials for all species. The addition to fresh cultures of one volume of support containing
Bactopeptone 11% Dextran 4% has provided excellent results for lyophilisation of certain fish
bacteria, but other formulae would be worth testing in many cases.
3. SEROLOGY
3.1. Production of rabbit antisera and polyclonal antibodies to fish viruses
There are various ways in which antibodies against fish viruses can be raised in rabbits. Titre and
specificity are influenced, however, by the inoculation programme used. The following
immunisation protocols may be used to produce antisera for use in the virus isolation and/or
identification procedures described later.
a) Antisera to infectious pancreatic necrosis virus
Intravenous injection with 50100 g of purified virus on day 0, followed by an identical
booster on day 21, and bleeding 57 days later. Rabbits may be reused if not bled completely.
b) Antisera to other viruses
The immunisation protocols alternate an intramuscular or intra-dermal injection with further
intravenous boosters:
Day 0: primary injection, 5001000 g of virus is mixed (v/v) with adjuvant (Freunds
incomplete or other
2
) giving a total volume of 1.2 ml. This antigen is delivered to the rabbit as
multipoint intradermal injections (20 points on each side) after the animal has been shaved.
Day 21: collect about 20 ml of blood and check for reactivity (neutralisation, fluorescence);
boost intravenously with the same amount of virus as in the primary injection.
Prior to the intravenous booster injection, the rabbit must be treated with promothazine
(12 mg intramuscularly) to prevent possible anaphylactic response.
Day 28: sample the blood, check the serum reactivity and bleed or boost according to the
results.
For rhabdoviruses, this immunisation procedure is well suited to production of antisera to be
used in immunofluorescence and enzyme-linked immunosorbent assay. However, a more
efficient method for production of neutralising antisera is regular intravenous injection without
adjuvant (0.2 ml) every 34 days (twice a week). As many as 15 injections may be necessary; 1
week after the last injection, a serum sample should be collected and tested.
3.2. Antisera to fish bacteria
It is still difficult to obtain antimicrobial sera in large amounts from commercial sources, and it
will often be necessary to prepare such antisera. The general methods are the same as for viruses.
Bacterial antigens are often used as crude preparations killed by heating or by formalin (0.35%
formalin). Rabbit injection at increasing doses can be either intramuscular, each week, with
adjuvant the first time, or intravenous at 34-day intervals without adjuvant. A booster injection is
often required after 15 days.
A special multipoint intradermal schedule has proven very efficient for anti-Renibacterium sera
production, and is also valuable for other bacteria of low antigenicity in rabbits.
The antigen is heat killed (60C, 45 minutes) and adjusted to 2 mg/ml. The flanks of the animals
are thoroughly shaved, and multipoint intradermal injections are performed using total amounts
of 1 mg bacteria/animal, according to the following schedule:
Day 1 1 mg complete Freunds adjuvant (CFA) (v/v)
Day 21 1 mg incomplete Freunds adjuvant (IFA) (v/v)
Day 42 1 mg IFA
Day 63 1 mg antigen
Day 68 Collecting of blood samples (about 30 ml)
Withdrawal injections and bleeding for serum collection may be repeated at 1-month intervals.
3.3. Processing and storage of immune sera

2 Use of Freunds complete adjuvants may be restricted on animal welfare grounds. Alternative synthetic adjuvants include
trehalose dimycolate and monophosphate lipid A.
After blood clotting, collect and centrifuge the serum at 20C and heat it for 30 minutes at 56C.
Filter the resulting heat-inactivated serum through a membrane filter (450 nm pore size) and
temporarily store it at 4C for the time necessary for the screening of its reactivity and specificity
and for checking that these properties are not affected by preservation conditions (e.g. freezing or
lyophilisation). Sterile rabbit sera can be kept for at least 2 months at 4C without any change in
their properties. Dispense (usually as small volumes) and freeze at 20C or lyophilise.
Immunoglobulins (Ig) may be extracted from antisera using conventional methods suitable for Ig
purification. Selective attachment to protein A constitutes a reliable and effective method. The
concentration of Ig solutions is adjusted to the values required for further conjugate preparation
or storage.
Preservation of Ig: Mix a solution of Ig of concentration 2 mg/litre with sterile pure glycerol (v/v)
and keep at 20C. Solutions of Ig with a higher concentration may also be prepared in glycerol.
3.4. Mouse monoclonal antibodies to fish viruses and bacteria
Monoclonal antibodies (MAbs) to most of the fish viruses have been raised over the past years.
Some of them, singly or as two or three associated MAbs, have given rise to biological reagents
suitable for the identification of virus groups (IPN, VHS, IHN). Other MAbs, taken individually
or as components of Ab panels, allow accurate typing of VHSV and IHNV. These MAbs can be
obtained from the Reference Laboratories listed at the end of this Aquatic Manual.
The production of MAbs to bacteria has also been described. It has resulted in the development
of commercial diagnostic kits for Renibacterium salmoninarum, but in most cases remains limited to
specialised laboratories.
In theory, mouse monoclonal IgGs can be processed and stored as for polyclonal IgGs. However,
the reactivity of certain MAbs may be impaired by processes such as enzymatic- or radio-labelling
or lyophilisation. It is thus necessary to test various MAbs for the conditions under which they
will be used.
3.5. Use of molecular techniques for confirmatory testing and diagnosis
Molecular techniques including DNA probes and the polymerase chain reaction (PCR) have been
developed for the identification of many pathogens of aquatic animals. However, as is the case
with several other diagnostic techniques, an advantage in sensitivity is frequently offset by
problems in interpretation or susceptibility to technical problems. Whereas methods based on
direct culture or serology are relatively robust, PCR can be quite dependent on the conditions
under which it is run and can be highly subject to laboratory contamination by previous PCR
products, yielding false-positive results. Thus, while several DNA probe and PCR protocols are
included in this version of this Aquatic Manual as diagnostic or confirmatory methods, where
possible, well-established techniques (e.g. virus isolation) are specified as standard screening
methods. Whenever these newer molecular techniques are used, they should be performed with
caution and with special attention to the inclusion of adequate positive and negative controls.
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*
* *


1. PRELIMINARY REMARK
World mollusc production is adversely affected by several diseases and, given their severe impact on
economic and socio-economic development in many countries, some diseases have become a primary
constraint to the growth and sustainability of this sector. Disease agent transfer via transfers of live
molluscs has been a major cause of disease outbreaks and epizootics. Eleven significant diseases of
molluscs are currently listed in the Aquatic Animal Health Code. Information relevant to these diseases
is provided in the following chapters of this Manual of Diagnostic Tests for Aquatic Animals. Recent
scientific information available in taxonomic affiliation of Mikrocytos roughleyi and its relationship with
Bonamia species has resulted in the re-definition of bonamiosis. Table 1. below shows how the chapters
on mollusc diseases in this edition of the Aquatic Manual correspond with those in the Aquatic Code.
Table 1. How the chapters on mollusc diseases in this edition of the Manual of Diagnostic Tests for
Aquatic Animals correspond with those in the Aquatic Animal Health Code
Aquatic Manual: Aquatic Code:
infection with Bonamia ostreae
infection with Bonamia exitiosus
Bonamiosis:
infection with Mikrocytos roughleyi
MSX disease: infection with Haplosporidium nelsoni
infection with Marteilia refringens Marteiliosis:
infection with Marteilia sydneyi
Mikrocytosis: infection with Mikrocytos mackini
infection with Perkinsus marinus Perkinsosis:
infection with Perkinsus olseni/atlanticus
SSO disease: infection with Haplosporidium costale
Withering syndrome of abalone: infection with Candidatus Xenohaliotis californiensis
2. SAMPLING
2.1. Sampling
A general approach to surveillance and sampling is given in Chapter 1.1.4. of this Aquatic Manual.
The sampling should be designed in order to enable detection, at a 95% confidence level, of
infected animals. The following section gives information relevant to sampling molluscs. Until
disease-specific details are included in the individual disease chapters in this Aquatic Manual, the
following table can be used to calculate sample size.
Chapter I.2. - Diseases of molluscs: General information
size of sample
At 5% prevalence,
size of sample
At 10% prevalence,
size of sample
50 50 35 20
100 75 45 23
250 110 50 25
500 130 55 26
1000 140 55 27
1500 140 55 27
2000 145 60 27
4000 145 60 27
10,000 145 60 27
100,000 or more 150 60 30
2.2. Specific recommendations for sampling molluscs
The timing and frequency of sampling should be determined by the cycle of infection by the
disease agent and the prepatent period. An adequate time period should be allocated when
sampling for seasonal diseases, for example infection with Marteilia refringens or Haplosporidium
nelsoni, to ensure optimal detection. As disease agents may increase in intensity of infection
associated with loss of host condition following spawning, post-spawning sampling is also
recommended. Sampling periods must also take account of the transfer of juveniles and spats into
outgrowing areas, and the transfer of adults for further fattening or relaying.
Samples should also cover a range of size groups, or target the most susceptible age group when
this is known. During sampling, any molluscs showing abnormalities (abnormal growth, gaping
valves, elevated or high mortality rates) should be selected. Diseases have subclinical stages of
development that can escape detection using routine screening methodology. However, the
probability of detection of infection may be increased by holding the bivalves in quarantine for a
long period and subjecting them to stress (crowding, handling, temperature and salinity changes,
etc.). To detect infection, species of molluscs that are more susceptible to infection should be
examined histologically. For example, members of the Arcidae (Arca, Barbatia), Malleidae
(Malleus), Isognomonidae (Isognomon), Chamidae (Chama) and Tridacnidae (Tridacna) tolerate high
prevalence of Perkinsus infection and are good indicators of the presence of Perkinsus.
Prevalence of infection is an important factor affecting the chance of detection. When molluscs
are to be moved from natural beds into a farm site or between natural beds in different zones,
large numbers of bivalves may be required to detect low prevalence of infection. For example, in
Western Australia, Marteilia sydneyi and Perkinsus sp. occur at 0.1% prevalence in isolated beds
untouched by humans. This level of sampling should be commensurate with the level of risk to
the receiving waters and the aquatic resources they support.
For each zone, a number of sampling sites must be selected in the most practical way so as to
maximise the chances of detecting disease agents. The number of sites must be increased for large
zones that contain several discrete areas of cultivation of the susceptible species. Account must be
taken of parameters that have an effect on the development of the disease agents, such as stocking
density, water flow, and the developmental cycle of the molluscs.
It is an important requirement that the screening techniques used be the optimum methods
available for detection of the disease agent in question, and that when the infection is present, it
can be detected. For screening methods, sensitivity and specificity should have been assessed. This
information has a strong influence on sample size.
3. SHIPMENT OF SAMPLES
All sampled molluscs must be delivered to the approved diagnostic laboratory within 24 hours of
sampling. The laboratory should be informed of the estimated time of arrival of the sample so the
required materials to process the molluscs can be prepared before reception of samples.
Mollusc samples must be packed in accordance with current standards in order to keep them alive. If
the sampling site is a long distance from the laboratory, moribund animals or those with foul-smelling
tissues may be of little use for subsequent examination. Required samples should be shipped as soon as
possible after collection from the water, in order to reduce air storage and possible mortality during
transportation, especially for moribund diseased molluscs.
For samples that cannot be delivered live to the diagnostic laboratory, due to advanced stages of
disease, long distance or slow transportation connections, etc., specimens should be fixed on site as
recommended in the following sections of this chapter or the individual disease chapters of this
Aquatic Manual. While this is suitable for, for example, subsequent histology or transmission electron
microscopy examination, other techniques, such as fresh smears, tissue imprints, routine bacteriology,
mycology or Rays fluid thioglycollate culture of Perkinsus spp., cannot be performed. Diagnostic needs
and sample requirements should be discussed with the diagnostic laboratory prior to collection of the
sample.
Samples should be accompanied with background information, including the reason for submitting the
sample (surveillance, abnormal mortality, abnormal growth, etc.), gross observations and associated
environmental parameters, approximate prevalence and patterns of mortality, origin and nature of the
molluscs (species, age, whether or not the samples are from local mollusc populations or stocks
transferred from another site, date of transfer and source location, etc.). This information should
identify possible changes in handling or environmental conditions that could be a factor in mortality in
association, or not, with the presence of infectious agents.
4. MACROSCOPIC EXAMINATION
The gross observation of molluscs should target, as far as possible, animal behaviour, shell surface,
inner shell and soft tissues.
It is often difficult to observe the behaviour of molluscs in open waters. However, observation of
molluscs in certain rearing facilities such as brood-stock in tanks and larvae in hatcheries can provide
useful indications of disease-related behavioural changes. If signs are noted (e.g. pre-settlement of
larvae on the bottom, food accumulation in tanks, signs of weakening, etc.), samples may be examined
for gross signs, including observation under a dissecting microscope for abnormalities and deformities,
fouling organisms, and fixed for further processing as recommended below. For adults and juveniles,
signs of weakening may include gaping, accumulation of sand, mud and debris in the mantle and on the
gills, mantle retraction away from the edge of the shell, decreased activity (scallop swimming, clam
burrowing, abalone grazing), etc. Open-water mortality should be monitored for patterns of losses and
samples collected for further analysis. Environmental factors pre- and post-mortality should be
recorded.
Even under culture conditions, the shells of molluscs may not be clean and fouling organisms are
normal colonists of mollusc shell surfaces. Organisms such as barnacles, limpets, sponges, polychaete
worms, bivalve larvae, tunicates, bryozoans, etc., do not normally threaten health of molluscs. Culture
systems, such as suspension and shallow water culture, can even increase exposure to fouling
organisms and shells may become covered by other animals and plants. This can affect the health
directly by impeding shell opening and closing or indirectly through competition for food resources.
Signs of weakening associated with heavy fouling should be a cause for concern rather than fouling
itself. Shell damages by boring organisms such as sponges and polychaete worms are usually benign,
but under certain conditions may reach proportions that make the shell brittle or pierce through to the
soft-tissues. This degree of shell damage can weaken the mollusc and render it susceptible to inter-
current pathogen infections. Shell deformities (shape, holes in the surface), fragility, breakage or repair
should be noted, but are not usually indicative of a disease concern. Abnormal coloration and smell,
however, may indicate a possible soft-tissue infection that may need to be examined at a laboratory.
The molluscs must be opened carefully so as not to damage the soft tissues, in particular the mantle,
gills, heart and digestive gland. The presence of fouling organisms on the inner shell surface is a clear
indication of weakness. The inner surface of the shell is usually smooth and clean due to mantle and
gill action. Perforation of the inner surface may occur, but can be sealed off by the deposition of
additional conchiolin and nacre. This may result in formation of mud- or water-filled blisters. Blisters
may also form over superficial irritants such as foreign bodies. The degree of shell perforation can be
determined by holding the shell up to a strong light. Where abnormalities occurring within the matrix
of the shell warrant further investigation, freshly collected specimens can be brought intact to the
laboratory or fixed for subsequent decalcification, as required. The appearance of the soft-tissues is
frequently indicative of the physiological condition of the animal. Soft tissues should be examined for
the presence of abscess lesions, pustules, tissue discoloration, pearls, oedema, overall transparency or
wateriness, gill deformities, etc., and, when found in association with weak or dying animals, these
abnormalities should be a cause for concern.
Abnormalities and lesions of the tissues should be noted and recorded, as well as any shell deformities,
shell-boring organisms and conspicuous mantle inhabitants. Levels of tissue damage should be
recorded and samples of affected and unaffected animals collected for laboratory examination as soon
as possible.
5. EXAMINATION OF STOCKS WHERE ABNORMAL MORTALITY OCCURS
Abnormal mortality of molluscs is usually recognised as a sudden sizeable mortality that occurs in a
short time between two observations or inspections of the stocks (for example, about 15 days in the
case of facilities located in inter-tidal zone). In a hatchery, abnormal mortality is the failure of
successive production of larvae coming from different brood-stock. Given the broad spectrum of
species, environments and culture conditions, these definitions should be adapted when and where
necessary.
Whenever abnormal mortality occurs in stocks of molluscs, an urgent investigation must be carried out
to determine the aetiology.
The samples taken must be consistent with the requirements for surveillance provided in the Aquatic
Manual. The samples should also be preserved or fixed and stored in accordance with the procedures
defined for histological and other appropriate presumptive and confirmatory methods.
Where and when available, unaffected or control molluscs should also be fixed for histological
comparison with abnormal tissues. Whatever the fixative, it is essential that the shell be removed to
allow easy ingress of the fixative. Bivalves and operculated gastropods can keep the shell shut against
fixative until autolysis begins.
6. DIAGNOSTIC METHODS
Techniques applicable to molluscan disease agents are limited, and most of the investigations are based
on histological and ultrastructural examinations. Classic serological methods cannot be used for
diagnostic purposes because molluscs do not produce antibodies. Immunoassays using monoclonal
antibodies or nucleic acid probes can be used for direct detection of certain disease agents. A number
of research teams and diagnostic laboratories have been engaged in developing DNA-based diagnostic
techniques for mollusc disease agents. Given the development and potential for widespread application
of these diagnostic techniques and the inherent problems currently associated with their use, the issue
of validation is of the utmost importance.
Three levels of examination procedures are proposed in the following sections. Screening (surveillance)
is routinely performed by histology. Histology is recommended as a standard screening method
because it provides a large amount of information. It is particularly important because macroscopic
examination usually gives no pathognomonic signs or solid indicative information. Also, mortality may
be due to several disease agents or physiological problems, such as loss of condition following
spawning, and this can only be determined using histology.
When abnormal mortality outbreaks occur, various presumptive diagnostic methods can be used in
addition to histology, among which, tissue imprints, Rays fluid thioglycollate medium (RFTM) culture
or polymerase chain reaction (PCR) may be used, as recommended in the individual disease chapters.
Such methods may provide advantages of quick and/or cheap procedures as an answer to suspicion of
infection with a given disease agent.
When a disease agent is encountered during screening or mortality outbreaks, electron microscopy
and/or molecular probes should be used for specific identification, if available. Some of the OIE listed
diseases for molluscs cover disease agents belonging to one or more species of the same genus. Specific
reagents designed to detect certain listed agents are recommended in the following chapters, to be used
to confirm histological examination results and/or to give a species-specific diagnosis where available.
6.1. Histological techniques
Because of the generic use of histology in diagnostic procedures for diseases of molluscs, a
detailed technical guideline is provided in this chapter.
Histology is a technique that is used to study the structure of cells and tissues under light
microscopy. Tissue preparation involves different steps, including tissue fixation, dehydration,
impregnation and embedding of samples, preparation of sections, staining and mounting of slides.
Live moribund animals or freshly dead (within minutes) animals provide the optimum conditions
under which to collect tissues. A standard section should be taken through the digestive gland, to
include the gills, mantle and palps, where possible. Alternatively for large specimens, several
sections should be taken to include all the important tissues.
6.1.1. Tissue fixation
The role of the fixative is to maintain the morphology of the tissues as close to in-vivo
morphology as possible and to prevent post-sampling necrosis. Recommended fixatives used
for the study of marine molluscs are Davidsons solution and Carsons solution for large
specimens. For smaller specimens, glutaraldehyde fixatives may be used and are compatible for
electron microscopy use. The ratio of fixative to tissue volume should be at least 10:1 to ensure
good fixation.
Davidsons solution:
Sea water 1200 ml
95% Alcohol 1200 ml
38% Formaldehyde 300 ml
Glycerol 400 ml
Glacial acetic acid 10% (add extemporaneously)
Carsons solution:
NaH
2
PO
4
.2H
2
O 23.8 g
NaOH 5.2 g
Distilled water 900 ml
40% Formaldehyde 100 ml
Adjust the pH to 7.27.4
There is no universal fixative and choice should be made taking into account later use of fixed
material as well as practical aspects of fixative use (price, component availability, etc.).
Davidsons solution is an excellent choice for preserving the structure of the tissues. In
addition, tissue sections fixed in Davidsons solution can be stained later by different
histochemical methods, as well as in-situ hybridisation with DNA probes. For this purpose,
over-fixation (over 2448 hours) should be avoided. Carsons solution may not be as good as
Davidsons solution for histological analysis. Nevertheless it does allow good preservation of
the ultrastructure and may be used to preserve samples for later study by electron microscopy.
Because electron microscopy may be a valuable adjunct in diagnosing or confirming infections
in molluscs, fixation of some samples (especially smaller samples) using glutaraldehyde, as
described in Section 6.2 of this chapter, may be considered. Otherwise, material fixed in
Carsons solution, and shown to contain adequate levels of targeted disease agents or
abnormalities, can be refixed in glutaraldehyde. It is recommended that part of the mollusc be
fixed in Davidsons solution while the other part be fixed in Carsons solution for further
investigation. This should be done in order to ensure fixation of all tissues/organs in the two
fixatives. If neither are available, 10% buffered formalin made up with filtered seawater is
adequate. Within each country, the molluscan aquaculture industry must agree on the most
effective way of ensuring adequate fixation.
6.1.2. Dehydration, impregnation and embedding of the samples
The embedding of the samples in paraffin requires several steps during which the water
contained in the tissues is progressively replaced, first by alcohol, then by xylene or equivalent
less toxic clearing solution, and lastly by paraffin.
After having fixed the samples in Carsons or Davidsons solution, they are transferred through
graded alcohols (7095 [v/v]) before final dehydration in absolute ethanol. The alcohol
contained in the tissues is next eliminated by immersing them in xylene. The tissues are then
impregnated with paraffin, which is soluble in xylene, at 60C. These steps may be all carried
out automatically using a machine.
Blocks are produced by letting the tissues cool in moulds filled with paraffin on a cooling table;
cooling and moisturising are essential to section cutting.
6.1.3. Preparation of the sections
After the blocks have been cooled on a cold plate, which allows the paraffin to solidify,
histological sections of about 23 m are cut using a microtome. The sections are recovered
on histological slides, drained and dried overnight at 60C. Drying the samples at this
temperature allows the excess moisture to be eliminated and thus the sections adhere to the
slides.
6.1.4. Staining and mounting the slides
Before staining, the paraffin is removed from the sections by immersing them in xylene or
equivalent less toxic clearing solution for 1020 minutes. This is repeated once and then the
solvent is eliminated by immersion in two successive absolute ethanol baths for 10-minute
periods each and rehydrated by immersion in a bath of tap water for 10 minutes. Different
topographical or histochemical staining techniques can then be performed.
When staining with haematoxylineosin (H&E) is used, (haematoxylin or equivalent) nuclear
and basophilic structures stain a blue to dark purple colour, the endoplasmic reticulum stains
blue, while the cytoplasm takes on a grey colour. The acid dye eosin stains the other structures
pink. This staining technique is simple and reproducible and, although it only allows a limited
differentiation of cell structures, it is possible to detect any abnormalities in tissue and cellular
structure. Other techniques may be applied to demonstrate particular structures or features as
required (e.g. trichrome for connective tissue and cytoplasmic granules).
6.2. Transmission electron microscopy methods
Because of the very frequent use of transmission electron microscopy in confirmatory
identification of disease agents in diagnostic procedures for diseases of molluscs, detailed technical
guidelines are provided in this chapter for indication.
Fixation for electron microscopy should be done immediately before fixation for histology. Only
samples taken rapidly from live animals will be of any use. The preparation of samples for electron
microscopy involves the following steps: tissue fixation, decalcification of the samples (when
necessary), dehydration, impregnation and embedding of the samples, preparation and
counterstaining of the sections.
6.2.1. Tissue fixation
For tissues that are to be examined by electron microscopy, it is important that the fixation be
performed correctly in order to cause as little damage as possible to the ultrastructure. The
specimens are cut such that their width does not exceed 12 mm. This small size allows the
various solutions to penetrate rapidly into the sample.
Fixation of the samples is carried out directly in 3% glutaraldehyde for 1 hour. Fixation for
longer periods leads to membranous artefacts. The samples are washed in buffer three times,
then fixed in 1% osmic acid and washed twice again in buffer. Various formulations of
glutaraldehyde fixative and buffers work equally well.
In order to cause as little damage as possible to the ultrastructure, the samples are treated with
solutions that have an osmolarity close to that of the tissues. Thus, mollusc tissues are treated
with solutions with an osmolarity of around 1000 mOsm. The osmolarity of the solutions is
adjusted with NaCl. As mollusc tissues are nearly iso-osmotic with seawater, it is possible to
make the glutaraldehyde up with 0.22 m filtered seawater, and use the filtered seawater for
subsequent washes.
Sodium cacodylate 0.4 M: 8.6 g in 100 ml of distilled water
Sodium chloride 10% in distilled water
Cacodylate buffer, pH 7.4:
1000 mOsm
Sodium cacodylate 50 ml from 0.4 M stock solution
NaCl 20 ml from 10% stock solution
Adjust the pH to 7.4
3% Gluteraldehyde:
1000 mOsm
25% gluteraldehyde 2.5 ml
0.4 M sodium cacodylate 5 ml
10% NaCl 3.5 ml
1% Osmic acid:
1000 mOsm
4% Osmic acid 1 volume
0.4 M sodium cacodylate 1 volume
NaCl 1 volume from 10% stock solution
Distilled water 1 volume
5% EDTA:
Disodium EDTA 5 g
Cacodylate buffer 100 ml
Dissolve by adding a few sulphur tablets. EDTA dissolves when the pH is above 8. When the
solution becomes clear adjust the pH to 7.4 by adding concentrated HCl.
If the samples have been previously fixed and stored in Carsons solution, they must be
washed several times in a bath of buffer before fixation with 3% glutaraldehyde.
6.2.2. Dehydration, impregnation and embedding of the samples
The samples are dehydrated in successive baths of ethanol: 70% ethanol once, 95% ethanol
twice, absolute ethanol three times. The dehydration is completed by two baths of propylene
oxide, which allows the subsequent impregnation with Epon or other resin.
The samples are impregnated progressively. After a first bath in a mixture of polypropylene
oxideEpon (50/50), the samples are placed in a bath of Epon. The longer the incubation, the
better the impregnation of the tissues.
Embedding is carried out by placing the samples in moulds filled with Epon resin. A label
identifying the sample is included in each block and the blocks are then placed at 60C (the
temperature at which Epon resin polymerises) for 48 hours.
6.2.3. Preparation of the sections and the counterstaining
The blocks are cut to appropriate sizes with a razor blade and the sections are then cut using
an ultramicrotome. Semi-thin sections (0.51 m) are cut and placed on glass slides. These will
be used to control the quality of the samples by light microscopy and to find the areas of
interest on the section.
The semi-thin sections are stained at 90100C with 1% toluidine blue solution. After drying,
the slides are mounted under cover-slips with a drop of synthetic resin and observed under the
light microscope.
Ultrathin sections 80100 nm thick are placed on mesh copper grids for electron microscopy
analysis. Uranyl acetate and lead citrate are used to counterstain the ultrathin sections.
KEY REFERENCES
1. AUSTIN B. & AUSTIN D.A. (1989). Methods for the Microbiological Examination of Fish and
Shellfish. Ellis Horwood, Chichester, UK.
2. BONDAD-REANTASO M.G., MCGLADDERY S.E., EAST I. & SUBASINGHE R.P. (2001). Asian
Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper, No. 402, supplement 2.
Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 240 pp.
3. BOWER S.M., MCGLADDERY S.E. & PRICE I.M. (1994). Synopsis of infectious diseases and parasites
of commercially exploited shellfish. Ann. Rev. Fish Dis., 4, 1199.
4. ELSTON R.A. (1999). Health Management, Development and Histology of Seed Oysters. World
Aquaculture Society. Baton Rouge, Louisiana, USA, 110 pp.
5. GALTSOFF P.S. (1964). The American oyster, Crassostrea virginica Gmelin. Fishery Bull., 64, 480 pp.
6. HOWARD D.H. & SMITH C.S. (1983). Histological techniques for marine bivalve molluscs. NOAA
Technical Memorandum NMFS-F/NEC, 25, 97 pp.
7. MIALHE E., BACHERE E., BOULO V., CADORET J.P., ROUSSEAU C., CEDENO V., SARAIVA E.,
CARRERA L., CALDERON J. & COLWELL R.R. (1995). Future of biotechnology-based control of
disease in marine invertebrates. Molec. Mar. Biol. Biotechnol., 4, 275283.
8. THOESON J.C. (1994). Suggested Procedures for the Detection and Identification of Certain Finfish
and Shellfish Pathogens, Fifth Edition. Bluebook, American Fisheries Society, Bethsada, USA.
9. WALKER P. & SUBASINGHE R.P. (2000). DNA-based Molecular Diagnostic Techniques. Research
needs for standardization and validation of the detection of aquatic animal pathogens and diseases.
FAO Fisheries Technical Paper, n395, 93 pp.
*
* *


1. DISEASES OF CRUSTACEANS LISTED BY THE OIE
Crustaceans are adversely affected by a number of diseases. This is especially evident in penaeid shrimp
from aquaculture. All of the crustacean diseases that have significant social or economic notoriety are
infectious diseases. The crustacean diseases and their aetiological agents that are included in the Aquatic
Animal Health Code (the Aquatic Code) have a restricted geographical range, have no therapeutic
remedies or treatments, are potentially excludable, and are of significant social and economic
importance. There are currently eight diseases of crustaceans listed by the OIE. Seven of these eight
crustacean diseases are listed because of the size and importance of the penaeid shrimp aquaculture
industry. Therefore, the principles and methods discussed in this chapter will, of necessity, emphasise
the penaeid shrimp.
The OIE listed crustacean diseases, the nature of their respective aetiological agents, and their principal
hosts are:
Diseases of crustaceans listed by the OIE:
Taura syndrome (viral/penaeid shrimp)
White spot disease (viral/penaeid shrimp and other decapod crustaceans)
Yellowhead disease (viral/penaeid shrimp)
Tetrahedral baculovirosis (Baculovirus penaei) (viral/penaeid shrimp)
Spherical baculovirosis (Penaeus monodon-type baculovirus) (viral/penaeid shrimp)
Infectious hypodermal and haematopoietic necrosis (viral/penaeid shrimp)
Crayfish plague (Aphanomyces astaci) (fungal/freshwater crayfish)
Spawner-isolated mortality virus disease (viral/penaeid shrimp)
2. DIAGNOSTIC METHODS
The methods available for diagnosis of the above-listed diseases include the traditional methods of
morphological pathology (direct light microscopy, histopathology, and electron microscopy), bioassay
methods with susceptible indicator hosts, and molecular methods (gene probes and polymerase chain
reaction [PCR]). While tissue culture is considered to be a standard tool in medical, veterinary, and fish
diagnostic laboratories, it has yet to be developed as a usable, routine diagnostic tool for crustacean
pathogens. Clinical chemistry has not become a routinely used diagnostic tool by crustacean
pathologists.
2.1. Diagnostic methods for diseases of crustaceans
As of the time of writing this section of the Aquatic Manual, the available diagnostic methods that
may be selected for diagnosis of the OIE listed crustacean diseases or detection of their
aetiological agents are based on:
Chapter I.3. - Diseases of crustaceans: General information
Gross and clinical signs
Direct bright-field, phase-contrast or dark-field microscopy with whole stained or
unstained tissue wet-mounts, tissue squashes, and impression smears; and wet-
mounts of faecal strands
Histology of fixed specimens
Bioassays of suspect or asymptomatic carriers using a highly susceptible host (life
stage or species) as the indicator for the presence of the pathogen
Transmission or scanning electron microscopy
Antibody-based tests for pathogen detection using immune sera polyclonal
antibodies (PAbs) or monoclonal antibodies (MAbs)
Molecular methods
DNA probes in dot-blot hybridisation assays directly with fresh tissue samples or with
extracted DNA
DNA probes or RNA probes for in situ hybridisation assays with histological sections of
fixed tissues
PCR and reverse-transcription (RT)-PCR for direct assay with fresh tissue samples or with
extracted DNA or RNA.
The detailed procedures for each of the available methods (screening, presumptive, and
confirmatory) for diagnosis of each of the OIE listed crustacean diseases are outlined in the
individual disease chapters of this Aquatic Manual.
There is a paucity of antibody-based diagnostic tests available for the pathogens that cause
crustacean diseases. As crustaceans do not produce antibodies, antibody-based diagnostic tests are
limited in their application to pathogen detection. While a number of antibody-based diagnostic
methods have been developed and are described in the literature, these were developed with
mouse or rabbit antibodies generated to viruses purified from infected hosts. Because crustacean
viruses cannot be routinely produced in tissue culture, purified virus from infected hosts must be
used to produce antibody. This has severely limited the development and availability of this
diagnostic tool. The recent application of MAb technologies to this problem has begun to provide
a few antibody-based tests. MAbs are available for three of the OIE listed crustacean diseases (for
Taura syndrome virus [TSV], infectious hypodermal and haematopoietic necrosis virus [IHHNV],
and white spot syndrome virus [WSSV]). Antibody-based diagnostic kits/reagents for TSV and
WSSV infections are currently available from commercial sources.
Molecular methods have been developed and some methods are in widespread use for the
detection of many of the viral, bacterial, and protozoan pathogens of the penaeid shrimp. DNA-
based detection methods are readily available from the literature and some are available in kit form
from commercial sources for the OIE listed pathogens TSV, WSSV, and yellowhead disease virus
(YHV/GAV), IHHNV, Penaeus monodon-type baculovirus (MBV), Baculovirus penaei (BP), and
spawner-isolated mortality virus (SMV). PCR or RT-PCR methods are available for several of
these viruses and some are in routine use by certain sectors of the crustacean aquaculture industry.
For other OIE listed viral pathogens, specific DNA probes tagged with nonradioactive labels are
either reported in the literature or available commercially for application in dot-blot formats with
haemolymph or tissue extracts, or for use with routine histological sections using in-situ
hybridisation.
Despite the growing dependence of the shrimp aquaculture industry on DNA-based diagnostic
methods, few of the tests that is available from commercial sources or reported in the literature
has been validated using controlled field trials. Likewise, there are few formal accreditation or
certification programmes yet in place to assure that test results from technicians and laboratories
are indeed accurate and the tests properly controlled. There is a growing need to standardise and
validate the DNA-based diagnostic methods and the laboratories that use them. Standardisation
of DNA-based diagnostic methods is almost inherent in the nature of the tests that is, a specific
DNA probe or a specific set of primers that is used to demonstrate the presence or absence of a
unique DNA or RNA sequence does not vary from batch to batch. Hence, with proper controls,
these DNA-based methods are readily standardised. The implementation of a formal programme
by appropriate international agencies or professional societies is needed to validate new diagnostic
methods and to periodically review the accreditation and certification of diagnosticians and
diagnostic laboratories. The establishment of regional reference laboratories for DNA-based
diagnostic methods of penaeid shrimp/prawn pathogens would fit well into such a programme
with the goal of making these methods uniform, reliable, and readily applicable to disease control
and management strategies for viral diseases of cultured penaeids.
3. SAMPLING
There are at least three purposes for which crustacean stocks may be sampled with regard to the OIE
listed crustacean pathogens. These are: 1) surveillance; 2) stock or facility certification; and 3) disease
diagnosis. The number and type of samples to be taken for analysis varies greatly according to which of
these purposes applies.
A general approach to surveillance and sampling is given in Chapter 1.1.4. of this Aquatic Manual. The
sampling should be designed in order to enable detection, at a 95% confidence level, of infected
animals. The following section gives information relevant to sampling crustaceans. Until disease-
specific details are included in the individual disease chapters in this Aquatic Manual, Table 1 can be
used to calculate sample size.
3.1. Diagnosis in disease situations
In clinical disease episodes, carefully selected quality specimens with representative lesions should
be obtained from live or moribund crustaceans. Every effort should be made to sample those
specimens for diagnosis that are representative of the disease(s) that is (are) affecting the
crustacean stock of interest, and that are moribund or clinically diseased. Collection of dead
specimens should be avoided. When cultured or wild crustacean stocks are presenting clinical
signs of an active disease that are consistent with, or suggestive of, any one of the OIE listed
crustacean diseases, care should be taken to ensure that the samples collected are preserved
appropriately for the anticipated diagnostic tests (see sample preservation section for
recommended methods).
The recommended minimum numbers of specimens to collect for diagnostic testing are 100 for
the larval stages of most crustaceans; 50 for the postlarval stages; and 10 for juveniles and adults.
Sample numbers may be greater if clinically diseased specimens are readily apparent and collected.
Nonetheless, these recommended minimum sample numbers are provided as guidelines, and it
must be emphasised that carefully selected, quality specimens are far more valuable (and cost-
effective) diagnostic specimens than dozens or hundreds of specimens taken at random to fill out
the sample.
3.2. Diagnosis in asymptomatic crustaceans
When samples are to be taken for surveillance, for testing of asymptomatic carriers of previous
disease epizootics, for certification of specific pathogen free (SPF) status, or for freedom of
particular disease within a country, zone, or facility the sample size to be taken should be
determined using a statistical table. The minimum sample size for each lot tested should provide a
95% level of confidence that infected specimens, if present, will be in the sample, assuming a
defined minimum prevalence of infection equal or greater than 2%, 5% or 10%. For surveillance
and certification purposes for OIE listed diseases, the samples taken for diagnostic tests at any
given aquaculture site or from wild stocks, should include the appropriate number of specimens
from each lot to be tested according to Table 1. For the OIE listed diseases it is highly
recommended that the scheduling of sampling be planned (i.e. by farm schedule, season, etc.) so
that the particular life-stage(s) are sampled at a time when the pathogen of concern is most likely
to be detected. This is especially important when the available diagnostic methods are dependent
on simple microscopy or histological methods and do not include molecular methods. For the
baculoviruses BP and MBV larval and early postlarval are the most appropriate samples; for TSV,
IHHNV, WSSV and YHV/GAV, juveniles and subadults provide the best samples; and for
crayfish plague, juveniles and adults are suitable samples.
Samples taken for molecular or antibody-based tests for OIE-listed crustacean diseases may be
combined as pooled samples of no more than five specimens per pooled sample.
3.3. Testing for verification or maintenance of freedom from specific diseases
Once a crustacean production facility has been recognised to be free of all or certain diseases
listed in the Aquatic Code after 2 years of surveillance with laboratory tests and in the absence of
any suspect clinical signs, twice-yearly inspections should continue. However, collection of
specimens for testing may be reduced to 30 crustaceans (shrimp), including especially broodstock.
Moribund shrimp observed during inspection visits must, however, be collected for further
laboratory examination
size of sample
At 5% prevalence,
size of sample
At 10% prevalence,
size of sample
50 50 35 20
100 75 45 23
250 110 50 25
500 130 55 26
1000 140 55 27
1500 140 55 27
2000 145 60 27
4000 145 60 27
10,000 145 60 27
100,000 or more 150 60 30
4. SAMPLE TYPE AND PRESERVATION
4.1. Samples for direct microscopy
Samples for direct microscopic examination should be examined as soon as possible after
collection. Use live specimens whenever possible, or use fresh, chilled, or 10% buffered formalin-
fixed specimens when live specimens are not practical. If an adequate field laboratory is available,
it should be used to process and examine samples near the site of collection.
4.2. Samples for histology
Collect shrimp by whatever means are available with a minimum of handling stress. Transport the
shrimp to the laboratory via a well oxygenated water-filled utensil. Supply adequate aeration to the
container if the shrimp are to be left for a short period of time before actual fixation. For the
study of presumably diseased shrimp, select those shrimp that are moribund, discoloured,
displaying abnormal behaviour, or otherwise abnormal, except in the case of intentional random
sampling for estimation of disease prevalence.
i) Have ready an adequate supply of fixative. A general rule is that a minimum of ten volumes
of fixative should be used for one volume of tissue sample (i.e. a 10 g sample of shrimp
would require 100 ml of fixative).
ii) Davidsons AFA (alcohol, formalin, acetic acid) fixative
Davidsons AFA fixative is recommended for most histological applications. The fixative is
rapid, reduces autolytic changes in tropical crustaceans (i.e. the penaeid shrimp), and its acidic
content decalcifies the cuticle. The formulation for Davidsons AFA is (for 1 litre):
330 ml 95% ethyl alcohol
220 ml 100% formalin* (a saturated 3739% aqueous solution of formaldehyde gas)
115 ml glacial acetic acid**
335 ml tap water (for marine crustaceans, sea water may be substituted)
Store the fixative in glass or plastic bottles with secure caps at room temperature.
* Do not use previously made 10% formalin to prepare Davidsons AFA because the
formalin content of the Davidsons AFA will be inadequate to provide satisfactory
fixation.
** Do not substitute other acids, such as HCl, for acetic acid. Histological sections
prepared from HCl-Davidsons solution are not suitable for routine haematoxylin and
eosin histological staining.
iii) Nonacidic R-F (RNA-friendly) fixative
For some applications where in situ assays are planned with MAbs or with cDNA probes for
RNA viruses (TSV or YHV), Davidsons fixative may be too acidic or too harsh. For such
specimens an alternative fixative, which has advantages over buffered formalin, has been
developed. R-F fixative is not recommended for routine use in crustacean histology. This is
because it does not penetrate or fix tissues rapidly, and neither does it decalcify the cuticle.
Hence, its use may result in poorly fixed tissues that are more difficult to section, stain and
evaluate for signs of disease than are tissues preserved correctly in Davidsons AFA fixative.
The formulation of R-F fixative is (for 1 litre):
407 ml 95% ethyl alcohol
349 ml 100% formalin
222 ml tap water (for marine crustaceans, sea water may be substituted)
22 ml ammonium hydroxide (2830% as NH
3
)
pH ~6.07.0
Store the fixative in glass or plastic bottles with secure caps at room temperature.
iv) Fixation procedures with Davidsons AFA or R-F fixative
For larvae and postlarvae that are too small to be easily injected with fixative using a tuberculin
syringe: Using a fine mesh screen or a Pasteur pipette, select and collect specimens.
Immerse shrimp selected for sampling directly in the fixative. Fix for 1224 hours in
fixative, then transfer to 5070% ethyl alcohol for storage.
For larger postlarvae and very small juveniles that are too small to be injected: Select and collect
specimens as described in Section 3. Use a needle or fine-pointed forceps to incise the
cuticle. Immerse shrimp selected for sampling directly in the fixative. Fix for 1224
hours in fixative, then transfer to 5070% ethyl alcohol for storage.
For larger postlarvae, juveniles, and adults: Inject fixative (use 510% volume: weight) via
needle and syringe (needle gauge dependent on shrimp size, i.e. 27 gauge needle for
postlarvae and small juveniles) into the living shrimp.
The hepatopancreas (HP) should be injected first and at two or more sites, with a
volume sufficient to change the HP to a white to orange colour; then inject fixative into
adjacent regions of the cephalothorax, into the anterior abdominal region, and into the
posterior abdominal region.
The fixative should be divided between the different regions, with the cephalothorasic
region, specifically the HP, receiving a larger share than the abdominal region.
A good guide to insure adequate fixation is to inject an equivalent of 510% of the
shrimps (or other crustaceans) body weight; all signs of life should rapidly cease, and
visible colour change should occur in the injected areas.
Immediately following injection, slit the cuticle, with dissecting scissors, from the sixth
abdominal segment to the base of the rostrum, being particularly careful not to cut
deeply into the underlying tissue. The incision in the cephalothorasic region should be
just lateral to the dorsal midline, while that in the abdominal region should be
approximately mid-lateral.
For shrimp (and most other crustaceans) larger than ~12 g: After injection of fixative, the
body should then be transversely bisected, at least once, just posterior to the
abdomen/cephalothorax junction, and (optional) again mid-abdominally.
For very large crustaceans and crabs: The organs of interest may be excised after injection
of fixative. Completion of fixation of these tissue samples is then handled as outlined
previously.
Following injection, incisions and bisection/trisection, or excision of key organs,
immerse the specimen in the fixative (use 10:1 fixative:tissue ratio).
Allow fixation to proceed at room temperature for 2472 hours depending on the size
of shrimp (or crustacean) being preserved. Longer fixation times in Davidsons AFA
may be used to thoroughly decalcify the shell of crabs, lobsters, crayfish, etc.
Following fixation, the specimens should be transferred to 70% ethyl alcohol, where
they can be stored for an indefinite period.
Record a complete history of the specimens at the time of collection: gross observations
on the condition of the shrimp (or other crustacean), species, age, weight, source (wild,
or if culture pond or tank number, stock number, etc.), and any other pertinent
information that may be needed at a later time.
The label should stay with the specimens in the same container during fixation, storage
and transport to the laboratory. Always use No. 2 soft-lead pencil on water-resistant
paper (plastic paper is recommended; never use ink or marking pens as the ink is
dissolved by alcohol).
v) Transport and shipment of preserved samples
Because large volumes of alcohol should not be posted or shipped, the following methods
are recommended: Remove the specimens from the 70% ethyl alcohol. For larvae,
postlarvae, or small juveniles, use leak-proof, screw-cap plastic vials if available; if glass vials
must be used, pack to prevent breakage. For larger specimens, wrap samples with white
paper towels to completely cover (do not use raw cotton). Place towel-wrapped specimens in
a sealable plastic bag and saturate with 70% ethyl alcohol. Insert the label and seal the bag.
Place the bag within a second sealable bag. Multiple small sealable bags can again be placed
within a sturdy, crush-proof appropriately labelled container for shipment (see Chapter 1.5.6
of the Aquatic Code for details).
4.3. Preservation of samples for antibody, DNA probe dot-blot tests, or polymerase chain
reaction
For routine diagnostic testing by PCR, RT-PCR or for dot-blot tests with DNA probes, samples
must be prepared to preserve the pathogens nucleic acid. Likewise, samples intended for testing
with antibody-based methods must be preserved to retain reactive antigenic sites for the
antibodies used.
4.3.1. Sample types
Samples selected for DNA-based or antibody-based diagnostic tests should be handled and
packaged (in new plastic sample bags or bottles) with great care to minimise the potential for
cross contamination among the sample set taken from different (wild or farmed) stocks, from
tanks, ponds, farms, etc. New plastic sample bags or bottles must be used. A water-resistant
label, with the appropriate data filled out in No. 2 pencil, should be placed within each package
or container for each sample set.
Some suitable methods for preservation and transport of samples taken for molecular or
antibody-based tests are:
Live specimens: These may be processed in the field or shipped to the diagnostic laboratory
for testing.
Haemolymph: This tissue is the preferred sample for certain molecular and antibody-based
diagnostic tests. Samples may be collected by needle and syringe by cardiac puncture,
from the haemocoel (i.e. the ventral sinus in penaeids), or from a severed appendage.
Iced or chilled specimens: This is for specimens that can be transported to the laboratory for
testing within 24 hours. Pack samples in sample bags surrounded by an adequate quantity
of wet ice around the bagged samples in a Styrofoam-insulated box and ship to the
laboratory.
Frozen whole specimens: Select live specimens according to the criteria listed in Section 3,
quick freeze in the field using crushed dry-ice, or freeze in the field laboratories using a
mechanical freezer at 20C or lower temperature. Prepare and insert the label into the
container with the samples, pack samples with an adequate quantity of dry-ice in a
Styrofoam-insulated box, and ship to the laboratory.
Alcohol-preserved samples: In regions where the storage and shipment of frozen samples is
problematic, 9095% ethanol may be used to preserve, store, and transport certain types
of samples. Whole crustaceans (any life stage provided the specimen is no larger than 2
3 g), excised tissues (i.e. pleopods) from large crustaceans, or haemolymph may be
preserved in 9095% ethanol, and then packed for shipment according to the methods
described in Section 4.2.v (see Chapter 1.5.6 of the Aquatic Code for details).
KEY REFERENCES
1. ALDAY DE GRAINDORGE V. & FLEGEL T.W. (1999). Diagnosis of Shrimp Diseases. Food and
Agriculture Organization of the United Nations and Mulitmedia Asia, Bangkok, Thailand.
2. BELL T.A. & LIGHTNER D.V. (1988). A Handbook of Normal Shrimp Histology. Special Publication
No. 1, World Aquaculture Society, Baton Rouge, Louisiana, USA.
3. BONDAD-REANTASO M.G., MCGLADDERY S.E., EAST I. & SUBASINGHE R.P. (2001). Asian
Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper, No. 402, supplement 2.
Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 240 pp.
4. BROCK J.A. & MAIN K. (1994). A Guide to the Common Problems and Diseases of Cultured Penaeus
vannamei. Published by the Oceanic Institute, Makapuu Point, P.O. Box 25280, Honolulu, Hawaii,
USA.
5. CHANRATCHKOOL P., TURNBULL J.F., FUNGE-SMITH S.J., MACRAE I.H. & LIMSUWAN C. (1998).
Health Management in Shrimp Ponds. Aquatic Animal Health Research Institute, Department of
Fisheries, Kasetsart University Campus, Jatujak, Bangkok, Thailand, 152 pp.
6. JOHNSON P.T. (1980). Histology of the Blue Crab, Callinectes sapidus. A Model for the Decapoda.
Prager, New York, USA, 440 pp.
7. JOHNSON S.K. (1995). Handbook of Shrimp Diseases. TAMU-SG-90-601(r). Texas A&M Sea Grant
College Program, Texas A&M University, College Station, Texas, USA, 26 pp.
8. LIGHTNER D.V. (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases
of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA. 304 p.
9. LIGHTNER D.V. (1996). The penaeid shrimp viruses IHHNV and TSV: epizootiology, production
impacts and role of international trade in their distribution in the Americas. Rev. sci. tech. Off. int.
Epiz., 15, 579601.
10. LIGHTNER D.V. & REDMAN R.M. (1998). Shrimp diseases and current diagnostic methods.
Aquaculture, 164, 201220.
11. LIGHTNER D.V. & REDMAN R.M. (1998). Strategies for the control of viral diseases of shrimp in the
Americas. Fish Pathol., 33, 165180.
12. LOTZ J.M. (1997). Special topic review: Viruses, biosecurity and specific pathogen-free stocks in
shrimp aquaculture. World J. Microbiol. Biotechnol., 13, 405413.
13. REDDINGTON J. & LIGHTNER D.V. (1994). Diagnostics and their application to aquaculture. World
*
* *
S E C T I ON 2 . 1 .

DI S E AS E S L I S T E D BY T HE OI E

C HA P T E R 2 . 1 . 1 .

E P I ZOOT I C HAE MAT OP OI E T I C NE CROS I S
SUMMARY
Epizootic haematopoietic necrosis (EHN) is a systemic iridovirus (Ranavirus) infection of redfin perch
(Perca fluviatilis), rainbow trout (Oncorhynchus mykiss), sheatfish (Silurus glanis) and catfish
(Ictalurus melas) (3, 4, 12, 14). The disease is caused by three similar viruses: epizootic haematopoietic
necrosis virus (EHNV), European sheatfish virus (ESV) and European catfish virus (ECV) (3, 7, 11,
14, 15). The geographical range of EHNV is currently restricted to Australia (10, 24). The ECV and
ESV agents have only been detected among fish in Europe (1, 3, 14). Although inducing similar diseases
in their respective hosts, EHNV, ECV and ESV are distinct agents distinguished by molecular
techniques (10, 13, 17).
EHN infection in redfin perch is generally lethal (19). Rainbow trout are relatively resistant and only a
small proportion of individuals become infected (2022). Infections with ESV and ECV can induce high
morbidity and mortality in their catfish hosts (3, 14). The disease caused by all three iridoviruses is
characterised by mortalities due to necrosis in the liver, spleen, haematopoietic tissue of the kidney and other
tissues. Experimental exposure studies have shown that rainbow trout can be infected with ECV and
ESV without showing morbidity and mortality (1).
The viruses do not induce differentiating neutralising antibodies in mammals or fish (10). The viruses can
be identified by immunofluorescence, enzyme-linked immunosorbent assays (ELISAs) or immunoelectron
microscopy (710, 16). EHNV has several antigens in common with ESV and ECV from Europe and
at least one antigen in common with the amphibian iridoviruses from North America (frog virus 3) and
Australia (Bohle iridovirus) (2, 7, 8). Antigen-detection tests with polyclonal antibodies against EHNV
detect each of these viruses. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE),
Western blotting and polymerase chain reaction (PCR) can differentiate to varying degrees between the
different viruses (10, 13).
The epidemiology of EHNV in rainbow trout is incompletely understood (2022). Due to their extreme
susceptibility, it is unlikely that redfin perch are a natural host (18, 19). Infection may recur annually at a
rainbow trout production site and this may be due to reinfection from wild redfin perch in the catchment
area. A carrier state in naturally infected rainbow trout appears to be very uncommon as neither EHNV
antigen nor anti-EHNV antibody are routinely detected in rainbow trout surviving an outbreak (20, 22).
However, the disease can occur at a very low frequency in an infected population, and mortalities may not
exceed the usual background rate. Thus infected fish could easily be included in batches of healthy
translocated fish and there is good evidence that this has occurred. No epidemiological data are available for
ESV or ECV.
The factors modulating the susceptibility of fish to EHNV, ESV and ECV infection are poorly
understood. Clinical outbreaks due to EHNV are associated with poor water quality. In rainbow trout,
Chapter 2.1.1. - Epizootic haematopoietic necrosis
infection occurs naturally at water temperatures from 11 to 17C and experimentally from 8 to 21C (20).
Disease in redfin perch does not occur at temperatures below 12C. The following fish species were found to
be susceptible to EHNV following bath exposure: redfin perch, rainbow trout, Macquarie perch
(Macquaria australasica), mosquito fish (Gambusia affinis), silver perch (Bidyanus bidyanus) and
mountain galaxias (Galaxias olidus) (11). Both juvenile and adult redfin perch may be affected in
outbreaks, but juveniles may be more susceptible to the disease. EHNV has now been detected in diseased
rainbow trout ranging from hatchery fry to table-sized fish, although mortalities are most often seen in 0
fish up to 125 mm fork-length.
The screening and diagnostic procedures for EHNV, ESV and ECV are based on isolation of virus in
cell culture, ELISA, indirect fluorescent antibody tests (IFAT), and electron microscopy (3, 5, 7, 9, 12,
14, 16). Antigen-capture ELISA has a sensitivity of 6080% depending on the stage of infection when
used to test tissues from both rainbow trout and redfin perch (9, 16, 20, 23). Antigen-capture ELISA is
the method of choice for confirming the cause of cytopathic effect in cell culture; IFAT and electron
microscopy are also useful. IFAT or immunoperoxidase staining may also be used for diagnosis on
formalin-fixed tissues. PCR may be used to detect Ranavirus DNA in infected tissues (6). Western blots,
SDS-PAGE and PCR sequencing can be used to specifically identify EHNV, ESV and ECV (10,
13).
DIAGNOSTIC PROCEDURES
The diagnosis of epizootic haematopoietic necrosis (EHN) is based on direct methods that are either the
isolation of virus (epizootic haematopoietic necrosis virus [EHNV], European sheatfish virus [ESV] and
European catfish virus [ECV]) in cell culture followed by its immunological identification (conventional
approach), or the immunological demonstration of virus antigen in infected fish tissues.
Due to insufficient knowledge of the serological responses of fish to virus infections, the detection of fish
antibodies to viruses has not thus far been recognised as a valuable diagnostic method for assessing the
virus status of fish populations. In the case of EHNV, preliminary investigations using enzyme-linked
immunosorbent assay (ELISA) in known infected rainbow trout populations have revealed up to 1%
prevalence of seropositive fish in adult age classes. Further investigation of the epidemiology of EHNV in
rainbow trout is needed before serological results can be interpreted.
Infected fish material suitable for virological examination is:
Clinically affected fish: whole alevin (body length 4 cm), viscera including kidney (4 cm body
length 6 cm) or, for larger size fish, kidney, spleen and liver.
Asymptomatic fish (apparently healthy fish): kidney, liver, spleen, heart, milt and ovarian fluid at
spawning time.
Sampling procedures: see Chapter I.1. Section B.
1. STANDARD SCREENING METHOD FOR EHNV, ESV AND ECV
1.1. Isolation of EHNV, ESV and ECV in cell culture
Cell line to be used: BF-2, CHSE-214 or EPC
BF-2 cells are preferred for EHNV and should be grown at 22C in a temperature-controlled
refrigerated incubator to ensure subsequent success in isolation of EHNV. Incubation at higher or
fluctuating temperatures may reduce the susceptibility of the cells to infection, regardless of the
incubation temperature after inoculation with virus. In the isolation of iridovirus of catfish (ECV),
the best results have been obtained using EPC cells. Each laboratory should confirm the
susceptibility of cell line(s) in current use and the suitability of cell lines other than those specified
here. The most sensitive cell line should always be used. Although EHNV growth is optimum at
22C, laboratories may need to determine optimum incubation temperatures to suit local
conditions, and for temperate regions and viruses other than EHNV (such as ESV and ECV) this
may be 15C. In all cases, appropriate positive and negative controls should be included with the
virus isolation tests.
a) Inoculation of cell monolayers
i) Make two additional tenfold dilutions of the 1/10 organ homogenate supernatants and
transfer an appropriate volume of each of the three dilutions on to 24-hour-old cell
monolayers. Inoculate at least 2 cm
2
of drained cell monolayer with 100 l of each
dilution.
ii) Inoculate directly or allow to adsorb for 0.51 hour at 2225C and, without withdrawing
inoculate, add cell culture medium buffered at pH 7.6 and supplemented with 2% fetal
calf serum (FCS) (1 ml/well for 24-well cell culture plates) and incubate at 22C using a
temperature-controlled refrigerated incubator to ensure successful isolation.
b) Monitoring incubation
i) Follow the course of infection in positive controls and other inoculated cell cultures by
daily microscopic examination at magnification 40100, for 14 days. The use of a phase-
contrast microscope is recommended.
ii) Maintain the pH of the cell culture medium at between 7.3 and 7.6 over the whole
incubation phase. This can be achieved by addition to the inoculated cell culture medium
of sterile bicarbonate buffer (for tightly closed cell culture flasks) or 2 M Tris buffer
solution (for cell culture plates) or, preferably, by using HEPES-buffered media (HEPES
= N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid).
iii) If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of
the tested homogenate supernatants, identification procedures have to be undertaken
immediately (see Section 1.2. below).
If a fish health surveillance/control programme is being implemented, provisions have to be taken to
suspend the approved health status of the production unit and/or the zone (if it was approved
previously) from which the virus positive sample originated. The suspension of approved status will be
maintained until it is demonstrated that the virus in question is not EHNV, ESV or ECV.
iv) If no CPE develops in the inoculated cultures (despite normal progression of CPE in the
virus controls), the inoculated cultures should be subcultured for a further 7 days and, if
no CPE has developed by then, tests for ESV and ECV can be declared negative.
However, for EHNV testing, the cultures should be subjected to two further blind
passages (giving a total of three passes, a primary culture and two subcultures). Should the
virus control fail to develop CPE, the process should be repeated with fresh susceptible
cells and new batches of organ samples.
c) Subcultivation procedures
i) Collect aliquots of cell culture medium from all monolayers inoculated with dilutions of
each supernatant of organ homogenates.
ii) Inoculate cell monolayers as described above (1.1.a.).
iii) Incubate and monitor as in 1.1.b.
1.2. Identification of EHNV, ESV and ECV
Note: All antibodies used in the tests described below have been generated against a defined strain
of EHNV at the OIE Reference Laboratories in Australia. The antibodies cross-react with ESV,
ECV and all other known Ranaviruses. Techniques including but not limited to indirect
fluorescent antibody staining, immunoperoxidase staining and antigen-capture ELISA can be used
to identify EHNV, ESV, ECV and other Ranaviruses in cell culture.
a) Neutralisation test
EHNV, ESV and ECV cannot be identified by neutralisation as the antisera generated by the
immunisation of rabbits have few neutralising antibodies.
b) Indirect fluorescent antibody test
This indirect fluorescent antibody test (IFAT) is to be conducted directly after virus isolation
in cell culture.
i) Prepare monolayers of susceptible cells in 2 cm
2
wells of cell culture plastic plates or on
cover-slips in order to reach around 80% confluency, which is usually achieved within
24 hours of incubation at 22C (seed six cell monolayers per virus isolate to be identified,
plus two for positive and two for negative controls). The FCS content of the cell culture
medium can be reduced to 24%. If numerous virus isolates have to be identified, the use
of Terasaki plates is strongly recommended.
ii) When the cell monolayers are ready for infection, i.e. on the same day or on the day after
seeding, inoculate the virus suspensions to be identified by making tenfold dilution steps
directly in the cell culture wells or flasks.
iii) Dilute the control virus suspension of EHNV, ESV or ECV in a similar way, in order to
obtain a virus titre of about 500010,000 plaque-forming units per ml in the cell culture
medium.
iv) Incubate at 22C for 24 hours.
v) Remove the cell culture medium, rinse once with 0.01 M phosphate buffered saline (PBS),
pH 7.2, then three times briefly with cold fixative. This fixative will be acetone (stored at
20C) for cover-slips or a mixture of acetone 30%/ethanol 70% (v/v) for plastic, also
stored at 20C.
vi) Let the fixative act for 15 minutes. A volume of 0.5 ml is adequate for 2 cm
2
of cell
monolayer.
vii) Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or
freeze at 20C.
viii) Prepare a solution of purified polyclonal antibody or serum to EHNV (also provided by
OIE Reference Laboratories) in 0.01 M PBS, pH 7.2, containing 2% skim milk, at the
appropriate dilution (which has been established previously or is given by the reagent
supplier).
ix) Rehydrate the dried cell monolayers by rinsing four times with PBS and remove this
buffer completely after the last rinsing.
x) Treat the cell monolayers with the antibody solution for 1 hour at 37C in a humid
chamber. The volume of solution to be used is 0.25 ml/2 cm
2
well.
xi) Rinse four times with PBS as above.
xii) Incubate the cell monolayers in appropriately diluted biotinylated anti-rabbit antibody (in
2% skim milk in PBS) at 37C for 1 hour.
xiii) Rinse four times with PBS as above.
xiv) Treat the cell monolayers for 1 hour at 37C with a solution of FITCstreptavidin
conjugate (FITC = fluorescein isothiocyanate).
xv) Rinse four times with PBS.
xvi) Examine the treated cell monolayers on plastic plates immediately, or mount the cover-
slips using glycerol saline at pH 8.5 prior to microscopic observation.
xvii) Examine under incident UV light using a microscope with 10 eye pieces and 20
40 objective lens having numerical aperture >0.65 and >1.3, respectively. Positive and
negative controls must be found to give the expected results prior to any other
observation. Positive results are indicated by a granular fluorescence within the cytoplasm,
fluorescent inclusion bodies are also evident. An overall fluorescence indicates
nonspecific reactivity.
c) Enzyme-linked immunosorbent assay
i) Purify rabbit anti-EHNV immunoglobulins by affinity chromatography with Protein A.
Coat the wells of microplates designed for ELISAs with 100 l of an appropriate dilution
of purified rabbit immunoglobulins in an alkaline coating buffer, pH 9.6, designed for
ELISA, such as borate or carbonate buffer.
ii) Incubate overnight at 4C.
iii) Rinse four times with 0.01 M PBS containing 0.05% Tween 20 (PBST).
iv) Block with skim milk (5% in PBST) or other blocking solution for 30 minutes at 22C.
v) Rinse four times with PBST.
vi) Dispense 100 l/well of the cell culture fluid containing the virus to be identified, and of
EHNV control virus, and allow to react with the coated antibody for 6090 minutes at
22C.
vii) Rinse four times with PBST. Collect waste fluids and autoclave. (Note: all waste materials
should be treated this way.)
viii) Add to the wells 100 l of an appropriate dilution of a second antibody, usually sheep
antibodies to EHNV (provided by OIE Reference Laboratories). These may or may not
be conjugated to biotin. Allow to react for 6090 minutes at 22C.
ix) Rinse four times with PBST.
x) Add to the wells 100 l of appropriate dilutions of either streptavidin-conjugated
horseradish peroxidase, or a commercial anti-sheep immunoglobulin conjugated to
horseradish peroxidase, as appropriate. Allow to react for 6090 minutes at 22C.
xi) Rinse four times with PBST.
xii) Add the substrate (H
2
O
2
) and chromogen (O-phenylenediamide or other approved
chromogen). Stop the course of the test when positive controls react, and read the results.
Note: Dilutions of second antibody and enzyme conjugate should be prepared in PBST to
which 0.1% (w/v) gelatin, ovalbumin or other suitable blocking agent is added.
2. DIAGNOSTIC METHODS FOR EHNV, ESV AND ECV
2.1. Virus isolation with subsequent identification
As in Section 1.1. and 1.2.
2.2. Indirect fluorescent antibody test for fish tissues
i) Bleed the fish thoroughly.
ii) Make kidney imprints on cleaned glass slides or at the bottom of the wells of a plastic cell
culture plate.
iii) Store the kidney pieces (as indicated in Chapter I.1. Section B.3.1.), together with the other
organs required for virus isolation in case this becomes necessary later.
iv) Allow the imprint to air-dry for 20 minutes.
v) Fix with acetone or ethanol/acetone and dry as indicated in Section 1.2.b. steps vvii.
vi) Rehydrate the above preparations (see Section 1.2.b. step ix) and block with 5% skim milk or
1% bovine serum albumin, in PBS for 30 minutes at 37C.
vii) Rinse four times with PBS.
viii) Treat the imprints with the solution of antibody to EHNV and rinse as indicated in Section
1.2.b.
ix) Block and rinse as previously in steps vi and vii.
x) Treat the imprints for 1 hour at 37C with a solution of FITCstreptavidin conjugate as in
Section 1.2.b.
xi) Rinse four times with PBS.
xii) Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips
using glycerol saline at pH 8.5 prior to microscopic observation.
xiii) Examine under incident UV light. Positive and negative controls must be found to give the
expected results prior to any other observation. Positive results are indicated by a granular
fluorescence within the cytoplasm, fluorescent inclusion bodies are also evident. An overall
fluorescence indicates nonspecific reactivity. If the immunofluorescence test is negative,
process the organ samples stored at 4C for virus isolation in cell culture as in Section 1.1.
2.3. Enzyme-linked immunosorbent assay for fish tissues
a) Microplate processing
As described in Section 1.2.c. of this chapter up to step iv (inclusive).
b) Sampling procedures
EHNV, and presumably ESV and ECV are highly resistant viruses and withstand freezing in
fish carcasses for prolonged periods. Fish may be frozen at the farm and dissected later in the
laboratory.
In order to detect EHNV in rainbow trout only dead or moribund fish should be examined as
it is almost impossible to find EHNV among healthy in-contact fish, even during an outbreak
of EHN.
See the following sections in Chapter I.1:
B.1. for the selection of fish specimens
B.2. for the selection of materials sampled.
c) Processing of organ samples
See the following sections in Chapter I.1.:
B.3.1. for transportation, note that organ samples can be frozen prior to transport to the
laboratory as Ranaviruses (e.g. EHNV) are resistant viruses.
B.3.2. for virus extraction and obtaining of organ homogenates.
d) The enzyme-linked immunosorbent assay procedure
i) Set aside an aliquot of 1/4 of each homogenate and store at 20C or 80C in case virus
isolation in cell culture is required.
ii) Treat the remaining part of the homogenate with 2% Triton X-100 (v/v) and 2 mM of
phenyl methyl sulfonide fluoride; mix gently.
iii) Complete the other steps of the procedure described in Section 1.2.c.
iv) The test has been validated only for EHNV in tissues from redfin perch and rainbow
trout. Positive results in ELISA from tissues of species other than redfin perch and
rainbow trout should be confirmed by virus culture, electron microscopy of fish tissues
and polymerase chain reaction (PCR).
2.4. Polymerase chain reaction amplification for fish tissues
a) Processing of organ samples
laboratory as Ranaviruses (e.g. EHNV) are resistant viruses.
B.3.2. for virus extraction and obtaining organ homogenates.
b) Polymerase chain reaction amplification and sequencing
Ranaviruses such as EHNV, ESV and ECV have a large double-stranded DNA genome of
approximately 125 kb. Two primers, a reverse primer (5-AAA-GAC-CCG-TTT-TGC-AGC-
AGC-AAA-C-3) and a forward primer (5-CGC-AGT-CAA-GGC-CTT-GAT-GT-3), are
used for amplification of the target capsid sequence (580 base pairs [bp]) of EHNV DNA by
PCR. This PCR procedure can be used for detection of iridoviruses from redfin perch,
rainbow trout, sheatfish, catfish, guppy fish (Poecilia reticulata), doctor fish (Labroides dimidatus)
and a range of amphibian Ranaviruses. Fish samples are prepared as per Gould et al. (6).
Nucleic acid (1 l) is added to Taq polymerase buffer containing 0.1 M of each primer, 2.5 U
Taq polymerase (Promega) and 2.5 mM MgCl
2
. The mixture is incubated in an automatic
thermal cycler programmed for 35 cycles at 95C for 60 seconds, 55C for 60 seconds, and
72C for 60 seconds, and finally held at 72C for 15 minutes. Amplified DNA (580 bp) is
analysed by agarose gel electrophoresis, excised and sequenced using the Cyclone Sequencing
Kit (Bresatec, Australia). Each viral species is identified by its unique DNA sequence.
Samples should be submitted to the OIE reference laboratory for specific identification.
c) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
EHNV, ESV and ECV can be differentiated from other iridoviruses by analyses of viral
proteins in reducing polyacrylamide gels (10%). The major coat protein of EHNV is always
slightly larger (approximately 51 kDa) compared with other iridoviruses such as ESV and ECV
(approximately 49 kDa).
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Dis., 9, 263268.
13. MAO J., HEDRICK R.P. & CHINCHAR V.G. (1997). Molecular characterisation, sequence analysis and
taxonomic position of newly isolated fish iridoviruses. Virology, 229, 212220.
14. POZET F., MORAND M., MOUSSA A., TORHY C. & DE KINKELIN P. (1992). Isolation and preliminary
characterization of a pathogenic icosahedral deoxyribovirus from the catfish (Ictalurus melas). Dis.
Aquat. Org., 14, 3542.
15. REDDACLIFF L.A. & WHITTINGTON R.J. (1996). Pathology of epizootic haematopoeitic necrosis virus
(EHNV) infection in rainbow trout (Oncorhynchus mykiss Walbaum) and redfin perch (Perca fluviatilis
L.). J. Comp. Pathol., 115, 103115.
16. STEINER K.A., WHITTINGTON R.J., PETERSEN R.K., HORNITZKY C. & GARNETT H. (1991).
Purification of epizootic haematopoietic necrosis virus and its detection using ELISA. J. Virol.
Methods, 33, 199209.
17. TAPIOVAARA H., OLESEN N.J., LINDEN J., RIMAILA-PARNANEN VON BONSDORFF C.-H. (1998).
Isolation of an iridovirus from pikeperch (Stizostedion lucioperca). Dis. Aquat. Org., 32, 185193.
18. WHITTINGTON R.J. & HYATT A.D. (1996). Contingency planning or control of epizootic
haematopoietic necrosis disease. Singapore Vet. J., 20, 7987.
19. WHITTINGTON R.J., KEARNS C., HYATT A.D., HENGSTBERGER S. & RUTZOU T. (1995). Spread of
epizootic haematopoietic necrosis virus (EHNV) in redfin perch (Perca fluviatilis) in southern Australia.
Aust. Vet. J., 73, 112114.
20. WHITTINGTON R.J., PHILBY A., REDDACLIFF G.L. & MACGOWN A.R. (1994). Epidemiology of
epizootic haematopoietic necrosis virus (EHNV) infection in farmed rainbow trout, Oncorhynchus
mykiss (Walbaum): findings based on virus isolation, antigen capture ELISA and serology. J. Fish Dis.,
17, 205218.
21. WHITTINGTON R.J. & REDDACLIFF G.L. (1995). Influence of environmental temperature on
experimental infection of redfin perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss) with
epizootic haematopoietic necrosis virus, an Australian iridovirus. Aust. Vet. J., 72, 421424.
22. WHITTINGTON R.J., REDDACLIFF L.A., MARSH I., KEARNS C., ZUPANOVIC Z. & CALLINAN R.B.
(1999). Further observations on the epidemiology and spread of epizootic haematopoietic necrosis
virus (EHNV) in farmed rainbow trout Onchorhynchus mykiss in southeastern Australia and a
recommended sampling strategy for surveillance. Dis. Aquat. Org., 35, 125130.
23. WHITTINGTON R.J. & STEINER K.A. (1993). Epizootic haematopoietic necrosis virus (EHNV):
improved ELISA for detection in fish tissues and cell cultures and an efficient method for release of
antigen from tissues. J. Virol. Methods, 43, 205220.
476 pp.
*
* *
C HA P T E R 2 . 1 . 2 .

I NF E CT I OUS HAE MAT OP OI E T I C NE CROS I S

SUMMARY
Infectious haematopoietic necrosis (IHN) is an infectious disease of rainbow or steelhead trout
(Oncorhynchus mykiss), Pacific salmon including chinook (O. tshawytscha), sockeye (O. nerka),
chum (O. keta), masou (O. masou), and coho (O. kisutch), and Atlantic salmon (Salmo salar).
Historically, the geographical range of IHN was limited to the western parts of North America, but the
disease, caused by a rhabdovirus (IHNV), has spread to continental Europe and the Far East via the
importations of infected fish and eggs. For more detailed reviews of the disease, see Bootland & Leong (3)
or Wolf (19).
The principal clinical and economic consequences of IHN occur at farms rearing rainbow trout in
freshwater; however, both Pacific and Atlantic salmon reared in freshwater or seawater can be severely
affected. Large mortalities have also been recorded among some wild stocks of Pacific salmon. Infection is
often lethal due to the impairment of osmotic balance, and occurs within a clinical context of oedema and
haemorrhage. Virus multiplication in endothelial cells of blood capillaries, haematopoietic tissues and
nephron cells, underlies the clinical signs. High levels of virus are shed from infected juvenile fish. Older fish
are increasingly resistant to infection, but adult fish at spawning may shed virus in sexual products.
Survivors of IHNV infection demonstrate a strong protective immunity with the synthesis of circulating
antibodies to the virus (12) and, in certain individuals, a covert carrier state (6).
On the basis of antigenic studies conducted with neutralising polyclonal rabbit antisera, IHNV isolates
form a single serogroup (7). However, mouse monoclonal antibodies have revealed a number of neutralising
epitopes on the glycoprotein (8, 14, 17), as well as the existence of a non-neutralising, group epitope borne
by the nucleoprotein (13). Variations in the virulence of IHNV strains have been recorded during both
natural cases of disease and in experimental infections (10).
The reservoirs of IHNV are clinically infected fish and covert carriers among cultured, feral or wild fish.
Virus is shed via faeces, urine, sexual fluids and external mucus, whereas kidney, spleen, encephalon and
the digestive tract are the sites in which virus is most abundant during the course of overt infection. The
transmission of IHNV between fish is primarily horizontal; however, cases of vertical or egg-associated
transmission have been recorded. Horizontal transmission is typically by direct exposure, but invertebrate
vectors have been proposed to play a role in some cases. Egg-associated transmission is significantly reduced
by the now common practice of surface disinfection of eggs with an iodophor solution, but is the only
mechanism accounting for the appearance of IHN in new geographical locations among alevins originating
from eggs that were incubated and hatched in virus-free water. Once IHNV is established in a farmed
stock or in a watershed, the disease may become established among carrier fish.
Among individuals of each fish species, there is a high degree of variation in susceptibility to IHNV. The
age of the fish appears to be extremely important: the younger the fish, the more susceptible to disease. As
with viral haemorrhagic septicaemia virus, good overall fish health condition seems to decrease the
susceptibility to overt IHN, while co-infections with bacterial diseases (e.g. bacterial coldwater disease),
handling and other types of stress frequently cause subclinical infections to become overt.
The most prominent environmental factor affecting IHN is water temperature. Clinical disease occurs
between 8C and 15C under natural conditions.
Chapter 2.1.2. - Infectious haematopoietic necrosis
The screening procedure for IHNV is based on virus isolation in cell culture. Confirmatory identification
may be achieved by use of immunological (neutralisation, indirect fluorescent antibody test or enzyme-linked
immunosorbent assay), or molecular (DNA probe or polymerase chain reaction) methods (1, 2, 4, 5, 9,
11, 18).
Control methods for IHN currently rely on avoidance of exposure to the virus through the implementation of
strict control policies and sound hygiene practices (15). The thorough disinfection of fertilised eggs and the
incubation of eggs and rearing of fry and alevins on virus-free water supplies in premises completely separated
from those harbouring possible virus carriers and free from possible contact with inanimate objects, are
critical for preventing the occurrence of IHNV in a defined fish production site. At present, vaccination is
at an experimental stage; however, several new vaccine preparations have shown substantial promise in both
laboratory and field trials (16).
The standard screening method for infectious haematopoietic necrosis (IHN) is based on the isolation of
IHN virus (IHNV) in cell culture followed by its immunological or molecular identification as described in
Section 1. Diagnosis of IHN during outbreaks of disease may be made via this conventional approach or
by more rapid immunological or molecular methods that detect IHNV antigens or nucleic acids in infected
fish tissues as described in Section 2. While the diagnostic methods listed in Section 2 can be used for
confirmation of overt infections in fish, they are not approved for use as screening methods for obtaining
approved IHN-free status.
Due to substantial variation in the serological responses of fish to virus infections, the detection of fish
antibodies to viruses has not thus far been accepted as a routine diagnostic method for assessing the viral
status of fish populations. In the future, validation of serological techniques for diagnosis of fish virus
infections could render the use of fish serology more widely acceptable for diagnostic purposes.
For general information, sampling procedures, specimen preparation, and materials required for
virological assays: see Chapter I.1.
1. STANDARD SCREENING METHOD FOR IHN
1.1. Isolation of IHNV in cell culture
Cell lines to be used: EPC and BF-2
i) Make an additional tenfold dilution of the 1/10 organ homogenate supernatants and
transfer an appropriate volume of each of the two dilutions on to 24-hour-old cell
2

dilution.
ii) Allow to adsorb for 0.51 hour and, without withdrawing the inoculate, add the cell
culture medium buffered at pH 7.6 and supplemented with 2% fetal calf serum (FCS)
(1 ml/well for 24-well cell culture plates), and incubate at 15C.
iii) If required, the inoculate may be pre-incubated with neutralising antiserum against
infectious pancreatic necrosis virus (IPNV) or other enzootic viruses as previously
described (see Chapter I.1. Section B.3.3.).
daily microscopic examination at 40100 magnification for 7 days. The use of a phase-
ii) Maintain the pH of the cell culture medium between 7.3 and 7.6 during incubation. This
can be achieved, especially in open plates, by using cell culture medium containing
sodium bicarbonate that is further buffered by addition of Tris or HEPES (see Chapter
I.1.C).
iii) If a cytopathic effect (CPE) appears in cell cultures inoculated with dilutions of the fluids
or homogenates, identification procedures must be undertaken immediately (see Section
1.2. below).
If a fish health surveillance/control programme is being implemented, steps may have to be taken to
suspend the approved health status of the production unit and/or the zone (if approved previously)
from which the suspected virus-positive sample originated. The suspension of approved status will be
maintained until it is demonstrated that the virus in question is not IHNV.
virus controls), the inoculated cultures should be subcultured for a further 7 days. Should
the virus control fail to develop CPE, the process should be repeated with fresh cell
cultures and new batches of samples.
i) Collect aliquots of cell culture medium from all monolayers inoculated with various
dilutions of fluid or organ homogenate samples.
ii) If required, repeat the neutralisation test to IPNV or other enzootic viruses as previously
described (see Section 1.1.a).
iii) Inoculate cell monolayers as described above in Section 1.1.a.
iv) Incubate and monitor as described in Section 1.1.b.
v) If no CPE occurs, the test may be declared negative.
1.2. Identification of IHNV
1.2.1. Neutralisation test
i) Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge it at
2000 g for 15 minutes at 4C to remove cell debris.
ii) In parallel, other neutralisation tests must be performed against:
a known isolate of IHNV (positive neutralisation test).
a heterologous virus (negative neutralisation test).
iii) If required, a similar neutralisation test may be performed using antibodies to IPNV or
other enzootic viruses to ensure that no contaminant has escaped the first assay.
a) The neutralisation test procedure
i) Dilute the virus-containing medium from 10
2
to 10
4
.
ii) Mix aliquots (for example 200 l) of each virus dilution with equal volumes of rabbit
polyclonal or mouse monoclonal antibody (MAb) against IHNV, and similarly treat
aliquots of each virus dilution with cell culture medium. (The neutralising antibody [Nab]
solution must have a 50% plaque reduction titre of at least 2000.)
iii) Incubate all the mixtures at 15C for 1 hour.
iv) Transfer aliquots of each of the above mixtures on to drained cell monolayers (inoculate
two cell cultures per dilution) and allow adsorption to occur for 0.51 hour at 15C; 24-
or 12-well cell culture plates are suitable for this purpose, using a 50 l inoculum.
v) When adsorption is complete, add cell culture medium, supplemented with 2% FCS and
buffered at pH 7.47.6, to each well and incubate at 15C.
vi) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
non-neutralised controls (cell monolayers being protected in positive neutralisation
controls). Results are recorded either after a simple microscopic examination (phase-
contrast preferable) or after discarding the cell culture medium and staining the cell
monolayers with a solution of 1% crystal violet in 10% buffered formalin.
vii) The tested virus is identified as IHNV when CPE is prevented or noticeably delayed in
the cell cultures that received the virus suspension treated with the IHNV-specific
antibody, whereas CPE is present in the untreated sample.
In the absence of significant neutralisation when IHNV is suspected, it is advisable to conduct
an indirect fluorescent antibody test (IFAT) as antigenic drift has been observed in the
neutralising epitopes on the IHNV glycoprotein, resulting in occasional failures of the
neutralisation test for certain strains of the virus.
1.2.2. Indirect fluorescent antibody test
a) Preparation of monolayers
i) Prepare monolayers of cells in 2 cm
2
wells of plastic cell culture plates or on glass cover-
slips to reach around 80% confluency within 24 hours of incubation at 22C. Seed six cell
monolayers per virus isolate to be tested, plus two for positive and two for negative
controls. The FCS content of the cell culture medium can be reduced to 24%.
ii) When the cell monolayers are ready for infection (i.e. on the same day or on the day after
seeding), inoculate the virus suspensions to be identified by making tenfold dilution steps
iii) Dilute the control virus suspension of IHNV in a similar way, in order to obtain a virus
titre of about 500010,000 plaque-forming units (PFU)/ml in the cell culture medium.
pH 7.2, then three times briefly with cold acetone (stored at 20C) for glass cover-slips
or a mixture of 30% acetone and 70% ethanol, also at 20C, for plastic wells.
2
of cell
monolayer.
freeze at 20C.
b) The indirect fluorescent antibody procedure
i) Prepare a solution of antibody against IHNV in PBS containing 0.05% Tween 80 (PBST)
at the appropriate dilution (established previously or given by the reagent supplier). The
antibody used should be able to bind to all isolates of the virus.
ii) Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, and
remove this buffer completely after the last rinsing.
iii) Treat the cell monolayers with the antibody solution for 1 hour at 37C in a humid
chamber and do not allow evaporation to occur. The volume of solution to be used is
0.25 ml/2 cm
2

well.)
iv) Rinse four times with PBST as above.
v) Treat the cell monolayers for 1 hour at 37C with a solution of fluorescein isothiocyanate
(FITC)-conjugated antibody to the immunoglobulin used in the first layer and prepared
according to the instructions of the supplier. These FITC antibodies are most often rabbit
or goat antibodies.
vi) Rinse four times with PBST.
vii) Examine the treated cell monolayers on plastic plates immediately, or mount the cover-
viii) Examine under incident UV light using a microscope with a 2040 objective lens having
a high numerical aperture. Positive and negative controls must be found to give the
expected results prior to any other observation.
1.2.3. Enzyme-linked immunosorbent assay
a) Preparation of microplates
i) Coat the wells of microplates designed for enzyme-linked immunosorbent assays
(ELISAs) with appropriate dilutions of immunoglobulins (Ig) specific for IHNV, in
0.01 M PBS, pH 7.2 (200 l/well). The Ig may be polyclonal or monoclonal, most often
from rabbit or mouse. For the identification of IHNV, MAbs specific for certain domains
of the nucleocapsid (N) protein are suitable.
iv) Block with skim milk (5% in PBST) or other blocking solution for 1 hour at 37C
(200 l/well).
b) The enzyme-linked immunosorbent assay procedure
i) Add 2% Triton X-100 to the virus suspension to be identified.
ii) Dispense 100 l/well of two- or four-step dilutions of the virus to be identified and of
IHNV control virus, and allow to react with the coated antibody to IHNV for 1 hour at
20C.
iii) Rinse four times with PBST.
iv) Add to the wells either biotinylated polyclonal rabbit antiserum to IHNV or biotinylated
mouse MAb to an N protein epitope different from the one recognised by the coating
MAb.
v) Incubate for 1 hour at 37C.
vi) Rinse four times with PBST.
vii) Add streptavidin-conjugated horseradish peroxidase to those wells that have received the
biotin-conjugated antibody, and incubate for 1 hour at 20C.
viii) Rinse four times with PBST.
ix) Add the substrate and chromogen. Stop the course of the test when positive controls
react, and read the results.
1.2.4. DNA probe
a) Infect cells with virus isolates
i) Trypsinise EPC (or other) cells and resuspend to 1.0 10
6
cells per ml with MEM-5-T
(5% FBS [fetal bovine serum], Tris buffer). Transfer 1 ml of cell suspension to each well
of a 24-well tissue culture plate and incubate the plate at an appropriate temperature.
ii) On the day after seeding cells, remove the fluid from each cell monolayer. Infect cells
with positive control (IHNV) and with the unknown fish virus isolates in 1 ml total
volume to provide a multiplicity of infection (m.o.i.) of 10 PFU per cell. Negative control
cells receive 1 ml MEM-5-T. Incubate for 2 hours at 1520C on a rocker platform.
iii) Incubate infected and control cells at 15C overnight. The desired product is the N gene
mRNA, and maximum production occurs at about 12 hours post-infection when
inoculated at high m.o.i. Thus, mRNA production is optimum when infected cells are at
an early stage of virus replication, and RNA can be extracted from cells at the first sign of
any CPE. Alternatively, use recently infected cells from the virus isolation procedure.
b) Make or purchase the following solutions
i) Prehybridisation buffer
Deionised water (DEPC [diethyl pyrocarbonate]-
treated; autoclaved)
69.5 ml
10 Denhardts solution 10 ml of 100 stock (step iv)
2 SSC (standard saline citrate) 10 ml of 20 stock (step vi)
1% SDS (sodium dodecyl sulfate) 10 ml of 10% stock (step viii)
0.1 mg/ml SSS (sonicated salmon sperm) DNA 0.5 ml stock (step v)
ii) Hybridisation solution
Prehybridisation buffer 10 ml (step i)
Biotinylated DNA probe 100 ng/ml
(Store at 20C; may re-use up to five times)
iii) Post-hybridisation solution
2 SSC 50 ml of 20 stock (step vi)
0.1% SDS 5 ml of 10% stock (step viii)
Deionised water (DEPC-treated; autoclaved) up to 500 ml
iv) 100 Denhardts solution
Bovine serum albumin 50 g
Polyvinylpyrrolidone 360 50 g
Ficoll 400 50 g
v) Sonicated salmon sperm DNA (SSS DNA at 20 mg/ml)
Transfer 0.5 ml of SSS DNA (20 mg/ml) into vials and place into boiling water for
10 minutes. Cool vials in crushed ice and store at 20C. When needed, add 0.5 ml to
prehybridisation buffer (see step i).
vi) 20 standard saline citrate (20 SSC)
NaCl 87.65 g
Citric acid 44.11 g
(Adjust to pH 7.0 with HCl, autoclaved)
vii) 10 standard saline citrate (10 SSC)
NaCl 43.82 g
Citric acid 22.05 g
(Adjust to pH 7.0 with HCl, autoclaved OR dilute 1/2 from 20 SSC using deionised
water, autoclaved)

viii) 10% sodium dodecyl sulfate (10% SDS)
Lauryl sulfate sodium salt 10.0 g
(Adjust to pH 7.2. Do not autoclave this solution!)
ix) Streptavidin/alkaline phosphate conjugate (SA/AP)
0.1 g/ml streptavidin/alkaline phosphatase conjugate. Prepare by diluting SA/AP
1/1000 in buffer A (step x). (May re-use this solution up to five times, store at 4C.)
x) Buffer A
0.1 M Tris, pH 7.5 50 ml of 1 M stock (step xiv)
0.1 M NaCl 10 ml of 5 M stock (step xii)
2 mM MgCl
2
1 ml of 1 M stock
0.05% Triton X-100 0.25 ml
xi) Buffer B
0.1 M Tris, pH 9.5 50 ml of 1 M stock (step xiii)
0.1 M NaCl 10 ml of 5 M stock (step xii)
50 mM MgCl
2
25 ml of 1 M stock
xii) 5 M NaCl
NaCl 146.1 g
(autoclave this solution)
xiii) 1 M Tris buffer, pH 9.5
Tris base 54.7 g
Tris HCl 7.6 g
(Adjust to pH 9.5, then autoclave)
xiv) 1 M Tris buffer, pH 7.5
Tris base 11.8 g
Tris HCl 63.5 g
(Adjust to pH 7.5, then autoclave)
xv) Chloroform
Chloroform (Store at 20C until needed)
xvi) Isopropyl alcohol
2 propanol (isopropyl alcohol). Use undiluted for precipitation of RNA.
xvii) Trizol
RNA isolation reagent, store at 28C in the dark. Invitrogen Catalog No. 15596-026.
Caution: Contains guanidinium isothiocyanate and phenol.
xviii) Alkaline phosphatase conjugate substrate kit
(NOTE: This product contains dimethylformamide. Use in area with good ventilation.)
Dissolve alkaline phosphatase (AP) colour development buffer in 1 litre volume of distilled
deionised water. Filter-sterilise then store at 4C. Immediately before use, add 0.4 ml of AP
colour reagent A and 0.4 ml AP colour reagent B to 39.2 ml colour development buffer at
room temperature.
c) Prepare biotinylated oligonucleotide probe
i) The biotinylated probe (5-CTT-GTT-TTG-GCA-GTA-TGT-GGC-CAT-CTT-GTC-3) is
made using the 30-nucleotide sequence identified by Deering et al. (4); however, three
nucleotides containing biotin can be conveniently added to the 5 end during DNA
synthesis rather than by a subsequent terminal transferase reaction at the 3 end. This
antisense probe is complementary to a conserved region in the middle of the IHNV
nucleoprotein gene mRNA. It should react only with IHNV and should recognise all
isolates of the virus. The final concentration of the biotinylated probe will be 0.1 g/ml in
hybridisation solution.
d) Extraction of mRNA from infected cells
i) Always wear protective gloves to avoid contaminating solutions with RNase from skin
and to avoid injury. Provide adequate ventilation, especially for RNA extraction steps
with Trizol and for dimethylformamide in colour development solutions.
ii) Remove culture medium from infected cells and add 1.0 ml Trizol to each well. Replace
lid and place plates on a rocker platform for 510 minutes at room temperature to digest
the cells. During incubation, load 100 l chloroform into siliconised 1.7 ml centrifuge
tubes, keep on ice.
iii) Triturate cell debris by pipetting with a sterile 1 ml pipette (5), then transfer solution
from each well into a separate tube. Vortex the tubes (about 3 seconds each), store on
crushed ice for 5 minutes to allow phase separation.
iv) Centrifuge the suspension at 12,500 g for 15 minutes at 4C. The RNA will remain in the
clear aqueous phase while the DNA and protein will be left in the lower red phenol
phase.
v) During step iv, load another set of tubes with 0.5 ml of absolute isopropyl alcohol, store
on ice.
vi) Carefully transfer the upper aqueous phase, which contains the RNA (about 0.5 ml), to a
tube that contains an equal volume (0.5 ml) of absolute isopropyl alcohol. Vortex tubes
for 1 second, and chill tubes on ice for 15 minutes to precipitate RNA.
vii) Centrifuge the mixture at 12,500 g for 15 minutes at 4C. Remove as much fluid from the
pellet as possible. Partially dry the pellet following the manufacturers instructions.
viii) Prepare a nitrocellulose membrane (0.45 m pore size): wet in distilled deionised water
for 1 minute, pour water off, then soak for at least 5 minutes in 10 SSC.
ix) Warm the prehybridisation buffer to 55C in a water bath.
x) Add 170 l distilled deionised, ribonuclease-free (DEPC-treated and autoclaved) or
molecular-biology grade water to the RNA pellets. Mix the contents and warm the tubes
in a heat block at 65C for 1520 minutes (RNA pellets should dissolve).
xi) Install the membrane in 96-well vacuum blotting device, attach vacuum pump hoses and
add 200 l of 10 SSC to each well to insure that the membrane is not dry when the
RNA is added.
xii) Heat IHNV PCR products (if used as positive control instead of RNA from IHNV-
infected cells) to 95C for 5 minutes to denature the double-stranded DNA, then put
tubes directly on ice.
xiii) Add 170 l of 20 SSC into microcentrifuge tubes containing the dissolved pellets of
RNA in 170 l of water (tubes now contain 340 l of 10 SSC). Store on ice.
xiv) Add 100 l of each RNA solution to wells of a blotting device that contain 200 l of 10
SSC. Blot positive controls last. Apply vacuum for about 1 minute until fluid is pulled
through the membrane. Remove membrane and transfer to thick filter paper wetted with
10 SSC.
xv) Cut the membrane into sections if required and label the membranes in one corner.
xvi) Place the membrane(s) between dry sheets of blotting paper and microwave for
60 seconds on high power to attach nucleic acids to the membrane. Include a beaker of
water in the microwave. (Ultraviolet radiation or other suitable methods may be used.)
e) Hybridisation of probe to RNA on nitrocellulose membrane
i) Place the membranes (spots up) in a hybridisation pouch, bottle or plastic bag. Add 10 ml
prehybridisation buffer to each membrane. Prehybridise for 1 hour at 55C in a shaker
water bath.
ii) Thaw the probe solution and add 100 l to prehybridisation buffer. React the membranes
in probe solution for 1 hour in shaker water bath at 55C.
iii) Remove probe solutions and store in tubes at 20C for re-use up to five times.
iv) Rinse the membranes with 40 ml of post-hybridisation solution. Discard the solution then
add a fresh 40 ml of post-hybridisation solution. Wash for 15 minutes on a rocker
platform at room temperature. Wash two more times (40 ml) for 15 minutes each at room
temperature on a rocker platform. Put the container with the membranes into a 55C
water bath with pre-warmed post-hybridisation solution (55C) for 15 minutes.
v) Rinse the membranes briefly with 40 ml of buffer A.
f) Colour development of biotinylated probe
i) Incubate the membranes in a solution containing 40 l streptavidin/alkaline phosphatase
in 40 ml buffer A for 30 minutes at room temperature on a rocker platform. This solution
can be re-used at least five times.
ii) Rinse the membranes briefly (40 ml) then wash twice using 40 ml buffer A incubated for
7 minutes at room temperature on a rocker platform.
iii) Wash twice in 40 ml buffer B incubated for 7 minutes at room temperature on a rocker
platform.
iv) Warm the colour development buffer prepared according to the manufacturers protocol.
Buffer should be filter-sterilised through a 0.2 m membrane and stored at 4C.
Immediately before use, add 0.4 ml alkaline phosphatase (AP) colour reagent A and
0.4 ml AP colour reagent B to 39.2 ml AP colour development buffer at room
temperature. Add 40 ml colour development solution containing nitro-blue tetrazolium
(NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to each container with the
membranes. Incubate at room temperature on a rocker platform and observe for colour
development (up to 15 minutes).
v) Wash the membranes in distilled deionised water for 10 minutes, change the water at least
once to remove excess colour development solution. Store membranes in distilled
deionised water until photographs can be taken (if desired).
1.2.5. Polymerase chain reaction
a) Viral RNA preparation
i) Collect aliquots of culture medium from cell monolayers exhibiting CPE and centrifuge at
ii) Release RNA from virus solution by diluting the sample 1/20 in sterile deionised water,
heating the tube at 95C for 2 minutes, then storing on ice. This simple procedure is
especially suitable for virus grown in cell culture where few PCR-inhibiting substances are
present. Alternatively, RNA can be extracted from a pellet of infected cells using methods
described in Section 1.2.4. or by use of other commercially available kits (18).
b) Reverse-transcription and first round PCR protocol
i) Prepare a master mix for the number of samples to be analysed. Work under a hood and
wear gloves.
ii) The master mix for one 50 l reverse-transcription PCR is prepared as follows: 23.75 l
ribonuclease-free (DEPC-treated) or molecular-biology grade water; 5 l 10 buffer; 5 l
25 mM MgCl
2
; 5 l 2 mM dNTP; 2.5 l (20 pmoles/l) Upstream Primer 5-TCA-AGG-
GGG-GAG-TCC-TCG-A-3; 2.5 l (20 pmoles/l) Downstream Primer 5-CAC-CGT-
ACT-TTG-CTG-CTA-C-3; 0.5 l Taq polymerase (5 U/l); 0.5 l AMV reverse
transcriptase (9 U/l); 0.25 l RNasin (39 U/l).
iii) Centrifuge the tubes briefly (10 seconds) to make sure the contents are at the bottom.
iv) Place the tubes in the thermal cycler and start the following cycles 1 cycle: 50C for
15 minutes; 1 cycle: 95C for 2 minutes; 25 cycles: 95C for 30 seconds, 50C for
30 seconds, 72C for 60 seconds; 1 cycle: 72C for 7 minutes and soak at 4C.
vi) Visualise the 786 bp PCR amplicon by electrophoresis of the product in 1.5% agarose gel
with ethidium bromide and observe using UV transillumination.
c) Second round PCR protocol
i) If the first round PCR provides insufficient amplified product, an internally nested set of
primers is used for additional DNA amplification. Prepare a master mix for the number
of samples to be analysed. Work under a hood and wear gloves.
ii) The master mix for one 50 l second round PCR is prepared as follows: 27.5 l
molecular-biology grade water; 5 l 10 buffer; 5 l 25 mM MgCl

; 5 l 2 mM dNTP;
2.5 l (20 pmoles/l) Upstream Primer 5-TTC-GCA-GAT-CCC-AAC-AAC-AA-3;
2.5 l (20 pmoles/l) Downstream Primer 5-GCG-CAC-AGT-GCC-TTG-GCT-3;
0.5 l Taq polymerase (5 U/l).
iii) Dispense 48 l of the master mix into each tube and add 2 l of first round PCR
template.
iv) Centrifuge the tubes briefly (10 seconds) to make sure the contents are at the bottom.
v) Place the tubes in a thermal cycler and start the following cycles 1 cycle: 95C for
2 minutes; 25 cycles: 95C for 30 seconds, 50C for 30 seconds, 72C for 60 seconds;
1 cycle: 72C for 7 minutes and soak at 4C.
vi) Visualise the 323 bp PCR amplicon by electrophoresis of the product in 1.5% agarose gel
with ethidium bromide and observe using UV transillumination.
The DNA probe procedure (Section 1.2.4. of this chapter) can be used to further confirm that
the amplicon is from IHNV. Alternatively, the amplicon can be sequenced (18).

NOTE: Other primer sets have been used for the successful amplification of a portion of the
N gene of IHNV (18); however, the primer sequences used here have been shown to be
conserved among all known isolates of IHNV and are not present in the N gene of the related
fish rhabdoviruses, viral hemorrhagic septicemia virus or hirame rhabdovirus.
2. DIAGNOSTIC METHODS FOR IHN
a) Sampling procedures
b) Processing of organ samples
B.3.1. for transportation
c) Virus isolation and identification
As in Sections 1.1. and 1.2.
2.2. Indirect fluorescent antibody test
culture plate.
iii) Store the kidney pieces (as indicated in Chapter I.1. Section B.3.1.) together with the other
b) Indirect fluorescent antibody procedure
i) Fix with acetone or ethanol/acetone and dry as indicated in Section 1.2.2. of this chapter.
ii) Rehydrate the above preparations and block with 5% skim milk or 1% bovine serum
albumin, in phosphate buffered saline containing 0.05% Tween 80 (PBST) for 30 minutes
at 37C.
iii) Rinse four times with PBST.
iv) Treat the imprints with the solution of antibody to IHNV and rinse as indicated in
Section 1.2.2.
v) Block and rinse as described previously.
vi) Reveal the reaction with suitable FITC-conjugated specific antibody, rinse and observe as
indicated in Section 1.2.2.
If the test is negative, process the organ samples stored at 4C for virus isolation in cell culture
as described in Section 1.1.
2.3. Enzyme-linked immunosorbent assay
c) Microplate processing
As described in Section 1.2.3. of this chapter.
i) Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell
culture is required.
ii) Treat the remaining part of the homogenate with 2% Triton X-100, as in Section 1.2.3. of
this chapter, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
iii) Complete the other steps of the procedure described in Section 1.2.3.
2.4. Polymerase chain reaction
c) Viral RNA preparation
ii) Release RNA from tissue homogenate by diluting sample 1/20 in sterile deionised water,
heating the tube at 95C for 2 minutes, then storing on ice. Alternatively, RNA can be
extracted from tissue homogenates using methods described in Section 1.2.4. or by use of
other commercially available kits (18).
d) The polymerase chain reaction procedure
Perform the remaining steps of the PCR procedure described in Section 1.2.5.
REFERENCES
1. ARAKAWA C.K., DEERING R.E., HIGMAN K.H., OSHIMA K.H., OHARA P.J. & WINTON J.R. (1990).
Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious
hematopoietic necrosis virus. Dis. Aquat. Org., 8, 165170.
2. ARNZEN J.M., RISTOW S.S., HESSON C.P. & LIENTZ J. (1991). Rapid fluorescent antibody tests for
infectious hematopoietic necrosis virus (IHNV) utilizing monoclonal antibodies to the nucleoprotein
and glycoprotein. J. Aquat. Anim. Health, 3, 109113.
3. BOOTLAND L.M. & LEONG J.C. (1999). Infectious hematopoietic necrosis virus. In: Fish Diseases
and Disorders, Volume 3: Viral, Bacterial and Fungal Infections, Woo P.T.K. & Bruno D.W., eds.
CAB International, Oxon, UK, 57121.
4. DEERING R.E., ARAKAWA C.K., OSHIMA K.H., OHARA P.J., LANDOLT M.L. & WINTON J.R.
(1991). Development of a biotinylated DNA probe for detection and identification of infectious
hematopoietic necrosis virus. Dis. Aquat. Org., 11, 5765.
5. DIXON P.F. & HILL B.J. (1984). Rapid detection of fish rhabdoviruses by the enzyme-linked
immunosorbent assay (ELISA). Aquaculture, 42, 112.
6. DROLET B.S., CHIOU P.P., HEIDEL J. & LEONG J.C. (1995). Detection of truncated virus particles in
a persistent RNA virus infection in vivo. J. Virol., 69, 21402147.
7. ENGELKING H.M., HARRY J.B. & LEONG J.C. (1991). Comparison of representative strains of
infectious hematopoietic necrosis virus by serological neutralization and cross-protection assays.
8. HUANG C., CHIEN M-S., LANDOLT M. & WINTON J.R. (1994). Characterization of the infectious
hematopoietic necrosis virus glycoprotein using neutralizing monoclonal antibodies. Dis. Aquat. Org.,
18, 2935.
9. JORGENSEN P.E.V., OLESEN N.J., LORENZEN N., WINTON J.R. & RISTOW S.S. (1991). Infectious
hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS): detection of trout antibodies
to the causative viruses by means of plaque neutralization, immunofluorescence, and enzyme-linked
immunosorbent assay. J. Aquat. Anim. Health, 3, 100108.
10. LAPATRA S.E., FRYER J.L. & ROHOVEC J.S. (1993). Virulence comparison of different
electropherotypes of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 16, 115120.
11. LAPATRA S.E., ROBERTI K.A., ROHOVEC J.S. & FRYER J.L. (1989). Fluorescent antibody test for the
rapid diagnosis of infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 1, 2936.
12. LAPATRA S.E., TURNER T., LAUDA K.A., JONES G.R. & WALKER S. (1993). Characterization of the
humoral response of rainbow trout to infectious hematopoietic necrosis virus. J. Aquat. Anim.
Health, 5, 165171.
13. RISTOW S.S. & ARNZEN J.M. (1989). Development of monoclonal antibodies that recognize a type 2
specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus. J.
14. RISTOW S.S. & ARNZEN DE AVILA J.M. (1991). Monoclonal antibodies to the glycoprotein and
nucleoprotein of infectious hematopoietic necrosis virus (IHNV) reveal differences among isolates of
the virus by fluorescence, neutralization and electrophoresis. Dis. Aquat. Org., 11, 105115.
15. WINTON J.R. (1991). Recent advances in the detection and control of infectious hematopoietic
necrosis virus (IHNV) in aquaculture. Ann. Rev. Fish Dis., 1, 8393.
16. WINTON J.R. (1997). Immunization with viral antigens: Infectious haematopoietic necrosis. Dev. Biol.
Stand., 90, 211220.
17. WINTON J.R., ARAKAWA C.K., LANNAN C.N. & FRYER J.L. (1988). Neutralizing monoclonal
antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus. Dis.
Aquat. Org., 4, 199204.
18. WINTON J.R. & EINER-JENSEN K. (2002). Molecular diagnosis of infectious hematopoietic necrosis
and viral hemorrhagic septicemia. In: Molecular Diagnosis of Salmonid Diseases. Cunningham C.O.,
ed. Kluwer, Dordrecht, The Netherlands. pp. 4979.
19. WOLF K. (1988). Infectious hematopoietic necrosis. In: Fish Viruses and Fish Viral Diseases. Cornell
University Press, Ithaca, New York, USA. pp. 83114.
*
* *
C HA P T E R 2 . 1 . 3 .

ONCORHYNCHUS MAS OU VI RUS DI S E AS E

SUMMARY
Oncorhynchus masou virus disease (OMVD) is an oncogenic and skin ulcerative condition among
salmonid fish in Japan, and probably in the coastal rivers of eastern Asia that harbour Pacific salmon.
Oncorhynchus masou virus (OMV), the causative virus, is also known as Yamame tumour virus
(YTV), Nerka virus Towada Lake, Akita and Amori prefecture (NeVTA), coho salmon tumour virus
(CSTV), Oncorhynchus kisutch virus (OKV), coho salmon herpesvirus (CSHV), rainbow trout
kidney virus (RKV), or rainbow trout herpesvirus (RHV). For a recent and more detailed review of the
condition, see refs 4, 12 and 13.
Fish species that are susceptible to OMV include: kokanee (sockeye) salmon (Oncorhynchus nerka),
masou salmon (O. masou), chum salmon (O. keta), coho salmon (O. kisutch) and rainbow trout
(O. mykiss) (6).
Clinically, the initial infection by OMV (taxonomically known as Salmonid herpesvirus 2; SalHV-2)
appears as a systemic and frequently lethal infection that is associated with oedema and haemorrhages.
Virus multiplication in endothelial cells of blood capillaries, haematopoietic tissue and hepatocytes underlies
the clinical signs (7, 11). Four months after this first clinical condition, a varying number of surviving fish
exhibit epithelioma occurring mainly around the mouth (upper and lower jaw) and, to a lesser extent, on the
caudal fin, operculum and body surface. These neoplasia may persist for up to 1 year post-infection. In the
case of coho salmon, 1-year-old infected fish in particular show ulcers on the skin, white spots on the liver
and neoplastic tissues around the mouth parts or body surface. In rainbow trout, the diseased fish exhibit
almost no external signs, although some fish manifest ulcerative lesions on the skin. Internally, intestinal
haemorrhage and white spots on the liver are observed (5, 15, 16).
Following the septicaemia phase of OMV infection, an immune response takes place that results in the
synthesis of neutralising antibodies to OMV. A carrier state frequently occurs that leads to virus shedding
via the sexual products at the time of spawning.
On the basis of antigenic studies conducted with neutralising polyclonal rabbit antisera, OMV differs from
Salmonid herpesvirus 1(SalHV-1), which is present in the western United States of America and is only
weakly pathogenic (3, 9, 12).
The reservoirs of OMV are clinically infected fish and covert carriers among groups of cultured, feral or wild
fish. Infectious virus is shed via faeces, urine, sexual products and probably skin mucus, while the kidney,
spleen, liver and tumours are the sites where virus is the most abundant during the course of overt infection.
The transmission of OMV is horizontal and possibly egg-surface associated. Horizontal transmission may
be direct or vectorial, water being the major abiotic factor. Animate vectors and inanimate objects also act in
OMV transmission. Disinfection of the eggs just after fertilisation and eyed stage is effective in preventing
OMV infection. OMV disease was not reported in alevins originating from disinfected eggs that had been
incubated and hatched in virus-free water (14).
Salmonids are the only fish species susceptible to OMV infection; the order of the fish species from the most
to the least susceptible is: kokanee salmon, chum salmon, masou salmon, coho salmon and rainbow trout.
The age of the fish is critical and 1-month-old alevins are the most susceptible target for virus infection (6).
The main environmental factor favouring OMV infection is low water temperature (below 14C) (10).
Chapter 2.1.3. - Oncorhynchus masou virus disease
The screening procedures for OMV are based on direct isolation of the virus in cell culture and co-culture of
neoplastic tissues with salmonid cell lines (8). Confirmatory testing is by immunological identification using
neutralisation or immunofluorescence tests, and virus-specific gene detection using polymerase chain reaction.
Control methods currently rely on the implementation of avoidance and hygiene practices in the operating of
salmonid husbandry. The thorough disinfection of fertilised eggs and the incubation of these eggs and rearing
of fry and alevins in premises completely separated from those harbouring virus carriers and free from contact
with inanimate objects are the key measures needed to decrease contamination of OMV in a defined fish
production site (14).
The screening for and diagnosis of Oncorhynchus masou virus disease (OMVD) is based on direct methods,
which are either the isolation of the Oncorhynchus masou virus (OMV) in cell culture followed by its
immunological identification (conventional approach), or the immunological demonstration of OMV
antigen in infected fish tissues (2, 5).
antibodies to viruses has not thus far been recognised as a valuable diagnostic method for assessing the
viral status of fish populations. However, the validation of some serological techniques for diagnosis of
certain fish virus infections could arise in the near future, rendering the use of fish serology more widely
acceptable for diagnostic purposes.
Clinically affected fish: Whole alevin (body length 4 cm), viscera including kidney (4 cm body
length 6 cm) or, for larger size fish, skin ulcerative lesions or neoplastic tissues, and kidney, spleen,
liver and encephalon.
Asymptomatic fish (apparently healthy fish): Kidney, spleen and encephalon (any size fish) and/or
ovarian fluid from broodfish at spawning time.
1. STANDARD SCREENING METHOD FOR OMV
1.1. Isolation of OMV in cell culture
Cell line to be used: RTG-2 or CHSE-214
2

dilution.
ii) Allow to adsorb for 0.51 hour at 1015C and, without withdrawing the inoculate, add
cell culture medium buffered at pH 7.6 and supplemented with 2% fetal calf serum (FCS)
(1 ml/well for 24-well cell culture plates), and incubate at 1015C.
ii) Maintain the pH of the cell culture medium at between 7.3 and 7.6 during incubation.
This can be achieved by the addition to the inoculated cell culture medium of sterile
bicarbonate buffer (for tightly closed cell culture flasks) or Tris buffer solution (for cell
culture plates) or, even better, by using HEPES-buffered medium (HEPES = N-2-
hydroxyethyl-piperazine-N-2-ethanesulfonic acid).
the tested homogenate supernatants, identification procedures have to be undertaken
If a fish health surveillance/control programme is being implemented, provisions may have to be
taken to suspend the approved health status of the production unit and/or the zone (if it was
approved previously) from which the virus-positive sample originated. The suspension of approved
status will be maintained until it is demonstrated that the virus in question is not OMV.
the virus controls fail to develop CPE, the process should be repeated with fresh
susceptible cells and new batches of samples.
ii) If required, repeat the neutralisation test to infectious pancreatic necrosis virus (IPNV)
and/or infectious haematopoietic necrosis virus (IHNV) as previously described (see
Chapter I.1. Section B.3.3.), with dilution of the above supernatant (1/1 to 1/100).
iv) Incubate and monitor as described above in Section 1.1.b.
d) Isolation of OMV from cultures of neoplastic cells
i) Collect neoplastic tissues, disinfect with iodophore, 50 parts per million for 20 minutes,
and wash three times with Hanks balanced salt solution.
ii) The tissues are left overnight in 0.25% trypsin in phosphate buffered saline (PBS) at 5C.
Then, 3.5 10
5
neoplastic cells/ml are seeded in a tissue culture flask and incubated with
culture medium containing 20% fetal bovine serum (FBS).
iii) Harvest the primary neoplastic cell culture and co-cultivate with RTG-2 or CHSE-214
cells.
iv) Incubate and monitor as described in Section 1.1.b.
1.2. Identification of OMV
i) Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge at
ii) Dilute the virus-containing medium from 10
2
to 10
4
.

iii) Mix aliquots (for example 200 l) of each virus dilution with equal volumes of an
antibody solution specific for OMV, and similarly treat aliquots of each virus dilution
with cell culture medium.
(The neutralising antibody [NAb] solution must have a 50% plaque reduction titre of at
least 2000.)
iv) In parallel, other neutralisation tests must be performed against:
a homologous virus strain (positive neutralisation test)
a heterologous virus strain (negative neutralisation test).
v) If required, a similar neutralisation test may be performed using antibodies to IPNV, to
ensure that no IPNV contaminant has escaped the first anti-IPNV test.
vi) Incubate all the mixtures at 15C for 1 hour.
vii) Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell
cultures per dilution) and allow adsorption to occur for 0.51 hour at 15C; 24- or 12-
well cell culture plates are suitable for this purpose, using a 50-l inoculum.
viii) When adsorption is completed, add cell culture medium supplemented with 2% FCS and
buffered at pH 7.47.6 into each well and incubate at 1015C.
ix) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
monolayers with a solution of 1% crystal violet in 20% ethanol.
x) The tested virus is identified as OMV when CPE is prevented or noticeably delayed in the
cell cultures that had received the virus suspension treated with the OMV-specific
antibody, whereas CPE is evident in all other cell cultures.
xi) In the absence of any neutralisation by NAb to OMV, it is mandatory to conduct an
indirect fluorescent antibody test (IFAT) with the suspect sample.
2
wells of cell culture plastic plates or on cover-slips in
order to reach around 80% confluency, which is usually achieved within 4 hours of
incubation at 22C (seed six cell monolayers per virus isolate to be identified, plus two for
positive and two for negative controls). The FCS content of the cell culture medium can
be reduced to 24%. If numerous virus isolates have to be identified, the use of Terasaki
plates is strongly recommended.
iii) Dilute the control virus suspension of OMV in a similar way, in order to obtain a virus
v) Remove the cell culture medium, rinse once with 0.01 M PBS, pH 7.2, then three times
briefly with cold acetone (stored at 20C) for cover-slips or a mixture of acetone
30%/ethanol 70%, also at 20C, for plastic wells.
2
of cell
monolayer.
freeze at 20C.
viii) Prepare a solution of purified antibody or serum to OMV in 0.01 M PBS, pH 7.2,
containing 0.05% Tween 80 (PBST), at the appropriate dilution (which has been
established previously or is given by the reagent supplier).
ix) Rehydrate the cell monolayers by four rinsing steps with the PBST solution, and remove
this buffer completely after the last rinsing.
0.25 ml/2 cm
2

well.
xi) Rinse four times with PBST as above.
xii) Treat the cell monolayers for 1 hour at 37C with a solution of fluorescein isothiocyanate
or goat antibodies.
xiii) Rinse four times with PBST.
xiv) Examine the treated cell monolayers on plastic plates immediately, or mount the cover-
xv) Examine under incident UV light using a microscope with 10 eye pieces and 20
observation.
(ELISAs) with appropriate dilutions of monoclonal antibody or purified
immunoglobulins (Ig) specific for OMV, in 0.01 M PBS, pH 7.2 (200 l/well).
(200 l/well).
vi) Add 2% Triton X-100 to the virus suspension to be identified.
vii) Dispense 100 l/well of a two- or four-step dilution of the virus to be identified and of
OMV control virus, and allow to react with the coated antibody to OMV for 1 hour at
20C.
ix) Add to the wells, biotinylated polyclonal antibody to OMV.
x) Incubate for 1 hour at 37C.
xii) Add streptavidin-conjugated horseradish peroxidase to those wells that have received the
xiv) Add the substrate and chromogen. Stop the course of the test when positive controls
react, and monitor the results.
d) Polymerase chain reaction method
i) Extract nucleic acid from cells infected with OMV strains and SalHV-1 using the
InstGene Matrix (Biorad).
ii) Pellet the virus-infected tissues or infected cultured cells by centrifugation at 19,000 g
(14,800 rpm) for 15 minutes.
iii) Wash the pellets twice with 1 ml PBS and mix with 200 l of chelating resin (Sigma).
iv) Incubate the mixture at 56C for 20 minutes in a water bath, vortex it, and then place it in
a boiling water bath for 8 minutes.
v) Vortex the samples and centrifuge at 8200 g (10,000 rpm) for 90 seconds.
vi) Subject the supernatant to PCR.
vii) The forward primer (F10) is 5-GTA-CCG-AAA-CTC-CCG-AGT-C-3, and the reverse
primer (R5) is 5-AAC-TTG-AAC-TAC-TCC-GGG-G-3.
viii) Incubate the specimens, primer sets and reaction mixtures for 30 cycles in an automatic
thermal cycler (GeneAmp PCR 9700, Applied Biosystems), with each cycle consisting of
denaturation at 94C for 30 seconds, annealing at 56C for 30 seconds and extension at
72C for 30 seconds.
ix) Analyse the amplified product for size and purity by electrophoresis (100 V for
30 minutes) in 2% agarose gel and stain with ethidium bromide.
x) A PCR using these primer sets amplified a 439 base-pair segment of DNA from OMV
strains isolated from masu salmon, coho salmon and rainbow trout, and liver, kidney,
brain and nervous tissues, and an 800 base-pair segment of DNA from SalHV-1. SalHV-1
and SalHV-2 could be distinguished by agarose gel profile of this amplified DNA (1).
2. DIAGNOSTIC METHODS FOR OMV
a) Indirect fluorescent antibody test method
culture plate.
iii) Store the kidney pieces (as indicated in Section B.3.1. in Chapter I.1.) together with the
other organs required for virus isolation in case this becomes necessary later.
vi) Rehydrate the above preparations (see Section 1.2.b. step ix) and block with 5% skim milk
or 1% bovine serum albumin (BSA), in PBST for 30 minutes at 37C.
vii) Rinse four times with PBST.
viii) Treat the imprints with the solution of antibody to OMV and rinse as indicated in Section
1.2.b.
ix) Block and rinse as described previously in steps vi and vii.
x) Reveal the reaction with suitable FITC-conjugated specific antibody, rinse and observe as
indicated in Section 1.2.b. steps xiixv.
ii) Treat the remaining part of homogenate with 2% Triton X-100, as in Section 1.2.c. step
vi, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
iii) Complete the other steps of the procedure described in Section 1.2.c.
2.4. Polymerase chain reaction method
a) DNA extraction
As described in Section 1.2.d. of this chapter up to step v (inclusive).
b) Primer set and PCR condition
As described in Section 1.2.d of this chapter, steps viviii. PCR using these primer sets
amplified a 439 base-pair segment of DNA from OMV. SalHV-1 and SalHV-2 could be
distinguished by agarose gel profile of this amplified DNA (1).
REFERENCES
1. ASO Y., WANI J., KLENNER D.A.S. & YOSHIMIZU M. (2001). Detection and identification of
Oncorhynchus masou virus (OMV) disease by polymerase chain reaction (PCR). Bull. Fish. Sci.
Hokkaido Univ., 52, 111116.
2. HAYASHI Y., IZAWA H., MIKAMI T. & KODAMA H. (1993). A monoclonal antibody cross-reactive
with three salmonid herpesviruses. J. Fish Dis., 16, 479486.
3. HEDRICK R.P., MCDOWELL T.S., EATON W., KIMURA R. & SANO T. (1987). Serological relationships
of five herpesvirus isolated from salmonid fishes. J. Ichthyol., 3, 8792.
4. KIMURA T. & YOSHIMISU M. (1989). Salmon herpesvirus: OMV, Oncorhynchus masou virus. In: Viruses
of Lower Vertebrates, Ahne W. & Kurstak E., eds. Springer-Verlag, Berlin, Germany, 171183.
5. KIMURA T., YOSHIMISU M. & TANAKA M. (1981). Studies on a new virus (OMV) from Oncorhynchus
masou II. Oncogenic nature. Fish Pathol., 15, 149153.
6. KIMURA T., YOSHIMISU M. & TANAKA M. (1983). Susceptibility of different fry stages of
representative salmonid species to Oncorhynchus masou virus (OMV). Fish Pathol., 17, 251258 (in
Japanese).
7. KIMURA T., YOSHIMISU M., TANAKA M. & SANNOHE H. (1981). Studies on a new virus (OMV)
from Oncorhynchus masou I. Characteristics and pathogenicity. Fish Pathol., 15, 143147.
8. LANNAN C.N., WINTON J.R. & FRYER J.L. (1984). Fish cells: Establishment and characterization of
nine cell lines from salmonids. In Vitro, 20, 671676.
9. ROIZMAN B. (1991). Family Herpesviridae. In: Classification and Nomenclature of Viruses, Francki
R.I., Fauque C.M., Knudson D.L. & Brown F., eds. Arch. Virol., (Suppl. 2). Springer, New York,
USA and Vienna, Austria, 103110.
10. SUZUKI S., KIMURA T. & SANEYOSHI M. (1986). Characterization of DNA polymerase induced by
salmon herpesvirus, Oncorhynchus masou virus. J. Gen. Virol., 67, 405408.
11. TANAKA M., YOSHIMISU M. & KIMURA T. (1984). Oncorhynchus masou virus: Pathological changes in
masu salmon (Oncorhynchus masou), chum salmon (O. keta) and coho salmon (O. Kisutch) fry infected
with OMV by immersion method. Bull. Jpn Soc. Scientif. Fisheries, 50, 431437.
476 pp.
13. YOSHIMIZU M., FUKUDA H., SANO T. & KIMURA T. (1995). Salmonid herpesvirus 2. Epizootiology
and serological relationship. Vet. Res., 26, 486492.
14. YOSHIMISU M., NOMURA T., EZURA Y. & KIMURA T. (1993). Surveillance and control of infectious
hematopoietic necrosis virus (IHNV) and Oncorhynchus masou virus (OMV) of wild salmonid fish
retruning to the northern part of Japan 19761991. Fisheries Res., 17, 163173.
15. YOSHIMISU M., TANAKA M. & KIMURA T. (1987). Oncorhynchus masou virus (OMV): Incidence of
tumor development among experimentally infected representative salmonid species. Fish Pathol., 22,
710.
16. YOSHIMISU M., TANAKA M. & KIMURA T. (1988). Histopathological study of tumours induced by
Oncorhynchus masou virus (OMV) infection. Fish Pathol., 23, 133138 (in Japanese).
*
* *
C HA P T E R 2 . 1 . 4 .

S P RI NG VI RAE MI A OF CARP

SUMMARY
Spring viraemia of carp (SVC) is a rhabdovirus infection (6) of several carp species and of some other
cyprinid and ictalurid fish species (5). Natural infections have been recognised in common carp and koi carp
(Cyprinus carpio), grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys
molitrix), bighead carp (Aristichthys nobilis), crucian carp (Carassius carassius), goldfish (C. auratus),
roach (Rutilus rutilus), ide (Leuciscus idus), tench (Tinca tinca) and sheatfish (Silurus glanis).
However, common carp is the most susceptible of these species and is the principal host. The geographical
range of SVC is currently limited to countries of the European continent that experience low water
temperatures during winter. For a detailed review of the condition, see Fijan (4) and Wolf (9).
As with other rhabdoviruses of fish, infection by SVC virus (SVCV) can be lethal due to the impairment
of the salt-water balance, which occurs in a clinical context of oedema and haemorrhages. Virus
multiplication, especially in endothelial cells of blood capillaries, haematopoietic tissue and nephron cells,
underlies the clinical signs.
Overcoming SVCV infection results in a strong protective immunity associated with the presence of
circulating antibodies detectable by methods such as virus neutralisation (VN), immunofluorescence or
enzyme-linked immunosorbent assay (ELISA). In certain individuals, this health status also results in a
covert virus carrier state.
On the basis of antigenic studies conducted with rabbit polyclonal neutralising antibodies, SVCV was
found, using the VN test, to present only one serotype, but both the indirect fluorescent antibody test and
the ELISA have revealed that SVCV shares common antigenic domain(s) with the pike-fry rhabdovirus
(7). Differences in virulence of virus strains have been recorded during both natural cases of disease and
experimental infections.
The reservoirs of SVCV are clinically infected fish and covert virus carriers from either cultured, feral or
wild fish. Virulent virus is shed via faeces, urine, gill and skin mucus and exudate of skin blisters or
oedematous scale pockets. However, liver, kidney, spleen, gill and encephalon are the organs in which
SVCV is most abundant during the course of overt infection (3).
The mode of transmission for SVCV is horizontal, but an egg-associated transmission (usually called
vertical) cannot be ruled out. Horizontal transmission may be direct or vectorial, water being the major
abiotic vector. Animate vectors and fomites are also involved in transmission of SVCV. Among animate
vectors, the parasitic invertebrates Argulus foliaceus (Crustacea, Branchiura) and Piscicola piscicola
(Annelida, Hirudinea) are able to transfer SVCV from diseased to healthy fish (1). Once SVCV is
established in pond stock or pond farm stock, it may be very difficult to eradicate without destroying all
kinds of life on the fish production site.
There is a high variability in the degree of susceptibility to SVC among individuals of the same fish species.
Apart from the physiological state of the fish, the role of which is poorly understood, age or age-related status
of innate immunity appears to be extremely important: the younger the fish, the higher the susceptibility to
overt disease, but even adult broodfish can be susceptible to infection. It is clear that the environmental factor
that is critical for virulence of an SVC infection is water temperature (5): in yearling or older fish overt
infection is not often observed above 17C whereas fry can be affected at temperatures as high as 2223C.
Chapter 2.1.4. - Spring viraemia of carp
Apart from the formerly cited cyprinid and ictalurid species, it seems that very young fish of various pond
fish species are susceptible to SVC under experimental conditions over a wide temperature range. The most
striking example is that of the pike (Esox lucius), which can be easily infected via the water route (2).
The implementation of hygiene measures and control policy rules are the only control methods currently
feasible. Vaccination is mostly still at the experimental stage.
The screening procedure for SVCV in asymptomatic (clinically healthy) fish is based on isolation of the
virus in cell culture as described in Section 1.1. below. Confirmation of SVCV is by immunological
identification using VN (Section 1.2.). However, rapid, presumptive identification of SVCV isolated in
tissue culture may be achieved using immunofluoresence or ELISA as described in Section 2. Here, a
positive test is sufficient to initiate fish health control measures while awaiting confirmation by VN test.
Diagnosis of spring viraemia of carp (SVC) in clinically affected fish may be achieved by virus isolation or
more rapidly by direct immunofluorescence (IF) tests (3) or enzyme-linked immunosorbent assays
(ELISAs) (8) on infected tissues (Section 2). Ideally, direct diagnosis by IF or ELISA should be confirmed
by virus isolation followed by a VN test. However, virus isolation may not be possible from decomposed
clinical samples (8), so the presence of signs of SVC disease and a positive direct IF test or ELISA may be
considered sufficient to initiate control measures.
antibodies to viruses has not thus far been accepted as a routine screening method for assessing the viral
status of fish populations. However, the validation of some serological techniques for certain fish virus
infections could arise in the near future, rendering the use of fish serology more widely acceptable for
health screening purposes.
Fish material suitable for virological examination is:
Asymptomatic fish (apparently healthy fish): Kidney, spleen, gill and encephalon (any size fish).
Clinically affected fish: Whole alevin (body length 4 cm), entire viscera including kidney and
encephalon (4 cm body length 6 cm) or, for larger size fish, liver, kidney, spleen and encephalon.
1. STANDARD SCREENING METHOD FOR SVC
1.1. Isolation of SVCV in cell culture
Cell line to be used: EPC or FHM
i) Make two serial tenfold dilutions of the 1/10 organ homogenate supernatants and
transfer an appropriate volume of each of these two dilutions on to 24-hour-old cell
2

dilution.
ii) Inoculate directly or allow to adsorb for 0.51 hour at 1015C and, without withdrawing
inoculate, add the cell culture medium buffered at pH 7.6 and supplemented with 2%
fetal calf serum (FCS) (1 ml/well for 24-well cell culture plates), and incubate at 20C.
bicarbonate buffer (for tightly closed cell culture flasks) or HEPES-buffered medium
(HEPES = N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid) or 2 M Tris/HCl
buffer solution (for cell culture plates).
the tested homogenate supernatants, identification procedures must be undertaken
previously) from which the suspected virus-positive sample originated. The suspension of approved
status will be maintained until it is demonstrated that the virus in question is not SVCV.
the virus control fail to develop CPE, the process should be repeated with fresh
ii) If required, repeat the neutralisation treatment for infectious pancreatic necrosis virus
(IPNV) as previously described (see Chapter I.1. Section B.3.3.), with dilution of the
above supernatant (1/1 to 1/100).
iv) Incubate and monitor as described above in Section 1.1.b.
1.2. Identification of virus isolated in cell culture
1.2.1. Rapid presumptive methods
a) Indirect fluorescent antibody test
2
order to reach around 80% confluency within 24 hours of incubation at 30C (seed six
cell monolayers per virus isolate to be identified, plus two for positive and two for
negative controls). The FCS content of the cell culture medium can be reduced to 24%.
If numerous virus isolates have to be identified, the use of Terasaki plates is strongly
recommended.
iii) Dilute the control virus suspension of SVCV in a similar way, in order to obtain a virus
pH 7.2, then three times briefly with cold acetone (stored at 20C) for cover-slips or a
mixture of acetone 30%/ethanol 70%, also at 20C, for plastic wells.
2
of cell
monolayer.
freeze at 20C.
viii) Prepare a solution of purified antibody or serum to SVCV in 0.01 M PBS, pH 7.2,
ix) Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, and
0.25 ml/2 cm
2

well.
or goat antibodies.
observation.
b) Enzyme-linked immunosorbent assay
i) Coat the wells of microplates designed for ELISAs with appropriate dilutions of purified
immunoglobulins (Ig) specific for SVCV, in 0.02 M carbonate buffer, pH 9.5
(200 l/well). Ig may be polyclonal or monoclonal Ig originating most often from rabbit
or mouse, respectively. For the identification of SVCV, monoclonal antibodies (MAbs)
specific for certain domains of the nucleocapsid (N) protein are suitable.
iv) Block with skim milk (5% in carbonate buffer) or other blocking solution for 1 hour at
37C (300 l/well).
vi) Add 2% non-ionic detergent (Triton X-100 or Nonidet P-40) to the virus suspension to
be identified.
vii) Dispense 100 l/well of two- or four-step dilutions of the virus to be identified, and of
the non-infected cell culture harvest (negative control), and the SVCV control virus
(positive control); allow to react with the coated antibody to SVCV for 1 hour at 20C.
ix) Add to the wells, 200 l of horseradish peroxidase (HRPO)-conjugated MAb or
polyclonal antibody to SVCV; or polyclonal IgG to SVCV or MAb to N protein specific
for a domain different from the one of the coating MAb and previously conjugated with
biotin.
xii) Add 200 l of HRPO-conjugated streptavidin or ExtrAvidin (Sigma) to those wells that
have received the biotin-conjugated antibody and incubate for 1 hour at 37C.
xiv) Add 200 l of the substrate and chromogen. Stop the course of the test when positive
controls react, and read the results.
xv) Alternatively, add substrate and chromogen to those wells containing the peroxidase-
conjugated antibody and proceed as above.
1.2.2. Confirmatory identification methods
2000 g for 15 minutes at 4C, or filter through a 0.45 m pore membrane to remove cell
debris.
2
to 10
4
.
iii) Mix aliquots of each dilution with equal volumes of an antibody solution against SVCV,
and similarly treat aliquots of each virus dilution with cell culture medium.
least 2000.)
v) If required, a similar neutralisation test may be performed using antibodies to IPNV, to
ensure that no IPNV contaminant has escaped the first anti-IPNV test.
vii) Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell
cultures per dilution) and allow adsorption to occur for 0.51 hour at 1520C; 24- or 12-
well cell culture plates are suitable for this purpose, using a 50 l inoculum.
viii) When adsorption is completed, add cell culture medium, supplemented with 2% FCS and
buffered at pH 7.47.6, to each well and incubate at 20C.
ix) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
x) The tested virus is identified as SVCV when CPE is prevented or noticeably delayed in
the cell cultures that received the virus suspension treated with the SVCV-specific
NOTE: In the absence of any neutralisation by NAb to SVCV, it is mandatory to conduct an
indirect fluorescent antibody test (IFAT) with the suspect isolate.
2. DIAGNOSTIC METHODS FOR CLINICALLY DISEASED FISH
2.1. Direct detection in fish tissues
culture plate.
v) Fix with acetone or ethanol/acetone and dry as indicated in Section 1.2.1.a. steps vvii.
vi) Rehydrate the above preparations (see Section 1.2.1.a. step ix) and block with 5% skim
milk or 1% bovine serum albumin, in PBST for 30 minutes at 37C.
viii) Treat the imprints with the solution of antibody to SVCV and rinse as indicated in
Section 1.2.1.a. steps viiixi.
indicated in Section 1.2.1.a. steps xiixv.
If the test is negative, process the organ samples stored at 4C, for virus isolation in cell culture
i) Microplate processing
As described in Section 1.2.1.b. of this chapter up to step iv (inclusive).
ii) Sampling procedures
iii) Processing of organ samples
iv) The enzyme-linked immunosorbent assay procedure
Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell
Treat the remaining part of the homogenate with 2% Triton X-100 or Nonidet P-40
and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
Complete the other steps of the procedure described in Section 1.2.1.b.
If the test is negative, process the organ samples stored at 4C, for virus isolation in
cell culture as described in Section 1.1.
2.2. Virus isolation in cell culture
As described in Section 1.1. of this chapter.
REFERENCES
1. AHNE W. (1985). Argulus foliaceus L. and Philometra geometra L. as mechanical vectors of spring
viraemia of carp virus (SVCV). J. Fish Dis., 8, 241242.
2. AHNE W. (1985). Viral infection cycles in pike (Esox lucius L). Z. Angew. Ichthyol., 2, 9095.
3. FAISAL M. & AHNE W. (1984). Spring viraemia of carp virus (SVCV): comparison of
immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of
antigen. J. Fish Dis., 7, 5764.
4. FIJAN N. (1999). Spring viraemia of carp and other viral disease agents of warm water fish. In: Fish
Diseases and Disorders, Volume 3; Viral, Bacterial and Fungal infections, Woo P.T.K. & Bruno
D.W., eds. CABI Publishing, UK, 177244.
5. FIJAN N. (1972). Infectious dropsy in carp a disease complex. In: Diseases of Fish, Mawdesley T.,
ed. Symposia of the Zoological Society of London. Academic Press, London, UK, 3951.
6. FIJAN N., PETRINEC Z., SULIMANOVIC D. & ZWILLENBERG L.O. (1971). Isolation of the causative
agent from the acute form of infectious dropsy of carp. Vet. Arch. Zagreb, 41, 125138.
7. VESTERGAARD-JORGENSEN P.E., OLESEN N.J., AHNE W. & LORENTZEN N. (1989). SVC and PFR
viruses. Serological examination of 22 isolates indicates close relationship between the two
rhabdoviruses. In: Viruses of Lower Vertebrates, Ahne W. & Kurstak E., eds. Springer-Verlag, Berlin,
Germany, 349366.
8. WAY K. (1991). Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased
carp by the enzyme-linked immunosorbent assay (ELISA). J. Appl. Ichthyol., 7, 95107.
476 pp.
*
* *
C HA P T E R 2 . 1 . 5 .

VI RAL HAE MORRHAGI C S E P T I CAE MI A

SUMMARY
Viral haemorrhagic septicaemia (VHS) is an infectious disease of rainbow trout (Oncorhynchus
mykiss), brown trout (Salmo trutta), grayling (Thymallus thymallus), whitefish (Coregonus sp.), pike
(Esox lucius), largemouth bass (Micropteus salmoides), Japanese flounder (Paralichthys olivaceus)
and turbot (Scophthalmus maximus). The disease is caused by viral haemorrhagic septicaemia virus
(VHSV, synonym: Egtved virus) (2), a virus belonging to the newly approved genus Novirhabdovirus
within the family Rhabdoviridae (23).
Until the mid-1980s VHS was regarded as a disease affecting only rainbow trout and a few other
freshwater fish species in aquaculture in continental Europe. In the past decades, however, VHSV has been
isolated from a large range of free-living marine fish species in the North American part of the Pacific and
Atlantic Oceans, in the North Atlantic, the North Sea and the Baltic Sea, and in the waters around
Japan. The number of susceptible wild marine species is still growing and, to date, at least 45 different
species (both freshwater and marine) have been found to test positive for the virus. Susceptible fish species
are found among the Salmoniformes (seven species), Esociformes (one species), Clupeiformes (four species),
Gadiformes (eleven species), Pleuronectiformes (seven species), Osmeriformes (three species), Perciformes (six
species), Scorpaeniformes (two species), Anguilliformes (one species), Cyprinodontiformes (one species) and
Gasterosteiformes (two species).
At present, the isolates from wild marine fish are not distinguishable from normal fresh water isolates by
serological means. Isolations of VHSV from fish from the North American waters and Japan, however,
have so far been associated with genetically characteristic virus strains (6, 8, 14). Four major genotypes
have been identified (21): Genotype I European freshwater VHSV isolates and a group of marine
isolates from the Baltic Sea, Genotype II a group of marine isolates circulating in the Baltic Sea,
Genotype III isolates from the North Sea, Skagerrak and Kattegat and Genotype IV North
American VHSV isolates. The genetic differences appear to be more strongly related to geographical
location than to year of isolation or host species (1, 2, 21, 22). The genetic characteristics of VHSV
isolates highly pathogenic for rainbow trout have not yet been defined. All examined isolates from wild
marine fish have shown low or no mortality in infection trials by immersion with rainbow trout (3, 4, 24).
Several marine isolates are pathogenic to turbot fry (9). Japanese flounder are highly susceptible to Japanese
VHSV isolates (5). The Pacific isolates are highly pathogenic to Pacific herring (10). This newly
discovered large host range and the significant differences in pathogenicity in different host species cause some
problems for VHS control programmes, which are based mainly on the protection of the significant rainbow
trout production industry. The main problem is whether the finding of marine VHSV in free-living fish in
an approved VHSV-free area should lead to the withdrawal of that status. It has been observed in a large
part of Europe that VHS in free-living fish in the marine environment affects an approved VHS-free
status in only very few cases.
VHS occurs in continental Europe and is important because of its clinical and economic consequences to
rainbow trout farming. For more detailed reviews of the condition, see Wolf (26) and Jorgensen (7).
Natural infections with VHSV have caused significant mortality in turbot and Japanese flounder in
aquaculture, and significant natural mortality in Pacific herring and pilchard along the Pacific coast of
Alaska and Washington, United States of America (USA), and Canada.
Chapter 2.1.5. - Viral haemorrhagic septicaemia
The infection of susceptible fish species is often lethal, due to the impairment of the osmotic balance, and
occurs within a clinical context of oedema and haemorrhages. Virus multiplication in endothelial cells of
blood capillaries, leukocytes, haematopoietic tissues and nephron cells, underlies the clinical signs.
Three neutralising subtypes of VHSV have been recognised using a panel of polyclonal and monoclonal
antibody preparations (20). Apart from the above variation, VHSV seems to share a VHS-group
neutralising epitope, and several non-neutralising epitopes located on the viral glycoprotein (G protein).
Variations in virus strain virulence have been recorded in both natural cases of disease and infection trials.
The reservoirs of VHSV are clinically infected fish and covert carriers among cultured, feral or wild fish.
Virulent virus is primarily shed in the urine, whereas kidney, spleen, and heart are the sites in which virus
is most abundant. Once VHSV is established in a farm stock and therefore in the water catchment system,
the disease becomes enzootic due to the virus carrier fish.
Several factors influence susceptibility to VHS. Among each fish species, there is individual variability in
susceptibility, and the age of the fish appears to be of some importance the younger the fish the higher the
susceptibility. In highly susceptible fish stocks, however, overt infection is seen in all sizes of fish.
Water temperature is an important environmental factor. Disease generally occurs at temperatures between
4C and 14C. Low water temperatures (15C) generally result in an extended disease course with low
daily mortality but high accumulated mortality. At high water temperatures (1518C), the disease
generally takes a short course with a modest accumulated mortality. VHS outbreaks occur during all
seasons, but are most common in spring when water temperatures are rising or fluctuating.
The screening procedure for VHS is based mainly on virus isolation in cell culture. Confirmatory testing is
by immunological virus identification, e.g. neutralisation, immunofluorescence, enzyme-linked immuno-
sorbent assay (ELISA), and immunoperoxidase staining or by reverse-transcription polymerase chain
reaction (RT-PCR)-based techniques. However, more rapid diagnostic methods for presumptive evidence of
viral antigen in infected organ imprints or homogenates (fluorescence, ELISA, immunohistochemistry, RT-
PCR) may be suitable for fish with overt disease. Fish serology (neutralisation, ELISA) could be of
importance to the detection of the carrier state among fish stocks, but has yet to be validated.
Control methods for VHS currently lie in official health surveillance schemes coupled with control policy
measures, such as stamping-out and fallowing procedures, and have resulted in the eradication of the disease
from several parts of Europe (2, 17). At present, genetic approaches using selection and intergeneric
hybridisation and vaccination are both at experimental stages.
The standard screening method for viral haemorrhagic septicaemia (VHS) is based on direct methods, i.e.
the isolation of VHS virus (VHSV) in cell culture followed by immunological identification or
identification by reverse-transcription polymerase chain reaction (RT-PCR) (25). Due to low sensitivity the
direct immunological demonstration of VHSV antigen in infected fish tissues can only be used when VHS
infection is suspected. PCR-based technology using direct identification of the VHSV genome in fish
tissue is currently being developed and validated. The technique can be used for confirmation of overt
infection in fish, but has not yet been validated for use in direct surveillance programmes for obtaining
approved VHS-free status.
Due to insufficient knowledge of the serology of virus infections of fish, the detection of fish antibodies to
viruses has thus far not been accepted as a routine diagnostic method for assessing the virus status of fish
populations (12).
Infected fish material suitable for virological examination is: whole alevin (body length < 4 cm), viscera
including kidney (4 cm < body length < 6 cm) or, for larger size fish, kidney and spleen (heart and/or
encephalon might be included), and ovarian fluid from broodfish at time of spawning.
1. STANDARD SCREENING METHODS FOR VHSV
1.1. Isolation of VHSV in cell culture
Cell line to be used: BF-2, RTG-2
Alternatively, EPC or FHM cells may be used, but are in general less susceptible than BF-2 and
RTG-2 (11, 19).
a) Virus extraction
Use the procedure described in Chapter I.1. (Section B.3.2.).
b) Inoculation of cell monolayers
i) Prior to inoculation of cells, the supernatant is mixed with equal parts of a suitably diluted
pool of antisera to the indigenous serotypes of infectious pancreatic necrosis virus
(IPNV) and incubated for a minimum of 1 hour at 15C or for 1218 hours at 4C. The
titre of the antiserum must be at least 1/2000 in a 50% plaque neutralisation test.
ii) Treatment of all inocula with antiserum to IPNV (a virus that in some parts of the world
occurs in 50% of fish samples) aims at preventing cytopathic effect (CPE) due to IPNV
from developing in inoculated cell cultures. This will reduce the duration of virological
examinations as well as the number of cases in which occurrence of CPE would have to
be considered potentially indicative of VHS.
iii) When samples come from production units that are considered to be free from IPNV,
treatment of inocula with antiserum to IPNV may be omitted.
iv) Transfer organ homogenate supernatant on to 24-hour-old monolayers overlaid with cell
culture medium containing 210% fetal calf serum (FCS) and suitable buffer (e.g. Tris or
HEPES [N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid]) in two dilutions, i.e. the
primary dilution and, in addition, a 1/10 dilution thereof, resulting in final dilutions of
tissue material in cell culture medium of 1/100 and 1/1000, respectively (in order to
prevent homologous interference). The ratio of inoculum size to volume of cell culture
medium should be about 1:10.
v) For each dilution a minimum of about 2 cm
2
cell area, corresponding to one well in a 24-
well cell culture tray, must be used. Use of cell culture trays is recommended, but other
units of similar or bigger growth area may be used instead.
c) Monitoring incubation
contrast microscope is recommended. If no positive controls are included, at least every
six months or if decreased cell susceptibility is suspected, titration of frozen stocks of
VHSV shall be performed to verify the susceptibility of the cell cultures to infection.
ii) Inoculated cell cultures shall be incubated at 15C for 710 days and be inspected
regularly (at least three times a week) for the occurrence of CPE at
40100 magnification.
iii) Maintain the pH of the cell culture medium at between 7.3 and 7.6 during incubation.
This is achieved by the addition of sterile bicarbonate buffer (for tightly closed cell culture
flasks) or 2 M Tris buffer solution (for cell culture plates) to the cell culture medium or,
preferably, by using HEPES-buffered medium.
iv) If CPE appears in cell cultures inoculated with dilutions of the homogenate, identification
procedures must be undertaken immediately (see Section 1.2. below).
previously) from which the suspected virus positive sample originated. The suspension of approved
status will be maintained until it is demonstrated that the virus in question is not VHSV.
v) If no CPE has developed after the primary incubation for 710 days, subcultivation is
performed to fresh cell cultures using a cell area similar to that of the primary culture.
d) Subcultivation procedures
i) Aliquots of medium (supernatant) from all cultures/wells constituting the primary culture
are pooled according to cell line 710 days after inoculation.
ii) The pools are then inoculated into homologous cell cultures undiluted and diluted 1/10
(resulting in final dilutions of 1/10 and 1/100, respectively, of the supernatant) as
previously described.
iii) The inoculation may be preceded by preincubation of the dilutions with IPN antiserum at
appropriate dilution as described in Chapter I.1. Section B.1.
iv) Alternatively aliquots of 10% of the medium constituting the primary culture are inoculated
directly into a well with fresh cell culture (well-to-well subcultivation).
v) The inoculated cultures are then incubated for 7 days with observation as described for
the primary inoculation.
vi) If no CPE occurs the test may be declared negative.
1.2. Virus identification
i) Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge it at
2000 g for 15 minutes at 4C, or filter through a 45-nm pore membrane to remove cell
debris.
ii) Dilute virus-containing medium from 10
2
to 10
4
.
iii) Mix aliquots (for example 200 l) of each dilution with equal volumes of a VHSV
antibody solution, and likewise treat aliquots of each virus dilution with cell culture
medium. (The neutralising antibody [NAb] solution must have a 50% plaque reduction
titre of at least 2000.)
v) If required, a similar neutralisation test may be performed using antibodies to IPNV.
vii) Transfer aliquots of each of the above mixtures on to 24-hour-old monolayers overlaid
with cell culture medium containing 10% FCS (inoculate two wells per dilution) and
incubate at 15C; 24- or 12-well cell culture plates are suitable for this purpose, using a
50 l inoculum.
viii) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
controls). Results are recorded either after a simple microscopic examination (phase
contrast preferable) or after discarding the cell culture medium and staining cell
ix) The tested virus is identified as VHSV when CPE is prevented or noticeably delayed in
the cell cultures that received the virus suspension treated with the VHSV-specific
x) In the absence of any neutralisation by NAb to VHSV, it is mandatory to conduct an
indirect fluorescent antibody test (IFAT), an immunoperoxidase test, an enzyme-linked
immunosorbent assay (ELISA) or RT-PCR using the suspect sample. Some cases of
antigenic drift of surface antigen have been observed, resulting in occasional failure of the
neutralisation test using NAb to VHSV.
Other neutralisation tests of proven efficiency may be used alternatively.
2
be reduced to 24%. If numerous virus isolates have to be identified, the use of Terasaki
plates is strongly recommended.
iii) Dilute the control virus suspension of VHSV in a similar way, in order to obtain a virus
pH 7.2, then three times briefly with a cold mixture of acetone 30%/ethanol 70% (v/v)
(stored at 20C).
2
of cell
monolayer.
freeze at 20C.
viii) Prepare a solution of purified VHSV antibody or serum in 0.01 M PBS, pH 7.2,
containing 0.05% Tween-80 (PBST), at the appropriate dilution (which has been
chamber and do not allow evaporation to occur, e.g. by adding a piece of wet cotton to
the humid chamber. The volume of solution to be used is 0.25 ml/2 cm
2
well.
(FITC)- or tetramethylrhodamine-5-(and-6-)isothiocyanate (TRITC)-conjugated antibody
to the immunoglobulin used in the first layer and prepared according to the instructions
of the supplier. These conjugated antibodies are most often rabbit or goat antibodies.
slips using, for example glycerol saline, pH 8.5 prior to microscopic observation.
40 objective lens having numerical aperture >0.65 and >1.3 respectively. Positive and
observation.
Other IFAT techniques of proven efficiency may be used alternatively.
immunoglobulins (Ig) or serum specific for VHSV, in 0.01 M PBS, pH 7.2 (200 l/well).
Ig may be polyclonal or monoclonal originating most often from rabbit or mouse,
respectively. For the identification of VHSV, monoclonal antibodies (MAbs) specific for
certain domains of the nucleocapsid protein (N) is suitable (13).
iii) Rinse four times with 0.01 M PBS containing 0.05% Tween-20 (PBST).
(200 l/well).
vii) Dispense 100 l/well of two- or four-step dilutions of the virus to be identified and of
VHSV control virus, and negative control (e.g. infectious haematopoietic necrosis virus)
and allow to react with the coated antibody to VHSV for 1 hour at 20C.
ix) Add to the wells either biotinylated polyclonal VHSV antiserum or MAb to N protein
specific for a domain different from the one of the coating MAb and previously
conjugated with biotin.
xiii) Rinse four times with PBST. Add the substrate and chromogen. Stop the course of the
test when positive controls react, and read the results.
xiv) Interpretations of the results is according to the optical absorbencies achieved by negative
and positive controls and must follow the guidelines for each test, e.g. absorbency at 450
nm of positive control must be minimum 510 A
450
of negative control. Absorbencies
> 3 A
450
of negative controls are regarded as positives.
The above biotinavidin-based ELISA version is given as an example (15). Other ELISA
versions of proven efficiency may be used instead (16, 18).
d) Reverse-transcription polymerase chain reaction
All work with RNA should be performed under a hood, using gloves.
i) Isolation of RNA: Total RNA from infected cells in suspension (cell culture medium) is
extracted using the phenol-chloroform method or by RNA affinity spin columns, e.g.
RNeasy Total RNA kit (Qiagen, Germany), according to the manufacturers instructions.
RNA must be resuspended in distilled RNAse-free water (e.g. water treated with 0.1%
diethyl pyrocarbonate.
ii) RT-PCR: The RT-PCR amplification can be performed in one or two step(s) using a
primer set that binds to a conserved region within the nucleocapsid (N): 5-GGG-GAC-
CCC-AGA-CTG-T-3 (forward primer) and 5-TCT-CTG-TCA-CCT-TGA-TCC-3
(reverse primer). The resulting amplicon is 811 base pairs (bp).
iii) One-step RT-PCR: 50 l single tube RT-PCR can be performed using Titan One Tube RT-
PCR System (Roche, Germany) according to the manufacturers instructions. Briefly, the
PCR reaction mixture consists of: 5 l of extracted viral RNA (approximately 0.52 g),
50 pmol of each primer, 1 l 10 mM dNTP, 10 l 5 RT-PCR reaction buffer (with 7.5
mM MgCl
2
and dimethyl sulfoxide), 2.5 l 100 mM DTT, and 1 l enzyme mix. The
following cycles are recommended: 50C for 30 minutes, 94C for 2 minutes, 35 cycles at
94C for 30 seconds, 52C for 30 seconds, 68C for 60 seconds; the RNA reaction is
finally held at 68C for 7 minutes.
iv) Two-step RT-PCR: The cDNA mixture consists of 5 l of the extracted viral RNA
(approximately 0.52 g), 50 pmol forward primer, 1 reverse transcript buffer (with
50 mM Tris/HCl, pH 8.3, 3 mM MgCl
2
, 75 mM KCl, 10 mM), 1 mM mixed dNTP, 5 U
reverse transcriptase (StrataScript RNase H
-
reverse transcriptase, Stratagene, USA) adjusted
with H
2
O to a final volume

of 20 l. Reverse transcription should be performed at 42C for
30 minutes, 94C for 5 minutes and cooled at 410C for 3 minutes.
v) The master mixture for subsequent PCR amplification is prepared on 5 l of the cDNA
reaction, 50 pmol of each primer, 1 Taq polymerase buffer (with 10 mM Tris/HCl,
pH 9, 1.5 mM MgCl
2
, 50 mM KCl, 0.01% gelatin, 0.1% Triton X-100), 1 mM mixed
dNTP, 1.5 U Taq polymerase and adjusted with H
2
O up to final volume of 50 l. The
subsequent PCR is performed by the following cycles: 94C for 2 minutes, 35 cycles at
94C for 30 seconds, 52C for 30 seconds, 68C for 60 seconds, and is finally held at
68C for 7 minutes.
vi) Quantity and specificity of the RT-PCR reactions can be evaluated by 1% agarose gel
electrophoresis of 1/10 reaction in 1.5% agarose gel with ethidium bromide and observed
using UV transillumination.
Please note: The thermal protocols might need optimisation, depending on the thermal cycler
in use.
2. DIAGNOSTIC METHODS FOR VHSV
culture plate.
vi) Rehydrate the above preparations (see Section 1.2.b. step ix) and block with 5% skim milk or
1% bovine serum albumin, in PBST for 30 minutes at 37C.
viii) Treat the imprints with the solution of antibody to VHSV and rinse as indicated in Section
1.2.b.
xi) If the test is negative, process the organ samples stored at 4C for virus isolation in cell
culture as described in Section 1.1.
2.3. Enzyme-linked immunosorbent assay (16)
c) The enzyme-linked immunosorbent assay procedure
i) Microplate processing is described in Section 1.2.c. of this chapter up to step iv
(inclusive).
ii) Set aside an aliquot of 1/4 dilution of each homogenate in case further virus isolation in
cell culture is required.
iii) Treat the remaining part of the homogenate with 2% Triton X-100 as in Section 1.2.c.
step vi, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
iv) Complete the other steps (viixiv) of the procedure described in Section 1.2.c.
v) If the test is negative, process the organ samples stored at 4C for virus isolation in cell
culture as described in Section 1.1.
The above biotinavidin-based ELISA version is given as an example (7). Other ELISA
versions of proven efficiency may be used instead (18).
REFERENCES
1. BASURCO B., VENDE P., MONNIER A.F., WINTON J.R., DE KINKELIN P. & BENMANSOUR A. (1995).
Genetic diversity and phylogenetic classification of viral hemorrhagic septicemia virus (VHSV). Vet.
Res., 26, 460463.
2. BENMANSOUR A., BASURCO B., MONNIER A.F., VENDE P., WINTON J.R. & DE KINKELIN P. (1997).
Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of
viral haemorrhagic septicaemia virus, a fish rhabdovirus. J. Gen. Virol., 78, 28372846.
3. DIXON P.F., FEIST S., KEHOE E., PARRY L., STONE D.M. & WAY K. (1997). Isolation of viral
haemorrhagic septicaemia virus from Atlantic herring Clupea harengus from the English Channel. Dis.
Aquat. Org., 30, 8189.
4. FOLLETT J.E., MEYERS T.R., BURTON T.O. & GEESIN J. L. (1997). Comparative susceptibilities of
salmonid species in Alaska to infectious hematopoietic necrosis virus (IHNV) and North American
viral hemorrhagic septicemia virus (VHSV). J. Aquat. Anim. Health, 9, 3440.
5. ISSHIKI T., NISHIZAWA T., KOBAYASHI T., NAGANO T. & MIYAZAKI T. (2001). An outbreak of
VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus
in Japan. Dis. Aquat. Org., 47, 8799.
6. JENSEN M.H. (1965). Research on the virus of Egtved disease. Ann. N.Y. Acad. Sci., 126, 422426.
7. JORGENSEN P.E.V. (1992). Recent advances in surveillance and control of viral haemorrhagic
septicaemia (VHS) of trout. Proceedings of the OJI International Symposium on Salmonid Diseases,
Hokkaido University Press, Sapporo, Japan, 6071.
8. EINER-JENSEN K., OLESEN N.J., LORENZEN N. & JORGENSEN P.E.V. (1995). Use of the
polymerase chain reaction (PCR) to differentiate serologically similar viral haemorrhagic septicaemia
(VHS) virus isolates from Europe and America. J. Vet. Res., 26, 464469.
9. KING J.A., SNOW M., SKALL H.F. & RAYNARD R.S. (2001). Experimental susceptibility of Atlantic
salmon Salmo salar and turbot Scopthtalmus maximus to European freshwater and marine isolates of
viral haemorrhagic septicaemia virus. Dis. Aquat. Org., 47, 2531.
10. KOCAN R., BRADLEY M., ELDER N., MEYERS T.R., BATTS W.N. & WINTON J.R. (1997). North
American strain of viral hemorrhagic septicemia virus is highly pathogenic for laboratory-reared
pacific herring. J. Aquat. Anim. Health, 9, 279290.
11. LORENZEN E., CARSTENSEN B. & OLESEN N.J. (1999). Inter-laboratory comparison of cell lines for
susceptibility to three viruses: VHSV, IHNV and IPNV. Dis. Aquat. Org., 37, 8188.
12. LORENZEN N. & LAPATRA S. (1999). Immunity to rhabdoviruses in rainbow trout: the antibody
response. Fish Shellfish Immunol., 9, 345360.
13. LORENZEN N., OLESEN N.J. & JORGENSEN P.E.V. (1988). Production and characterization of
monoclonal antibodies to four Egtved virus structural proteins. Dis. Aquat. Org., 4, 3542.
14. NISHIZAWA T., IIDA H., TAKANO R., ISSHIKI T., NAKAJIMA K. & MUROGA K. (2002). Genetic
relatedness among Japanese, American and European isolates of viral haemorrhagic septicaemia virus
(VHSV) based on partial G and P genes. Dis. Aquat. Org., 48, 143148.
15. MOURTON C., BEAZOTTI-LE BERRE M., PIECHACZYK M., PADUCEI F., PAU B., BASTIDE J.M. & DE
KINKELIN P. (1990). Antigen capture ELISA for viral haemorrhagic septicaemia virus serotype 1. J.
Virol. Methods, 29, 325334.
16. MOURTON C., ROMESTAND B., DE KINKELIN P., JEFFROY J., LE GOUVELLO R. & PAU B. (1992).
Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicaemia which uses anti-
nucleocapsid monoclonal antibodies. J. Clin. Microbiol., 30, 23382345.
17. OLESEN N.J. (1998). Sanitation of viral haemorrhagic septicaemia (VHS). J. Appl. Ichthyol., 14, 173
177.
18. OLESEN N.J. & JORGENSEN P.E.V. (1991). Rapid detection of viral haemorrhagic septicaemia virus
in fish by ELISA. J. Appl. Ichthyol., 7, 143186.
19. OLESEN N.J. & JORGENSEN P.E.V. (1992). Comparative susceptibility of three fish cell lines to
Egtved virus, the virus of viral haemorrhagic septicaemia (VHS). Dis. Aquat. Org., 12, 235237.
20. OLESEN N.J., LORENZEN N. & JORGENSEN P.E.V. (1993). Serological differences among isolates of
viral haemorrhagic septicaemia virus detected by neutralizing monoclonal and polyclonal antibodies.
Dis. Aquat. Org., 16, 163170.
21. SNOW M., CUNNINGHAM C.O., MELVIN W.T. & KURATH G. (1999). Analysis of the nucleoprotein
gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine
environment. Virus Res., 63, 3544.
22. STONE D.M., WAY K. & DIXON P.F. (1997). Nucleotide sequence of the glycoprotein gene of viral
haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in
farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). J. Gen. Virol., 78,
13191326.
23. WALKER P.J., BENMANSOUR A., DIETZGEN R., FANG R.-X., JACKSON A.O., KURATH G., LEONG
J.C., NADIN-DAVIES S., TESH R.B. & TORDO N. (2000). Family Rhabdoviridae. In: Virus Taxonomy,
Seventh Report of the International Committee on Taxonomy of Viruses, van Regenmortel M.H.V.,
Fauquet C.M., Bishop D.H.L., Carstens E.B., Estes M.K., Lemon S.M., Maniloff J., Mayo M.A.,
McGeoch D.J., Pringle C.R. & Wickner R.B., eds. Academic Press, San Diego, USA.
24. WINTON J.R., BATTS W.N., DEERING R.E., BRUNSON R., HOPPER K., NISHIZAWA T. & STEHR C.
(1991). Characteristics of the first North American isolates of viral hemorrhagic septicemia virus.
Proceedings of the Second International Symposium on Viruses of Lower Vertebrates, 4350.
25. WINTON J.R. & EINER-JENSEN K. (2002). Molecular diagnosis of infectious hematopoietic necrosis
and viral hemorrhagic septicemia. In: Molecular Diagnosis of Salmonid Diseases. Cunningham C.O.,
ed. Kluwer, Dordrecht, The Netherlands, 4979.
26. WOLF K. (1988). Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca, New York,
USA, 217249.
*
* *
C HA P T E R 2 . 1 . 6 .

CHANNE L CAT F I S H VI RUS DI S E AS E

SUMMARY
Channel catfish virus disease (CCVD) is caused by a herpesvirus designated Ictalurid herpesvirus 1 by the
International Committee on Taxonomy of Viruses, but the commonly used name is channel catfish virus
(CCV). CCV affects channel catfish (Ictalurus punctatus) in the United States of America. For more
detailed reviews of the condition, see Wolf (22) or Plumb (15).
CCVD is of importance because of its clinical and economic consequences in channel catfish farming.
CCVD results in high mortality rates in populations of fry and juvenile catfish. Diseased fish demonstrate
ascites, exophthalmia and haemorrhage in fins and musculature. Histologically the most remarkable
damage occurs in the kidney with extensive necrosis of renal tubules and interstitial tissue.
In survivors, CCVD results in a strong protective immunity, the synthesis of circulating antibodies to the
virus and, a covert latent carrier state. During this latent carrier state the virus is undetectable by
traditional culture or antigen-detection means, even when adults are immunosuppressed during spawning.
On the basis of antigenic studies conducted with polyclonal rabbit antibodies, CCV isolates form a
homogeneous group. However, the use of monoclonal antibodies shows some variation in antigenic
determinants between isolates (1). Some variation in the virulence of CCV strains has been recorded during
natural outbreaks of disease and has been demonstrated experimentally. Additionally, molecular data
indicate genetic variation within this species (7, 18).
Reservoirs of CCV are clinically infected fish and covert carriers. Infectious CCV can be detected in the
water from tanks of experimentally infected fish, but the route of shedding has not been determined. The
sites where the virus is most abundant during the course of overt infection are posterior kidney, skin, gills,
spleen and intestine, respectively, in decreasing magnitude (12, 13). The transmission of CCV is horizontal
and vertical. Horizontal transmission may be direct or vectorial with water being the main abiotic vector.
The virus has been shown to readily adsorb to pond sediments (6) and interaction with suspended clay
particles in pond water may influence horizontal transmission. Animate vectors and inanimate objects could
also act in CCV transmission. Vertical transmission is thought to be common, but the mechanism of
vertical transmission is not known, as infectious virus has not been detected on the skin or in the sexual
products of spawning adults. Once CCVD occurs in a fish population, survivors of the disease become
covert carrier fish.
Channel catfish and the closely related blue catfish (Ictalurus furcatus) have been the only fish found to be
infected with CCV, and variations in susceptibility to CCV have been recorded depending on fish strain.
The age of the fish is extremely important for overt infection. Although experimental data suggest that older
fish are susceptible to natural outbreaks of acute CCVD (11), the disease occurs almost exclusively in fish
that are less than 1 year of age, and generally less than 4 months of age. Water temperature is the critical
environmental factor. The mortality rate is high above 27C, but readily decreases and ceases below 18C.
Diagnosis of CCVD is based on virus isolation in cell culture. Confirmatory testing is by immunological
identification by neutralisation, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or
polymerase chain reaction (PCR). Rapid techniques by immunofluorescence tests or ELISA are suitable
mainly for diagnosis in clinically infected fish. Because virus proteins or infectious virus is not produced,
Chapter 2.1.6. - Channel catfish virus disease
culture methods or antigen-based testing is of little use for carrier screening. Instead, detection of neutralising
antibodies in a population of fish and, more recently, the use of PCR to detect latent CCV genomic DNA
is of more use.
Control methods currently rely on maintaining relatively low stocking densities and avoiding stressful
handling of young fish during the summer months. Also, control policies and hygiene practices have been
used, where practical, in catfish husbandry. The incubation of eggs and rearing of fry and juveniles in
facilities separated from carrier populations are critical to preventing the occurrence of CCVD in a CCV-
free fish production site. Because virus is only detected during active outbreaks, defining CCV-free status
has been done largely from historical data or identifying populations that are seronegative to the virus.
Recent use of PCR and hybridisation probes to detect latent CCV genomic DNA suggests that CCV is
present in many populations that have no history of the disease (2, 5, 9, 21). Vaccination, although
experimentally promising (19, 20, 23, 24), is not in use at this time.
The diagnosis of channel catfish virus disease (CCVD) is generally based on the isolation of channel
catfish virus (CCV) in cell culture followed by its immunological or nucleic-acid-based identification
(conventional approach). Alternatively, the immunological demonstration of CCV antigen in infected fish
tissues can be used. The conventional approach is most common because the virus produces rapid
cytopathic effect (CPE) in cell culture and there are no commercial sources of CCV-specific antiserum,
and custom-produced antisera to CCV is often of low titre or has cross-reaction with fish tissue.
Due to insufficient knowledge of the serology of fish virus infections, the detection of fish antibodies to
viruses has not yet been recognised as a valuable diagnostic method for assessing the viral status of fish
populations. However, the use of direct culture or detection of viral antigen is of little use in detecting
carrier fish. Therefore, the identification of antibodies to CCV has more merit in screening carrier
populations (8, 14). The antibody titres in carrier populations vary seasonally, with the lowest titres
occurring in the late winter and early spring (3). The validation of some serological techniques for
diagnosis of certain fish virus infections could arise in the near future, rendering the use of fish serology
more widely acceptable for diagnostic purposes.
Clinically affected fish: Whole fry or small juveniles (body length 3 cm), viscera including kidney
(3 cm body length 6 cm) or, for larger size fish, kidney, and spleen.
There is no definitive test for asymptomatic fish (apparently healthy fish): Cell culture or antigen
detection of latent virus is not possible. Occasional recrudescence of latent infection has been detected
in adult catfish after spawning (16) or during winter months or after dexamethazone treatment (4), but
these methods are not reliable enough for inspection purposes. Detection of latent virus DNA can be
done using polymerase chain reaction (PCR) on kidney, fin or gill tissue (2, 5, 10). However, these tests
were developed on experimentally infected fish and have not been applied to production systems.
Therefore, their use or reliability in detecting CCV in populations or predicting the potential for vertical
transmission has not been documented.
1. STANDARD SCREENING METHOD FOR CCVD
1.1. Isolation of CCV in cell culture
Cell line to be used: CCO
2
of cell monolayer with 100 l of each dilution.
ii) Allow virus to adsorb for 0.51 hour at 2530C. Then (without withdrawing the
inoculum), add the cell culture medium buffered at pH 7.6 and supplemented with 10%
fetal calf serum (FCS) (1 ml/well for 24-well drained cell culture plates), and incubate at
2530C.
contrast microscope is recommended. CPE is extensive and rapidly progressing in
cultures from overtly diseased individuals. CPE consists of cell fusion (syncytium)
formation and contraction leaving cytoplasmic spindles irradiating from the syncytium to
points on the flask surface where the cells were originally attached.
This can be achieved by the addition to the inoculated cell culture medium of bicarbonate
buffer (for tightly closed cell culture flasks or in a CO
2
incubator), 2 M Tris buffer
solution or by using 25 mM HEPES-buffered medium (HEPES = N-2-hydroxyethyl-
piperazine-N-2-ethanesulfonic acid).
iii) If CPE appears in those cell cultures inoculated with the dilutions of the tested
homogenate supernatants, identification procedures must be undertaken immediately (see
Section 1.2. below).
approved previously) from which the virus-positive sample originated. The suspension of approved
status will be maintained until it is demonstrated that the virus in question is not CCV.
the sample inoculated with the virus control fail to develop CPE, the process should be
repeated with fresh susceptible cells and new batches of samples.
ii) Inoculate cell monolayers as described above in Section 1.1.a.
iii) Incubate and monitor as described in Section 1.1.b.
iv) Perform a second (and last) subcultivation step if the first one remains virus-negative.
1.2. Identification of CCV
a) Neutralisation test (Note: when developing antisera for CCV most researchers have found a weak
neutralising antibody response in rabbits; cross-reaction with cellular components also often occurs)
ii) Dilute virus-containing medium from 10
2
to 10
4
.
iii) Mix aliquots (for example 200 l) of each virus dilution with equal volumes of an
antibody solution specific for CCV, and similarly treat aliquots of each virus dilution with
cell culture medium.
least 2000.)
a homologous virus (positive neutralisation test)
a heterologous virus (negative neutralisation test).
v) Incubate all the mixtures at 25C for 1 hour.
vi) Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell
cultures per dilution) and allow adsorption to occur for 0.51 hour at 25C; 24- or 12-well
cell culture plates are suitable for this purpose, using a 50 l inoculum.
vii) When adsorption is complete, add the cell culture medium, supplemented with 2% FCS
and buffered at pH 7.37.6, into each well and incubate at 2530C.
viii) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
monolayers with a solution of 1% crystal violet in ethanol 20%.
ix) The tested virus is identified as CCV when CPE is abolished or noticeably delayed in the
cell cultures that had received the virus suspension treated with the CCV-specific
x) In the absence of any neutralisation by NAb to CCV, conduct an indirect fluorescent
antibody test (IFAT) with the suspect sample, perform an enzyme-linked immunosorbent
assay (ELISA) or use a CCV-specific nucleic-acid-based assay.
2
positive and two for negative controls).
iii) Dilute the control virus suspension of CCV in a similar way, in order to obtain a virus
2
of cell
monolayer.
freeze at 20C.
viii) Prepare a solution of purified antibody or serum to CCV in 0.01 M PBS, pH 7.2,
established previously, there is no commercial source for CCV-specific antiserum).
2
well.
or goat antibodies.
40 objective lens having a high numerical aperture. Positive and negative controls must be
found to give the expected results prior to any other observation.
immunoglobulins (Ig) or serum specific for CCV, in 0.01 M PBS, pH 7.2 (200 l/well). Ig
may be polyclonal or monoclonal Ig originating most often from rabbit or mouse,
respectively.
(200 l/well).
vii) Dispense 100 l/well of a two- or four-step dilution of the virus to be identified and of
CCV control virus, and allow to react with the coated antibody to CCV for 1 hour at
20C.
ix) Add biotinylated polyclonal antibody to CCV to the wells.
xiv) Add the substrate and chromogen. Stop the course of the test when positive controls
react, and monitor the results.
d) Polymerase chain reaction
There are several published PCR assays for CCV (2, 5, 10). As stated in Chapter I.1. Section
C.3.5., PCR is very susceptible to false-positive and false-negative results. Therefore each assay
and tissue extraction should include a negative control to rule out contamination. PCR reaction
set-up should be done in a separate physical location from where the PCR products are
evaluated. PCR is a useful method for identifying the virus after isolation in culture. The
following is a modification of the method of Boyle & Blackwell (5) and uses a modified target
as an internal control (12). The 53 sequence of the upper primer is TCA-TCC-GAA-TCC-
GAC-AAC-TGA and that of the lower primer is CCA-AGA-TCG-CGG-AGA-AAC. To
minimise the potential for contamination, all sample preparation and reaction set-up should be
done with aerosol-preventing pipette tips.
Sample preparation
i) Collect 10.5 ml of cell culture supernatant from affected wells into a 1.5 ml
microcentrifuge tube.
ii) Centrifuge at maximum speed in microfuge (18,00020,000 g) for 30 minutes.
iii) Discard supernatant and resuspend the pellet in 10 l proteinase K buffer (50 mM KCl,
15 mM Tris-HCl pH 8.3 and 0.5% Nonidet P-40) containing 0.5 mg/ml proteinase K and
incubate at 55C for 1 hour.
iv) Heat inactivate the proteinase K at 95C for 10 minutes. Centrifuge at maximum speed
for 10 seconds. Use 3 l in the PCR reaction.
Other standard DNA extraction methods may be used.
PCR set-up
The mastermix should be made-up in a separate location from areas where diagnostic samples
are prepared and PCR product are analysed.
i) Make-up the mastermix. Prepare at least one extra aliquot for each 10 reactions planned.
Include a positive and a negative (water blank) control for every 10 samples.
Per sample: distilled water 33.6 l
10 buffer 5.0 l
Upper primer (50 pmole/l) 0.4 l
Lower primer (50 pmole/l) 0.4 l
dNTPs (2.5 mM each) 2.0 l
25 mM MgSO
4
5.0 l
Internal control (0.025 pg/l) 0.3 l
Taq polymerase (5 U/l) 0.3 l
ii) Add 47 l per reaction tube.
iii) Add 3 l of sample or water (negative control).
iv) If using a thermocycler without a hot lid, add 50 l of mineral oil.
v) Run the reaction. Use 30 cycles of 93C for 30 seconds, 60C for 30 seconds, and 72C
for 30 seconds.
vi) Electrophorese 10 l of the product on a 10% polyacrylamide gel with a 100 bp size
ladder. Stain the gel with ethidium bromide. Observe and photograph under UV
transillumination (17).
Evaluate results
The internal control produces a 149 bp product. CCV produces a 136 bp product. Expect a
149 bp band within the negative control and on negative samples. Expect a 136 bp product
and a 149 bp product on CCV-positive samples. If CCV is present at a high level, the 149 bp
band may be missing. If no bands are present, the PCR reaction did not work indicating a
failure of one of the PCR components or an inhibitor in the sample. If the negative control
shows a 136 bp product, then there is a contaminant in the PCR set-up and the assay must be
redone. If an aberrant size band is produced or to confirm that the PCR product is from CCV,
the PCR can be redone using no internal standard and the product can be directly sequenced.
The sequence can then be evaluated using BLAST on the National Center for Biological
Information internet site (http://www3.ncbi.nlm.nih.gov/BLAST/) to identify sequences with
high homology. The PCR amplifies the region from 107827 to 107962 of the CCV genome
(GenBank accession M75136) representing a portion of open reading frame 73 within ORF
(9). Expect at least 95% identity to this sequence.
TCA-TCC-GAA-TCC-GAC-AAC-TGA-CGC-GTC-GGT-AGC-CCG-ACC-GAT-CCG-
TAT-GTT-ACG-GGT-GCG-GGG-GTC-GAC-ACC-GTG-CTC-GCC-GCG-ATG-AGG-CTG-
ACC-GCG-GAC-ACG-GGG-GGT-CCC-CCT-CGT-TTC-TCC-GCG-ATC-TTG-G
Figure 1. Sequence of CCV PCR product. Italic underlined portions indicate primer sequences.
2. DIAGNOSTIC METHODS FOR CCV
Test procedure
culture plate.
iii) Store the kidney pieces (as indicated in Section B.3.1. in Chapter I.1.) together with the
other organs required for virus isolation in case this becomes necessary later.
or 1% bovine serum albumin, in PBST for 30 minutes at 37C.
viii) Treat the imprints with the solution of antibody to CCV and rinse as indicated in Section
1.2.b.
ii) Treat the remaining part of the homogenate with 2% Triton X-100 as described in
Section 1.2.c. step v, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
Complete the other steps of the procedure described in Section 1.2.c.
As stated in Chapter I.1. Section C.3.5., PCR is very susceptible to false-positive and false-negative
results. Therefore each assay and tissue extraction should include a negative control to rule out
contamination. PCR reaction set-up should be done in a separate physical location from that
where the PCR products are evaluated. Thus the use of nested PCR protocols for inspection
purposes is not recommended. The PCR assay given in Section 1.2.d. can be modified for direct
detection of CCV in overtly infected fish. This is done by centrifuging 1.5 ml of a 1/10 dilution of
the homogenate at 8000 g for 5 minutes. The supernatant is then used the same way as the cell
culture supernatant in Section 1.2.d. If this assay is used to detect carrier fish, the DNA must be
purified from the tissue (using a commercially available kit such as the Puregene DNA isolation
kit, Gentra Systems, Minneapolis Minnesota, USA). To increase the detection limit to that needed
to detect a carrier state, use 0.5 g of DNA, leave out the internal control DNA, and use 35
40 cycles. In performing PCR on tissue extracts, it is recommended that a parallel sample be run
that has been spiked with dilute purified CCV DNA or cloned target fragment (such as the
internal standard used above) as a positive control to rule out the presence of inhibitors giving
false-negative results.
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* *
C HA P T E R 2 . 1 . 7 .

VI RAL E NCE P HAL OP AT HY AND RE T I NOP AT HY

SUMMARY
Viral encephalopathy and retinopathy (VER), or viral nervous necrosis (VNN) has been reported as a
serious disease of larval and juvenile and sometimes older marine fish that occurs world-wide except for
Africa (23). To date, the disease has been reported in at least 30 fish species, with the greatest impact being
in sea bass (Lates calcarifer [10] and Dicentrarchus labrax [2]), groupers (Epinephelus akaara [22],
E. fuscogutatus [3], E. malabaricus [6], E. moara [25], E. septemfasciatus [9], E. tauvina [4], E.
coioides [19] and Cromileptes altivelis [34]), jack (Pseudocaranx dentex [21]), parrotfish
(Oplegnathus fasciatus [33]), puffer (Takifugu rubripes [25]), and flatfish (Verasper moseri [32],
Hippoglossus hippoglossus [11], Paralichthys olivaceus [26], Scophthalmus maximus [1]).
Virus particles of about 2530 nm in diameter have been visualised in affected fish, and the agents in
striped jack, barramundi, and European sea bass have been characterised and placed in the genus
Betanodavirus, family Nodaviridae (5, 21). Immunological studies have shown relationships between
striped jack nervous necrosis virus (SJNNV, the type species of the genus Betanodavirus) and the other
betanodaviruses. Genomic classification of betanodaviruses has shown close relationships, with major
groupings being SJNNV-type, tiger puffer nervous necrosis virus (TPNNV)-type, barfin flounder nervous
necrosis virus (BFNNV)-type and red-spotted grouper nervous necrosis virus (RGNNV)-type (27).
Complete nucleotide sequences of RNA1 and RNA2 of SJNNV and GGNNV (a grouper
betanodavirus) have been reported (15, 29).
All diseases are characterised by a variety of neurological abnormalities, such as erratic swimming behaviour
(spiral, whirling or belly-up at rest) and vacuolation of the central nervous tissues. Usually there is also
vacuolation of the nuclear layers of the retina. In general, younger fish have more severe lesions; older fish
have less extensive lesions and these may show a predilection for the retina (23). Intracytoplasmic inclusions
have been described in brain cells of European sea bass, barramundi, Japanese parrotfish, and brownspotted
grouper. Neuronal necrosis has been described in most species.
Interesting differences with regard to the occurrence and severity of the diseases are shown in Table 1. There
are considerable variations in the age at which disease is first noted and the period over which mortality
occurs. In general, the earlier the signs of disease occur, the greater is the rate of mortality. Although disease
occurrence at the juvenile stages in some species is very rare, mass mortalities often occur at juvenile to young
stages in the other fish species, but usually do not reach 100%, indicating the age-dependence of
susceptibility (23). Mortalities have been reported in production-size European sea bass (18) and grouper
(9), but even in these cases mortalities were greatest in younger fish.
It has been demonstrated that vertical transmission of the causative agent occurs in Pseudocaranx dentex
and this fact is reflected by the early occurrence of clinical disease. This finding led to the successful control of
VNN of larval striped jack, where elimination of virus-carrying broodstock by reverse-transcription
polymerase chain reaction and disinfection of fertilised eggs by ozone were applied (20, 24). Paradoxically,
ovarian infection has also been reported in Dicentrarchus labrax in which disease is usually not seen until
about 30 days post-hatch. The mode of transmission/introduction of the viruses, other than in gametes and
by cohabitation, has not been demonstrated, but the possibilities include influent water, juvenile fish held on
the same site, and carriage on utensils, vehicles, etc. It is possible that these small viruses are quite resistant
to environmental conditions (8) and therefore readily translocated by commercial activities. Vaccination
using a recombinant capsid protein is at the experimental stage (14, 30).
Chapter 2.1.7. - Viral encephalopathy and retinopathy
Table 1. Important features of VER/VNN of larval and juvenile fish
Earliest
occurrence of
disease
Usual onset
of disease
Latest occurrence
of new outbreaks
Usual
mortality rate
Highest
mortality rate
Lates calcarifer 9 days
post-hatch
1518 days
post-hatch
24 days post-hatch 50100%/
month
100% in
<1 month
Dicentrarchus
labrax
10 days
post-hatch
2540 days
post-hatch
Body weight
400580 g
10%/month
Oplegnathus
fasciatus
625 mm
total length
<40 mm total length Up to 100%
Epinephelus
akaara
14 days post-
hatch (78 mm
total length)
910 mm
total length
<40 mm total length 80% Up to 100%
Epinephelus
malabaricus

2050 mm
total length

5080%

Pseudocaranx
dentex
1 day
post-hatch
14 days
post-hatch
<20 days post-hatch
(8 mm total length)
100%
Scophthalmus
maximus
<21 days
post-hatch
Body weight
50100 mg
Up to 100%
Presumptive diagnosis of viral encephalopathy and retinopathy (VER) or viral nervous necrosis (VNN)
can be made on the basis of the appearance of vacuoles in the brain, spinal cord and/or retina as seen by
light microscopy. However, individual fish with the presence of only a few vacuoles in the nervous tissues
pose a difficult diagnostic problem.
Virus particles can be visualised in affected brain and retina by both positive and negative staining. In
positively stained material, the virus is mainly associated with vacuolated cells and, especially, any
inclusions. The reported particles vary in size from 22 to 34 nm arranged intracytoplasmically in crystalline
arrays, or as aggregates and single virions both intra- and extracellularly. The virus is nonenveloped and
icosahedral in shape.
All betanodaviruses can be detected by the indirect fluorescent antibody test (IFAT) or
immunohistochemistry with a rabbit anti-SJNNV serum (12, 16). Most can be detected by reverse-
transcription polymerase chain reaction (RT-PCR) with a single primer set designed to amplify the T4
region (427 bases) of SJNNV coat protein gene (27, 28) (this primer set can be used for detection of all the
genotypic variants of betanodaviruses with only one exception [31]). Although other immunological
methods, such as the enzyme-linked immunosorbent assay (ELISA) or neutralisation test, are available for
virus identification, they can only be used for some of betanodaviruses because of limited serological
information.
The betanodaviruses can be cultured in a fish cell line, SSN-1, which is derived from striped snakehead (7,
16). A clonal cell line, E-11, derived from the SSN-1 cell line is useful for qualitative and quantitative
analyses of all the betanodaviruses (17). It is notable that both SSN-1 and E-11 cells are infected by a
spontaneously productive C-type retrovirus designated SnRV (13, 17).
status of fish populations.
Asymptomatic fish (apparently healthy fish): whole larvae or small juveniles; brain, spinal cord, and
eyes for larger size fish and/or ovarian fluid from broodfish at spawning time.
Clinically affected fish: whole larvae or small juveniles; brain, spinal cord, and eyes for larger size fish.
1. STANDARD SCREENING METHOD FOR VER/VNN
1.1. Isolation of betanodaviruses in cell culture
Cell line to be used: SSN-1 or E-11 (a cell clone of SSN-1)
i) Fish samples are homogenised with nine volumes of Hanks balanced salt solution
(HBSS), centrifuged and filtered (0.2 m membrane filter).
ii) Tenfold dilutions of the virus filtrate are inoculated in monolayers of cells cultured using
Leibovitz L-15 medium or other medium supplemented with 5% fetal bovine serum
(FBS). Inoculate at least 2 cm
2
of drained cell monolayer with 100 l of each dilution.
iii) Allow to adsorb for 1 hour at room temperature, add the medium supplemented with 5%
FBS and incubated at 2025C.
Note: Optimal growth temperatures are different among the four genotypic variants: 2530C
for RGNNV-type, 2025C for SJNNV-type, 20C for TPNNV-type, and 1520C for
BFNNV-type (16, 17).
daily microscopic examination for 10 days.
ii) If cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of
the tested homogenate supernatants, betanodavirus identification procedures must be
undertaken (see Section 1.2. below).
Note: CPE in SSN-1 or E-11 cells is characterised by thin or rounded, refractile, granular
cells with vacuoles, the monolayer then partially or completely disintegrates (7, 16).
iii) If no CPE occurs after 10 days of incubation, subcultivation of the inoculated cell
cultures must be performed.
i) Collect aliquots of cell culture medium from all monolayers inoculated with organ
homogenates.
ii) Inoculate cell monolayers as described in Section1.1.a.
1.2. Identification of betanodavirus isolated in cell culture
2
wells of cell culture plastic plates or on cover-slips
(or chamber slides) in order to reach around 70% confluency, which is usually achieved
within 24 hours of incubation at 25C.
ii) Inoculate the virus suspensions to be identified by making tenfold dilution steps directly
in the cell culture wells or flasks.
iii) Incubate at 20C or 25C for 4872 hours (See Section 1.1.a. Note).
iv) Remove the culture medium, rinse once with PBS, then fix with methanol for 10 minutes.
v) Allow the cell monolayers to air-dry.
vi) Treat the cell monolayers with a rabbit anti-betanodavirus serum for 30 minutes at 37C
in a humid chamber, and rinse four times with PBSTween 80 (PBST).
vii) Treat the cell monolayers for 30 minutes at 37C with commercially available fluorescein
isothiocyanate-conjugated anti-rabbit Ig antibody, and rinse with PBST.
viii) Examine the treated cell monolayers on plates immediately, or mount the cover-slips
using glycerol saline, pH 8.5, prior to microscopic observation.
b) Reverse-transcription polymerase chain reaction
i) Total RNA is extracted from virus-inoculated cells by a commercially available RNA
extraction kit according to the manufacturers instructions.
ii) There are several published primers for RT-PCR amplification of betanodaviruses.
Primers, R3 (5-CGA-GTC-AAC-ACG-GGT-GAA-GA-3) and F2 (5-CGT-GTC-AGT-
CAT-GTG-TCG-CT-3), designed to amplify the T4 region (427 bases) of SJNNV coat
protein gene, are available for all genotypic variants (27).
i) Fish samples fixed in 10% buffered formalin are dehydrated and embedded in paraffin
wax. Deparaffinise sections and rehydrate to PBS.
ii) The sections are treated with 0.1% trypsin in PBS at 37C for 30 minutes.
iii) After washing with cold PBS, proceed as described in Section 1.2.a. steps vi to viii.
iv) Specific fluorescence is observed in the cytoplasm of the affected cells in brain, spinal
cord, or retina.
b) Immunohistochemistry (avidin-biotin-alkaline phosphatase technique) (12)
i) Prepare paraffin sections as described above in Section 2.1.a.
ii) Add a blocking solution, for example 5% bovine serum albumin (BSA) in Tris-buffered
saline (TBS) for 20 minutes.
iii) Incubate with the anti-betanodavirus rabbit serum for 30 minutes, and wash in TBS.
iv) Add biotinylated goat anti-rabbit Ig for 30 minutes, and wash in TBS.
v) Add streptavidin alkaline phasphatase complex, incubate for 30 minnutes, and wash in
TBS.
vi) Add fuchsin chromogen reagent for 5 minutes, and wash in tap water
vii) Counterstain with haematoxylin.
viii) A positive result is indicated by red colour.
c) Reverse-transcription polymerase chain reaction
i) The fish sample is homogenised with distilled water treated with 0.1% diethyl
pyrocarbonate.
ii) Centrifuge at 10,000 g for 10 minutes.
iii) Using the supernatant, continue as described in Section 1.2.b.
See Section 1.1.
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a picornavirus-like agent. Dis. Aquat. Org., 10, 6570.
2. BREUIL G., BONAMI J.R., PEPIN J.F. & PICHOT Y. (1991). Viral infection (picorna-like virus)
associated with mass mortalities in hatchery-reared sea-bass (Dicentrarchus labrax) larvae and juveniles.
3. CHI S.C., LO C.F., KOU G.H., CHANG P.S., PENG S.E. & CHEN S.N. (1997). Mass mortalities
associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper,
Epinephelus fuscogutatus and E. akaara (Temminck & Schlegel). J. Fish Dis., 20, 185193.
4. CHUA F.H.C., LOO J.J. & WEE J.Y. (1995). Mass mortality in juvenile greasy grouper, Epinephelus
tauvina, associated with vacuolating encephalopathy and retinopathy. In: Diseases in Asian
Aquaculture II, Schariff M., Arthur J.R. & Subasinghe R.P., eds. Fish Health Section, Asian Fisheries
Society, Manila, the Philippines, 235241.
5. COMPS M., PEPIN J.F. & BONAMI J.R. (1994). Purification and characterization of two fish
encephalitis viruses (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture, 123, 110.
6. DANAYADOL Y., DIREKBUSARAKOM S. & SUPAMATTAYA K. (1995). Viral nervous necrosis in
brownspotted grouper, Epinephelus malabaricus, cultured in Thailand. In: Diseases in Asian
Aquaculture II, Schariff M., Arthur J.R. & Subasinghe R.P., eds. Fish Health Section, Asian Fisheries
Society, Manila, the Philippines, 227233.
7. FRERICHS G.N., RODGER H.D. & PERIC Z. (1996). Cell culture isolation of piscine neuropathy
nodavirus from juvenile sea bass, Dicentrarchus labrax. J. Gen. Virol., 77, 20672071.
8. FRERICHS G.N., TWEEDIE A., STARKEY W.G. & RICHARDS R.H. (2000). Temperature, pH, and
electrolyte sensitivity, and heat, UV and disinfectant inactivation of sea bass (Dicentrarchus labrax)
neuropathy nodavirus. Aquaculture, 185, 1324.
9. FUKUDA Y., NGUYEN H.D., FURUHASHI M. & NAKAI T. (1996). Mass mortality of cultured
sevenband grouper, Epinephelus septemfasciatus, associated with viral nervous necrosis. Fish Pathol., 31,
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with mass mortalities in larval barramundi, Lates calcarifer Bloch. J. Fish Dis., 13, 245249.
11. GROTMOL S., TOTLAND G.K., THORUD K. & HJELTNES B.K. (1997). Vacuolating encephalopathy
and retinopathy associated with a nodavirus-like agent: a probable cause of mass mortality of cultured
larval and juvenile Atlantic halibut Hippoglossus hippoglossus. Dis. Aquat. Org., 29, 8597.
12. GROTMOL S., BERGH O & TOTLAND G.K. (1999). Transmission of viral encephalopathy and
retinopathy (VER) to yolk-sac larvae of the Atlantic halibut Hippoglossus hippoglossus: occurrence of
nodavirus in various organs and a possible route of infection. Dis. Aquat. Org., 36, 95-106.
13. HART D., FRERICHS G.N., RAMBAUT A. & ONIONS D.E. (1996). Complete nucleotide sequence and
transcriptional analysis of the snakehead fish retrovirus. J. Virol., 70, 36063616.
14. HUSGARD S., GROTMOL S., HJELTNES B., RODSETH O.M. & BIERING E. (2001). Immune response
to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus
maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV.
Dis. Aquat. Org., 45, 33-44.
15. IWAMOTO T., MISE K., MORI K., ARIMOTO M., NAKAI T. & OKUNO T. (2001). Establishment of an
infectious RNA transcription system for striped jack nervous necrosis virus, the type species of the
Betanodavirus. J. Gen. Virol., 82, 26532662.
16. IWAMOTO T., MORI K., ARIMOTO M. & NAKAI T. (1999). High permissivity of the fish cell line SSN-
1 for piscine nodaviruses. Dis. Aquat. Org., 39, 3747.
17. IWAMOTO T., NAKAI T., MORI K., ARIMOTO M. & FURUSAWA I. (2000). Cloning of the fish cell
line SSN-1 for piscine nodaviruses. Dis. Aquat. Org., 43, 8189.
18. LE BRETON A., GRISEZ L., SWEETMAN J. & OLIEVIER F. (1997). Viral nervous necrosis (VNN)
associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax (L.). J. Fish Dis., 20, 145
151.
19. LIN L., HE J., MORI K., NISHIOKA T., WU J.L., WENGS S., MUSHIAKE K., ARIMOTO M. & NAKAI T.
(2001). Mass mortalities associated with viral nervous necrosis in hatchery-reared groupers in the
Peoples Republic of China. Fish Pathol., 36, 186188.
20. MORI K., MUSHIAKE K. & ARIMOTO M. (1998). Control measures for viral nervous necrosis in
striped jack. Fish Pathol., 33, 443444.
21. MORI K., NAKAI T., MUROGA K., ARIMOTO M., MUSHIAKE K. & FURUSAWA I. (1992). Properties
of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with
nervous necrosis. Virology, 187, 368371.
22. MORI K., NAKAI T., NAGAHARA M., MUROGA K., MEKUCHI T. & KANNO T. (1991). A viral disease
in hatchery-reared larvae and juveniles of redspotted grouper. Fish Pathol., 26, 209210.
23. MUNDAY B.L., KWANG J. & MOODY N.(2002) Betanodavirus infections of teleost fish: a review. J.
Fish Dis., 25, 127-142.
24. MUSHIAKE K., NISHIZAWA T., NAKAI T., FURUSAWA I. & MUROGA K. (1994). Control of VNN in
striped jack: Selection of spawners based on the detection of SJNNV gene by polymerase chain
reaction (PCR). Fish Pathol., 29, 177182.
25. NAKAI T., NGUYEN H.D., NISHIZAWA T., MUROGA K., ARIMOTO M. & OOTSUKI K. (1994).
Occurrence of viral nervous necrosis in kelp grouper and tiger puffer. Fish Pathol., 29, 211212.
26. NGUYEN H.D., MEKUCHI T., IMURA K., NAKAI T., NISHIZAWA T. & MUROGA K. (1994).
Occurrence of viral nervous necrosis (VNN) in hatchery-reared juvenile Japanese flounder
Paralichthys olivaceus. Fisheries Sci., 60, 551554.
27. NISHIZAWA T., FURUHASHI M., NAGAI T., NAKAI T. & MUROGA K. (1997). Genomic classification
of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl. Environ.
Microbiol., 63, 16331636.
28. NISHIZAWA T., MORI K., NAKAI T., FURUSAWA I. & MUROGA K. (1994). Polymerase chain reaction
(PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Dis. Aquat. Org., 18,
103107.
29. TAN C., HUANG B., CHANG S.F., NGOH G.H., MUNDAY B.L., CHEN S.C. & KWANG J. (2001).
Determination of the complete nucleotide sequence of RNA1 and RNA2 from greasy grouper
(Epinephelus tauvina) nervous necrosis virus, Singapore strain. J. Gen. Virol., 82, 647653.
30. TANAKA S., MORI K., ARIMOTO M., IWAMOTO T. & NAKAI T. (2001) Protective immunity of
sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis.
J. Fish Dis., 24, 15-22.
31. THIERY R., ARNAULD C. & DELSERT C. (1999). Two isolates of sea bass, Dicentrarchus labrax L.,
nervous necrosis virus with distinct genomes. J. Fish Dis., 22, 201207.
32. WATANABE K., YOSHIMIZU M., ISHIMA M., KAWAMATA K. & EZURA Y. (1999). Occurrence of viral
nervous necrosis in hatchery-reared barfin flounder. Bull. Fac. Fish. Hokaido Univ., 50, 101113.
33. YOSHIKOSHI K. & INOUE K. (1990). Viral nervous necrosis in hatchery-reared larvae and juveniles of
Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). J. Fish Dis., 13, 6977.
34. ZAFRAN, KOESHARYANI I., JOHNNY F., YUASA K., HARADA T. & HATAI K. (2000). Viral nervous
necrosis in humpback grouper Cromileptes altivelis larvae and juveniles in Indonesia. Fish Pathol., 35,
9596.
*
* *
C HA P T E R 2 . 1 . 8 .

I NF E CT I OUS P ANCRE AT I C NE CROS I S

SUMMARY
Infectious pancreatic necrosis (IPN) is a highly contagious viral disease of young fish of salmonid species
held under intensive rearing conditions (14, 32, 33). The disease most characteristically occurs in rainbow
trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), brown trout (Salmo trutta),
Atlantic salmon (Salmo salar), and several Pacific salmon species (Oncorhynchus spp.). Susceptibility
generally decreases with age, with resistance to clinical disease in salmonid fish usually being reached at
about 1500 degree-days (value obtained by multiplying the age in days by the average temperature in degrees
centigrade during the lifespan) (11), except for Atlantic salmon smolts, which can be affected after transfer
from fresh water to seawater (29). The first sign of an outbreak in salmonid fry is frequently a sudden and
usually progressive increase in daily mortality, particularly in the faster growing individuals. Clinical signs
include darkening pigmentation, a pronounced distended abdomen and a corkscrewing/spiral swimming
motion. Cumulative mortalities may vary from less than 10% to more than 90% depending on the
combination of several factors, such as virus strain (17) and quantity (24), host and environment (10). For
further details see reviews by Hill (14), Reno (27) and Wolf (32).
The disease is transmitted both horizontally via the water route and vertically via the egg (3, 4, 12).
Surface disinfection of eggs is not entirely effective in preventing vertical transmission (6).
The disease has a wide geographical distribution, occurring in most major salmonid-farming countries of
North and South America, Europe and Asia. Oceania is free of the disease.
IPN virus (IPNV), or viruses showing serological relatedness to IPNV, have been reported to cause
diseases in some farmed marine fish species, such as yellowtail (Seriola quinqueradiata) (21), turbot
(Scophthalmus maximus) (7, 20, 22), dab (Limanda limanda) (25), halibut (Hippoglossus
hippoglossus) (20, 28) and Atlantic cod (Gadus morhua). Subclinical covert infections have been
detected in a wide range of estuarine and freshwater fish species, such as loach (Misgurnus
anguillicaudatus) (8), pike (Esox lucius) (2) and numerous other species in the families Anguillidae,
Atherinidae, Bothidae, Carangidae, Cotostomidae, Cichlidae, Clupeidae, Cobitidae, Coregonidae,
Cyprinidae, Esocidae, Moronidae, Paralichthydae, Percidae, Poecilidae, Sciaenidae, Soleidae and
Thymallidae (27).
The causative agent, IPNV, is a bi-segmented double-stranded RNA virus belonging to the family
Birnaviridae (see review by Dobos & Roberts [10]). Isolates display wide antigenic diversity (11, 16, 19,
23) and fall into two serogroups that do not cross-react in serum neutralisation tests (3, 25, 31), with the
majority of strains belonging to serogroup A, which comprises at least nine serotypes (16). Isolates also show
marked differences in degrees of virulence (14, 15, 17).
Control methods currently rely on the implementation of control policies and of hygiene practices in salmonid
husbandry, through the avoidance of the introduction of fertilised eggs originating from IPNV-carrier
broodstock, and the use of a protected water supply (e.g. spring or borehole pond) where the ingress of fish,
particularly possible virus carriers, is prevented. In outbreaks, a reduction in the population density
(thinning out) may help to reduce the overall mortality. No treatment or entirely effective vaccine is
available at present.
Chapter 2.1.8. - Infectious pancreatic necrosis
The screening procedure for infectious pancreatic necrosis (IPN) is based on IPN virus (IPNV) isolation
tests in cell culture (1) followed by immunological identification, either by serum neutralisation (16) or
enzyme-linked immunosorbent assay (ELISA) (9), of virus isolated. Diagnosis of clinical cases is normally
based on histology (18) and/or immunological demonstration of IPNV antigen in infected tissues (13),
confirmed by isolation and immunological identification of IPNV in tissue culture, as for screening.
status of fish populations. However, the validation of some serological techniques for certain fish virus
infections could arise in the near future, rendering the use of fish serology more widely acceptable for
health screening purposes.
Asymptomatic fish (apparently healthy fish): liver, kidney and spleen (any size fish) and/or ovarian
fluid from broodfish at spawning time.
Clinically affected fish: whole alevin (body length 4 cm), entire viscera including kidney (4 cm
body length 6 cm) or, for larger size fish, liver, kidney and spleen.
1. STANDARD SCREENING METHOD FOR IPN
1.1. Isolation of IPNV in cell culture
Cell line to be used: BF-2 or CHSE-214 or RTG-2
transfer an appropriate volume of each of the two dilutions on to 24-hour cell
2
dilution.
ii) Allow to adsorb for 0.51 hour at 1015C and, without withdrawing the inoculate, add
the cell culture medium buffered at pH 7.6 and supplemented with 2% fetal calf serum
(FCS) (1 ml/well for 24-well cell culture plates) and incubate at 15C.
bicarbonate buffer (for tightly closed cell culture flasks) or 2 M Tris buffer solution (for
cell culture plates) or, even better, by using HEPES-buffered medium (HEPES = N-2-
hydroxyethyl-piperazine-N-2-ethanesulfonic acid).
the homogenate, IPNV identification procedures must be undertaken immediately (see
Section 1.2. below).
iv) If no CPE occurs after 7 days of incubation (except in positive control cell cultures),
subcultivation of the inoculated cell cultures must be performed.
i) Subject cell culture monolayers to one freezethaw cycle. Pool aliquots of the
supernatants from all cell monolayers inoculated with dilutions of organ homogenates.
ii) Dilute 1/20 and 1/100 and inoculate cell monolayers as described above in Section 1.1.a.
iii) Incubate at 15C and monitor as described in above Section 1.1.b.
iv) If no CPE occurs, the test may be declared negative.
v) If CPE occurs, the IPNV identification procedures must be undertaken immediately.
1.2. Identification of IPNV isolated in cell culture
i) Dilute the virus-containing medium from 10
2
to 10
4
.
ii) Mix aliquots of each dilution with equal volumes of a neutralising antibody (NAb)
solution against the indigenous serotypes of IPNV known in the region. If IPNV is not
known to be indigenous to the region, mix aliquots of each dilution with equal volumes
of a pooled NAb solution against all IPNV serotypes (16). Similarly, treat aliquots of each
virus dilution with cell culture medium. In parallel, a neutralisation test must be
performed against homologous IPNV (positive neutralisation test).
(The NAb solution must have a 50% plaque reduction titre of at least 2000.)
iii) In parallel, other neutralisation tests must be performed against:
iv) Incubate all the mixtures at 15C for 1 hour.
v) Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell
cultures per dilution).
vi) Incubate at 15C.
vii) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
viii) The tested virus is identified as IPNV when CPE is prevented or noticeably delayed in
the cell cultures that received the virus suspension treated with the IPNV-specific
ix) If CPE develops in the presence of antisera against the indigenous serotypes of IPNV,
the neutralisation test must be repeated using pooled antibody against the non-indigenous
serotypes of IPNV.
2
be reduced to 24%.
iii) Dilute the control virus suspension of IPNV in a similar way, in order to obtain a virus
2
of cell
monolayer.
freeze at 20C.
viii) Prepare a solution of purified antibody or serum to IPNV in 0.01 M PBS, pH 7.2,
0.25 ml/2 cm
2
well.
(FITC)-conjugated antibody to the immunoglobulin (Ig) used in the first layer and
prepared according to the instructions of the supplier. These FITC antibodies are most
often rabbit or goat antibodies.
40 objective lens having high numerical aperture. Positive and negative controls must be
found to give the expected results prior to any other observation.
(ELISAs) with appropriate dilutions of purified Ig specific for IPNV, in 0.02 M carbonate
buffer, pH 9.5 (200 l/well). Ig may be polyclonal or monoclonal Ig originating most
often from rabbit or mouse, respectively. For the identification of IPNV, monoclonal
antibodies (MAbs) specific for certain domains of the capsid protein are suitable.
iii) Rinse twice with 0.01 M PBS.
iv) Block with bovine serum albumin (BSA) (1.5% in carbonate buffer) or other blocking
solution for 1 hour at 37C (300 l/well).
v) Rinse four times with PBS containing 0.05% Tween 20 (PBST).
vi) Add an equal volume of PBST to the virus suspension to be identified.
vii) Dispense 100 l/well of two- or four-step dilutions of the suspension of virus to be
identified and of the non-infected cell culture harvest (negative control), and the IPNV
control virus (positive control); allow to react with the coated antibody to IPNV for
30 minutes at 37C.
viii) Rinse once with PBST followed by three washes, allowing to soak for 3 minutes between
washes.
ix) Add to the wells, horseradish peroxidase (HRPO)-conjugated MAb or polyclonal
antibody to IPNV; or polyclonal IgG to IPNV or MAb to capsid protein specific for a
domain different from the one of the coating MAb and previously conjugated with biotin.
x) Incubate for 30 minutes at 37C.
xi) Rinse and wash with PBST as in step viii above.
xii) Add 200 l of HRPO-conjugated streptavidin or ExtrAvidin (Sigma) to those wells that
have received the biotin-conjugated antibody and repeat steps x and xi.
xiii) Add 200 l of the substrate and chromogen. Stop the course of the test when positive
controls react, and read the results.
xiv) Alternatively, add substrate and chromogen to those wells containing the peroxidase-
conjugated antibody and proceed as above.
(The procedure described above is just one example of an ELISA. Other procedures that use
smaller volumes [50100 l] of reagents and/or that use alkaline phosphatase as the enzyme
are also acceptable.)
culture plate.
or 1% BSA, in PBST for 30 minutes at 37C.
viii) Treat the imprints with the solution of antibody to IPNV and rinse as indicated in Section
1.2.b.
i) Microplate processing
As in Section 1.2.c. of this chapter up to step iv (inclusive).
ii) Sampling procedures
iii) Processing of organ samples
iv) Enzyme-linked immunosorbent assay procedure
Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell
Add a volume of 100 mM phenyl methyl sulphonide fluoride (PMSF) to the
homogenate to a final concentration of 2 mM PMSF, and mix gently.
Complete the other steps of the procedure described in Section 1.2.c.
c) Co-agglutination test
IPNV may be detected by a rapid co-agglutination test method (30). The test comprises two
test solutions an anti-IPNV solution, consisting of Staphylococcus aureus cells (Phadebact
coating kit, Boule Diagnostics AB, Sweden) coated with rabbit-anti-IPNV antibody, according
to the manufacturers instructions, and a negative control solution of S. aureus cells coated with
normal rabbit serum and cardboard slides, a stomacher plastic bags and PBS.
The procedure for the test is as follows:
i) Use 0.51 g tissue material, preferably kidney, but if small fry are to be examined, all of
the visceral organs or whole fry except head and tail may be used. Homogenise the tissue
sample with 0.51 ml of PBS.
ii) Centrifuge the homogenate at 2000 g for 10 minutes, remove the supernatant and dilute
in an equal volume of PBS.
iii) Add one drop of the anti-IPNV solution and one drop of the negative control solution
separately to a cardboard slide. Add one drop of the diluted homogenate (see step ii)
separately to the drop containing anti-IPNV and to the drop containing negative control
solution. Use a bacterial loop or syringe point to mix the contents of the drops.
iv) A positive reaction is shown as agglutination in the sample where anti-IPNV antibody is
present, but not in the negative control solution.
d) Immunohistochemistry
i) Fixation: The tissues for examination have to be fixed in aldehyde fixatives (10%
formaldehyde, 3% paraformaldehyde, 2.5% glutaraldehyde) for 48 hours or in absolute
alcohol or 96% alcohol. Tissues for freeze sections are frozen in Freon gas or in liquid
nitrogen as fast as possible. Such tissues must be kept at 70C prior to and after
sectioning.
ii) Pretreatment of tissue samples: After fixation, the sample is processed through a histokinette
according to normal histological procedures and embedded in liquid paraffin. Freeze
sections are thawed, dried by a fan without heat and fixed in acetone, acetone/methanol,
ethanol or other similar solvents.
iii) Histolological sections are put on slides coated with poly-L-lysine. (Poly-L-lysine glass is
prepared in the following manner: 0.5% poly-L-lysine is dissolved in distilled water to
reach a user concentration of 0.05%. The slide is left in poly-L-lysine solution for
30 minutes, and then rinsed for 5 minutes in running tap water. The slides are air dried
over night. Note: Poly-L-lysine-coated slides are also commercially available.)
iv) The sections are then deparaffinised and hydrated in the following way:
Heat the chamber for 25 minutes at 5658C. The temperature must not exceed
60C. Sections are then placed in the following xylene/ethanol baths in a fume-
hood:
5 minutes in xylene,
5 minutes in xylene,
5 minutes in absolute alcohol,
5 minutes in absolute alcohol,
5 minutes in 96% alcohol,
5 minutes in 70% alcohol,
5 minutes in running tap water.
v) Blocking of endogenous peroxidase activity can be done by two different methods:
H
2
O
2
/methanol (30% H
2
O
2

diluted in 3% methanol) in staining jars. The sections
are kept in this solution for 20 minutes and then washed in PBS.
Phenylhydrazine diluted in PBS to a final concentration 0.05%. The PBS must be
preheated to 37C before phenylhydrazine is added. Mix well. The sections are
covered with this solution and incubated, in a humid chamber, at 37C for
40 minutes and then washed in PBS.
vi) Pretreatment of sections to unmask antigens: As antigens may be masked by methylene bridges
from the formalin fixation, it is necessary to break the cross-bindings. This can be done
by pretreatment in a microwave oven, or by trypsin treatment or by boiling under
pressure/autoclaving.
vii) Immunohistochemical procedure
After deparaffinisation and unmasking procedures are completed, the sections are
overlaid with 5% (w/v) BSA for 20 minutes. The solution is then blotted off the slides
(without washing) and the specific polyclonal or MAbs against IPNV are incubated at
concentrations/dilutions that will give an optimal signal to noise ratio. After washing for
5 minutes in Tris-buffered saline (TBS, pH 7.4), the secondary antibody, biotinylated anti-
rabbit, diluted from 1/300 to 1/500, or anti-mouse immunoglobulin, is incubated for 30
minutes. After washing in TBS, streptavidin alkaline phosphatase (Boehringer Mannheim
GmbH 1089-161) diluted 1/1000 (30 minutes incubation),
avidin/biotin/alkaline/phosphatase (Dakopatts, D 365) or avidin/biotin/peroxidase
(ABC-PO, Vector PK-4001) (the two latter complexes diluted according to the
manufacturers instructions) should be incubated for 3045 minutes at room temperature.
After washing, the specimens are incubated with either Fast Red in the case of alkaline
phosphatase or with AEC (3-amino-9-ethylcarbazole, 0.27 g/litre, Sigma A-5754) in the
case of peroxidase. Fast Red TR salt (Sigma F 1500) (1 g/litre) should be dissolved in
TBS, and naphthol AS-MX phosphate (0.2 g/litre), N,N-dimethylformamide (20 ml/litre)
(Sigma D-4254) and 1 mM levamisole are then added. Levamisole should be added as an
inhibitor of endogenous alkaline phosphatase. The sections are incubated for 20 minutes.
AEC is dissolved in N,N-dimethylformamide (67 ml/litre) in 0.1 M acetate buffer, pH
5.2, and 0.03% H
2
O
2
is added. The sections should be incubated for 15 minutes. The
sections are then washed in running tap water for 10 minutes and counterstained with
Mayers haematoxylin for 2 minutes. Finally, the sections are washed in tap water for
another 2 minutes and cover-slips are applied with an aqueous medium (Aquamount,
BDH-Laboratory Supplies, UK, 36086). All incubations, unless otherwise stated, are
performed at room temperature in a humidified chamber.
Performance controls should include application of a non-immune serum (normal rabbit
serum) at the same dilution as the immune serum or of heterologous MAbs (of the same
isotype) at almost identical protein concentrations. Tissue sections from non-infected fish
(same species as subject to analysis) should be incubated with immune and non-immune
serum-specific or heterologous MAbs. Estimation of end-point dilution value (the highest
dilution giving a positive reaction that was discernible from background) must be performed
for antibody solutions using a 60-minute incubation at 37C to validate the method.
See Section 1.1.
REFERENCES
1. AGIUS C., MANGUNWIRYO H., JOHNSON R.H. & SMAIL D.A. (1982). A more sensitive technique for
isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri
Richardson. J. Fish Dis., 5, 285292.
2. AHNE W. (1978). Isolation and characterisation of infectious pancreatic necrosis virus from pike
(Esox lucius). Arch. Virol., 58, 6569.
3. AHNE W., JORGENSEN P.E.V., OLESEN N.J., FISCHER-SCHERL T. & HOFFMANN R. (1989). Aquatic
Birnaviruses: virus of the serogroup II isolated from an IPN outbreak in brook trout (Salvelinus
fontinalis). Bull. Eur. Ass. Fish Pathol., 9, 1416.
4. AHNE W. & NEGELE R.D. (1985). Studies on transmission of infectious pancreatic necrosis virus via
eyed eggs and sexual products of salmonid fish. In: Fish and Shellfish Pathology, Ellis A.E., ed.
Academic Press, London, UK, 261269.
5. BRANTZAEG P. (1982). Tissue preparation methods for immunohistochemistry. In: Techniques in
Immunocytochemistry, Bullock G.R. & Petrusz P., eds. Academic Press, London, UK, 275.
6. BULLOCK G.L., RUCKER R.R., AMEND D., WOLD K. & STUCKEY H.M. (1976). Infectious pancreatic
necrosis: transmission with iodine-treated and non-treated eggs of brook trout (Salvelinus fontinalis). J.
Fish. Res. Board Can., 33, 11971198.
7. CASTRIC J., BAUDIN-LAURENCIN F., COUSTANS M.F. & AUFFRET M. (1987). Isolation of infectious
pancreatic necrosis virus, Ab serotype, from an epizootic in farmed turbot, Scophthalmus maximus.
8. CHOU H.Y., LO C.F., TUNG M.C., WANG C.H., FUKUDA H. & SANO T. (1993). The general
characteristics of a birnavirus isolated from cultured loach (Misgurnus anguillicaudatus) in Taiwan. Fish
Pathol., 28, 17.
9. DIXON P.F. & HILL B.J. (1983). Rapid detection of infectious pancreatic necrosis virus (IPNV) by the
enzyme-linked immunosorbent assay. J. Gen. Virol., 64, 321330.
10. DOBOS P. & ROBERTS T.E. (1983). The molecular biology of infectious pancreatic necrosis virus: a
review. Can. J. Gen. Microbiol., 29, 377384.
11. DORSON M. & TORCHY C. (1981). The influence of fish age and water temperature on mortalities of
rainbow trout, Salmo gairdneri Richardson, caused by a European strain of infectious pancreatic
necrosis virus. J. Fish Dis., 4, 213221.
12. DORSON M. & TORCHY C. (1985). Experimental transmission of infectious pancreatic necrosis virus
via the sexual products. In: Fish and Shellfish Pathology, Ellis A.E., ed. Academic Press, London,
UK, 251260.
13. EVENSEN O. & RIMSTAD E. (1990). Immunohistochemical identification of infectious pancreatic
virus in paraffin-embedded tissues of Atlantic salmon (Salmo salar). J. Vet. Diagn. Invest., 2, 288293.
14. HILL B.J. (1982). Infectious pancreatic necrosis and its virulence. In: Microbial Diseases of Fish
(Special Publication of the Society for General Microbiology), Roberts R.J., ed. Academic Press,
London, UK, 91114.
15. HILL B.J. (1992). Impact of viral diseases of salmonid fish in the European Community. In: Salmonid
Diseases, Kimura T., ed. Hokkaido University Press, Sapporo, Japan, 4859.
16. HILL B.J. & WAY K. (1995). Serological classification of infectious pancreatic necrosis (IPN) virus
and other aquatic birnaviruses. Ann. Rev. Fish Dis., 5, 5577.
17. MCALLISTER P.E. & OWENS W.J. (1995). Assessment of the virulence of fish and molluscan isolates
of infectious pancreatic necrosis virus for salmonid fish by challenge of brook trout, Salvelinus
fontinalis (Mitchill). J. Fish. Dis., 18, 97103.
18. MCKNIGHT I.J. & ROBERTS R.J. (1976). The pathology of infectious pancreatic necrosis. I. The
sequential histopathology of the naturally occurring condition. Br. Vet. J., 132, 7685.
19. MELBY H.P., CASWELL-RENO P. & FALK K. (1994). Antigenic analysis of Norwegian aquatic
birnavirus isolates using monoclonal antibodies with special reference to fish species, age and health
status. J. Fish Dis., 17, 8591.
20. MORTENSEN S.H., HJELTNES B., RODSETH O., KROGSRUD J. & CHRISTIE K.E. (1990). Infectious
pancreatic necrosis virus, serotype N1, isolated from Norwegian halibut (Hippoglossus hippoglossus),
turbot (Scophthalmus maximus) and scallops (Pecten maximus). Bull. Eur. Ass. Fish Pathol., 10, 4243.
21. NAKAJIMA K., MAENO Y., ARIMOTO M., INOUYE K. & SORIMACHI M. (1993). Viral deformity of
yellowtail fingerlings. Fish Pathol., 28, 125129.
22. NOVOA A., FIGUERAS A., PUENTES C.F., LEDO A. & TORANZO A.E. (1993). Characterization of a
birnavirus isolated from diseased turbot cultured in Spain. Dis. Aquat. Org., 15, 163169.
23. OKAMOTO N., SANO T., HEDRICK R.P. & FRYER J.L. (1983). Antigenic relationships of selected
strains of infectious pancreatic necrosis virus and European eel virus. J. Fish Dis., 6, 1925.
24. OKAMOTO N., TANIGUCHI N., SENO Y. & SANO T. (1984). The relation between the change of
quantities of infectious pancreatic necrosis virus in infected rainbow trout fry and the disease process.
Fish Pathol., 19, 14.
25. OLESEN N.J., JORGENSEN P.E.V., BLOCH B. & MELLERGAARD S. (1988). Isolation of an IPN-like
virus belonging to the serogroup II of the aquatic birnaviruses from dab (Limanda limanda). J. Fish
Dis., 11, 449451.
26. POLAK J.M. & VAN NOORDEN S., eds. (1986). Immunocytochemistry. Modern Methods and
Applications. John Wright, Bristol, UK, pp 736.
27. RENO P.W. (1999). Infectious pancreatic necrosis virus and its virulence. In: Fish Diseases and
Disorders. Vol. 3: Viral, Bacterial and Fungal Infections, Woo P.T.K. & Bruno D.W., eds. CABI
Publishing, Wallingford, UK, 155.
28. RODGER H.D. & FRERICHS G.N. (1997). Clinical infectious pancreatic necrosis virus infection in
farmed halibut in the United Kingdom. Vet. Rec., 140, 401402.
29. SMAIL D.A., BRUNO D.W., DEAR G., MCFARLANE L.A. & ROSS K. (1989). Infectious pancreatic
necrosis (IPN) virus Sp serotype in farmed Atlantic salmon, Salmo salar L., post-smolts associated
with mortality and clinical disease. J. Fish Dis., 15, 7783.
30. TAKSDAL T. & THORUD K.E. (1999). Evaluation of a rapid co-agglutination (COA) test for the
detection of infectious pancreatic necrosis virus (IPNV) in tissue samples of atlantic salmon (Salmo
salar). J. Fish Dis., 22, 117124.
31. UNDERWOOD B.O., SMALE C.J., BROWN F. & HILL B.J. (1977). Relationship of a virus from Tellina
tenuis to infectious pancreatic necrosis virus. J. Gen. Virol., 36, 93109.
32. WOLF K. (1988). Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca, NY, USA,
476 pp.
33. WOLF K., SNIESZKO S.F., DUNBAR C.E. & PYLE E. (1960). Virus nature of infectious pancreatic
necrosis virus in trout. Proc. Soc. Exp. Biol. Med., 104, 105108.
*
* *
C HA P T E R 2 . 1 . 9 .

I NF E CT I OUS S AL MON ANAE MI A

SUMMARY
Infectious salmon anaemia (ISA) is an infectious disease of Atlantic salmon (Salmo salar) (28) caused by
an orthomyxo-like virus (7, 15). Initially reported in Norway in the mid-1980s, ISA has to date been
reported to occur in Canada (New Brunswick and Nova Scotia), the United Kingdom (Scotland and the
Shetland Islands), the Faroe Islands and USA (Maine) (1, 12, 21, 24) and the causal virus has been
isolated from samples from Coho salmon from Chile (9) and from rainbow trout in Ireland. For more
detailed information on the condition see refs 3, 5, 8, 26 and 27. Atlantic salmon is the only susceptible
fish species known to develop the disease, but, following experimental infection, the ISA virus (ISAV) may
survive and replicate in sea trout and brown trout, (Salmo trutta) and in rainbow trout (Oncorhynchus
mykiss) (16, 17, 19), which thus may act as carriers of the virus for an unknown period of time.
Clinically, the infection appears as a systemic and lethal condition that is characterised by anaemia, ascites,
congestion and enlargement of the liver (dark in colour) and spleen, as well as peritoneal petechiae.
Haemorrhages in the eyes may also be seen. Hepatocellular degeneration and necrosis, tubular necrosis and
haemorrhages in the kidneys are consistent histopathological findings in typical outbreaks. The infection is
mainly observed in fish held in sea water or in fish exposed to sea water, but indications of disease
outbreaks in fish held in fresh water has also been reported (18). The infection spreads slowly and the virus
is considered to be of relatively low virulence. However, mortality may ensue in some outbreaks of the
disease.
The infective agent, ISAV, is an enveloped virus, 100130 nm in diameter, with morphological,
biochemical and genomic properties consistent with those of the Orthomyxoviridae (7, 11, 15). The genome
of ISAVconsists of eight single-stranded RNA segments with negative polarity (15).
The reservoirs of ISAV are not known, but spread of the disease has occurred as a result of the purchase of
subclinically infected Atlantic salmon smolts, from farm to farm, and from fish slaughterhouses or industries
where organic material (especially blood and processing water) from ISA-infected fish has been discharged
directly into sea water without further treatment. Transfer of ISA by salmon lice (Lepeophtheirus
salmonis) has been demonstrated under experimental conditions (20). Evidence for the presence of ISAV
in wild salmonids is increasing (22).
Few environmental factors have been identified that can be directly linked to outbreaks of the disease. In a
latent carrier population, various stress factors, such as treatment against salmon lice, cestodes or other
infectious diseases may be followed by disease outbreaks some 23 weeks later.
Screening of potential carriers for ISAV have hitherto been made by polymerase chain reaction (PCR)-
based technology, or by virus isolation in cell culture followed by confirmatory testing by immunological
methods or by PCR (4, 22, 23). The current diagnostic procedures for ISA are based on clinical,
pathological, histopathological and haematological changes, detection of ISAV by means of virus isolation
or detection of the virus in tissues by means of an indirect immunofluorescent antibody test. PCR may be a
useful diagnostic tool in cases with inconclusive disease signs. The incidence of ISA may be greatly reduced
by implementation of general legislatory measures regarding the movement of fish, mandatory health control,
and slaughterhouse and transport regulations, as well as specific measures including restrictions on affected,
suspected and neighbouring farms, epizootiological studies, enforced sanitary slaughtering, generation
Chapter 2.1.9. - Infectious salmon anaemia
segregation (all in/all out), and disinfection of offal and wastewater, etc., from fish slaughterhouses and
fish processing plants.
The diagnosis of infectious salmon anaemia (ISA) was initally based on clinical and patholgoical findings,
but direct methods for detection of virus have recently been established. These include isolation of virus in
cell culture followed by immunological identification (2, 6), immunological demonstration of ISA virus
(ISAV) antigen in tissues (6) and polymerase chain reaction (PCR) techniques (4, 13, 15).
The first isolation of ISAV was performed in the salmonid cell line SHK-1 (3), but other salmonid cell
lines such as CHSE-214, AS and ASK may also support virus propagation (4, 10, 25, 29). A monoclonal
antibody (MAb) (3H6F8) against ISAV that reacts with the viral haemagglutinin has been produced for
confirming a diagnosis of ISA (6, 7).
Sampling procedures: See Chapter I.1. Section B.
1. STANDARD SCREENING METHOD FOR ISAV
Detection of latent carriers of ISAV has been demonstrated by PCR, but detection of carriers by virus
isolation has also been reported. It is generally assumed that PCR is a more sensitive technique than
isolation in cell culture for detection of carriers or subclinical cases (14), but relatively few validation
studies of the different methods have been performed. A recently reported reverse-transcription PCR
(RT-PCR) procedure has proved to be at least ten times more sensitive than isolation in cell culture
(13). The standard procedures for virus isolation and PCR are described in Sections 2.2.1. and 2.3.2.,
respectively.
2. DIAGNOSTIC METHODS FOR ISA
Fish affected with ISA may show a range of signs from none to severe, depending on factors such as
infective dose, temperature, age, immune status, virus strain and pathogenicity, seasonal variation, etc.
A pathological diagnosis can be made from identification of typical pathological changes. None of the
described lesions is pathognomonic to ISA. The following changes have been described to be
consistent with ISA: exophthalmus, distended abdomen (ascites), anaemic gills, sometimes skin
haemorrhages and scale oedema, pale heart, dark liver, congested spleen and kidneys, and petechia in
perivisceral fat and internal organs. Liver pathology and anaemia were the central and classical
diagnostic criteria.
2.1. Diagnosis based on clinical signs and post-mortem findings
The following requirements should be fulfilled when making a pathological diagnosis:
macroscopic findings, histological findings and haematological findings consistent with the
descriptions given for ISA as detailed below.
2.1.1. Macroscopic findings
Dark livers Not necessarily present in all individuals, but there must be some
fish with dark livers. Alternatively, livers may become yellow with
haemorrhagic spots or may be pale
Pale gills and heart Result of anaemia
Ascites Always present, early sign
Enlarged spleen Always present, early sign
Visceral fat petechiae Usually present
Dark foregut Sometimes present
All of these findings are typical of ISA, although some variation will occur in their severity.
Confidence in the diagnosis increases with the increased observation of the occurrence of
these signs. Dark livers are an essential sign because this finding is most specific to ISA. (Dark
livers are also seen in cardiomyopathic syndrome [CMS], which is distinguished from ISA by
typical gross and histological heart lesions.) Furthermore, pale livers with nonhaemorrhagic
necroses have been observed both in ISA and winter ulcers.
2.1.2. Histological findings
The following are typical histological findings:
Multifocal haemorrhagic hepatic necroses that may become confluent to give the changes
a zonal appearance, leaving areas around large veins intact (late stage of disease
development);
Focal congestion and dilatation of hepatic sinusoids, sometimes with distribution as
described for the necroses (early stage). Rupture of sinusoidal endothelium with the
presence of erythrocytes within the space of Disse (early sign);
Kidney lesions are characterised by either or both acute tubular necrosis with eosinophilic
casting and moderate sinusoidal congestion and interstitial haemorrhages;
In the spleen, moderate to severe sinusoidal congestion and erythrophagia may be seen.
Findings described as supportive are present in the early stages of disease development at
haematocrit values of 1525. The ISA-typical liver changes are observed at haematocrit values
below 10. Comparable, although not identical, liver changes (usually without rupture of
endothelium) may be seen in advanced stages of CMS.
ISA is diagnosed most often in spring. Mortality may be low for months following the
introduction of ISA, until an outbreak occurs. Necropsy and examination of a large number
of diseased or dead fish increase the probability of detecting ISA.
Always suspect ISA when extremely dark livers are observed.
2.1.3. Haematological findings
The following are typical haematological findings:
Haematocrit <10;
Blood smears with degenerate and vacuolised erythrocytes and the presence of
erythroblasts with irregular nuclear shape. A reduction in the proportion of leukocytes
relative to erythrocytes, with the largest reduction being among lymphocytes and
thrombocytes. A reduction in several plasma parameters, except for some enzymes
indicating liver damage, and for electrolytes (fish in sea water).
Other evidence of anaemia e.g. pale gills and heart.
A haematocrit value below 10 is not a unique finding for ISA. Fish with ulcerations and fish
with erythrocytic inclusion body syndrome, may regularly demonstrate haematocrit values this
low.
2.2. Isolation of virus in cell culture
Infected material suitable for virological examination is: spleen, heart, liver, and preferably kidney
from clinically infected fish.
2.2.1. Isolation of ISA virus in cell culture
Cell line to be used: SHK-1 or other susceptible cell line
SHK-1 or other susceptible cell lines, such as TO, ASK and CHSE-214, may be used, but
strain variability and the ability to replicate in different cell lines should be taken into
consideration. The SHK-1 cells seem to support growth of the hitherto known virus isolates
(14).
The SHK-1 cells are grown at 20C in Leibovitzs L-15 cell culture medium supplemented
with fetal bovine serum (5%), L-glutamine (4 mM), gentamicin (50 g/ml) and 2-
mercaptoethanol (40 M) (may be omitted).
For virus isolation, cells grown in 25 cm
2
tissue culture flasks or multi-well cell culture plates,
which may be sealed with parafilm to stabilise the pH of the medium, may be used. Cells
grown in 24-well plates may not grow very well into monolayers, but this trait may vary
between laboratories and according to the type of cell culture plates used. Serially diluted
ISAV-positive controls should be inoculated in parallel with the tissue samples as a test for cell
susceptibility to ISAV.
Inoculate actively growing monolayers (13-day-old cultures) with a small volume of tissue
homogenate supernatant (e.g. 1 ml per well in a six-well plate) after removal of the growth
medium. Dilute tissue supernatant with L-15 medium without serum to final dilutions of tissue
material of 1/50 and/or higher. Allow 34 hours incubation at 15C followed by removal of
the inoculate and addition of fresh, fully supplemented growth medium. Using this procedure,
cytotoxicity is seldom observed even at the lowest dilution of supernatant. Alternatively, a
1/1000 dilution and direct inoculation without medium replacement can be used.
When samples come from production sites where infectious pancreatic necrosis virus (IPNV)
is regarded as ubiquitous, the tissue homogenate supernatant should be incubated (for a
minimum of 1 hour at 15C) with a pool of antisera to the indigenous serotypes of IPNV prior
to inoculation.
Inoculated cell cultures (kept at 15C) are examined at regular intervals (at least every 7 days)
for the occurrence of a cytopathic effect (CPE). Typical CPE due to ISAV appears as
vacuolated cells that subsequently round up and loosen from the growth surface. If a CPE
appears that is consistent with that described for ISAV or IPNV, remove an aliquot of the
medium for virus identification as described below (Section 2.2.2.). In the case of an IPNV
infection, re-inoculate cells with tissue homogenate supernatant that has been incubated with a
lower dilution of IPNV antisera. If no CPE has developed after 14 days, subcultivate to fresh
cell cultures.
c) Subcultivation procedure
i) Aliquots of medium (supernatant) from the primary cultures are collected 14 days (or
earlier when obvious CPE appears) after inoculation. Supernatants from wells inoculated
with different dilutions of identical samples may be pooled.
ii) The supernatants are inoculated into fresh cell cultures as described for the primary
inoculation: remove growth medium, inoculate monolayers with a small volume of diluted
supernatants (1/5 and higher dilutions) for 34 hours before addition of fresh medium.
Alternatively, add supernatants (final dilutions 1/10 and higher) directly to cell cultures
with growth medium.
iii) The inoculated cell cultures are incubated for at least 14 days and examined at regular
intervals as described for the primary inoculation. At the end of the incubation period, or
earlier if obvious CPE appears, medium is collected for virus identification as described
below (Section 2.2.2.). Cell cultures with no CPE should always be examined for the
presence of ISAV by indirect fluorescent antibody test (IFAT), haemadsorption (7) or by
PCR because virus replication may occur without the development of apparent CPE.
2.2.2 Virus identification
i) Prepare monlayers of cells in appropriate tissue culture plates (e.g. 96-well or 24-well
plates), in slide flasks or on plastic cover-slips dependent on the type of microscope
available (inverted microscope equipped with UV light is necessary for monolayers grown
on tissue culture plates). The SHK-1 cells grow rather poorly on glass cover-slips. Include
the necessary monolayers for negative and positive controls.
ii) Inoculate the monolayers with the virus suspensions to be identified in tenfold dilutions,
two monolayers for each dilution. Add positive virus control in dilutions known to give
good staining reaction.
iii) Incubate inoculated cell cultures at 15C for 7 days or, if CPE appears, for shorter times.
iv) Remove cell culture medium and rinse once with 80% acetone. Add 80% acetone and let
the fixative act for 20 minutes at room temperature. Remove the fixative and air dry for
1 hour. The fixed cell cultures may be stored dry for less than a week at 4C or at 20C
for longer storage.
v) Prepare a solution of antibody against ISAV (MAb 3H6F8) at an appropriate dilution in
phosphate buffered saline (PBS) containing 0.5% skimmed dry milk. Treat the cell
monolayers with the antibody solution for 1 hour at room temperature or 30 minutes at
37C.
vi) Rinse twice with PBS/0.05% Tween 20. Keep the monolayers in the dark for 12 minutes
before removal of the last washing solution.
vii) Treat the cell monolayers with fluoescein isothiocyanate (FITC)-conjugated goat anti-
mouse immunoglobulin (or if antibody raised in rabbits is used as the primary antibody,
use FITC-conjugated antibody against rabbit immunoglobulin) according to the
instructions of the supplier. Dilute the conjugate with PBS to appropriate working
dilution and incubate for 1 hour at room temperature or 30 minutes at 37C in the dark.
viii) Rinse once with PBS/0.05% Tween 20 as described above.
ix) To stain the nuclei (red colour) add propidiumiodid (100 g/ml in aqua dest.) and
incubate for 12 minutes at room temperature in the dark followed by rinsing once with
PBS/0.05% Tween 20.
x) If the plates cannot be examined immediately, add a solution of 1,4-diazabicyclooctane
(DABCO 2.5% in PBS, pH 8.2) or similar reagent as an anti-fade solution.
xi) Examine under UV light immediately or store the stained monolayers in the dark at 4C
for 23 days before exmination (some reduction in the fluorescent signal may be
observed by storage).
b) Polymerase chain reaction
As an alternative to identification of isolated virus by IFAT, infected cell monolayers may be
examined by RT-PCR according to the procedure described in Section 2.3.2.
2.3. Direct detection of virus in clinical material
2.3.1. Indirect fluorescent antibody test
An IFAT using anti-ISAV MAb (3H6F8) on frozen tissue sections of kidney, heart and liver or
on kidney imprints has given positive reactions in both experimentally and naturally infected
Atlantic salmon. Cases suspected from pathological signs are verified with a positive IFAT.
a) Preparations of tissue imprints
A small piece of the mid-kidney is briefly blotted against absorbant paper to remove excess
fluid, and several imprints are made on poly-L-lysine-coated microscope slides. The imprints
are air-dried, fixed in chilled 100% acetone for 10 minutes and stored either at 4C for a few
days or at 80C until use.
b) Preparations of cryosections
Tissue samples from kidney, liver and heart are collected from moribund fish, frozen in
isopentane, chilled in liquid nitrogen, and stored at 80C. Sections are cut on a cryostat,
placed on poly-L-lysine-coated slides, fixed in chilled 100% acetone for 10 minutes and stored
at 80C until use.
c) Staining procedure
After blocking with 5% nonfat dry milk in PBS for 30 minutes, the preparations are incubated
for 1 hour with diluted (e.g. 1/100) anti-ISAV MAb supernatant, followed by three washes.
For the detection of bound antibodies, the preparations are incubated with FITC-conjugated
anti-mouse Ig for 1 hour. PBS with 0.1% Tween 20 is used for washing. All incubations are
performed at room temperature.
2.3.2. Polymerase chain reaction
Amplification of a 155 bp fragment of ISAV cDNA is performed using primers derived from
sequences of segment 8: 5-GGC-TAT-CTA-CCA-TGA-ACG-AAT-C-3 (forward primer)
and 5-GCC-AAG-TGT-AAG-TAG-CAC-TCC-3 (reverse primer) (13, 14). The RT reaction
and the PCR are performed in two separate steps. This method may be regarded as a basic
procedure for detection of ISAV. Improved methods and methods using other sets of primers
have recently been reported (14).
a) Isolation of RNA
Total RNA from tissue homogenate or cell culture is extracted using, for example, the phenol-
chloroform method. RNA is dissolved in distilled water treated with 0.1% diethyl
pyrocarbonate (DEPC) and preheated at 55C for 10 minutes (2 g RNA in a total volume of
11 l).
b) RT reaction
For cDNA synthesis (RT reaction), the following reagents are added giving a final volume of
20 l (all concentrations are final): 50 mM Tris/HCl, pH 8.3, 75 mM KCl, 3 mM MgCl
2
,
1 mM
(each) of four deoxynucleoside triphosphates (dNTP), 5 mM dithiothrietol (DTT), 10 mM of
the RNase inhibitor ribonucleoside vanadyl complex (RVC), 4.5 M random hexamers primers
and 200 U of murine leukaemia virus (M-MLV) reverse transcriptase. The mixture is incubated
at 37C for 1 hour.
c) PCR
Following centrifugation, 5 l of the mixture from the RT-reaction is transferred to 45 l PCR
mix consisting of (final concentrations in a total volume of 50 l): 20 mM Tris/HCl, pH 8.4,
50 mM KCl, 1.5 mM MgCl
2
, 0.02% polyoxyethylene ether, 0.2 mM of each dNTP, 25 pmol of
each forward and reverse ISAV-specific primers and 1.25 U Taq DNA polymerase. This
mixture is incubated in an automatic thermal cycler programmed for one cycle at 94C for
5 minutes, 35 cycles at 94C for 30 seconds, 55C for 15 seconds, 72C for 30 seconds, and is
held finally at 72C for 7 minutes. Amplified DNA (155 bp) is analysed by agarose gel
electrophoresis or by spectrophotometer if real-time PCR is used. The optimal incubation
times at the different temperatures must be adjusted to the thermal cycler in use.
An optimised version of this method has recently been described where the RT reaction and
the PCR reaction are carried out in one tube (13).
Alternatively, the RT-PCR method described by Devold et al. (4) may be used. In this method
the primers are also derived from genomic segment 8 and the size of the amplified product is
estimated to be 211 bp when the primer set FA-3/RA-3 is used.
3. CRITERIA FOR SUSPICION AND VERIFICATION OF ISA
3.1. General principles for the diagnosis of ISA
Reasonable grounds for fish to be suspected of being infected with ISAV are outlined in Section
3.2. below. The Competent Authorities shall ensure that, following the suspicion of fish on a farm
being infected with ISAV, an official investigation to confirm or rule out the presence of the
disease be carried out as quickly as possible applying inspection and clinical examination, as well as
the collection and selection of samples and the methods for laboratory examination as described
in Section 2.
3.2. Suspicion of infection with ISA
The presence of ISA should be suspected if the criteria listed in points a) or b) or c) or d) or e)
below are met.
a) The presence of post-mortem findings consistent with ISA, as detailed in Section 2.1., with or without
clinical signs.
b) Isolation and identification of ISAV in cell culture, as described in Section 2.2., from a single sample
from any fish on the farm.
c) Reasonable evidence of the presence of ISAV from two independent laboratory methods, such as IFAT
(see Section 2.3.1.) and RT-PCR (see Section 2.3.2.).
d) Transfer of live fish into a farm where there are reasonable grounds to suspect that ISA was present at the
time of the fish transfer.
e) Where an investigation reveals other substantial epidemiological links to farms suspected or confirmed to be
infected with ISA.
3.3. Excluding suspicion of infection with ISA
Suspicion of ISA can be officially excluded if continual investigations involving at least 1 clinical
inspection per month for a period of 6 months reveals no further significant evidence of the
presence of ISA.
3.4. Confirmation of infection with ISA
The presence of ISA is officially confirmed if the criteria in Sections 3.4.1. or 3.4.2. or 3.4.3. are
met:
3.4.1. Clinical signs and post-mortem findings of ISA in accordance with Sections 2.1.1., 2.1.2.
and 2.1.3. are detected, and ISAV is detected by one or more of the following methods:
a) Isolation and identification of ISAV in cell culture as described in Section 2.2.
b) Detection of ISAV in tissues or tissue preparations by means of specific antibodies against ISAV (e.g.
IFAT on kidney imprints) as described in Section 2.3.1.
c) Detection of ISAV by RT-PCR using the methods described in Section 2.3.2.
3.4.2. Isolation and identification of ISAV in two samples from one or more fish at the farm
tested on separate occasions using the methods described in Section 2.2.
3.4.3. Isolation and identification of ISAV from at least one sample from any fish on the farm
using the methods described in Section 2.2. with corroborating evidence of ISAV in tissue
preparations from any fish on the farm using either IFAT (Section 2.3.1.) or RT-PCR (Section
2.3.2.).
REFERENCES
1. BOUCHARD D.A., BROCKWAY K., GIRAY C., KELEHER W. & MERRILL P.L. (2001). First report of
infectious salmon anemia (ISA) in the United States. Bull. Eur. Assoc. Fish Pathol., 21, 8688.
2. DANNEVIG B.H., FALK K. & NAMORK E. (1995). Isolation of the causal virus of infectious salmon
anemia (ISA) in a long-term cell line from Atlantic salmon head kidney. J. Gen. Virol., 76, 13531359.
3. DANNEVIG B.H., FALK K. & SKJERVE E. (1994). Infectivity of internal tissues of Atlantic salmon,
Salmo salar L, experimentally infected with the aetiological agent of infectious anaemia (ISA). J. Fish
Dis., 17, 613622.
4. DEVOLD M., KROSSOY B., ASPEHAUG V. & NYLUND A. (2000). Use of RT-PCR for diagnosis of
infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection.
Dis. Aquat. Org., 40, 918.
5. EVENSEN O., THORUD K.E. & OLSEN Y.A. (1991). A morphological study of the gross and light
microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar). Res. Vet. Sci., 51, 215222.
6. FALK K., NAMORK E. & DANNEVIG B.H. (1998). Characterization and application of a monoclonal
antibody against infectious salmon anaemia virus. Dis. Aquat. Org., 34, 7785.
7. FALK K., NAMORK E., RIMSTAD E., MJAALAND S. & DANNEVIG B.H. (1997). Characterization of
infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar
L). J. Virol., 71, 90169023.
8. HOVLAND T., NYLUND A., WATANABE K. & ENDRESEN C. (1994). Observations of infectious
salmon anaemia virus in Atlantic salmon, Salmo salar L. J. Fish Dis., 17, 291296.
9. KIBENGE F.S.B., GARATE O.N., JOHNSON G. ARRIAGADA R. KIBENGE M.J.T. & WADOWSKA D.
(2001). Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in
Chile. Dis. Aquat. Org., 45, 918.
10. KIBENGE F.S.B., LYAKU J.R., RAINNIE D. & HAMMELL K.L. (2000). Growth of infectious salmon
anaemia virus in CHSE-214 cells and evidence for phenotypic differences between virus strains. J.
Gen. Virol., 81, 143150.
11. KOREN C.W.R. & NYLUND A. (1997). Morphology and morphogenesis of infectious salmon anaemia
virus replicating in the endothelium of Atlantic salmon Salmo salar. Dis. Aquat. Org., 29, 99109.
12. LOVELY J.E., DANNEVIG B.H., FALK K., HUTCHIN L., MACKINNON A.M., MELVILLE K.J.,
RIMSTAD E. & GRIFFITHS S.G. (1999). First identification of infectious salmon anaemia virus in
North America with haemorrhagic kidney syndrome. Dis. Aquat. Org., 35, 145148.
13. MIKALSEN A.B., TEIG A., HELLEMAN A.-L., MJAALAND S. & RIMSTAD E. (2001). Detection of
infectious salmon anaemia virus (ISAV) by RT-PCR after cohabitant exposure in Atlantic salmon.
Dis. Aquat. Org., 47, 175181.
14. MJAALAND S., RIMSTAD E. & CUNNINGHAM C.O. (2002). Molecular diagnosis of infectious salmon
anaemia. In: Molecular Diagnosis of Salmonid Diseases, Cunningham C.O., ed. Kluwer Academic
Publishers, Dordrecht, The Netherlands, 122.
15. MJAALAND S., RIMSTAD E., FALK K. & DANNEVIG B.H. (1997). Genomic characterisation of the
virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L): an orthomyxo-like virus in
a teleost. J. Virol., 71, 76817686.
16. NYLUND A., ALEXANDERSEN S., ROLLAND B.J. & JAKOBSEN P. (1995). Infectious salmon anaemia
virus (ISAV) in brown trout. J. Aquat. Anim. Health, 7, 236240.
17. NYLUND A. & JAKOBSEN P. (1995). Sea trout as a carrier of infectious salmon anaemia virus. J. Fish
Biol., 47, 174176.
18. NYLUND A., KROSSOY B., DEVOLD M., ASPHAUG V., STEINE N.O. & HOVLAND T. (1998).
Outbreak of ISA during first feeding of salmon fry (Salmo salar). Bull. Eur. Assoc. Fish Pathol., 19, 71
74.
19. NYLUND A., KVENSETH A.M., KROSSOY B. & HODNELAND K. (1997). Replication of the infectious
salmon anaemia virus (ISAV) in rainbow trout Oncorhynchus mykiss, (Wallbaum), 1792. J. Fish Dis., 20,
275279.
20. NYLUND A., WALLACE C. & HOVLAND T. (1993). The possible role of Lepeophtheirus salmonis
(Kryer) in the transmission of infectious salmon anaemia. In: Pathogens of Wild and Farmed Fish:
Sea Lice, Boxhall G. & Defaye D., eds. Ellis Horwood, Chichester, UK, 367373.
21. OFFICE INTERNATIONAL DES EPIZOOTIES (2000). Disease Information, 14 (12), OIE, Paris, France.
22. RAYNARD R.S., MURRAY A.G. & GREGORY A. (2001). Infectious salmon anaemia virus in wild fish
from Scotland. Dis. Aquat. Org., 46, 93100.
23. RIMSTAD E., FALK K., MIKALSEN A.B. & TEIG A. (1999). Time course distribution of infectious
salmon anaemia virus in experimentally infected Atlantic salmon Salmo salar. Dis. Aquat. Org., 36,
107112.
24. RODGER H.D., TURNBULL T., MUIR F., MILLAR S. & RICHARDS R. (1998). Infectious salmon
anaemia (ISA) in United Kingdom. Bull. Eur. Assoc. Fish Pathol., 18, 115116.
25. SOMMER A.I. & MENNEN S. (1997). Multiplication and haemadsorbing activity of infectious salmon
anaemia virus in the established Atlantic salmon cell line. J. Gen. Virol., 78, 18911895.
26. SPEILBERG L., EVENSEN O. & DANNEVIG B.H. (1995). A sequential study of the light and electron
microscopical liver lesions of infectious anemia in Atlantic salmon (Salmo salar L). Vet. Pathol., 32,
466478.
27. THORUD K.E. (1991). Infectious salmon anaemia. Transmission trials, haematological, clinical
chemical and morphological investigations. Thesis for the degree of Doctor Scientarum, Norwegian
College of Veterinary Medicine, Norway.
28. THORUD K.E. & DJUPVIK H.O. (1988). Infectious salmon anaemia in Atlantic salmon (Salmo salar
L). Bull. Eur. Assoc. Fish Pathol., 8, 109111.
29. WERGELAND H.I. & JAKOBSEN R.A. (2001) A salmonid cell line (TO) for production of infectious
salmon anaemia virus (ISAV). Dis. Aquat. Org., 44, 183190.
*
* *
C HA P T E R 2 . 1 . 1 0 .

E P I ZOOT I C UL CE RAT I VE S YNDROME

SUMMARY
Epizootic ulcerative syndrome (EUS) is a seasonal epizootic condition of great importance in wild and
farmed freshwater and estuarine fish. It was first reported in farmed ayu (Plecoglossus altivelis) in Japan
in 1971 (6). It was later reported in estuarine fish, particularly grey mullet (Mugil cephalus) in eastern
Australia in 1972 (13). The outbreak has extended its range through Papua New Guinea into South-
East and south Asia, and recently into west Asia, where it has now reached Pakistan (8, 14). Outbreaks
of ulcerative disease in menhaden (Brevoortia tyrannus) in the United States of America (USA) have
been shown to be very similar to EUS in Asia (1).
EUS is also known as red spot disease (RSD), and mycotic granulomatoses (MG). The fungus involved in
EUS is also known variously as Aphanomyces invadans, A. piscicida, A. invaderis and ERA
(EUS-related Aphanomyces). Rhabdoviruses have also been associated with particular outbreaks, and
secondary Gram-negative bacteria invariably infect EUS lesions.
Region-wide, over 50 species of fish have been confirmed by histological diagnosis to be affected by EUS (8),
but some important culture species, including tilapia, milk fish and Chinese carp, have been shown to be
resistant.
EUS occurs mostly during periods of low temperatures and after periods of heavy rainfall (2). These
conditions favour sporulation of Aphanomyces invadans (12), and low temperatures have been shown to
delay the inflammatory response of fish to fungal infection (3, 5).
Control of EUS in natural waters is probably impossible. In outbreaks occurring in small, closed water-
bodies, liming water and improving water quality, together with removal of infected fish, is often effective in
reducing mortalities.
Diagnosis of epizootic ulcerative syndrome (EUS) is based on clinical signs and confirmed by
histopathology.
1. CLINICAL SIGNS
EUS outbreaks have been associated with mass mortality of various species of freshwater fish in the
wild (including rice-fields, estuaries, lakes and rivers) and in farms.
The early signs of the disease include loss of appetite, and fish become darker. Infected fish may float
below the surface of the water, and become hyperactive with a very jerky pattern of movement. Red
spots may be observed on the body surface, head, operculum or caudal peduncle. Large red or grey
shallow ulcers, often with a brown necrosis, are observed in the later stages. Large superficial lesions
occur on the flank or dorsum. Most species other than striped snakeheads and mullet will die at this
stage.
Chapter 2.1.10. - Epizootic ulcerative syndrome
Diagnostic Manual for Aquatic Animal Diseases 2003 163
In highly susceptible species, such as snakehead, the lesions are more extensive and can lead to
complete erosion of the posterior part of the body, or to necrosis of both soft and hard tissues of the
cranium, so that the brain is exposed in the living animal.
2. CLINICAL PATHOLOGY
Nonseptate hyphae of Aphanomyces invadans (1225 m in diameter) can be observed in muscle squash
preparations of the infected area around the lesion.
Lesion scrapes generally show secondary fungal, bacterial and/or parasitic infections.
3. HISTOPATHOLOGY
Histology is required for a confirmatory diagnosis.
3.1. Sampling procedure
i) Sample only live or moribund specimens of fish with clinical lesions.
ii) Take samples of skin/muscle (<1 cm
3
), including the side of the lesion and the surrounding
tissue.
iii) Fix the tissues immediately in 10% formalin. The amount of formalin should be 1520 times
the volume of the tissue to be fixed.
iv) Gently agitate the fixative 23 times during the first hour after adding the tissue.
3.2. Histological procedure
Processing of the fixed tissue involves dehydration through ascending alcohol grades, clearing in a
wax-miscible agent and impregnation with wax (4). The blocks of fish tissue are cut at about 5 m
and mounted on a glass slide. Before staining, the section must be completely de-waxed and
stained in haematoxylin and eosin (H&E) (4).
3.3. Confirmatory diagnosis
H&E and general fungus stains (e.g. Grocotts) will demonstrate typical granulomas and invasive
hyphae.
Early EUS lesions are erythematous dermatitis with no obvious fungal involvement. Aphanomyces
invadans hyphae are observed growing in skeletal muscle as the lesion progresses from a mild
chronic active dermatitis to a severe locally extensive necrotising granulomatous dematitis with
severe floccular degeneration of the muscle. The fungus elicits a strong inflammatory response
and granulomas are formed around the penetrating hyphae.
IDENTIFICATION
1. FUNGAL ISOLATION
The following are two methods of isolation of A. invadans fungus adapted from refs 7 and 15.
1.1. Moderate, pale, raised, dermal lesions are most suitable for fungal isolation attempts. Remove the
scales around the periphery of the lesion and sear the underlying skin with a red-hot spatula so as
to sterilise the surface. Using a sterile scalpel blade and sterile fine-pointed forceps, cut through
stratum compactum underlying the seared area and, by cutting horizontally and reflecting
superficial tissues, expose the underlying muscle. Ensure the instruments do not make contact
with the contaminated external surface and thereby contaminate the underlying muscle. Using
aseptic technique, carefully excise pieces of affected muscle, approximately 2 mm
3
, and place on a
Petri dish containing Czapek Dox agar with penicillin G (100 units/ml) and oxolinic acid
(100 g/ml). Seal plates, incubate at room temperature and examine daily. Repeatedly transfer
emerging hyphal tips on to fresh plates of Czapek Dox agar until cultures are free of
contamination.
1.2. Lesions located on the flank or tail of fish <20 cm in length can be sampled by cutting the fish in
two using a sterile scalpel, and slicing a cross-section through the fish at the edge of the lesion.
Flame the scalpel until red-hot and use this to sterilise the exposed surface of the muscle. Use a
small-bladed sterile scalpel to cut out a circular block of muscle (24 mm
3
) from beneath the
lesion and place it in a Petri dish of GP (glucose/peptone) medium (see Table 1) with
100 units/ml penicillin-K and 10 g/ml oxolinic acid. Instruments should not contact the
contaminated external surface of the fish. Incubate inoculated media at approximately 25C and
examine under a microscope (preferably an inverted microscope) within 12 hours. Repeatedly
transfer emerging hyphal tips to plates of GP medium with 12 g/litre technical agar, 100 units/ml
penicillin-K and 10 g/ml streptomycin sulphate until axenic cultures are obtained. They may
then be maintained at 10C on GP agar and subcultured at intervals of no greater than 7 days.
2. FUNGAL IDENTIFICATION
Aphanomyces invadans does not produce any sexual structures and should thus not be diagnosed by
morphological criteria alone. However the fungus can be identified to the genus level by inducing
sporogenesis and demonstrating typical asexual characteristics of Aphanomyces as described in ref. 8.
Aphanomyces invadans is characteristically slow growing in culture and fails to grow at 37C on GPY
agar (Table 1). Detailed temperature growth profiles are given in ref. 11. Two procedures that can use
to confirm the A. invadans are experimental fish infection and molecular technique. The experimental
infection can be made by injecting intramuscularly a 0.1 ml suspension of 100+ motile zoospores in
EUS-susceptible fish (preferably Channa striata) at 20C, and demonstrating histologically growth of
aseptate hyphae 1225 m in diameter in muscle of fish sampled after 7 days, and of typical mycotic
granulomas in muscle of fish sampled after 14 days. For the molecular technique, this pathogen is
readily identified by RAPD-PCR or sequencing of the internal spacer region (ITS) region of nuclear
rDNA. RAPD-PCR is rapid and does not require DNA sequencing, but it is necessary to isolate the
pathogen in pure culture for reliable results. Details regarding DNA preparation, PCR primers for
amplification can be found in refs 9 and 10.
All Aphanomyces invadans isolates found so far belong to a single genotype, which facilitates
identification. Alternatively, sequencing of the internal spacer region can be performed and the result
compared with the sequence deposited in public gene data banks. A. invadans is different to A. astaci
etiological agent of Crayfish plague (Chapter 4.1.7). As A. invadans does not have a sexual reproductive
stage. Both pathogenic fungi can also be differentiated using RAPD-PCR.
3. INDUCING SPORULATION IN APHANOMYCES INVADANS CULTURES
The induction of asexual reproductive structures is necessary in order to identify fungal cultures as
members of the genus Aphanomyces. To induce sporulation, place an agar plug (34 mm in diameter) of
actively growing mycelium in a Petri dish containing GPY broth and incubate for 4 days at
approximately 20C. Wash the nutrient agar out of the resulting mat by sequential transfer through five
Petri dishes containing autoclaved pond water, and leave overnight at 20C in autoclaved pond water.
After about 12 hours, the formation of achlyoid clusters of primary cysts and the release of motile
secondary zoospores should be apparent under the microscope. Media for inducing sporulation are
shown in Table 1.
Diagnostic Manual for Aquatic Animal Diseases 2003 165
Table 1. Media for isolation, growth and sporulation of Aphanomyces invadans cultures
GP (glucose/peptone) broth
3 g/litre glucose
1 g/litre peptone
0.128 g/litre MgSO
4
.7H
2
O
0.014 g/litre KH
2
PO
4

0.029 g/litre CaCl
2
.2H
2
O
2.4 mg/litre FeCl
3
.6H
2
O
1.8 mg/litre MnCl
2
.4H
2
O
3.9 mg/litre CuSO
4
.5H
2
O
0.4 mg/litre ZnSO
4
.7H
2
O
GPY (glucose/peptone/yeast) broth
GP broth + 0.5 g/litre yeast extract
GPY agar
GPY broth + 12 g/litre technical agar
Autoclaved pond water
Sample pond/lake water known to support fungal growth. Filter through Whatman 541 filter
paper. Combine one part pond water with two parts distilled water and autoclave. pH to 67.
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13. MCKENZIE R.A. & HALL W.T.K. (1976). Dermal ulceration of mullet (Mugil cephalus). Aust. Vet. J.,
52, 230231.
14. TONGUTHAI K. (1985). A preliminary account of ulcerative fish diseases in the Indo-Pacific region (a
comprehensive study based on Thai experiences). National Inland Fisheries Institute, Bangkok,
Thailand, 39 pp.
15. WILLOUGHBY L.G. & ROBERTS R.J. (1994). Improved methodology for isolation of the Aphanomyces
fungal pathogen of epizootic ulcerative syndrome (EUS) in Asian fish. J. Fish Dis., 17, 541543.
*
* *
C HA P T E R 2 . 1 . 1 1 .

BACT E RI AL KI DNE Y DI S E AS E
( Re n i b a c t e r i u m s a l mo n i n a r u m)

SUMMARY
Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum occurs in most parts of the
world where salmonid fish are cultured or occur in the wild (25, 26). Salmonids vary in their susceptibility
to BKD, and Pacific salmon species of the genus Oncorhynchus are generally considered to be the most
susceptible (21, 26, 49, 52). BKD can cause serious mortality in juvenile salmonids in both fresh water
and seawater, and also in prespawning adults. The disease has been described throughout North America
and in many countries in Europe, as well as Japan, Chile, and Iceland (2, 21, 25, 27). Most recorded
outbreaks of BKD have occurred in fish culture facilities, and the spread of BKD has followed the
expansion of salmonid culture (25). Clinical BKD has also been reported in feral fish (5, 36, 39, 45),
including naturally spawning populations that have never been supplemented with hatchery fish (22, 51).
Whereas the chronic nature of the disease has hindered accurate estimates of fish losses, particularly in feral
fish populations, BKD remains one of the most important bacterial diseases affecting cultured salmonids.
Losses as high as 80% in stocks of Pacific salmon and 40% in stocks of Atlantic salmon (Salmo salar)
have been reported (25).
Renibacterium salmoninarum is a small (0.30.1 m by 1.01.5 m), nonmotile, nonspore-forming,
nonacid-fast, Gram-positive diplobacillus (27). It typically causes a slowly progressing systemic infection,
with overt disease rarely evident until fish are 612 months old (21). Fish with severe R. salmoninarum
infections may show no obvious external signs, or may exhibit one or more of the following: lethargy; skin
darkening; abdominal distension due to ascites; pale gills associated with anaemia; exophthalmos;
haemorrhages around the vent; and cystic cavities in the skeletal muscle. Internal examination usually
reveals the presence of focal to multifocal grayish-white nodular lesions in the kidney, and sometimes in the
spleen and liver. In addition, there may be turbid fluid in the abdominal cavity, haemorrhages on the
abdominal wall and in the viscera, and a diffuse white membranous layer (pseudomembrane) on one or more
of the internal organs. In tissue sections of BKD lesions, R. salmoninarum is frequently observed within
phagocytic cells, particularly macrophages. The bacterium appears to survive and perhaps replicate within
these cells (4, 10, 31, 57).
The kidney disease bacterium can be transmitted both horizontally from infected fish sharing the water
supply (6, 38), and vertically in association with eggs from infected parents (23, 43). As with other
infectious diseases of salmonids that are difficult or impossible to treat, avoidance is recommended for the
control of BKD in cultured salmonid stocks (1, 21). Because R. salmoninarum is often enzootic in wild
salmonid populations (9, 22, 34), measures to control losses from BKD may be defeated by constant
exposure of hatchery fish to waterborne bacteria shed into the water supply by wild fish residing upstream
from the hatchery (33, 38). Salmonids reared in seawater present special problems because it is difficult to
ensure adequate separation of groups of fish to prevent horizontal transmission, and because of the
possibility that other marine species might serve as reservoirs for R. salmoninarum (7, 14, 50, 54).
To reduce the probability of vertical transmission of R. salmoninarum in cultured salmonids, brood stock
segregation or culling is now used to select egg lots to retain as a source of juvenile fish for hatchery rearing
(30, 43, 55) The selection process is aimed at rearing egg lots from mating pairs with undetectable or very
low levels of R. salmoninarum. This requires the use of sensitive BKD detection methods for testing the
prevalence and levels of R. salmoninarum in each parent fish. Elliott & Barila (16) believed that the
membrane-filtration fluorescent antibody test (MF-FAT) would provide the sensitivity and quantification
Chapter 2.1.11. - Bacterial kidney disease (Renibacterium salmoninarum)
necessary to investigate the relationship between the levels of R. salmoninarum in the female parent and the
probability of transmitting the disease to the progeny. Pascho et al. (43) later demonstrated the usefulness of
brood stock segregation for controlling losses from BKD by using the MF-FAT in conjunction with the
enzyme-linked immunosorbent assay to segregate egg lots from chinook salmon parents infected with either
very low or very high levels of R. salmoninarum. These researchers reported that the losses from BKD
among the progeny of parents with very low levels of R. salmoninarum were significantly less than those
among the progeny of parents with very high infection levels. The aquaculturist must be aware, however, that
brood stock segregation may not completely eliminate BKD from an affected population. Because the
broodstock used for commercial fish farming should be free of the kidney disease bacterium, it may be
necessary to repopulate a contaminated facility with brood fish from a BKD-free population.
Crucial to the success of any BKD control programme is the application of reliable diagnostic methods that
can detect low levels of R. salmoninarum in a variety of sample types. For that reason, fish health
specialists and researchers have long been interested in developing methods for more rapid and reliable
detection of R. salmoninarum infections (29, 41, 42, 48, 56). As each new test has been developed,
however, there has been a tendency to reject older techniques. Nevertheless, no single ideal diagnostic test has
yet been developed for the evaluation of multiple samples for the presence of BKD.
Bacteriological culture remains the benchmark method for determining the viability of Renibacterium
salmoninarum in a sample, and may also be used to quantify the number of bacteria in samples.
Immunodiagnostic procedures, however, have become the most widely used for screening large numbers
of fish in aquaculture facilities or elsewhere in the field. The two principal immunodiagnostic methods are
the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT). Nucleic-acid-
based diagnostic tests, such as the polymerase chain reaction (PCR), are now acceptable for confirmatory
identification of R. salmoninarum in bacteriological cultures and fish tissue or body fluid samples.
Sampling procedures: see Chapter I.1. Sections B and C.
1. STANDARD SCREENING METHODS FOR RENIBACTERIUM SALMONINARUM
Screening for and diagnosis of bacterial kidney disease (BKD) should be based on the ELISA and the
FAT. Confirmation of R. salmoninarum should be done by bacteriological culture on a KDM-2 medium
(kidney disease medium), or by the PCR. Sample preparation and processing procedures are detailed
below.
2. DIAGNOSTIC METHODS FOR RENIBACTERIUM SALMONINARUM
2.1. Enzyme-linked immunosorbent assay for testing tissue, plasma, and coelomic fluid
The recommended procedure is based on the method of Pascho & Mulcahy (44) as modified by
Pascho et al. (43). Other ELISA systems are also available commercially (46).
a) Supplies and reagents
i) Sample tubes
Recommended sample tubes are sterile, 2 ml microcentrifuge tubes with screen-printed
graduations, a writing space, and a gasketed screw cap.
ii) Test sample and control preparation diluent
Phosphate buffered saline (PBS), pH 7.4, supplemented with 0.05% (v/v) Tween 20
(PBST), and 0.01% (w/v) thimerosal as a preservative. For 1 litre: 8.00 g NaCl, 0.20 g
KH
2
PO
4
, 1.09 g Na
2
HPO
4
, 0.20 g KCl, 0.10 g thimerosal, confirm pH = 7.4, and 0.5 ml
Tween 20.
iii) 96-well microplates
For the ELISA a 96-well microplate that is designed for use in immunoassays must be
used. The performance of these plates will vary, and for a given ELISA, microplates from
several manufacturers should be tested to determine which is most suitable.
iv) Positive control antigen
Renibacterium salmoninarum cells (0.5% [w/v] wet packed cells) in PBS, pH 7.4, with 0.01%
(w/v) thimerosal. The bacterial cells can be lyophilised for long-term storage; lyophilised
bacteria normally contain a carrier compound, such as dextrose, for stability. Rehydrate
lyophilised bacteria with 1.0 ml reagent-grade water and prepare necessary dilutions,
1/100, 1/500, 1/1000, and 1/5000 (v/v), in PBST and store at 70C.
v) Coating buffer
Prepare sodium carbonate or purchase a commercial coating solution.
Sodium bicarbonate, pH 9.6: store at room temperature and discard after 30 days. For
1 litre: 1.59 g Na
2
CO
3
, 2.93 g NaHCO
3
, and 0.10 g thimerosal, confirm pH = 9.6.
Commercial coating solution concentrate: store and dilute according to the
manufacturers instructions.
vi) Wash solution
PBST, or a commercial wash solution that contains Tween 20. Prepare fresh wash
solution for each ELISA. Either make PBST as described above, or dilute commercial
wash solution concentrates according to the manufacturers instructions.
vii) Conjugate diluent
Prepare fresh for each ELISA. Use PBST as described above, or 2% (w/v) nonfat dry
milk may be substituted for the Tween 20. Commercial products are also available as
concentrates, often marketed as diluents or blocking solutions.
viii) ABTSperoxidasechromogen substrate
Commercial products are available, and often are provided as a two-part system: the
ABTS chromogen 0.6 g/litre ABTS (2,2-azino-di-[3-ethyl-benzthiazoline]-6-sulphonic
acid) in a glycine buffer, and a hydrogen peroxide substrate 0.02% hydrogen peroxide in
a citric acid buffer.
ix) Stop solution
5% (v/v) sodium dodecyl sulphate (SDS). Prepare in reagent-grade water immediately
before use.
b) Coating antibody and conjugate
i) Coating antibody
Affinity purified immunoglobulin to R. salmoninarum: rehydrate lyophilised coating
antibody by first mixing 1.0 ml glycerol + 1.0 ml reagent-grade water that contains 0.2%
(w/v; 2) thimerosal, and then transferring 1.0 ml of the 50% glycerol solution to each
product vial. Rehydrate the contents of a sufficient number vials to test the anticipated
number of fish that will be sampled for a given spawning season. Pool the contents of all
vials, then dispense into several cryovials and store at 70C.
ii) Conjugate
Affinity purified immunoglobulin to R. salmoninarum labelled with horseradish peroxidase
(HRPO): rehydrate lyophilised coating antibody by first mixing 1.0 ml glycerol + 1.0 ml
reagent-grade water that contains 0.2% (w/v; 2) thimerosal, and then transferring 1.0 ml
of the 50% glycerol solution to each product vial. Rehydrate the contents of a sufficient
number of vials to test the anticipated number of fish that will be sampled for a given
spawning season. Pool the contents of all vials, then dispense into several cryovials and
store at 70C.
iii) Working concentrations of the coating antibody and conjugate
Working concentrations for the purified goat anti-R. salmoninarum antibody (coating
antibody), and the HRPO-conjugated goat anti-R. salmoninarum antibody must first be
determined by checkerboard titration with the coating antibody and the HRPO-conjugate.
NOTE: The anti-R. salmoninarum is typically applied to the microplate wells at 1 g per
ml; the dilution is made from a concentrated antibody preparation at 1 mg/ml, and the
working dilution of the HRPO-conjugated antibody is normally about 1/2000 (v/v).
c) Sample collection and preparation
i) Adult fish
Kidney tissue, 1/4 (w/v): one part tissue + three parts PBST. Collect 25 g total; when
sampling the kidney, it is recommended that this sample consist of a pool of small tissue
pieces from the anterior, mid, and posterior kidney.
Ovarian fluid, 1/2 (v/v): one volume ovarian fluid + one volume PBST. Collect
approximately 1 ml.
Plasma, 1/5 (w/v): one volume whole blood + four volumes PBST. Collect blood in a
syringe or capillary tube, then dispense the correct volume of blood into the appropriate
volume of PBST, remove the cellular fraction by low-speed centrifugation, decant and
then test the supernatant.
Samples are usually taken following spawning. Care should be exercised to avoid cross-
contamination between fish and contamination of tissue samples from body fluids. Keep
tissue and body fluid samples on ice during collection. Store at 70C.
ii) Juvenile fish
Kidneyspleen tissue pool, recommend 1/4 (w/v) dilution, but can use 1/8 (w/v): one
part tissue + seven parts PBST. Juvenile fish are often collected as whole fish and
dissected on return to the laboratory. Remove the entire kidney and spleen from each
fish. It is preferable to test tissues from individual fish, but a tissue pool may be made if
the fish are too small. Two-fish pools are recommended when an insufficient amount of
tissue is recovered from an individual fish. Store at 70C.
Plasma, recommend 1/5 (w/v) dilution, but can use 1/10 (w/v): one volume whole
blood + nine volumes PBST. Collect blood in a syringe or capillary tube, then dispense
the correct volume of blood into the appropriate volume of PBST, remove the cellular
fraction by low-speed centrifugation, decant the supernatant, store fluid or heat at 100C
for 15 minutes, and test the supernatant. Store at 70C.
d) Sample processing for the ELISA
i) Prepare fish tissue or body fluid samples for the ELISA as described above.
ii) Heat each sample at 100C for 15 minutes, then centrifuge at from 8000 to 10,000 g for
10 minutes at 4C. If the samples were prepared earlier and frozen, thaw before heating.
iii) Store processed and heated samples at 4C, or freeze at 70C for later testing.
e) ELISA day 1
NOTE: Before beginning the ELISA, users should review the controls and appropriate
reagent applications described in Table 1.
i) Dilute the concentrated goat antibody to R. salmoninarum in coating buffer: carbonate/
bicarbonate coating buffer, pH 9.6, or a commercial coating solution. When using the
coating solution, make a fresh preparation for each ELISA. The carbonate/bicarbonate
buffer is normally discarded after 30 days. Use water of reagent grade or equivalent.
ii) The conjugate control and substrate/chromogen control wells receive no coating
antibody. Place 200 l of coating buffer in each of these wells.
iii) The blanks, negative controls wells, positive control wells, and the wells designated for
the test samples receive 200 l of coating antibody.
iv) Seal each plate with an adhesive plate sealer after addition of the buffer or coating
antibody. Place each plate in a humid chamber and incubate at 4C for 16 hours.
Table 1. Controls for the ELISA to detect Renibacterium salmoninarum
Step in the ELISA
Control
group
Purpose Coating
antibody
Sample
addition
Conjugate Substrate/
chromogen
Stop
solution
Blank Background
absorbance levels in
the absence of a test
sample
Yes PBST only Yes Yes Yes
Reference
or positive
controls
Internal control to
insure predictable
absorbance by certain
levels of antigen
Yes Yes Yes Yes Yes
Negative
control
Absorbance produced
by sample from a
negative control fish
Yes Yes Yes Yes Yes
Conjugate
control
To ensure that there
is no nonspecific
binding of the
conjugate to well
surfaces or to the
coating antibody
Coating buffer
only
PBST only Yes Yes Yes
Substrate/
chromogen
control
Test for
nonenzymatic
production of the
colour reaction
Coating buffer
only
PBST only Diluent
only
Yes Yes
f) ELISA day 2
i) Prepare PBST wash solution or dilute a commercial wash solution concentrate in reagent-
grade water, and store overnight at 4C. The wash buffer should remain at room
temperature during the ELISA.
ii) Remove the unbound coating antibody by washing each plate five times, with a 30-
second soak each time the wells are refilled. Shake excess wash buffer out of each plate
after the five washes are completed. Wash plates in numerical order.
iii) Place aliquots of controls and test samples into microplate wells. The following control
wells receive 200 l of the test sample diluent (PBS, pH 7.4, supplemented with 0.05%
[v/v] Tween 20): Blank, conjugate control, substrate/chromogen control. Control tissue
wells receive the appropriate tissue or body fluid.
iv) Place 200 l aliquots of each positive control in the appropriate wells.
v) Place 200 l aliquots of each test sample in the appropriate wells.
vi) Cover each plate with an adhesive plate sealer and incubate for 3 hours at 25C in a
humid chamber. Wash each microplate five times as described previously.
vii) Apply 200 l of the diluted conjugate to the appropriate wells. Substrate/chromogen
control wells receive an equivalent amount of diluent without conjugated antibody.
viii) Seal each plate with an adhesive plate sealer and incubate in a humid chamber for 2 hours
at 25C.
ix) Wash microplates five times as described previously.
x) Substrate/chromogen reaction.
The timing of the substrate/chromogen reaction is critical. The reaction must be stopped
after exactly 20 minutes. The volume of stop solution is 50 l to insure that the wells are
not over-filled. The SDS stop solution is prepared from a 5% (v/v) concentrate as
follows: four parts concentrate + one part water. Apply 200 l of substrate/chromogen
solution to each well. Fill all plates in numerical order. Immediately after all the wells have
received the substrate/chromogen solution, seal the plate and put it in the humid
chamber at 37C.
xi) Stop the substrate/chromogen reaction and measure the absorbance. Begin to apply the
stop solution immediately after the incubation period is complete. Wells should receive 50
l of stop solution in the exact sequence used to apply the substrate/chromogen solution.
Wipe any condensation off the bottom of the plate and immediately measure the
absorbances at 405 nm.
2.2. Standard indirect and direct fluorescent antibody technique for testing tissue samples and
bacteriological isolates
In general, there are two types of staining procedures that use fluorescein isothiocyanate (FITC)-
labelled antibodies: the indirect and direct fluorescent antibody techniques (IFAT and DFAT,
respectively). The principle and techniques are similar, but the indirect uses a second antibody that
is often biotinylated for increased sensitivity. The direct FAT is used more commonly for bacterial
corroboration testing and staining of R. salmoninarum.
There are three basic steps for DFAT: preparing and fixing bacterial cultures or kidney tissue on
multiple-well glass slides; staining the slides with antibody reagents; and reading and interpreting
the slides.
a) Preparing the slides
i) Pure bacterial cultures (corroboration testing of pure isolates R. salmoninarum): pure
isolates of bacteria are diluted in sterile PBS and applied to a glass microscope slide. Air-
dry and fix in absolute methanol for 510 minutes.
ii) Kidney smears from fish DFAT for R. salmoninarum: after the tissue has completely
dried, slides are fixed in methanol for approximately 510 minutes.
b) Staining
i) Positive and negative controls: slides containing both a non-cross-reacting bacterial
species and a known positive control can be prepared in quantity and stored refrigerated
for use as controls for DFAT staining. Positive controls are always used in corroboration
testing to correctly identify morphology and fluorescence of the bacteria in question. The
negative control is important in determining overall staining technique, background
debris, and nonspecific fluorescence.
ii) Place slides in dark, humidified chamber, and place one drop of specific FITC-conjugated
antibody on each slide.
iii) Incubate for 60 minutes at room temperature.
iv) Gently rinse the slides with PBS, pH 7.1 (see Section 2.3.).
v) Counterstain in Evans blue for 34 minutes for R. salmoninarum. (A counterstain is not
necessary for corroboration testing of pure bacterial cultures, although Rhodamine can be
used if desired.)
vi) Rinse with PBS, pH 7.1.
vii) Air-dry completely, add mounting medium and a cover-slip.
c) Reading and rating
Slides are read at 1000 magnification on a compound fluorescence microscope (refer to the
microscope manufacturer for correct wavelength and filters required for FITC epifluoresence
microscopy). The positive and negative control slides are read first. This has two purposes:
(1) quality assurance for the staining process (positive control should have myriad numbers of
fluorescing bacteria, the negative control should have no fluorescence); and (2) to familiarise
the reader with the correct bacterial size and shape, and the magnitude of the bacterias
fluorescent halo in the positive control. The reader can refer back to the positive control as a
reference, if needed, to confirm suspect bacteria in the sample wells.
i) Bacterial corroboration testing: positive bacterial isolates will fluoresce strongly and have
the same morphology and size as the positive control.
ii) Kidney smears tested for R. salmoninarum: bacterium will have a distinctly apple-green
fluorescent cell wall; be the appropriate size (1 m long by 0.5 m); and be the proper
shape (bean shaped or pear shaped). Compare any suspect bacteria with the control slide
to be sure all three of the above criteria are met for R. salmoninarum.
d) Fluorescent antibody technique rating guide
(Based on those described in Fish Pathology Section Laboratory Manual, Meyers, T.R., ed.
Special Publication 11, Alaska Dept Fish and Game, P.O. Box 25526, Juneau, AK 99802
USA.) Standard rating criteria for interpretation of the FAT on tissue smears are based on a
minimum of 60 fields examined at 1000 magnification.
i) Negative (): no organisms seen in thirty fields examined.
ii) Plus/minus (): organisms observed with questionable fluorescence or morphology not
typical of the target organisms. Total of one typical organism observed but suspected of
not originating from the sample examined, i.e. wash over from a high-level positive
sample.
iii) One plus (1+): one to five organisms observed. If only one organism is found,
examination of up to 100 fields continues in an attempt to find a second organism that
would confirm the 1+ status. If no other organism is detected the final or 1+
interpretation is at the discretion of the individual reader.
iv) Two plus (2+): 6 to 50 organisms observed in which some fields will be negative and
some will typically contain several organisms.
v) Three plus (3+): 51 to 150 organisms observed with a typical field containing a dozen or
more organisms.
vi) Four plus (4+): Greater than 150 organisms with no more than 200 organisms in an
average field.
vii) Clinical (C5+): Greater than 200 organisms in an average field. Gross lesions are likely to
be observed in the sampled kidneys from this category.
2.3. Membrane-filtration FAT for testing coelomic fluid
This procedure is based on the method of Elliott & Barila (16) as modified by Elliott &
McKibben (17).
a) Reagents
i) 0.01 M PBS, pH 7.1
8.50 g NaCl, 1.07 g Na
2
HPO
4
(anhydrous), 0.34 g Na
2
H
2
PO
4
H
2
O (monohydrate), and
distilled water to 1 litre. Preserve the solution by adding 0.01% (w/v) thimerosal.
ii) PBS/Triton
5.0 ml Triton X-100 (Bio-Rad), and PBS, pH 7.1, to 1 litre. Filter through a 0.2 m bottle
filter.
iii) Trypsin solution
1.0 g Trypsin powder (Difco) 1/250, and DH
2
O to 1 litre. Mix trypsin with water at 4C.
Clarify the solution by filtration through Whatman No. 1 filter paper (or by centrifugation
at 4000 g, followed by filtration through a 0.2 m filter). Dispense in small aliquots and
freeze at 20C or colder. (Trypsin will retain activity after storage at 20C for 1 or 2
months; longer storage should be at 70C.) Thaw new aliquots of trypsin each day as
needed; do not re-freeze.
iv) Eriochrome black T counterstain
1/2000 stock suspension
0.25 g Eriochrome black T, and PBS, pH 7.1, to 500 ml. Filter through Whatman No. 1,
then Whatman No. 42 filter papers (the latter is optional) to remove large chunks of stain.
Store the counterstain in a dark or foil-covered bottle.
NOTE: There are variations in dye content among lots of the compound. Eriochrome
black T dilutions (w/v in PBS) ranging from 1/2000 to 1/20,000 may be required for
appropriate staining, depending on the lot of stain used.
Working suspension
Check the stain quality on test filters. The prepared counterstain suspension should be a
medium purple colour, and the filters should show a definite purple colour after
counterstaining, but should not be so heavily counterstained that they are nearly black.
v) Glycerol mounting medium
0.01 M PBS, pH 7.4
3.36 ml solution A: 0.5 M KH
2
PO
4
, (anhydrous, 68.04 g/litre), 16.0 ml solution B: 0.5 M
K
2
HPO
4
, (anhydrous, 87.09 g/litre), 8.5 g NaCl, and distilled water to 1 litre.
Mounting medium
90 ml glycerol, 2.5 g 1,4-diazobicyclo-(2,2,2)-octane (DABCO), and 10 ml 0.01 M PBS,
pH 7.4. Add the DABCO to the glycerol and dissolve by heating the mixture gently in a
water bath. Then, add the PBS. Adjust to pH 8.69.0 by adding 0.1 N HCl or 0.1 N
NaOH as necessary. Store at room temperature in a dark or foil-covered bottle.
b) Sample preparation
i) Mix 0.5 ml of ovarian (coelomic) fluid with 0.5 ml of PBS/Triton and 0.5 ml of trypsin
solution in a small centrifuge tube. Mix vigorously for 2030 seconds.
ii) Heat the mixture at 50C for 10 minutes.
iii) Withdraw the sample from the centrifuge tube with a 3 ml syringe equipped with a needle
(22 g 1.5 inch or similar). Triturate the sample a few times with a syringe to break up
any remaining clumps of material.
c) Membrane filtration
i) Attach the syringe containing the sample to a Swinney-type filter holder or a disposable
pop-top holder. The filter holder should contain a 13 mm diameter, 0.2 m pore size
polycarbonate filter (Whatman Nuclepore, Cambridge, Massachusetts, USA) and a
supporting 13 mm diameter, 5.0 m (or larger) pore size nylon membrane filter
(Osmonics, Minnetonka, Minnesota, USA).
NOTE: The polycarbonate filter should be placed shiny side up (toward the syringe) in
the filter holder. Polycarbonate filters are thin, and frequently develop ridges identical to
the ridges on the support screens of the filter holders. Placing a thicker nylon filter
between the polycarbonate filter and the support screen helps to reduce the formation of
these ridges and therefore makes the filter surface flatter for easier observation and
counting of bacteria.
ii) Force the sample through the filter.
iii) Rinse each filter with 3 ml of PBS/Triton (force through the filter with a syringe; use a
separate syringe for each sample).
iv) Leave the filter in the holder, and drop on 100 l of FITC-conjugated anti-
R. salmoninarum serum at the optimum working dilution. Cover the top of the filter
holders with paraffin film or foil (alternatively, place them in a humid chamber), and
incubate upright in the dark at room temperature for 1 hour.
v) After incubation, rinse each filter with 3 ml of PBS/Triton by forcing the rinse through
the filter with a syringe.
vi) Counterstain by forcing 1 ml of Eriochrome black T suspension through the filter with a
syringe.
vii) Remove the polycarbonate filters from the holders and place them on microscope slides
to air-dry. Discard the support filters. When the filters are dry, place a drop or two of
glycerol mounting medium, pH 9, on the centre of each filter, and mount with cover-
slips. Examine at 1000 magnification with a microscope equipped for FITC
epifluorescence.
viii) Filter counts of the number of R. salmoninarum in a given number of microscope fields
can be converted to cells/ml of the original ovarian fluid sample according to the
formula:
Cells/ml = (conversion factor) (dilution factor) (total number of cells counted)
Total number of fields examined
Where the conversion factor is the filtering surface area divided by the area of a single
field at the magnification used. One can calculate the theoretical sensitivity of the
technique for any desired number of fields to be examined by entering 1 in the equation
for the total number of bacteria counted. In general, 50 or more fields are examined per
filter.
ix) To confirm MF-FAT results, a procedure such as a nested PCR for R. salmoninarum can
be used (11, 41).
2.4. Bacteriological culture
a) Sampling
Tissue samples for diagnosis and identification of R. salmoninarum should be taken aseptically
from kidney lesions or from lesions in other organs. When no lesions are present, the kidney is
the preferred organ for sampling. In mature females, the coelomic fluid may also represent
convenient material.
For routine controls for detecting infected individuals in a population, a sufficient number of
fish must be sampled (see Chapter I.1., Section B.1.).
b) Isolation
Renibacterium salmoninarum is a fastidiously growing organism that requires prolonged
incubation (23 weeks, sometimes more, at 15C) to produce colonies. L-Cysteine and serum
or serum substitutes are requisite factors, and different media or ingredients have been
proposed to improve its growth or reduce the development of associated microorganisms. The
following two special media are currently used:
Kidney disease medium enriched with serum (KDM-2) or charcoal (KDM-C) (15, 19):
0.1 g L-cysteine (chlorhydrate), 1 g tryptone, 0.05 g yeast extract, 1.5 g agar and 100 ml
distilled water. Adjust the pH to 6.56.8 with NaOH, distribute into flasks or tubes and
autoclave for 20 minutes at 120C. Can be stored for 1 month at 4C. Regenerate prior to
use and add 510% fetal calf serum (FCS), or 0.1% activated charcoal.
Selective kidney disease medium (SKDM-2) (3): 0.1 g L-cysteine (chlorhydrate), 0.005 g
cycloheximide, 1 g tryptone, 0.05 g yeast extract, 1 g agar and 100 ml distilled water.
Adjust the pH to 6.8 with NaOH and autoclave for 20 minutes at 120C. Cool to
approximately 48C, and add 10% FCS and the following components, previously filter
sterilised (0.22 m): 0.00125 g D-cycloserine, 0.00025 g oxolinic acid and 0.0025 g
polymyxin B sulfate (all final concentrations). Dishes with isolation medium are dried at
room temperature for 2448 hours, inoculated by streaking a 0.10.2 ml drop of
infectious material across the agar surface, and incubated at 15C in plastic bags or humid
chambers when the absorption is complete. The effect of storage of SKDM-2 plates on
the effectiveness of the antimicrobial supplements has not been reported. It is
recommended that the SKDM-2 dishes before stored at 4C and used shortly after
preparation
It seems that serum and charcoal are more likely to act as detoxifying agents than as sources of
essential nutrients, and their efficacy has been proved comparable (15), and even optional (53).
Supplementing KDM-2 medium with antibiotics may reduce the problem of fast-growing
organisms (bacteria and fungi), but there is some evidence that they may also inhibit
R. salmoninarum itself (40). Another possibility is to inspect the dishes regularly at intervals of
23 days, and to remove aseptically the colonies produced by fast-growing organisms. In order
to maintain the viability of the R. salmoninarum, Evelyn (20) recommends the preparation of
tissue suspensions in isotonic saline enriched with peptone (0.1 % [w/v]).
When a stock culture of R. salmoninarum is already available, it is possible to take advantage of
the satellitism phenomenon described by Evelyn et al. (24) for accelerating the growth of the
isolates. A heavy suspension of the laboratory feeding strain is dropped on to the centre of the
plate, and the samples to be tested are inoculated in the periphery. The growth rate and the
colony size of the isolates are noticeably increased. Growth enhancement may also be achieved
by adding 1.5% (v/v) sterile spent KDM-2 broth to the medium (24).
c) Characteristics (28, 35)
After a sufficiently long incubation period on KDM-2, KDM-C or SKDM-2, R. salmoninarum
produces white or creamy, shiny, smooth, round, raised, entire colonies that are pinpoint to
2 mm in size. Bacteria from diseased fish will produce visible colonies after 23 weeks on
average, however up to 8 weeks have been reported for initial growth on KDM-2, and
12 weeks on the selective medium SKDM-2. Old cultures may achieve a granular or crystalline
appearance. Transverse sections through such colonies will reveal the presence of Gram-
positive rods in a crystalline matrix. The crystalline material is thought to be cysteine
precipitated from the medium. Growth does not occur on blood agar medium without cysteine
supplement or on trypticaseyeast agar. For some strains a uniformly turbid growth occurs in
broth, but for others a sediment may develop. Renibacterium salmoninarum appears as small
(0.31.5 0.11 m) Gram-positive, PAS-positive, asporogenous, nonmotile, nonacid-fast
rods, frequently in pairs, short chains or pleomorphic forms as Chinese letters, especially in
fish tissue.
Renibacterium salmoninarum is catalase positive and oxidase negative. Its phenotypic
characteristics have been established using API-Zym systems and conventional tests (Table 2).
The API-Zym profile for R. salmoninarum is: + + + + + + + (Austin
& Austin 1999). However, the slow growth of the organism does not render such tests very
useful in practice, and serological methods are more usually employed to confirm the identity
of the isolated strains.
Table 2. Characteristics of Renibacterium salmoninarum (18, 28)
Characteristic Response Characteristic Response
Production of: Degradation of (continued):
Acid phosphatase

DNA
Alkaline phosphatase

Elastin
Butyrate esterase Gelatin
Caprylate esterase

Guanine
Catalase

Hyaluronic acid
Chymothrypsinase Hypoxanthine
Cystine arylamidase Lecithin
-fucosidase
RNA
-galactosidase
Starch
-galactosidase
Testosterone
-glucosaminidase
Tributyrin

-glucosidase
Tween 40

-glucosidase
Tween 60

-glucoronidase
Tween 80
Leucine arylamidase

Tyrosine
-mannosidase
Xanthine
Myristate esterase Acid production from sugars
Oxidase Growth on/at:
Trypsinase

pH 7.8

Valine arylamidase Bile salts, 0.025%, (w/v)
Nitrate reduction Crystal violet, 0.0001% (w/v)

Degradation of: Methylene blue, 0.001% (w/v)

Adenine Nile blue, 0.00001% (w/v)

Aesculin Phenol, 0.025% (w/v)
Arbutin Potassium thiocyanate, 1% (w/v)
Casein

Sodium chloride, 1%( w/v)
(poor)
Chitin Sodium selenite, 0.01% (w/v)
Chondroitin Thallous acetate, 0.001% (w/v)

Table 2 (cont.). Characteristics of Renibacterium salmoninarum (18, 28)
Characteristic Response Characteristic Response
Use of:: Use of::
4-umbelliferyl (4MU)-acetate

4PU-heptanoate

4MU-butyrate

4PU-laurate

4MU--D-cellobiopyranoside
monohydrate
4PU-nonanoate

4MU-elaidate 4MU-oleate

4MU--L-arabinopyranoside
4MU-palmitate
4MU-2-acetamido-2-deoxy--D-
galactopyranoside
4MU-propionate

4MU--L-fucopyranoside

2.5. Nested polymerase chain reaction for testing tissue samples, whole blood, and coelomic
fluid
This procedure is based on the nested PCR method described by Chase & Pascho (11), and
Pascho et al. (41). The nested design must only be used with rigorous controls to ensure the
accuracy of each diagnosis, and to avoid cross-contamination between samples. Other designs
may prove desirable when this can be difficult to achieve, as false-positive results may have serious
consequences such as the unnecessary culling of an entire broodstock. Other molecular detection
methods have also been reported for diagnosing R. salmoninarum infections (8, 13, 32, 37, 47).
a) General considerations
i) DNA extractions should be done in an area that is free from amplified PCR product to
reduce the risk of contamination.
ii) To prevent false-positive results from carry-over of amplified DNA sequences, first
round reaction mixtures should also be set up in a separate area, or under a UV hood.
iii) Each work area should include a separate set of supplies, reagents and pipetting devices.
iv) For each PCR analysis include multiple reagent controls without DNA to test for
contamination, and select a weak positive control that will be indicative of PCR
performance.
b) Primer design
i) Two pairs of oligonucleotide primers are used in the nested PCR protocol. The primers
were designed from the published sequence of the p57 protein of R. salmoninarum (12).
ii) The primers used in the first round were: forward 7593 (5-AGC-TTC-GCA-AGG-
TGA-AGG-G-3; P3) and reverse 438458 (5-GCA-ACA-GGT-TTA-TTT-GCC-GGG-
3; M21). Primer location number corresponds to the nucleotide sequence of the open
reading frame.
iii) The primers used in the second round of amplification reaction were: forward 95119 (5-
ATT-CTT-CCA-CTT-CAA-CAG-TAC-AAG-G-3, P4) and reverse 394415 (5-CAT-
TAT-CGT-TAC-ACC-CGA-AAC-C-3; M38). Primer location number corresponds to
the nucleotide sequence of the open reading frame.
c) DNA extraction and purification
To purify nucleic acids from tissue use a DNA recovery kit and follow the manufacturers
instruction for DNA recovery from tissue. DNA extraction protocols typically do not account
for the rigid cell wall of a Gram-positive bacterium, and the bacteria could remain intact during
the lysis steps. Treatment of tissue and body fluid samples with lysozyme, however, has been
reported to be effective and essential for recovery of high-quality bacterial DNA and rRNA
(11, 47). After the initial cell lysis, add 50 l of 4 lysozyme buffer and incubate at 37C for 1
hour. Consult the manufacturer for specific instructions regarding this lysis step.
Manufacturers of DNA recovery kits from tissue include: DNeasy Tissue Kit from Qiagen,
Chatsworth, California (CA), USA; Nucleospin Tissue Kit from Clonetech Palo Alto, CA,
USA, or Clinipure Genomic DNA Kit from GeneMate, Klaysville, Utah, USA.
i) Kidney tissue: cut 2550 mg of tissue into small pieces and place in a 1.5 ml microfuge
tube. Add tissue lysis buffer and incubate according to the manufacturers instructions.
After the initial tissue lysis, add 50 l of 4 lysozyme buffer (80 mg/ml lysozyme, 80 mM
Tris/HCl, pH 8.0, 8 mM EDTA [ethylene diamine tetra-acetic acid]; 4.8% Triton) and
incubate for 1 hour at 37C. Continue with DNA purification as instructed.
ii) Ovarian fluid: pippette 50 l of ovarian fluid into a 1.5 ml microfuge tube, add tissue lysis
buffer and incubate according to the manufacturers instructions. After the initial tissue
lysis, add 50 l of 4 lysozyme buffer and incubate for 1 hour at 37C. Continue with
DNA purification as instructed.
iii) Whole blood: pippette 50 l of whole blood into a 1.5 ml microfuge tube, add tissue lysis
buffer and incubate according to the manufacturers instructions. After the initial tissue
lysis, add 50 l of 4 lysozyme buffer and incubate for 1 hour at 37C. Continue with
DNA purification as instructed.
iv) Renibacterium salmoninarum cells: Pellet bacteria solution by centrifugation at 7000 g for 15
minutes. Pour off supernatant, resuspend pellet in lysis buffer and incubate according to
the manufacturers instructions. After the initial tissue lysis, add 50 l of 4 lysozyme
buffer and incubate for 1 hour at 37C. Continue with DNA purification as instructed.
d) Determination of yield and purity of nucleic acid samples
On the basis of their absorbance value at 260 nm, adjust the DNA samples with molecular
biology-quality water to a concentration between 0.01 and 0.1 ng/l. Determine the purity of
each DNA sample by calculating the ratio of the readings at 260 nm and 280 nm. Pure DNA
samples have an A
260
/A
280
ratio of 1.82.0.
e) First round PCR protocol
i) Preparation of first round PCR reaction mixture.
Total volume of the first round PCR is 50 l: 10 l of the nucleic acid sample and 40 l of
the reaction mixture. To prepare the reaction mixture, combine 0.2 mM of each
nucleotide, 50 mM KCl, 10 mM Tris/HCl, pH 8.3, 1.5 mM MgCl
2
, 0.2 mM each of
primers P3 and M21 and 2 U of Taq polymease.
ii) Add the PCR reagents except the template DNA into the Master Mix tube.
iii) In the PCR tubes, aliquot 40 l of Master Mix and overlay samples with one drop of
mineral oil. Close the cap tightly after each addition.
iv) Add 10 l of extracted DNA to the PCR tubes.
v) In each reaction well of the thermal cycler, add two drops of mineral oil. Load the
samples into the thermal cycler.
vi) Programme the thermal cycler for 30 cycles as follows: denaturing at 94C for 30 seconds;
annealing at 60C for 30 seconds; and extending at 72C for 1 minute.
f) Second round PCR protocol
i) Preparation of the second round PCR reaction mixture.
Total volume of the reaction mixture is 50 l; this will include 1 l of amplified DNA
from the first round as template DNA. Use the same reaction mixture as described for
the first round except that primers P4 and M38 are used.
ii) Add the PCR reagents except the template DNA into the Master Mix tube.
iii) In nested PCR tubes, aliquot 49 l of Master Mix and overlay the samples with one drop
of mineral oil. Close the cap tightly after addition.
iv) Add 1 l of first round PCR product to the nested PCR tubes.
v) In each reaction well of the thermal cycler, add two drops of mineral oil. Load the
samples into the thermal cycler.
vi) Programme the thermal cycler for 30 cycles as follows: denaturing at 94C for 30 seconds;
annealing at 60C for 30 seconds; and extending at 72C for 1 minute.
g) Visualisation of amplified DNA
i) Use 10 l of the second round PCR product for gel electrophoresis on a 2% agarose gel.
Each electrophoresis gel should include a 1 kb DNA ladder.
ii) Stain gels for 30 minutes in a solution of 5 g/ml ethidium bromide, 0.02 M
hydroxymethyl aminomethane, 0.02 M glacial acetic acid, and 0.5 mM EDTA.
iii) Examine the gels under UV transillumination. Samples are considered positive for
R. salmoninarum if the anticipated 320 base pair product is observed.
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*
* *
C HA P T E R 2 . 1 . 1 2 .

E NT E RI C S E P T I CAE MI A OF CAT F I S H
( E d wa r d s i e l l a i c t a l u r i )

SUMMARY
Enteric septicaemia of catfish (ESC) is caused by the bacterium Edwardsiella ictaluri, which belongs to
the Enterobacteriaceae family (11). ESC is one of the most important infectious disease problems in the
commercial catfish industry in the United States of America (USA). Most reported cases of disease caused
by E. ictaluri are in channel catfish (Ictalurus punctatus), but the bacterium has been isolated from
related North American catfish including blue catfish (I. furcatus), white catfish (Ameiurus catus), and
brown bullhead (A. nebulosus) (11). ESC has also been reported from Clarias batrachus in Thailand
(12) and from several ornamental species (13, 37). The susceptibility of other species including salmonids
has been shown experimentally (4). Edwardsiella ictaluri should not be confused with E. tarda, another
member of the same genus that is frequently found in aquatic animals and is responsible for opportunistic
infections in fish and mammals, including humans.
Several studies have shown that E. ictaluri is a very biochemically and antigenically homogeneous species
(5, 21, 33, 38).
Acute outbreaks of ESC occur within a limited temperature range, from 18 to 28C. This critical
temperature window makes spring and autumn the most common periods for outbreaks in regions where
channel catfish are normally cultured. However, low-level mortality due to ESC can occur in carrier
populations outside of this temperature range. Other environmental factors (poor water quality, high
stocking density and other stressors) predispose the host to ESC. Edwardsiella ictaluri is considered to be
a true obligate pathogen.
Two clinical forms of ESC occur in channel catfish, a chronic encephalitis and an acute septicaemia (20,
22, 30). In the chronic form the bacterium infects the olfactory sacs, and migrates along the olfactory nerves
to the brain, generating granulomatous inflammation. This meningo-encephalitis causes abnormal behaviour,
with alternating listlessness and chaotic swimming. In late stages of this disease, swelling develops on the
dorsum of the head as the inflammatory process erodes the connective tissue in this region. This swelling
ulcerates exposing the brain. This has lead to the term hole in the head disease, used in the industry. In
the acute form of ESC the bacterium is thought to infect through the intestinal mucosa (3), and then to
establish a bacteraemia. The affected fish display petechial haemorrhages around the mouth, on the throat,
the abdomen and at the base of the fins. Multifocal distinct 2 mm diameter raised haemorrhagic cutaneous
lesions that progress to depigmented ulcers also occur. Anaemia, moderate gill inflammation and
exophthalmia are common signs. Internally, haemorrhages and necrotic foci are scattered in the liver and
other internal organs. Haemorrhagic enteritis, systemic oedema, accumulation of ascitic fluid in the body
cavity and enlargement of the spleen are nonspecific signs. Histological examination reveals a systemic
infection of all organs and skeletal muscles, with the most severe changes being diffuse interstitial necrosis of
the anterior and posterior kidney. Focal necrosis in the liver and spleen are also generally seen.
Fish from a population that has recovered from the disease are considered to be carriers. These fish will have
protective immunity and may have high levels of E.-ictaluri-specific antibodies. Occasional losses due to
recurrent ESC will occur in these populations, especially after a stress is induced. Edwardsiella ictaluri
has been detected in the kidney of such fish well over 4 months after exposure (2, 14), suggesting that
carrier fish act as the natural reservoir for the organism. It is believed that shedding with faeces is the main
means of dissemination into the environment. The pathogen persistence and the common practice of continual
Chapter 2.1.12. - Enteric septicaemia of catfish (Edwardsiella ictaluri)
partial harvest and stocking within a production pond have contributed to the success of this pathogen and
the prevalence of ESC in the industry. Moreover, the agent can survive in pond sediments for an extended
period of time (27), and this may be another important factor in disease recurrence in given areas.
Researchers have found the bacterium in the gut of fish-eating birds by performing fluorescent antibody tests
on ingesta, but generally no E. Ictaluri could be cultured indicating that the bacteria were not viable (34,
40). This suggests that birds are not an important means of disseminating this pathogen.
ESC may be controlled through chemotherapy and/or prophylactic measures. The most common
antimicrobial treatments are oral application of potentiated sulfonamide sulfadimethoxine ormethoprim or
oxytetracycline, but plasmid-mediated resistance to these antibiotics does occur (6). Many producers are now
focusing on alternative methods to reduce losses. This relies on management to reduce stress in fish, the
cessation of feeding when ESC-induced losses are detected (41) and on vaccination.
1. IDENTIFICATION OF THE AGENT
The identification of Edwardsiella ictaluri is based on the isolation of the causative agent and
characterisation by biochemical tests. Edwardsiella ictaluri can easily be differentiated from E. tarda by
its inability to produce indole and hydrogen sulfide (E. tarda produces both). Additionally, the two
species do not cross-react serologically.
1.1. Isolation and bacteriological identification
a) Sampling and isolation of the agent
Bacteriological samples from freshly dead or moribund fish should be taken aseptically from
the brain and kidney tissue. The samples should be streaked for isolation on to blood agar
plates, brainheart infusion (BHI) agar or nutrient agar plates. The bacterium grows slowly but
does not require special nutrients. In mixed cultures E. ictaluri can be overgrown by more
rapidly growing bacteria, but E. ictaluri is present in very high numbers in fish affected by ESC.
A selective medium (EIM) has been developed (31) that may prove useful when samples are
taken from heavily contaminated environments, but it is not essential under normal conditions.
For detecting a carrier state in a healthy population, kidney tissue has been homogenised in
0.5% triton-X 100, filtered on to 0.45 m nitrocellulose and grown on EIM agar medium (8)
or homogenised kidney tissue was cultured overnight in liquid EIM and 100 l of this sample
was plated on to BHI agar plates (14). Intraperitoneal administration of suspect carrier fish
with 0.8 mg/g of Kenalog (triamcinolone acetonide) 2 weeks before attempted culture
enhances the detection of the bacterium (2). Optimal temperature for incubation is 2830C.
For routine sampling of fish populations, see Chapter I.1. Section B.1.
b) Characteristics
Following incubation for 3648 hours, E. ictaluri appears as smooth, circular (12 mm
diameter), slightly convex nonpigmented colonies with entire edges. It is a Gram-negative rod,
measuring 0.752.5 m, and is weakly motile by means of a peritrichous flagellation and is
cytochrome oxidase negative. This bacterium grows slowly or not at all at 37C.
After isolation, the bacterium should be identified by biochemical and serological
characteristics (10, 11). Table 1 shows some of the characteristics of the species and biogroups
of the genus Edwardsiella and similar bacteria that can be isolated from fish as given in Bergeys
Manual of Determinative Bacteriology (9). The optimal growth temperature 2830C should be
used for evaluating biochemical characteristics. Edwardsiella ictaluri is biochemically less active
than the other Edwardsiella species, but it appears to be homogeneous (38). A clear-cut biotype
variation is not detected. Edwardsiella ictaluri and E. tarda may be differentiated from each
other biochemically by the production of indole and hydrogen sulfide (E. tarda produces both,
while E. ictaluri does not). Also E. tarda, Yersinia ruckeri, Hafnia alvei and E. hoshinae grow well
at 37C whereas E. ictaluri does not. Edwardsiella ictaluri degrades chondroitin sulfate and this
may be an important virulence factor (7, 32, 38).
Table 1. Differentiation of the species and biogroups of the genus Edwardsiella
and other Enterobacteriaceae found in fish*
Characteristic Yersinia
ruckeri
Hafnia alvei E. tarda E. hoshinae E. ictaluri
Acid production from: Wildtype Biogroup 1
D-Mannitol

Sucrose

Trehalose

L-Arabinose

()
Malonate utilisation
d

Indole production

Hydrogen sulfide production in
triple sugar iron (agar)

Motility

**
Citrate (Christensens) + + +
()

* Bergeys Manual of Determinative Bacteriology.
** Weakly motile at 28C Hawke, 1979.
*** Citrate may be negative on rapid test strips (API 20E). Assay should be run at 25C.
1.2. Detection of the bacterial antigen by serological methods
In addition to well defined biochemical characteristics, there is antigenic homogeneity of the
species and serology can easily differentiate E. ictaluri from other Enterobacteriacae (5, 26, 29).
Slide agglutination with specific antisera against E. ictaluri, fluorescent antibody techniques
(FATs), enzyme-linked immunostaining and enzyme-linked immunosorbent assays (ELISAs) have
been used to provide confirmatory diagnosis. Characterised specific monoclonal antibodies
(MAbs) are generally used in these assays, but polyclonal antisera obtained using formalin-killed
bacterins to immunise rabbits according to classical standards may also be used (1, 16, 28).
a) Agglutination test
Isolated colonies are gently mixed into a drop of sterile saline on a clean glass slide, and a drop
of antiserum is added. The agglutination of the bacteria can be evaluated by comparison with a
similar suspension in normal rabbit serum (control). The appropriate dilution of reacting
serum will have been previously determined by testing a control E. ictaluri strain in twofold
dilutions of this serum.
b) Specific immunofluorescence
The indirect fluorescent antibody test (IFAT) may be employed on bacterial smears, or smears
from infected organs, for rapid confirmation of a clinical diagnosis (28). Smears are air-dried
and heated for 2 minutes at 60C before being flooded and incubated for 5 minutes with
specific rabbit antibody. They are washed in phosphate buffered saline (PBS), pH 7.2, flooded
for 5 minutes with the rabbit-Ig-specific secondary antibody conjugated with fluorescein
isothiocyanate (FITC). After rinsing, the slides are mounted with cover-slips using phosphate-
buffered mounting medium and observed microscopically for bright green fluorescence under
blue epi-illumination. Use undiluted cell culture supernate when using MAbs that are produced
from cell culture in the first step, and a commercially available FITC-conjugated mouse-Ig-
specific secondary antibody at the suggested working concentration (1). Smears of bacterial
suspensions must be very thin. Positive and negative controls (such as E. tarda) should be
stained on separate slides.
c) Enzyme-linked immunostaining
An enzyme-linked immunostaining technique to directly identify E. ictaluri in tissue smears
from infected fish has been described (28). Smears are prepared as for IFAT the first steps
are similar, but the second incubation step uses heterospecific immunoglobulin against rabbit
antiserum, conjugated to horseradish peroxidase. A third incubation step with a substrate
(DMOB, Sigma) is performed for 10 minutes, and after washing and drying, the smears are
mounted in buffered glycerine and observed microscopically under normal trans-illumination.
If smears are too thick, they may produce nonspecific retention of the stain. Rinsing the smear
again for 1 or 2 minutes in 1 N HCl can solve this problem.
1.3. Nucleic-acid-based diagnosis
Assays based on PCR amplification of structural RNA sequences from bacterial colonies and
direct sequencing the products are being adapted by several diagnostic bacteriology laboratories
and some of these assays are commercially available (MicroSeq, Applied Biosystems). Species
confirmation can be done by amplifying and sequencing the 16S portion of the ribosomal RNA
operon and comparing the sequence with GenBank accession AF310622.
2. STANDARD SCREENING METHODS FOR ESC
2.1. Detection of the agent
The techniques are the same as described in Section 1. Unless performed on bacteria isolated and
purified first, any positive result obtained using a detection method will have to be confirmed,
preferably by plating and isolation of the bacteria.
2.2. Serological tests
Although antibody detection tests are rarely used for routine diagnostic purposes and are not yet
approved as official procedures, they could be of value for the mass screening of large numbers of
fish that is required with the development of health control policies. The specificity of the
bacterium and the demonstration of circulating antibodies against E. ictaluri in the serum of fish
recovering from the disease support this hypothesis.
a) Microagglutination test
Direct microagglutination, performed in 96-well round-bottom microplates as described for
other bacterial pathogens (19) can provide quick quantitative data at minimal cost when high
sensitivity is not required. All that is needed is a formalin-killed bacterial suspension prepared
according to the usual techniques (i.e. formalin [0.35% (v/v)] overnight) and adjusted to an
optical density of 0.8 at 525 nm (about 5 10
8
colony-forming units/ml). Twofold dilutions of
the sera are made in isotonic saline, so that the final volume is 25 l/well. Antigen is added (75
l/well) and the plates are incubated for 2 hours at 37C and overnight at 4C before being
read. Controls include a rabbit standard serum of previously established titre and antigen
incubated in saline.
b) Passive haemagglutination
This technique has been described using E. ictaluri lipopolysaccharide (1 mg/ml in PBS, pH
7.2) passively coated on human Group O red blood cells at 4% (29). Tested sera must be
heated for 30 minutes at 45C to inactivate complement, and absorbed with Group O human
red blood cells to remove nonspecific agglutinins before dilutions are done in buffered saline.
Coated blood cells adjusted to 1% concentration are used. Incubation is for 6 hours at room
temperature and overnight at 4C. Controls include coated and uncoated red cells in buffer
and coated cells in serum.
c) Indirect enzyme-linked immunosorbent assay
ELISA methods have been developed to detect catfish antibodies to E. ictaluri and are the
most widely used assays in research (15, 39). The first method uses whole heat-killed bacteria
at 4 10
8
cells/ml PBS, 50 l/well to coat poly-L-lysine-treated ELISA plates (39). The plates
are washed with PBS and then blocked by the addition of 100 l of 100 mM glycine and 1%
bovine serum albumin in PBS for 30 minutes. The plates are then incubated for 30 minutes
with dilutions of the sera to be tested, washed, and incubated with anti-fish-species
immunoglobulin serum (MAb can be used), washed, and incubated with an antibody conjugate
(either horseradish peroxidase or alkaline phosphatase) specific to the secondary antibody.
Then the plate is washed and the chromogenic enzyme substrate is added, the colour is
allowed to develop and the plate is read. The second method is similar but uses a soluble major
antigen (16) obtained by sonicating the bacteria, or merely by dialysing the supernatants of 24-
hour broth cultures then concentrating the sample to 25 g/ml of protein content.
Approximately 100 l is used to coat the wells of the microplates. Optimal working
concentrations must first be determined for each reagent used in the test. These techniques
have proven useful for investigating the immune response of channel catfish to E. ictaluri, and
may be applicable for screening populations of fish for previous exposure.
REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
The possibility of protecting channel catfish populations by vaccination has been reviewed by Plumb in
1988 (25) and Thune et al. in 1997 (36). Edwardsiella ictaluri is known to induce an antibody response after
natural disease or immunisation. A commercial vaccine consisting of an inactivated bacterin was
provisionally licensed in the USA against ESC and marketed in 1991. The effectiveness of this vaccine was
low and it is no longer marketed. Age-related factors and the induction of a cellular immune response
could be of critical importance in inducing strong anti-E.-ictaluri defences. Recent studies by Petrie-
Hanson and Ainsworth show that catfish fry under 3 weeks of age are immunologically unresponsive to E.
ictaluri (23), and this is thought to be due to poorly developed lymphoid organs in the young fish (24).
New attenuated agents show promise in inducing more effective immune responses (7, 17, 18, 35). One
such mutant is commercially available (17) and when administered at a high dose to 12-day-old fry, persists
long enough to induce protective immunity (42). Additional research in developing genetically resistant
strains of catfish show promise (43) and may help to reduce losses caused by this important disease.
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antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella
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8. EARLIX D., PLUMB J.A. & ROGERS W.A. (1996). Isolation of Edwardsiella ictaluri from channel catfish
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Baltimore, Maryland, USA, 486491.
10. HAWKE J.P. (1979). A bacterium associated with the disease of pond cultured channel catfish,
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sp. nov., the causative agent of enteric septicaemia of catfish. Int. J. Syst. Bacteriol., 31, 396400.
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Clarias batrachus L., in Thailand. J. Fish Dis., 10, 137138.
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Health News, 2, ii.
14. KLESIUS P.H. (1992). Carrier state of channel catfish infected with Edwardsiella ictaluri. J. Aquat.
15. KLESIUS P. (1993). Rapid enzyme-linked immunosorbent tests for detecting antibodies to
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Immunopathol., 36, 359368.
16. KLESIUS P.H. & HORST M.N. (1991). Characterisation of a major outer-membrane antigen of
Edwardsiella ictaluri. J. Aquat. Anim. Health, 3, 181187.
17. KLESIUS P.H. & SHOEMAKER C.A. (1998). Development and use of modified live Edwardsiella ictaluri
vaccine against enteric septicemia of catfish. In: Advances in Veterinary Medicine, Volume 41:
Veterinary Vaccines and Diagnostics, Schultz R.D., ed, Academic Press, San Diego, California USA,
523537.
18. LAWRENCE M.L., COOPER R.K. & THUNE R.L. (1997). Attenuation, persistence and vaccine
potential of Edwardsiella ictaluri purA mutant. Infect. Immun., 65, 46424651.
19. MAISSE G. & DORSON M. (1976). Production dagglutinines anti Aeromonas salmonicida par la truite
arc-en-ciel. Influence de la temprature, dun adjuvant et dun immunodpresseur. Ann. Rech. Veter.,
7, 307313.
20. MIYAZAKI T. & PLUMB J.A. (1985). Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus
punctatus (Rafinesque). J. Fish Dis., 8, 389392.
21. NEWTON J.C., BIRD R.C., BLEVINS W.T., WILT G.R. & WOLFE L.G. (1988). Isolation,
characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri. Am. J.
Vet. Res., 49, 18561860.
22. NEWTON J.C., WOLFE L.G., GRIZZLE J.M. & PLUMB J.A. (1989). Pathology of experimental enteric
septicaemia in channel catfish, Ictalurus punctatus (Rafinesque), following immersion exposure to
Edwardsiella ictaluri. J. Fish Dis., 12, 335347.
23. PETRIE-HANSON L. & AINSWORTH A.J. (1999). Humoral immune responses of channel catfish
(Ictalurus punctatus) fry and fingerlings exposed to Edwardsiella ictaluri. Fish Shellfish Immunol., 9, 579
589.
24. PETRIE-HANSON L. & AINSWORTH A.J. (2001). Ontogeny of channel catfish lymphoid organs. Vet.
Immunol. Immunopathol., 81, 11327.
25. PLUMB J.A. (1988). Vaccination against Edwardsiella ictaluri. In: Fish Vaccination, Ellis A.E., ed.
Academic Press, USA, 152161.
26. PLUMB J.A. & KLESIUS P. (1988). An assessment of the antigenic homogeneity of Edwardsiella ictaluri
using monoclonal antibody. J. Fish Dis., 11, 499510.
27. PLUMB J.A. & QUINLAN E.E. (1986). Survival of Edwardsiella ictaluri in pond water and bottom mud.
Progress. Fish Cult., 48, 212214.
28. ROGERS W.A. (1981). Serological detection of two species of Edwardsiella infecting catfish. In:
International symposium on fish biologics: serodiagnostics and vaccines. Dev. Biol. Stand., 49, 169
172.
29. SAEED M.O. & PLUMB J.A. (1987). Serological detection of Edwardsiella ictaluri Hawke
lipopolysaccharide antibody in serum of channel catfish, Ictalurus punctatus Rafinesque. J. Fish Dis., 10,
205209.
30. SHOTTS E.B., BLAZER V.S. & WALTMAN W.D. (1986). Pathogenesis of experimental Edwardsiella
ictaluri infection in channel catfish (Ictalurus punctatus). Can. J. Fish. Aquat. Sci., 43, 3642.
31. SHOTTS E.B. & WALTMAN W.D. (1990). A medium for the selective isolation of Edwardsiella ictaluri.
J. Wildl. Dis., 26, 214218.
32. STANLEY L.A., HUDSON J.S., SCHWEDLER T.E. & HAYASAKA S.S. (1994). Extracellular products
associated with virulent and avirulent strains of Edwardsiella ictaluri from channel catfish. J. Aquat.
33. STARLIPER C.L., SCHILL JR W.B., SHOTTS E.B. & WALTMAN W.D. (1988). Isozyme analysis of
Edwardsiella ictaluri. Microbios Lett., 37, 8187.
34. TAYLOR P.W. (1992). Fish-eating birds as potential vectors of Edwardsiella ictaluri. J. Aquat. Anim.
Health, 4, 240243.
35. THUNE R.L., FERNANDEZ D.H. & BATTISTA J.R. (1999). An aroA mutant of Edwardsiella ictaluri is
safe and efficacious as a live, attenuated vaccine. J. Aquat. Anim. Health, 11, 358372.
36. THUNE R.L., HAWKE J.P., FERNANDEZ D.H., LAWRENCE M.L. & MOORE M.M. (1997).
Immunization with bacterial antigens: edwardsiellosis. In: Fish Vaccinology, Gudding R., Lillehaug
A., Midtlyng P.J. & Brown P.J., eds. Dev. Biol. Stand., Karger, Basel, Switzerland, 90, 125134.
37. WALTMAN W.D., SHOTTS E.B. & BLAZER V.S. (1985). Recovery of Edwardsiella ictaluri from Danio
(Danio devario). Aquaculture, 46, 6366.
38. WALTMAN W.D., SHOTTS E.B. & HSU T.C. (1986). Biochemical characteristics of Edwardsiella ictaluri.
39. WATERSTRAT P., AINSWORTH J. & CAPLEY G. (1989). Use of an indirect enzyme linked
immunosorbent assay (ELISA) in the detection of channel catfish, Ictalurus punctatus (Rafinesque),
antibodies to Edwardsiella ictaluri. J. Fish Dis., 12, 8794.
40. WATERSTRAT P.R., DORR B., GLAHN J.F. &TOBIN M.E. (1999). Recovery and viability of
Edwardsiella ictaluri from great blue herons Ardea herodias fed E. ictaluri-infected channel catfish
Ictalurus punctatus fingerlings. J. World Aquacult. Soc., 30, 115122.
41. WISE D.J. & JOHNSON M.R. (1998). Effect of feeding frequency and Romet-medicated feed on
survival, antibody response and weight gain of fingerling channel catfish (Ictalurus punctatus) after
natural exposure to Edwardsiella icaluri. J. World Aquacult. Soc., 29, 170176.
42. WISE D.J. & TERHUNE J.S. (2001). The relationship between dose and efficacy in channel catfish
Ictalurus punctatus vaccinated fry with a live attenuated strain of Edwardsiella ictaluri (RE-33). J. World
Aquacult. Soc., 32, 177183.
43. WOLTERS W.R. & JOHNSON M.R. (1994). Enteric septicaemia resistance in blue catfish and three
channel catfish strains. J. Aquat. Anim. Health, 6, 329334.
*
* *
C HA P T E R 2 . 1 . 1 3 .

P I S CI RI CKE T T S I OS I S
( P i s c i r i c k e t t s i a s a l mo n i s )

SUMMARY
Piscirickettsiosis is a disease of salmonids caused by Piscirickettsia salmonis that was first reported in
farmed coho salmon (Oncorhynchus kisutch). The disease was initially described in 1989 from fish in
Chile. Piscirickettsia salmonis is a Gram-negative, highly fastidious, intracellular bacterial pathogen of
fish. It is distantly related to the genera Coxiella and Francisella and is grouped with the gamma
subdivision of the Proteobacteria.
The identification of P. salmonis is based on isolation of the causative agent with subsequent testing for
characteristics of this intracellular pathogen. Piscirickettsia salmonis occurs in cytoplasmic vacuoles in the
host cell. The organism may be distinguished from Chlamydia as it does not possess the characteristic
chlamydial developmental cycle, and does not contain the group-specific lipopolysaccharide chlamydial
antigen. The identity is confirmed by means of serological tests or polymerase chain reaction (PCR).
For a rapid result, the identity of P. salmonis isolated in cell culture or observed in smears from diseased
tissue may be confirmed by means of the fluorescent antibody test or immunohistochemical methods with
polyclonal or monoclonal antibodies, or by PCR with P.-salmonis-specific primers.
The implementation of hygienic measures and management policy are the only control methods currently
available. Antibiotics have been used, but their value is questionable. Intensive efforts are underway by
various groups to develop an effective vaccine.
INTRODUCTION
Piscirickettsiosis is a septicaemic condition of salmonids. The causative agent of the disease is Piscirickettsia
salmonis (ATCC VR-1361), and the type strain is LF-89 (13, 15). Thus far the disease has been described
from Chile (5, 12), Ireland (22, 23), Norway (21), and both the West (6, 11) and East (16) coasts of
Canada.
Piscirickettsia salmonis has been detected in coho salmon (Oncorhynchus kisutch), chinook salmon
(O. tshawytscha), sakura salmon (O. masou), rainbow trout (O. mykiss), pink salmon (O. gorbuscha) and
Atlantic salmon (Salmo salar). Coho salmon are believed to be most susceptible (13). Mortality in seawater
net pens was reported to be 3090% among coho salmon reared in Chile (5). Piscirickettsia-salmonis-like
organisms have recently been isolated from non-salmonid fish (79). The relationships of most of these
organisms to P. salmonis have not been fully elucidated, but the organism isolated from white sea bass
(Atractoscion nobilis) (7) is genetically indistinguishable from P. salmonis.
The mechanisms of transmission are still under investigation. The disease has been primarily reported in
marine fish farms, and has also been observed in freshwater facilities (4, 14). Horizontal transmission
occurs in saltwater and freshwater (2, 10). Transmission by vectors remains a consideration, and the role
of vertical transmission is obscure.
Chapter 2.1.13. - Piscirickettsiosis (Piscirickettsia salmonis)
Although antibacterial treatment provides some benefit, it is not entirely effective as a means of
controlling the disease. Currently, oxalinic acid appears to be the drug of choice. Eggs may be disinfected
as part of good hatchery practice.
The disease is a chronic, systemic infection that generally affects salmonids reared in sea water. All ages are
susceptible, from smolts to market size fish. Signs of the disease both external and internal are well
documented (10, 12, 21, 23). The disease begins approximately 1 month after fish are introduced into the
seawater net pens, and the organism was thought to be a marine bacterium.
A range of gross signs of infection may be present in salmonids infected with P. salmonis. Severely affected
fish are dark, anorexic and lethargic. They often swim near the surface or edges of the cages. Fish with
milder infections often show no abnormal external signs. Infections of the brain may cause erratic
swimming behaviour (25). Skin lesions, appearing as small white patches that can progress to shallow
ulcers, may also be present on some fish. Perhaps the most consistent external signs observed during
P. salmonis infections are pale gills resulting from a significant anaemia, but this is not pathognomonic for
the disease.
Consistent with many systemic, chronic inflammatory diseases of salmonids, the internal signs are a
swollen and discoloured kidney and an enlarged spleen. Ascites in the peritoneum may be present and
haemorrhages on the visceral fat, stomach, swim bladder, and body musculature can also occur (10, 24).
Hallmark internal lesions of the disease are found in the liver, which may exhibit large, whitish or yellow,
multifocal, coalescing, pyogranulomatous nodules. These lesions often rupture, resulting in shallow crater-like
cavities in the liver. Whereas these liver lesions are somewhat unique to piscirickettsiosis, many fish with the
disease do not exhibit them.
The most prominent histological changes are found in the liver, kidney, spleen and intestine, but
pathological changes in the brain, heart, ovary and gill can also be observed (3, 7, 10, 22, 24). Multifocal
necrosis of hepatocytes, accompanied by a chronic inflammatory infiltrate of mononuclear cells, is
observed in the liver. Vascular and perivascular necrosis are also evident in the liver, and intravascular
coagulation resulting in fibrin thrombi within major vessels is a common finding. The focal areas of
necrosis underlie the pale circular lesions observed grossly in more chronically infected fish. In more acute
infections, the coalescence of areas of necrosis results in a more mottled appearance to the organ rather
than discrete nodules. Granulomatous inflammation also occurs in the interstitium and parenchyma of the
kidney and spleen, respectively. Vascular changes similar to those in the liver may also be observed in the
kidney and spleen. Meningitis, endocarditis, peritonitis, pancreatitis, and branchitis may be observed with
accompanying chronic inflammatory and vascular changes similar to those in the liver and haematopoietic
organs. The ovary was reported to be involved in certain infections in coho salmon (10).
High magnification examination of lesions reveals aggregates of the organism in the cytoplasm of degenerated
hepatocytes and in macrophages. Infected macrophages are usually hypertrophied and replete with cellular
debris. In tissue sections stained with haematoxylin and eosin (H&E), the organism appears as basophilic or
amphophilic spheres, about 1 m in diameter.
Gross and microscopic changes resulting from piscirickettsiosis are not unique enough to allow for
definitive diagnosis of the disease. Therefore, screening for and diagnosis of piscirickettsiosis is based on
detection of the causative agent. Presumptive diagnosis can be achieved by the visualisation of the
causative agent within macrophages or hepatocytes in histological sections or tissue imprints.
Confirmatory diagnosis is achieved by isolation of Piscirickettsia salmonis in cell culture, but it does not
grow on any known artificial bacteriological media. Confirmation of P. salmonis in culture may be made by
indirect fluorescent antibody test (IFAT) or polymerase chain reaction (PCR) assay.
PCR assays can also be conducted directly on tissues (19, 20), and thus PCR assays on tissues along with
the observation of suspect organisms within macrophages or hepatocytes are also suitable methods for
confirmatory diagnosis. Alternatively, P. salmonis can be detected with Giemsa-stained tissue smears,
followed by IFAT for positive identification. An enzyme-linked immunosorbent assay for detecting
P. salmonis is commercially available, although there is no published information using this method.
Piscirickettsia-salmonis-infected fish tissues suitable for examination in cell culture, PCR, tissue imprints and
histology are kidney, liver and blood, collected from diseased fish during either overt or covert infections
(18). Due to sensitivity of P. salmonis to antibiotics in vitro, none should be used in media during collection
of tissue or the culture of cells.
Sampling procedures: See Chapter I.1 Section B.
1. STANDARD MONITORING METHODS FOR PISCIRICKETTSIOSIS
1.1. Isolation of Piscirickettsia salmonis in cell culture (18)
Cell line to be used: CHSE-214 or EPC (without antibiotics added)
a) Preparation of tissue
i) The kidney must be aseptically removed and transferred to a sterile container. Antibiotics
must not be used at any step in the isolation procedure. Tissues must be kept at 4C or on
ice until processed, and must not be frozen.
ii) Kidney tissue should be homogenised at 1/20 in antibiotic-free balanced salt solution
(BSS), and then, without centrifugation, further diluted 1/5 and 1/50 in antibiotic-free
BSS for inoculation on to cell cultures. Final dilutions for use are 10
2
and 10
3
.
b) Inoculation of cell monolayers
i) A 10
2
and 10
3
dilution of the organ homogenates should be inoculated on to cultured
cell monolayers and maintained in antibiotic-free medium.
ii) The diluted homogenate can be inoculated directly (0.1 ml/culture) into the antibiotic-
free culture medium overlaying the cells.
iii) The cell cultures must be incubated at 1518C for 28 days and observed for the
appearance of cytopathic effect (CPE). The P. salmonis CPE consists of plaque-like
clusters or rounded cells. With time, the CPE progresses until the entire cell sheet is
destroyed.
iv) If CPE does not occur (except in positive controls), cultures should be incubated at 15
18C for an additional 14 days.
1.2. Giemsa stain and fluorescent antibody test of cell culture supernatant
Fluid from cell cultures showing extensive CPE can be spotted directly on to microscope slides,
and stained with Giemsa or tested in the IFAT (17) as described in the following section.
2. DIAGNOSTIC METHODS FOR PISCIRICKETTSIOSIS
2.1. Giemsa stain
i) Preparations of tissue culture supernatant, smears or impressions of the kidney, liver, and
spleen should be prepared, air-dried, and fixed for 5 minutes in absolute methanol.
ii) Immerse slides in a working solution of Giemsa stain for 30 minutes.
Stock solution: 0.4 (w/v) in buffered methanol solution, pH 6.9 (commercially available).
Working solution: diluted 1/10 in phosphate buffer pH 6.0 (0.074 M NaH
2
PO
4
, 0.009 M
Na
2
HPO
4
).
iii) Destain with tap water.
iv) Observe slides under oil immersion. Tissue smears from infected organs show darkly stained
pleiomorphic organisms occurring in coccoid or ring forms, frequently in pairs, with a
diameter of 0.51.5 m.
2.2. Fluorescent antibody test of tissue smear (17)
i) The positive identity of P. salmonis isolated in cell culture or observed in Giemsa-stained
smears may be determined by serological methods, for example IFAT.
ii) Smears or impressions of the kidney, liver, and spleen should be prepared, air-dried, and
fixed for 5 minutes in absolute methanol.
iii) Tissues smears to be examined by IFAT must be freshly prepared or stored at 20C.
iv) The sample is first incubated with anti-P.-salmonis polyclonal or monoclonal antibody, then
washed and incubated with a secondary antibody conjugated with fluorescein isothiocyanate.
v) Following washing, apply glycerol-based mounting media and cover-slip, then examine under
a fluorescence microscope.
2.3. Histology
i) Preserve visceral organs in formalin-based fixative and process for routine histology.
ii) Stain histological slides with H&E or Giemsa.
iii) Examine macrophages within the kidney interstitium, spleen or blood, or hepatocytes within
liver lesions, for the presence of multiple, spherical, basophilic or amphophilic bodies (by
H&E) or dark blue (by Giemsa), approximately 1 m in diameter in the cytoplasm.
2.4. Immunohistochemistry of tissue section (1)
i) Sections (5 m) of formalin-fixed, paraffin-embedded tissues are deparaffinised, and treated
to eliminate endogenous peroxidase activity.
ii) The tissue is first incubated with anti-P.-salmonis polyclonal or monoclonal antibody, then
washed and incubated with a secondary antibody conjugated with horseradish peroxidase.
iii) Following washing, the tissue is exposed to a chromogen, counterstained with haematoxylin,
dehydrated and prepared for examination under a light microscope.
2.5. Polymerase chain reaction amplification (20)
A nested PCR has been developed to detect genomic DNA of P. salmonis using general bacterial
16S rDNA primers in the first amplification and P.-salmonis- specific primers in a second reaction.
The two general eubacterial primer sequences are EubA (1518R) 5-AAG-GAG-GTG-ATC-
CAN-CCR-CA-3 and EubB (27F) 5-AGA-GTT-TGA-TCM-TGG-CTC-AG-3. The two
specific P. salmonis primers PS2S (223F) and PS2AS (690R) have the following sequences: 5-
CTA-GGA-GAT-GAG-CCC-GCG-TTG-3 and 5-GCT-ACA-CCT-GAA-ATT-CCA-CTT-3,
respectively. These primers yield a specific 467 bp product. The PS2AS primer sequence has been
updated to correspond to corrections in the target DNA sequence (GenBank accession number
U36941).
a) Preparation of infected cell culture supernatant or tissue
Use of a commercially available spin column to purify DNA from cell culture supernatant or
tissue is recommended for PCR sample preparation. In addition to following the
manufacturers instructions on the use of the columns, initial digestion of the sample in lysis
buffer (20 mM Tris/HC, pH 8.0, 2 mM EDTA (ethylene diamine tetra-acetic acid), 1.2%
Triton X-100, 4 mg/ml lysozyme) at 37C for 30 minutes is suggested.
b) Nested polymerase chain reaction
i) Add 5 l of the DNA preparation to the 45 l of reaction mixture consisting of PCR
buffer (10 mM Tris/HCl, pH 9, 1.5 mM MgCl
2
, 50 mM KCl, and 0.1% Trition X-100),
200 M each of dATP, dCTP, dTTP and dGTP, 1 M EubA primer, 1 M EubB primer,
and 2.5 units Taq DNA polymerase. The mixture is covered with 50 l of mineral oil.
Denature the mixture at 94C for 2 minutes and then amplify by 35 cycles at 94C for
1 minute, 50C for 2 minutes, and 72C for 3 minutes.
ii) The second amplification is performed by adding 2 l of the first PCR products to 48 l
of reaction mixture containing 2 M each of the PS2S and PS2AS primers instead of
EubA and EubB under the following reaction conditions: denature the mixture at 94C
for 2 minutes and then amplify by 35 cycles at 94C for 1 minute, 65C for 2 minutes and
72C for 3 minutes.
Amplified DNA (476 bp) (10 l of the PCR reaction mixture) is analysed by
electrophoresis in 2% agarose TAE gel (40 mM Tris acetate/1 mM EDTA) containing
1 mg per 50 ml ethidium bromide. To confirm the identification, 10 l aliquots of the
PCR amplification mixture can be digested with EcoR1 and Pst1 according to the
manufacturers instructions (expected products: EcoR1 994, 546; Pst1 541, 519, 480). If a
product is obtained with the P.-salmonis-specific primers but does not cut as expected,
further confirmation of the isolate should be obtained by sequencing.
c) Direct polymerase chain reaction
Add 5 l of the DNA preparation to the 45 l of reaction mixture consisting of PCR buffer
(10 mM Tris/HCl, pH 9, 1.5 mM MgCl
2
, 50 mM KCl, and 0.1% Trition X-100), 200 M each
of dATP, dCTP, dTTP and dGTP, 2 M each of PS2S primer and PS2AS primer, and
2.5 units Taq DNA polymerase. The mixture is covered with 50 l of mineral oil if the
thermocyler does not have a hot bonnet. The direct PCR is performed under the same reaction
conditions as for the second step of the nested PCR.
Other PCR assays have been developed to detect P. salmonis (19). These primer sequences and
reaction conditions would also be suitable for confirmation of the presence of P. salmonis.
REFERENCES
1. ALDAY-SANZ V., RODGER H., TURNBULL T., ADAMS A. & RICHARDS R.H. (1994). An
immunohistochemical diagnostic test for rickettsial disease. J. Fish Dis., 17, 189191.
2. ALMENDRAS F.E., FUENTEALBA I.C., JONES S.R.M., MARKHAM F. & SPANGLER E. (1997).
Experimental infection and horizontal transmission of Piscirickettsia salmonis in freshwater-raised
Atlantic salmon, salmo salar L. J. Fish Dis., 20, 409418.
3. BRANSON E.J. & NIETO DIAZ-MUNOZ D. (1991). Description of a new disease condition occurring
in farmed coho salmon, Oncorhynchus kisutch (Walbaum), in South America. J. Fish Dis., 14, 147156.
4. BRAVO S. (1994). Piscirickettsiosis in freshwater. Bull. Eur. Assoc. Fish. Pathol., 14, 137138.
5. BRAVO S. & CAMPOS M. (1989). Coho salmon syndrome in Chile. AFS/FHS Newsletter, 17, 3.
6. BROCKLEBANK J.R., SPEARE D.J., ARMSTRONG R.D. & EVELYN T. (1992). Septicemia suspected to
be caused by a rickettsia-like agent in farmed Atlantic salmon. Can. Vet. J., 33, 407408.
7. CHEN M.F., YUN S., MARTY G.D., MCDOWELL T.S., HOUSE M.L., APPERSEN J.A., GUENTHER T.A.,
ARKUSH K.D. & HEDRICK R.P. (2000). A Piscirickettsia salmonis-like bacterium associated with
mortality of white seabass Atractoscion nobilis. Dis. Aquat. Org., 43, 117126.
8. CHEN S.C., TUNG M.C., CHEN S.P., TSAI J.F., WANG P.C., CHEN R.S., LIN S.C. & ADAMS A.
(1994). Systemic granulomas caused by a rickettsia-like organism in Nile tilapia, Oreochronuis niloticus
(L.), from southern Taiwan. J. Fish Dis., 17, 591599.
9. CHEN S.C., WANG P.C., TUNG M.C., THOMPSON K.D. & ADAMS A. (2000). A Pisicirickettsia salmonis-
like organism in grouper, Epinephelus melanostigma, in Taiwan. J. Fish Dis., 23, 415418.
10. CVITANICH J.D., GARATE N.O. & SMITH C.E. (1991). The isolation of a rickettsia-like organism
causing disease and mortality in Chilean salmonids and its confirmation by Kochs postulate. J. Fish
Dis., 14, 121145.
11. EVELYN T.P.T. (1992). Salmonid rickettsial septicemia. In: Diseases of Seawater Netpen-reared
Salmonid Fishes in the Pacific Northwest, Kent M.L., ed. Can. Spec. Pub. Fish. Aquat. Sci. 116.
Dept. Fisheries and Oceans, Nanaimo, British Colombia, Canada, 1819.
12. FRYER J.L., LANNAN C.N., GARCES L.H., LARENAS J.J. & SMITH P.A. (1990). Isolation of a
rickettsiales-like organism from diseased coho salmon Oncorhynchus kisutch in Chile. Fish Pathol., 25,
107114.
13. FRYER J.L., LANNAN C.N., GIOVANNONI S.J. & WOOD N.D. (1992). Piscirickettsia salmonis gen. nov.,
sp. nov., the causative agent of an epizootic disease in salmonid fishes. Int. J. Syst. Bacteriol., 42, 120
126.
14. GAGGERO A., CASTRO H. & SANDINO A.M. (1995). First isolation of Piscirickettsia salmonis from
coho salmon, Oncorhynchus kisutch (Walbaum), and rainbow trout, Oncorhynchus mykiss (Walbaum),
during the freshwater stage of their life cycle. J. Fish Dis., 18, 277279.
15. GARCES L.H., LARENAS J.J., SMITH P.A., SANDINO S., LANNAN C.N. & FRYER J.L. (1991).
Infectivity of a rickettsia isolated from coho salmon (Oncorhynchus kisutch). Dis. Aquat. Org., 11, 93
97.
16. JONES S.R.M., MARKHAM R.J.F., GROMAN D.B. & CUSACK R.R. (1998). Virulence and antigenic
characteristics of a cultured Rickettsiales-like organism isolated from farmed Atlantic salmon Salmo
salar in eastern Canada. Dis. Aquat. Org., 33, 2531.
17. LANNAN C.N., EWING S.A. & FRYER J.L. (1991). A fluorescent antibody test for detection of the
rickettsia causing disease in Chilean salmonids. J. Aquat. Anim. Health, 3, 229234.
18. LANNAN C.N. & FRYER J.L. (1991). Recommended methods for inspection of fish for the salmonid
rickettsia. Bull. Eur. Assoc. Fish Pathol., 11, 135136.
19. MARSHALL S., HEATH S., HENRIQUEZ V. & ORREGO C. (1998). Minimally invasive detection of
Piscirickettsia salmonis in cultivated salmonids via the PCR. Appl. Environ. Microbiol., 64, 30663069.
20. MAUEL M. J., GIOVANNONI S.J. & FRYER J.L. (1996). Development of polymerase chain reaction
assays for detection, identification, and differentiation of Piscirickettsia salmonis. Dis. Aquat. Org., 26,
189195.
21. OLSEN A.B., MELBY H.P., SPEILBERG L., EVENSEN O. & HASTEIN T. (1997). Piscirickettsia salmonis
infection in Atlantic salmon Salmo salar in Norway epidemiological, pathological and
microbiological findings. Dis. Aquat. Org., 31, 3548.
22. PALMER R., RUTTLEDGE M., CALLANAN K. & DRINAN E. (1997). A Piscirickettsiosis-like disease in
farmed Atlantic salmon in Ireland isolation of the agent. Bull. Eur. Assoc. Fish Pathol., 17, 6872.
23. RODGER H.D. & DRINAN E.M. (1993). Observation of a rickettsia-like organism in Atlantic salmon,
Salmo salar L., in Ireland. J. Fish Dis., 16, 361369.
24. SCHAFER J.W., ALVARADO V., ENRIQUEZ R. & MONRAS M. (1990). The coho salmon syndrome
(CSS): a new disease in Chilean salmon, reared in sea water. Bull. Eur. Assoc. Fish Pathol., 10, 130.
25. SKARMETA A.M., HENRIQUEZ, V., ZAHR M., ORREGO C. & MARSHALL S.H. (2000). Isolation of a
virulent Piscirickettsia salmonis from the brain of naturally infected coho salmon. Bull. Eur. Assoc. Fish
Pathol., 20, 261264.
*
* *
C HA P T E R 2 . 1 . 1 4 . 1
2
GYRODACT YL OS I S 3
( Gy r o d a c t y l u s s a l a r i s ) 4
5
SUMMARY 6
Gyrodactylus salaris (Monogenea: Gyrodactylidae) is a highly pathogenic parasite of wild and farmed 7
Atlantic salmon parr (Salmo salar) and smolt. Several other salmonids are susceptible to G. salaris, such 8
as rainbow trout (Oncorhynchus mykiss), Arctic char (Salvelinus alpinus), North American brook 9
trout (S. fontinalis), grayling (Thymallus thymallus), North American lake trout (Salvelinus 10
namaycush) and brown trout (Salmo trutta) (in declining order of susceptibility). Gyrodactylus salaris 11
may be present in farmed salmonids for years, especially rainbow trout, without any signs of clinical disease. 12
Single parasite infestations are common and demand a special procedure when monitoring for the occurrence 13
of Gyrodactylus specimens. The whole surface of a fish, including gills and mouth cavity, must be 14
examined under a dissecting microscope. Identification of G. salaris is based on morphology and 15
morphometry of hooks and bars in the opisthaptor, or by DNA analysis. 16
INTRODUCTION 17
Gyrodactylosis of Atlantic salmon (Salmo salar), is caused by the viviparous freshwater monogenean 18
Gyrodactylus salaris (4). The presence of the parasite in Swedish hatcheries has been known since the 19
beginning of the 1950s, but it was regarded as harmless until its introduction into Norway in the mid- 20
1970s. 21
Gyrodactylus salaris is restricted in its distribution to Europe. It has been found in farmed Atlantic salmon 22
or farmed rainbow trout (Oncorhynchus mykiss) in several (mainly northern) European countries. The 23
parasite has been found in wild salmonids, mainly Atlantic salmon parr, from rivers in Russia, Sweden and 24
Norway. Gyrodactylus salaris is much more common in farmed rainbow trout than previously thought, and 25
is likely to be present in more countries than those currently known. Great Britain and Ireland appear to 26
be free from the parasite. 27
In experiments, Atlantic salmon from a Scottish river have shown equal susceptibility to G. salaris as 28
Norwegian salmon, while Atlantic salmon from the River Neva in the Baltic have shown significant 29
resistance to the parasite. 30
Gyrodactylus salaris survives and reproduces in several salmonids, such as rainbow trout (Oncorhynchus 31
mykiss), Arctic char (Salvelinus alpinus), North American brook trout (S. fontinalis), grayling (Thymallus 32
thymallus), North American lake trout (Salvelinus namaycush) and brown trout (Salmo trutta) (in declining 33
order of susceptibility). 34
Gyrodactylus salaris has spread between rivers and farms mainly by the transport/restocking of live fish. 35
Fish swimming through brackish water can also cause the parasite to be spread between rivers. Although 36
G. salaris is a freshwater parasite, it has an almost normal reproduction at 5% salinity. The survival time at 37
higher salinity may be significant, for example G. salaris survives for up to 240 and 42 hours at 10% and 38
20% salinity, respectively. 39
Chapter 2.1.14. - Gyrodactylosis (Gyrodactylus salaris)
SAMPLING PROCEDURES 40
Gyrodactylus specimens may die or detach from the host if the water quality/chemistry is changed. 41
Furthermore, Gyrodactylus specimens die rapidly if not covered with water, and often leave a host soon 42
after it dies. When fish are sent to a laboratory for Gyrodactylus examination, the sampling procedures used 43
must be somewhat different from those described in Chapter I.1. 44
Fish must be alive when collected and should be transported in the same water as that in which they lived. 45
Because excretory (and other) products from the fish may change the waters quality, the number of 46
fish/fish volume should be relatively small compared with the volume of water in the container. 47
Transportation time should also be considered. The container should carry one fish species only because 48
experiments and experience have shown that mixing fish species may initiate detachment of Gyrodactylus 49
specimens from their hosts. 50
Dead fish, transported on ice, are not acceptable for Gyrodactylus examination, even if the fish are sent 51
separately in plastic bags, etc. The parasites soon die if not covered in water, and as these parasites do not 52
have an exoskeleton, dead parasites disintegrate as quickly as the mucus and epidermis cells of the fish. If 53
such dead fish are rinsed in water, Gyrodactylus specimens may be found in the bottom sediment. However, 54
if specimens are not found in the sediment, it cannot be concluded that the fish were uninfected. 55
A good alternative to examination of live fish, is examination of formaldehyde-fixed or ethanol-preserved 56
fish. The concentration of formaldehyde after fixation should not be lower than 4% (10% formalin). The 57
formaldehyde concentration should be 810% (2025% formalin) before adding the fish, because water is 58
freed from the fish during fixation. The concentration of ethanol after conservation should be about 70%. 59
If the concentration is lower, the mucus and epidermis may disintegrate and Gyrodactylus specimens, even 60
if they are preserved, may drop off. Because water is released from the fish during preservation, the 61
concentration of ethanol may be as high as 96% before the fish are added. Gyrodactylus specimens are 62
more easily detected on formaldehyde-preserved compared with ethanol-preserved fish because with the 63
former, the parasites become more whitish and easier to detect. Also, for the safety of laboratory staff, 64
formaldehyde is a better preservative than pure ethanol with no additives. However, if examination of 65
DNA probes is to be used for identification of Gyrodactylus specimens, preservation in pure ethanol is (at 66
present) a prerequisite. 67
The fish should be fixed or preserved in relatively large bottles that provide excess space and fixative (5 68
10 fold). The opening to the bottles should be wide to avoid the possibility of scraping off Gyrodactylus 69
specimens when the fish are taken out for examination. Bottles should be stored in a horizontal position, 70
as should the fish, during fixation/preservation. This makes later examination of the fish easier. When fish 71
are fixed/preserved, the bottles should be stored in a vertical position after 23 days. 72
DIAGNOSTIC PROCEDURES 73
When present on wild and farmed Atlantic salmon parr and smolt, G. salaris usually causes gyrodactylosis. 74
Disease outbreaks may occur at any time and at any water temperature, but are most common in spring 75
and in periods when the water temperature is 717C. The fins, especially the dorsal and pectoral fins, are 76
most commonly infested, but parasite specimens may occur all over the epidermis of the host, including 77
the nostrils, the gills and mouth cavity. Gyrodactylus specimens are usually present in scrapings from gills, 78
skin or fins of fish with gyrodactylosis. However, if the number of Gyrodactylus specimens is low, the 79
chances of detecting the parasites by this method are limited. 80
Gyrodactylus salaris may be present in very low numbers on fish without any signs of clinical disease. This 81
occurs occasionally with Atlantic salmon, but commonly with other salmonids, especially rainbow trout. 82
Single parasite infestations are common, and the single parasite may be attached to any site on the fish 83
surface. A special procedure is therefore required when monitoring for the occurrence of Gyrodactylus 84
specimens on asymptomatic fish (healthy fish). 85
1. SCREENING AND PRESUMPTIVE DIAGNOSTIC METHODS FOR THE PRESENCE OF GYRODACTYLUS SPECIMENS 86
Fish should be examined individually under a binocular dissecting microscope with good illumination. 87
The fish should be placed in a box and completely covered in transport water. Living parasites are 88
more easily detected by their movements, so disturbing light refraction on the skin of the fish should 89
be avoided. Fish should be killed using a needle to the brain to avoid blood leaking into the water. If 90
the dissecting microscope is illuminated from above, the bottom of the box should be black. This will 91
increase the contrast and the parasites will be detected more easily. The whole surface of the fish, 92
including gills and mouth cavity, must be examined. It is best to use two forceps for this process. Each 93
fish should be examined for at least 5 minutes. The fins of relatively small, unfixed fish, which are 94
usually less than 10 cm, can also be studied using illumination through the bottom of the box. This 95
way, Gyrodactylus specimens on the fins can usually be easily observed. 96
Fixed or preserved fish should be studied in a similar way under a dissection microscope with 97
illumination from above. Gyrodactylus specimens turn almost white when fixed in formaldehyde, while 98
ethanol-preserved specimens are usually only slightly opaque. Before examination, the fish should be 99
rinsed in tap water. This can be done by placing the transport container under a tap and letting the 100
water flow slowly (gently) through the container for a couple of hours. Before rinsing, however, the 101
fixative/preservative, including the bottom sediment in the container, should be examined separately 102
for the presence of Gyrodactylus specimens that may have washed off during fixation and 103
transportation. For protection purposes, the dissecting microscope should be placed on a suction 104
bench with a downwards outlet to avoid inhalation of evaporated fixative/preservative. 105
2. CONFIRMATORY DIAGNOSTIC METHODS FOR THE IDENTIFICATION OF GYRODACTYLUS SALARIS 106
2.1. Morphology and morphometry of sclerites in the attachment organ 107
Identification of Gyrodactylus species is based on morphology and morphometry of marginal 108
hooks, anchors (hamuli) and bars in the opisthaptor (the attachment organ). Good preparation of 109
specimens is a prerequisite for discrimination of species (6). 110
Malmbergs ammonium picrate glycerine (APG) method for preparing whole mounts of small 111
haptor worms (Monogenea) by means of APG (5) is superior to other methods, especially when 112
the temporal relation is considered. According to this method, a drop of water is placed on a slide 113
(76 26 mm), a worm is transferred to the water and a cover-slip (18 18 mm) is gently placed 114
on top. Using a piece of filter paper, as much water as possible is absorbed at the edge of the 115
cover-slip so that the worm is very depressed but not squashed. A small drop of APG is added to 116
the edge of the cover-slip. The parasite will be fixed as the yellow APG solution penetrates the 117
space between the slide and the cover-slip. The slide should be labelled with the host species, 118
location, locality, date of collection and water temperature. If the slide is to be transported, a small 119
drop of nail-polish or a similar substance should be added to each corner of the cover-slip for 120
permanent attachment to the slide. The worm should be kept in vivo during preparation, but a 121
fixed/preserved worm may be used, although as the latter is harder to depress, species 122
identification may be difficult. 123
The APG solution is made by mixing one part saturated ammonium picrate solution and one part 124
glycerine/glycerol (puriss). Identification of G. salaris should be in accordance with refs 5, 79. 125
Morphology and morphometry in G. salaris are presented in Table 1 and Figures 1 and 2, 126
respectively. 127
Gyrodactylus salaris is morphologically very similar to G. teuchis from brown trout, Atlantic salmon, 128
rainbow trout and North American brook trout, and to G. thymalli from grayling. The species can 129
be differentiated on the basis of the shape of the marginal hook sickle. Gyrodactylus teuchis has a 130
longer and more constantly curved sickle blade while G. thymalli has a small angle on the shaft of 131
the sickle. 132
Table 1. Total range of variation in 14 characters measured on marginal hooks, anchors, and ventral bars of 133
Gyrodactylus salaris on Atlantic salmon parr and rainbow trout. The water temperature at the time of measuring 134
varied between 0.0C and 20.0C. All measurements are in m (n = number of specimens measured) 135
Character measured n Range of variation
Total length of marginal hook 783
33.046.6
Length of marginal hook handle 807
26.038.5
Length of marginal hook sickle 849
7.09.5
Total length of anchor 872
58.085.0
Length of anchor shaft 872
42.863.9
Length of anchor point 878
27.844.2
Length of anchor root 875
15.032.1
Maximal distance between processes of ventral bar 700
18.533.2
Length of ventral bar 747
19.532.0
Total basal width of ventral bar 715
20.336.5
Basal width of ventral bar 759
7.118.7
Total median width of ventral bar 720
17.035.5
Median width of ventral bar 778
5.015.5
Length of ventral bar membrane 736
12.523.0
136
137
138
Figure 1. Morphological variability/variation in opisthaptoral hand parts of Gyrodactylus salaris. 139
a) Marginal hooks; b) Anchors; c) Ventral bar. Scale bars: 40 m, 50 m and 30 m, respectively. 140
141
Figure 2. Fourteen characters measured on marginal hooks, anchors and ventral bars 142
of Gyrodactylus salaris. 143
lmh = total length of marginal hook lvb = length of ventral bar
lh = length of marginal hook handle tbwvb = total basal width of ventral bar
lsi = length of marginal hook sickle bwvb = basal width of ventral bar
la = total length of anchor tmwvb = total median width of ventral bar
las = length of anchor shaft mwvb = median width of ventral bar
lap = length of anchor point lvbm = length of ventral bar membrane
lar = length of anchor root (tmwvb = mwvb + lvbm)
mdpvd = maximum distance between processes of ventral bar
2.2. DNA analysis of Gyrodactylus specimens (14) 144
a) Preparation of samples 145
Individual specimens should be removed from ethanol, blotted to remove excess ethanol and 146
placed in individual 0.5 ml microfuge tubes containing 7.5 l lysis solution (0.45% NP40, 147
0.45% Tween 20, 60 g/ml Proteinase K). Tubes should be incubated at 65C for 20 minutes 148
and then at 95C for 10 minutes. This lysate is used as the DNA template in the polymerase 149
chain reaction (PCR), without further purification. 150
b) Examination of the small subunit ribosomal RNA (srRNA) gene V4 region 151
PCR amplification of the V4 region 152
Microfuge tubes should be prepared containing all PCR reagents except template and Taq 153
polymerase. These reagents are: 1 Buffer, 1.5 mM MgCl
2
, 200 M of each dNTP, 1 M of 154
each primer (5-CTA-TTG-GAG-GGC-AGT-CT-3 and 5-CTT-TTC-AGG-CAA-CAA-GG- 155
3) and dH
2
O. Reagents should be overlaid with mineral oil. An aliquot of 2.5 l lysate is added 156
to the reaction mixture and the tubes are incubated at 95C for 5 minutes. Then 1 unit Taq 157
polymerase is added and the tubes are subjected to 28 cycles of 94C for 1 minute, 50C for 158
30 seconds and 72C for 30 seconds. 159
A 4-l aliquot is examined by agarose gel electrophoresis and the presence of a 358 bp PCR 160
product should be confirmed. Any remaining PCR solution is decanted into a sterile microfuge 161
tube and stored at 4C. If this PCR product is to be used in hybridisation with DNA probes, it 162
should be applied to membranes within 2 days of PCR amplification. 163
c) DNA probe detection of Gyrodactylus salaris 164
Probe labelling and detection are carried out according to the protocols in the DIG System 165
Users Guide using DIG labelling and detection kits. 166
i) Control PCR products amplified from G. salaris, G. derjavini and G. truttae should be 167
included on all membranes. Denature the DNA before applying to the membranes. 168
Denaturation is achieved by heating 10-l aliquots of each PCR reaction in 0.5 ml 169
microfuge tubes in a boiling water bath or a dry block-heater at 99C for 10 minutes, and 170
placing on ice at once. 171
Immediately spot 1 l of each solution on to each of three positively charged nylon 172
membranes already marked to allow identification of samples. Bake membranes at 120C 173
for 30 minutes and store at room temperature until hybridisation is carried out. The 174
remainder of the PCR solution should be stored at 4C until diagnosis has been made. 175
ii) Label probes GsV4B (5-GTG-AAT-TGA-TTT-CGG-A-3), GdV4 (5-GGG-TTT- 176
CGG-CCT-TGT-3) and GtV4 (5-GTC-TAC-ACT-TTC-GGA-3) with dioxigenin-11- 177
ddUTP using the reagents and protocol supplied with the DIG Oligonucleotide 3-end 178
Labelling Kit. Estimate the yield of labelled oligonucleotide by comparing the signal 179
intensities with those produced by labelled control DNA. 180
iii) Place replicate membranes in separate hybridisation bottles and incubate in 20 ml 181
hybridisation buffer for 1 hour at 30C in a hybridisation oven. Dilute labelled 182
oligonucleotides in hybridisation buffer to a concentration of 10 pmol/ml. Discard 183
prehybridisation solution and replace with 6 ml of probe solution (10 pmol/ml). Each 184
membrane is incubated with a different probe at 30C for 3.5 hours. Following 185
hybridisation, decant the probe solutions and store at 20C for re-use. Wash membranes 186
at room temperature twice for 5 minutes each in 2 SSC (standard saline citrate), 0.1% 187
(w/v) SDS (sodium dodecyl sulfate), and twice for 15 minutes each in 0.1 SSC, 0.1% 188
(w/v) SDS at 30C. 189
iv) Chemiluminescent detection of bound probe is carried out using reagents and the 190
protocol given in DIG Chemiluminescent Detection Kit and Wash and Block Buffer Set. 191
v) Species are identified by comparing the signals from diagnostic samples with signals from 192
control samples of G. salaris, G. derjavini and G. truttae. Gyrodactylus salaris, G. teuchis and 193
G. thymalli will all react positively with the probe GsV4B and thus cannot be distinguished 194
by this method. 195
d) srRNA gene V4 region sequencing 196
Amplified V4 DNA prepared as in Section 2.2.b above may be sequenced and this sequence is 197
compared with those of G. salaris, G. derjavini, G. truttae and G. teuchis EMBL nucleotide 198
database accession numbers Z26942, Z35128, Z35129 and AJ249349, respectively. 199
e) Examination of the ribosomal RNA gene internal transcribed spacer region 200
i) PCR amplification of the internal transcribed spacer (ITS) 201
Microfuge tubes should be prepared containing all PCR reagents except template and Taq 202
polymerase. These reagents are: 1 Buffer, 1.5 mM MgCl
2
, 200 M of each dNTP, 1 M 203
of each primer (5-TTT-CCG-TAG-GTG-AAC-CT-3 and 5-TCC-TCC-GCT-TAG- 204
TGA-TA-3) and dH
2
O. Reagents should be overlaid with mineral oil. An aliquot of 205
2.5 l lysate is added to the reaction mixture and the tubes are incubated at 95C for 206
5 minutes. Then 1 unit Taq polymerase is added and the tubes are subjected to 30 cycles 207
of 94C for 1 minute, 50C for 1 minute and 72C for 1 minute. 208
A 4-l aliquot is examined by agarose gel electrophoresis and the presence of a 1300 bp 209
PCR product should be confirmed. 210
ii) Restriction enzyme digestion of ITS 211
An aliquot of 715 l PCR product (approximately 100400 ng) is digested with 212
approximately 2 units HaeIII enzyme. Tubes containing DNA, enzyme, and appropriate 213
buffer are incubated at 37C for at least 1.5 hours. The digested DNA is then subjected to 214
agarose gel electrophoresis in a 1.5% (w/v) gel alongside controls of HaeIII-digested ITS 215
from G. salaris, G. derjavini, G. truttae and G. teuchis. ITS from G. salaris produces HaeIII 216
fragments of 513, 399, 234 and 154 bp. 217
f) ITS region sequencing 218
Amplified ITS prepared as in Section 2.2.e.i. above may be sequenced and this sequence 219
compared with those of G. salaris, G. derjavini, G. truttae and G. teuchis EMBL nucleotide 220
database accession numbers Z72477, AJ132259, AJ132260 and AJ249350, respectively. 221
REFERENCES 222
1. CUNNINGHAM C.O. (1997). Species variation within the internal transcribed spacer (ITS) region of 223
Gyrodactylus (Monogenea; Gyrodactylidae) ribosomal RNA genes. J. Parasitol., 83, 215219. 224
2. CUNNINGHAM C.O., MCGILLIVRAY D.M., MACKENZIE K. & MELVIN W.T. (1995). Identification of 225
Gyrodactylus (Monogenea) species parasitizing salmonid fish using DNA probes. J. Fish Dis., 18, 539 226
544. 227
3. CUNNINGHAM C.O., MCGILLIVRAY D.M., MACKENZIE K. & MELVIN W.T. (1995). Discrimination 228
between Gyrodactylus salaris, G. derjavini and G. truttae (Platyhelminthes: Monogenea) using restriction 229
fragment length polymorphisms and an oligonuleotide probe within the small subunit ribosomal 230
RNA gene. Parasitology, 111, 8794. 231
4. CUNNINGHAM C.O., MO T.A., COLLINS C.M., BUCHMANN K., THIERY R., BLANC G. & LAUTRAITE 232
A. (2001). Redescription of Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 233
(Monogenea: Gyrodactylidae), a species identified by ribosomal RNA sequence. Syst. Parasitol., 48, 234
141150. 235
5. MALMBERG G. (1957). Om frekomsten av Gyrodactylus p svenska fiskar. Skr. sd. Sver. FiskFr., 236
(rsskr.) 1956, 1976. (In Swedish, species descriptions and summary in English). 237
6. MALMBERG G. (1970). The excretory systems and the marginal hooks as a basis for the systematics 238
of Gyrodactylus (Trematoda, Monogenea). Ark. Zool. Ser. 2, 23, 1235. 239
7. MO T.A. (1991). Seasonal variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 240
(Monogenea: Gyrodactylidae) on parr of Atlantic salmon Salmo salar L. in the River Batnfjordselva, 241
Norway. Syst. Parasitol., 19, 231240. 242
8. MO T.A. (1991). Variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 243
(Monogenea: Gyrodactylidae) on rainbow trout Oncorhynchus mykiss (Walbaum, 1792) in a fish farm, 244
with comments on the spreading of the parasite in south-eastern Norway. Syst. Parasitol., 20, 19. 245
9. MO T.A. (1991). Variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 246
(Monogenea: Gyrodactylidae) on parr of Atlantic salmon Salmo salar L. in laboratory experiments. 247
Syst. Parasitol., 20, 1119. 248
* 249
* * 250
C HA P T E R 2 . 1 . 1 5 .

RE D S E A BRE AM I RI DOVI RAL DI S E AS E

SUMMARY
Red sea bream iridoviral disease (RSIVD) is a significant cause of mortality among cultured marine fish
(1). The causative agent is the red sea bream iridovirus (RSIV) (1, 4, 12). Overt infections have been
recognised not only in red sea bream (Pagrus major), but also among other cultured marine fish including
yellowtail (Seriola quinqueradiata), sea bass (Lateolabrax sp.) and Japanese parrotfish (Oplegnathus
fasciatus) (5). Currently, RSIVD has only been reported from cultured marine fish in Japan, though a
serologically and genetically related iridovirus has been isolated from brown-spotted grouper (Epinephelus
malabaricus) from Thailand. This latter isolate did not induce disease in experimentally exposed red sea
bream (2, 6, 7).
The first outbreak of an RSIVD was recorded in cultured red sea bream in Shikoku Island, Japan in
1990 (1). Since 1991, the disease has produced mass mortalities in cultured fish populations in the western
part of Japan, mainly among juvenile red sea bream (5). However, mortality of market-sized fish has also
been reported.
Affected fish are lethargic, exhibit severe anaemia, petechiae of the gills, and enlargement of the spleen (1, 2,
7). The disease is characterised by the appearance of enlarged cells stained deeply with Giemsa solution on
microscopic observation of tissue sections of the spleen, heart, kidney, liver and gills of infected fish (1).
A similar disease has also seriously damaged stocks of more than 20 species of cultured marine fish in
18 prefectures located in south-western part of Japan. The infected fish include fish belonging to the orders
Perciformes, Pleuronectiformes and Tetradontiformes (5).
The principal mode of transmission of RSIVD is by horizontal means via the water.
The RSIV showed some weak cross-reactivity in indirect fluorescent antibody tests (IFAT) using polyclonal
rabbit anti-RSIV serum with the systemic Ranaviruses resembling epizootic haematopoietic necrosis virus
(EHNV) or FV-3 (11). However, monoclonal antibodies (MAbs) against RSIV (13) do not recognise
the systemic ranaviruses by IFAT, and two members of the systemic Ranavirus group (EHNV and
European catfish virus) did not infect red sea bream after experimental challenges (7).
Diagnostic methods, such as the observation of stained impression smears or tissue sections, an
immunofluorescence test with an MAb, and a polymerase chain reaction, have been reported for RSIV (3,
8, 11, 14, 15).
Control methods currently rely on the implementation of hygiene practices at the farm. A commercial vaccine
may soon be available for RSIVD in red sea bream (911). Vaccination for other marine fish species is at
the experimental stage.
The diagnosis of red sea bream iridoviral disease (RSIVD) is based on direct methods, which are either the
isolation of red sea bream iridovirus (RSIV) in cell culture followed by its identification with anti-RSIV
monoclonal antibodies (MAbs) or direct demonstration of RSIV antigens in infected fish tissue using
MAbs.
Chapter 2.1.15. Red sea bream iridoviral disease
Clinically affected fish: The kidney and spleen tissues from fish of any size suffering characteristic
signs of the disease.
Asymptomatic fish (apparently healthy fish): Kidney and spleen tissues.
1. STANDARD SCREENING METHOD FOR RSIVD
1.1. Isolation of RSIV in cell culture
Cell line to be used: GF cells
Cells should be grown at 25C in a temperature-controlled incubator to ensure subsequent success
in the isolation of RSIV.
i) Make an additional tenfold dilution of the 1/10 spleen homogenate supernatants and
2
dilution.
ii) Allow to adsorb for 0.51 hour at 25C and, without withdrawing the inoculate, add the
cell culture medium buffered at pH 7.6 and supplemented with 2% fetal calf serum (FCS)
(1 ml/well for 24-well cell culture plates), and incubate at 25C using a temperature-
controlled refrigerated incubator to ensure successful isolation.
daily microscopic examination at from 40 to 100 magnification for 10 days. The use of
a phase-contrast microscope is recommended.
ii) If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of
tested homogenate supernatants, identification procedures have to be undertaken
immediately (see below).
approved previously) from which the virus positive sample originated. The suspension of approved
status will be maintained until it is demonstrated that the virus in question is not RSIV.
iii) If no CPE develops in the inoculated cultures (despite normal progression of CPE in the
the virus control fail to develop CPE, the process should be repeated with fresh
1.2. Identification of RSIV
RSIV cannot be identified by neutralisation tests as the antisera generated by the immunisation of
rabbits have few neutralising antibodies.
The indirect fluorescent antibody tests (IFAT) is to be conducted directly after virus isolation
in cell culture
i) Prepare monolayers of cells on cover-slips in order to reach around 80% confluency,
which is usually achieved within 24 hours of incubation at 25C. The FCS content of cell
culture medium can be reduced to 24%.
ii) When the cell monolayers are ready for infection inoculate the virus suspension to be
identified by making tenfold dilution steps directly in the cell culture wells or flasks.
iii) Incubate at 25C for 2472 hours.
iv) When CPE appears, remove the cell culture medium, rinse three times with phosphate
buffered saline (PBS). Air-dry the infected cells, then fix with cold acetone (stored at
20C) for 10 minutes.
v) Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or
freeze at 20C.
vi) Prepare a solution of MAb (M10) to RSIV.
vii) Treat the cell monolayers with the solution of antibody to RSIV for 30 minutes at 37C in
a humid chamber.
viii) Rinse the cells three times for 5 minutes with PBS.
ix) Incubate with a suitable FITC-conjugated specific antibody for 30 minutes at 37C in
darkness in a humid chamber (FITC = fluorescein isothiocyanate).
x) Rinse three times for 5 minutes with PBS.
xi) Examine the treated cell monolayers on plastic plates immediately, or mount the cover-
xii) Examine under incident UV light using a microscope with 10 eye pieces and 20 to 40
objective lens having numerical aperture >0.65 and >1.3, respectively. Positive and
observation. Positive results are indicated by diffuse fluorescence throughout the
cytoplasm.
2. DIAGNOSTIC METHODS FOR RSIVD
As in Section 1.1 and 1.2.
ii) Make spleen imprints on the cleaned glass.
iii) Store the spleen pieces together with the other organs required for virus isolation in case this
becomes necessary later.
iv) Allow the imprints to air-dry for 20 minutes.
v) Fix with cold acetone.
vi) Treat the imprints with the solution of MAb (M10) to RSIV for 30 minutes at 37C.
vii) Rinse three times with PBS.
viii) Treat the imprints for 30 minutes at 37C with a solution of FITC-conjugated antibody to
the immunoglobulin used in step vi and prepared according to the instructions of the
supplier. These FITC antibodies are most often rabbit or goat antibodies.
ix) Rinse three times with PBS.
x) Mount the cover-slips using glycerol saline prior to microscopic observation.
xi) Examine under indirect UV light using a microscope with 10 eye pieces and 20 to 40
objective lens having high numeral aperture. Positive and negative controls must be found to
give the expected results prior to any other observation.
2.3. Polymerase chain reaction amplification
laboratory.
b) Polymerase chain reaction amplification and sequencing
RSIV has a large double-stranded DNA genome. Two primers, a forward primer (5-CGG-
GGG-CAA-TGA-CGA-CTA-CA-3) and reverse primer (5-CCG-CCT-GTG-CCT-TTT-
CTG-GA-3) are used for the amplification of the gene (568 bases) sequence of RSIV DNA by
polymerase chain reaction (PCR).
Fish samples are prepared as described in Kurita et al. (3). Nucleic acid (1 l) is added to Taq
polymerase buffer containing 1 mM of each primer, 200 mM of deoxynucleotide triphosphate,
1.25 U of ExTaq DNA polymerase in 20 mM Mg
2+
PCR buffer. The mixture is incubated in
an automatic thermal cycler programmed for 30 cycles at 94C for 30 seconds, 58C for
60 seconds, and 72C for 60 seconds, and finally held at 72C for 5 minutes. Amplified DNA
(568 bp) is analysed by agarose gel electrophoresis.
REFERENCES
1. INOUYE K., YAMANO K., MAENO Y., NAKAJIMA K., MATSUOKA M., WADA Y. & SORIMACHI M.
(1992). Iridovirus infection of cultured red sea bream, Pagrus major. Fish Pathol., 27, 1927.
2. JUNG S., MIYAZAKI T., MIYATA M., DANAYADOL Y. & TANAKA S. (1997). Pathogenicity of
iridovirus from Japan and Thailand for the red sea bream Pagrus major in Japan, and histopathology of
experimentally infected fish. Fisheries Sci., 63, 735740.
3. KURITA J., NAKAJIMA K., HIRONO I. & AOKI T. (1998). Polymerase chain reaction (PCR)
amplification of DNA of red sea bream iridovirus (RSIV) Fish Pathol., 33, 1723.
4. KUSUDA R., NAGATO K. & KAWAI K. (1994). Characteristics of an iridovirus isolated from red sea
bream, Pagrus major. Suisanzoshoku, 42, 151156.
5. MATSUOKA S., INOUYE K. & NAKAJIMA K. (1996). Cultured fish species affected by red sea bream
iridoviral disease from 1991 to 1995. Fish Pathol., 31, 233234.
6. MIYATA M., MATSUNO K., JUNG S.J., DANAYADOL Y. & MIYAZAKI T. (1997). Genetic similarity of
iridoviruses from Japan and Thailand. J. Fish Dis., 20, 127134.
7. NAKAJIMA K. & MAENO Y. (1998). Pathogenicity of red sea bream iridovirus and other fish
iridoviruses to red sea bream. Fish Pathol., 33, 143144.
8. NAKAJIMA K., MAENO Y., FUKUDOME M., FUKUDA Y., TANAKA S., MATSUOKA S. & SORIMACHI
M. (1995). Immunofluorescence test for the rapid diagnosis of red sea bream iridovirus infection
using monoclonal antibody. Fish Pathol., 30, 115119.
9. NAKAJIMA K., MAENO Y., HONDA A., YOKOYAMA K., TOORIYAMA T. & MANABE S. (1999).
Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test. Dis. Aquat. Org.,
36, 7375.
10. NAKAJIMA K., MAENO Y., KURITA J. & INUI Y. (1997). Vaccination against red sea bream iridoviral
disease in red sea bream. Fish Pathol., 32, 205209.
11. NAKAJIMA K., MAENO Y., YOKOYAMA K., KAJI C. & MANABE S. (1998). Antigen analysis of red sea
bream iridovirus and comparison with other fish iridoviruses. Fish Pathol., 33, 7378.
12. NAKAJIMA K. & SORIMACHI M. (1994). Biological and physicochemical properties of the iridovirus
isolated from cultured red sea bream, Pagrus major. Fish Pathol., 29, 2933.
13. NAKAJIMA K. & SORIMACHI M. (1995). Production of monoclonal antibodies against red sea bream
iridovirus. Fish Pathol., 30, 4752.
14. OSHIMA S., HATA J., HIRASAWA N., OHTAKA T., HIRONO T., AOKI T. & YAMASHITA S. (1998).
Rapid diagnosis of red sea bream iridovirus infection using the polymerase chain reaction. Dis. Aquat.
Org., 32, 8790.
15. OSHIMA S., HATA J., SEGAWA C., HIRASAWA N. & YAMASHITA S. (1996). A method for direct DNA
amplification of uncharacterized DNA viruses and for development of a viral polymerase chain
reaction assay: Application to the red sea bream iridovirus. Anal. Biochem., 242, 1519.
*
* *
C HA P T E R 2 . 1 . 1 6 .

WHI T E S T URGE ON I RI DOVI RAL DI S E AS E

SUMMARY
The white sturgeon iridoviral disease (WSIVD) is a significant cause of mortality among farm-raised
juvenile white sturgeon (Acipenser transmontanus) in North America and among Russian sturgeon
(A. guldenstadi) in Europe (1, 2, 4). White sturgeon are the host from which the causative agent, the
white sturgeon iridovirus (WSIV) was first isolated. Lake sturgeon (A. fluvescens) have been
experimentally infected with the WSIV, but the susceptibility of other sturgeon is currently unknown (3).
The original description of white sturgeon iridovirus was among the first hatchery-raised white sturgeon in
North America. The source of the virus was assumed to originate from captive wild sturgeon adults collected
from the Sacramento River in California, United States of America (USA) for use as broodstock (2). The
WSIV has also been detected in cultured white sturgeon from the lower Columbia River in Oregon and
Washington states of the USA, the Snake River in the southern region of the State of Idaho and the
Kootenai River in northern Idaho, USA (4, 5). These cultured white sturgeon were all progeny originating
from captured wild adult white sturgeon. The virus has been detected among wild juvenile white sturgeon
collected from the lower Columbia River and the virus is potentially enzootic in wild white sturgeon
populations throughout the Pacific Northwest of North America (4). An iridovirus similar to WSIV has
been identified in Russian sturgeon from Northern Europe where it may be enzootic among cultured
populations of several species of sturgeon (1).
The WSIV is an epitheliotropic virus infecting the skin, gills, and upper alimentary tract. Infections of the
oral mucosa and olfactory organ epithelium are presumed causes of the cessation of feeding that leads to a
progressive emaciation or starvation of the fish the principal external sign of the disease (6). Cumulative
mortality of up to 95% has been reported among groups of infected fish in the hatchery and secondary
infections with external protozoa or bacteria often contribute to the overall mortality (2). Infected fish with
moderate to severe emaciation began dying 23 weeks following exposure to the virus at water temperatures
of 1719C (7). Haemorrhages on the abdomen and the ventral scuta may be present, but these are not
specific for WSIVD. There are no specific internal signs of infection as the virus does not invade
systemically. Viral infection is evident on microscopic observation of stained tissue sections from infected fish.
Areas of the integument and particularly the skin may show a focal to diffuse hyperplasia with characteristic
amphophilic to basophilic enlarged Malpighian cells (6). These cells are filled with virus particles as
demonstrated by electron microscopy. The virus can be isolated, but with some difficulty, from infected fish
using sturgeon cell lines.
The modes of transmission of WSIV are not completely understood but horizontal transmission via the
water has been demonstrated in the hatchery and experimentally in the laboratory (3). There is strong
circumstantial evidence from epidemiological investigations at the hatchery that the virus is transmitted
vertically from adult broodstock, but the virus has never been isolated or observed in adult fish.
There appears to be little antigenic relationship of WSIV to the systemic iridoviral agents represented by
epizootic haematopoietic necrosis virus or the red sea bream iridovirus. The larger size and inner membrane
structure of WSIV virion, host cell-line specificity, type of cytopathic effect, and location of target host cells
(epitheliotropic and not systemic) distinguish the agent from other groups of fish iridoviruses. Although
lymphocystivirus (LCDV) has a similar virion morphology, LCDV infects fibroblasts and not
Malpighian cells as in WSIV infections, and the cell line specificity and types of cytopathic effects are clearly
different between the two agents.
Chapter 2.1.16. White sturgeon iridoviral disease
The principal diagnostic methods for WSIV include microscopic observation of characteristic infected cells in
stained tissue sections of the oral mucosa, gills and skin, or isolation of the virus in sturgeon cell lines (3).
Neutralising polyclonal antibodies and binding monoclonal antibodies recognise WSIV, but not the systemic
iridoviral agents. These antibodies can also be used in indirect immunofluorescence tests or for
immunohistochemical staining of infected cells in tissue culture and sections of infected tissues.
Control methods currently rely on avoidance of the agent where possible. Because there are currently no
methods for detecting the virus in adult broodstocks, quarantine and investigation of juveniles suffering
mortality are the principal means to detect WSIV in young fish. Methods to detect the virus in broodstock
are currently under development.
The diagnosis of white sturgeon iridovirus (WSIV) is based on the observation of pathognomonic infected
cells in stained tissue sections from infected fish and isolation of the virus using sturgeon cell lines.
Confirmation of WSIV infection relies on neutralisation of the isolated virus with polyclonal antibodies.
The specific binding of monoclonal antibodies (MAbs) to viral antigens in infected cell cultures or
impression smears from infected fish tissues are under investigation as alternatives to cell culture isolation
and virus neutralisation for identification of WSIV.
Clinically affected fish: Fish <6 cm: 1) for virus isolation gill arches and skin from the fleshy
portions of the mouth (oral flap) and fins; 2) for histology a sagittal section to include gills and the
epidermis of the head region, also include larger fins. For larger fish portions of the gill and fleshy
part of the oral flap and punches or portions from fleshy part of larger fins. These tissues can be used
for both virus isolation and histological examinations.
Asymptomatic fish (apparently healthy fish): As above.
1. STANDARD SCREENING METHOD FOR WSIV
1.1. Isolation of WSIV in cell culture
Cell lines to be used: WSS-2 (white sturgeon spleen) or WSSK-1 (white sturgeon skin) cells
Cells should be grown at 20C in a temperature-controlled incubator using standard minimal
essential medium (MEM) supplemented with 10% fetal calf serum (FCS) that can be reduced to
5% after virus inoculation.
i) Remove a portion of the gill, oral flap, and abdomen and prepare a supernatant from a
homogenate to yield a final tissue dilution of 1/50. Prepare a second dilution representing
1/100 (w/v). Transfer an appropriate volume of each of the two dilutions on to 24-hour-
old cell monolayers. Inoculate at least 2 cm
2
dilution.
ii) Allow to adsorb for 0.51 hour at 20C and, without withdrawing the inoculate, add the
cell culture medium buffered at pH 7.6 and supplemented with 5% FCS (2 ml/well for
12-well cell culture plates), and incubate at 20C using a temperature-controlled
refrigerated incubator to ensure successful isolation.
i) Follow the course of infection in inoculated and control cell cultures by daily microscopic
examination at 40 to 100 magnification for 30 days.
ii) If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of
tested homogenate supernatants, identification procedures have to be undertaken
approved previously) from which the virus positive sample originated. The suspension of approved
status will be maintained until it is demonstrated that the virus in question is not WSIV.
iii) If no CPE develops in the inoculated cultures (despite normal progression of CPE in the
virus controls), the inoculated cultures should be subcultured for a further 15 days.
Should the virus control fail to develop CPE, the process should be repeated with fresh
each supernatant of tissue homogenates.
iii) Incubate and monitor as described above in Section 1.1.b.
1.2. Identification of WSIV
1
to 10
3
.
iii) Mix aliquots (for example 200 l) of each virus dilution with equal volumes of rabbit anti-
WSIV serum diluted 1/100, and similarly treat aliquots of each virus dilution with cell
culture medium.
iv) Incubate all mixtures at 20C for 1 hour.
v) Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell
cultures per dilution) and allow adsorption to occur for 0.51 hour at 20C; 24- or 12-well
culture plates are suitable for this purpose, using a 50 l inoculum.
vi) When adsorption is completed, add cell culture medium supplemented with 5% FCS and
buffered to pH 7.47.6 into each well and incubate at 20C.
This indirect fluorescent antibody test (IFAT) is to be conducted directly after virus isolation
in cell culture.
i) Prepare monolayers of WSS-2 cells in 2 cm
2
wells of cell culture plastic plates or on
cover-slips in order to reach around 80% confluency. The FCS content of the cell culture
medium can be reduced to 5%.
ii) When the cell monolayers are ready for infection, inoculate the virus suspensions to be
identified by making tenfold dilution steps directly in the cell culture wells or flasks.
iii) Dilute the control virus suspension of WSIV in a similar way, in order to obtain a virus
titre of about 1000 TCID
50
(50% tissue culture infective dose) per ml in the cell culture
medium.
iv) Incubate at 20C for 7 days
pH 7.2, then three times briefly with cold fixative. This fixative will be acetone (stored at
20C) for cover-slips or a mixture of acetone 30%/ethanol 70% (v/v) for plastic, also
stored at 20C.
2
of cell
monolayer.
vii) Allow the cell monolayers to air dry for at least 30 minutes and process immediately or
freeze at 20C.
viii) Prepare a solution of MAbs to WSIV in 0.01 M PBS, pH 7.2, containing 2% skim milk, at
the appropriate dilution.
ix) Rehydrate the dried cell monolayers by four rinsing steps with PBS and remove this
buffer completely after the last rinsing.
2

well.
xi) Rinse four times with PBS as above.
xii) Incubate the cell monolayers in appropriately diluted biotinylated anti-mouse antibody (in
2% skim milk in PBS) at 37C for 1 hour.
xiii) Rinse four times with PBS as above.
xiv) Treat the cell monolayers for 1 hour at 37C with a solution of FITC conjugate (FITC =
fluorescein isothiocyanate).
xv) Rinse four times with PBS.
xvi) Examine the treated cell monolayers on plastic plates immediately, or mount with cover-
xvii) Examine under incident UV light using a microscope with 10 eye pieces and 20
observation. Positive results are indicated by diffuse fluorescence throughout the
cytoplasm.
2. DIAGNOSTIC METHODS FOR WSIV
Confirmation of WSIV outbreaks depends on the observation of clinical signs of disease among
affected sturgeon, presence of pathognomonic infected cells in stained tissue sections or isolation of
the virus in cell culture followed by its identification in a neutralisation test. Immunostaining of cells in
target tissues with antibodies to WSIV can replace the need to conduct electron microscopy to view the
virioins of WSIV.
2.1. Virus isolation and subsequent identification
2.2. Observation of infected cells in stained tissue sections
a) For fish <6 cm in length, preserve the entire fish in histological fixative
i) Prepare sagittal sections (off midline to include gills) of entire fish for hematoxylin and
eosin staining.
ii) Observe the epithelium of the gills, fins (pectoral and caudal) and body including the
mouth and esophagus, for evidence of enlarged cells with an amphophilic to basophilic
staining cytoplasm and a hypertrophic irregular nucleus.
b) For fish >6 cm in length, remove one gill arch and a 1 cm
2
portion of the fleshy section of mouth (oral
flap), and a punch sample and a similar sized piece of skin from the abdomen.
Fix tissues and prepare stained tissue sections for observation of characteristic infected cells as
described above.
2.3. Immunohistochemical test
Begin with tissues fixed in 10% neutral buffered formalin used for histological examinations
above.
i) Deparaffinise sections and rehydrate
a. Xylene-1 for 5 minutes
b. Xylene-2 for 5 minutes
1

c. 100% EtOH for 3 minutes
2
. Ring tissue area with a PAP pen
3

d. 95% EtOH for 3 minutes
e. 70% EtOH for 3 minutes
f. Rinse in deionised water for 3 minutes
ii) Inactivate endogenous peroxidase by soaking slides in 0.3% H
2
O
2
in methanol for
30 minutes at room temperature.
iii) Rinse slide with water then gently wash with PBS.
iv) Block for 30 minutes in PBS with 10% goat serum.
v) Shake off block solution and add 20 l of biotinylated IIIA11 or IIC7 mouse anti-WSIV
MAb in PBS (3 g/ml) to the section and incubate for 60 minutes at room temperature.
vi) Wash three times.
vii) Add peroxidase-streptavidin (1/1000) in PBS for 10 minutes.
viii) Wash three times.
ix) Stain with AEC
4
(aminoethyl carbazole) for 515 minutes at 25C. Monitor the level of
development by viewing the slide on a microscope. Stop development by immersing in
PBS.
x) Wash three times.
xi) Counterstain with Mayers haematoxylin
5
for 5 minutes at room temperature.
xii) Wash three times.
xiii) Mount slide while wet with Crystal/Mount
TM
aqueous/dry mounting medium
6
. (AEC
substrate is soluble with toluene or organic based mounting media.) After drying
overnight, a cover-slip may be mounted on the section using an organic solvent-based
mounting media such as Krystalon Mounting Media
7
.

1 At the end of a staining session, discard the first depariffinisation xylene. For the following session use xylene 2 as the initial
xylene and use fresh xylene in the second step.
2 DAKO protocol calls for 3 minutes 2 changes for each alcohol bath. Tech service indicates that this is excessive. Other
protocols call for 13 changes for 13 minutes each.
3 PAP Pen order from The Binding Site Inc., San Diego, CA. 1-800-633-4484.
4 AEC Substrate. Zymed AEC Substrate Kit.
5 Mayers haematoxylin solution. 0.1% soln. Sigma diagnostics, Cat#MHS-1.
6 Crystal/Mount aqueous/dry mounting medium. Biomeda Corp. Cat # MO3.
7 Krystalon Mounting Media, EM Science, 64969/95.
PBS 2 litres of 10
1. 160 g. NaCl (0.137 M)
2. 4.0 g. KCl (0.003 M)
3. 28.8 g. Na
2
HPO
4
(0.01 M)
4. 4.8 g. KH
2
PO
4
(0.002 M)
5. pH to 7.27.4 and Q.S. to 2 litres with d.d. H
2
O.
From Antibodies A Laboratory Manual. Harlow & Lane, 1988.
Tris buffered saline (TBS)

1 litre 1 2 litres 10
50 mM Tris base 6.07 g 121.1 g.
1 mM EDTA 0.409 g 8.18 g
150 mM NaCl 8.7 g 174 g.
pH to 8.0 with HCl ~2.5 ml conc. ~50 ml conc.
TTBS
To make TTBS add 0.1% Tween-20 (1.0 ml/litre).
REFERENCES
1. ADKISON M.A., CAMBRE M. & HEDRICK R.P. (1998). Identification of an iridovirus in Russian
sturgeon Acipenser guldenstadi from Northern Europe. Bull. Eur. Assoc. Fish Pathol., 18, 2932.
2. HEDRICK R.P., GROFF J.M., MCDOWELL T.S. & WINGFIELD W.H. (1990). An iridovirus infection of
the integument of the white sturgeon Acipenser transmontanus. Dis. Aquat. Org., 8, 3944.
3. HEDRICK R.P, MCDOWELL T.S., GROFF J.M., YUN S. & WINGFIELD W.H. (1992). Isolation and
properties of an iridovirus-like agent from white sturgeon Acipenser transmontanus. Dis. Aquat. Org. 12,
7581.
4. LAPATRA S.E., GROFF J.M., JONES, G.R., MUNN B., PATTERSON T.L., HOLT R.A., HAUCK A.K. &
HEDRICK R.P. (1994). Occurrence of white sturgeon iridovirus infections among cultured white
sturgeon in the Pacific Northwest. Aquaculture, 126, 201210.
5. LAPATRA S.E., IRELAND S., GROFF J.M., CLEMENS K. & SIPLE J. (1999). Adaptive disease
management strategies for the endangered population of Kootenai River white sturgeon Acipenser
transmontanus. Fisheries, 24, 613.
6. WATSON L.R., GROFF J.M. & HEDRICK R.P. (1998). Replication and pathogenesis of white sturgeon
iridovirus (WSIV) in experimentally infected white sturgeon Acipenser transmontanus juveniles and
sturgeon cell lines. Dis. Aquat. Org., 32, 173184.
7. WATSON L.R., MILANI A. & HEDRICK R.P. (1998). Effects of water temperature on experimentally-
induced infections of juvenile white sturgeon (Acipenser transmontanus) with the white sturgeon
iridovirus (WSIV). Aquaculture, 166, 213228.
*
* *
S E C T I ON 3 . 1 .


C HA P T E R 3 . 1 . 1 .

BONAMI OS I S
( Bo n a mi a e x i t i o s u s , B. o s t r e a e
a n d Mi k r o c y t o s r o u g h l e y i )
GENERAL INFORMATION
Bonamiosis here refers only to the diseases in oysters caused by Bonamia ostreae in the Northern
Hemisphere, and by Bonamia exitiosus and Mikrocytos roughleyi in the Southern Hemisphere. If
detected outside the known range of Mikrocytos roughleyi and Bonamia spp., electron microscopy or
DNA-based assays, when available, must be used to identify and distinguish the detected organism from
other microcell species (Bonamia ostreae, Bonamia exitiosus, Mikrocytos mackini and
M. roughleyi). The presence of these pathogens in any bivalve should be regarded as potentially serious and
the OIE Reference Laboratory should be consulted.
Bonamiosis is caused by the haplosporidians Bonamia ostreae, B. exitiosus and Mikrocytos roughleyi
(35, 8, 11, 17, 21). Bonamiosis is also known as microcell disease, haemocyte disease of flat oysters (B.
ostreae), haemocyte disease of dredge oysters (Bonamia exitiosus) or winter mortality (Mikrocytos
roughleyi).
Bonamia ostreae occurs naturally in Ostrea edulis and O. conchaphila (= O. lurida), and can infect
O. puelchana, O. angasi and Ostrea chilensis (= Tiostrea chilensis, T. lutaria) when moved into
endemic zones (12, 13, 20). Bonamia exitiosus occurs in O. angasi, O. denselammellosa and
T. chilensis (8). Species of Ostrea and Tiostrea and other ostreids (Crassostrea rivularis =
C. arakensis) (6) should be considered to be potentially susceptible to Bonamia spp. Mikrocytos
roughleyi infects the Sydney rock oyster Saccostrea glomerata (commercialis).
The geographical distribution of B. exitiosus is: New Zealand (around South Island and lower North
Island) (9) and Australia (Western Australia, Victoria and Tasmania). Bonamia ostreae has been
reported from France, Ireland, Italy, the Netherlands, Spain, the United Kingdom (excluding Scotland),
and the United States of America (California, Maine and Washington State) (2, 10, 18, 19, 22, 23),
but is now thought to be absent from Denmark. Mikrocytos roughleyi occurs in New South Wales,
Australia.
Bonamiosis is a lethal infection of the haemocytes of flat oysters, sometimes accompanied by yellow
discoloration and extensive lesions on the gills and mantle (21). However, most of the infected oysters
appear normal. Lesions occur in the connective tissue of the gills, mantle, and digestive gland. These
intrahaemocytic protistans quickly become systemic with overwhelming numbers of parasites coinciding with
the death of the oysters. In highly susceptible hosts, bonamiosis is a lethal infection of the haemocytes. Some
evidence suggests that the pathology caused by Bonamia spp. depends on which host species and population
is infected. For example, B. exitiosus in oysters from Tasmania were highly epitheliotropic and associated
with focal abscesses in some populations without evidence of systemic spread. Mikrocytos roughleyi is
Chapter 3.1.1. Bonamiosis (Bonamia exitiosus, B. ostreae, Mikrocytos roughleyi)
associated with focal abcess-type lesions in the gill, connective and gonadal tissues and alimentary tract (11).
It induces a systemic intracellular infection in the haemocytes (but never in the connective tissue cells).
Bonamia spp. may occur throughout the year, but prevalence and intensity of infection tend to increase
during the warm season (7, 12, 15). There is a seasonal variation in infection by B. ostreae with the
highest prevalence occurring in September in the Northern Hemisphere. In the Southern Hemisphere,
B. exitiosus shows the highest prevalence from January to April with the parasite being barely detectable in
September and October. The prepatent period for B. ostreae and B. exitiosus is usually 34 months but
can be up to 5 months. In the Southern Hemisphere, the disease caused by M. roughleyi is associated with
low temperatures and high salinities. It can kill up to 70% of mature Sydney rock oysters in their third
winter before marketing, and has a prepatent period of 2.5 months (11).
Bonamiosis can be transmitted experimentally by cohabitation or inoculation (14).
Examination of stained tissue sections and tissue imprints of susceptible organs are the recommended
methods for screening (1, 24). For diagnosis, the recommended guidelines for sampling are those stated in
Chapter 1.1.4 and Chapter I.2. of this Aquatic Manual. However, the disease may be not detectable by
the usual methods during the first 5 months of infection.
EXAMINATION PROCEDURES
1. SCREENING METHODS
1.1. Histology
General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. A transversal
steak is cut dorso-ventrally through the soft tissues and then immediately placed in fixative
medium, or (depending on the size of the animal) the whole animal, removed from the shell, can
be fixed with a fixative, such as Davidsons solution, to provide optimal parasite definition.
Several nonspecific stains, e.g. haematoxylineosin (H&E), enable Bonamia and Mikrocytos to be
visualised. It is recommended that a section through the cardiac cavity, digestive gland and gills be
examined per oyster.
Bonamia spp. (25 m in size) occurs within the haemocytes or freely in connective tissue or
sinuses of the gill, gut or mantle epithelium. However, the parasite has to be observed inside the
haemocytes for a positive diagnosis to be made to avoid false-positive results.
In abscesses, Mikrocytos roughleyi may be observed within the haemocytes as 12 m organisms
with spherical nucleus greater than 1 m containing bipolar or eccentric nucleolar structures.
When present, a cytoplasmic vacuole may displace the nucleus to the periphery of the cell.
Microcytos roughleyi differs from M. mackini in that all stages of M. roughleyi are present in the
haemocytes, and M. mackini occurs usually in the vesicular connective tissue cells on the periphery
of the lesions or in myocytes of the adductor muscle and occasionally within haemocytes.
1.2. Cytological examination: tissue imprints
Make impression smears of oyster spat or heart tissue (preferably ventricle) on a histological slide.
The slides are air-dried and fixed in methanol. The prepared imprints are stained using a
commercially available blood-staining kit, in accordance with the manufacturers instructions.
After staining, the slides are rinsed in tap water, allowed to dry completely in cold or warm air, and
mounted with a cover-slip using an appropriate synthetic resin.
The parasite (25 m in size) has basophilic cytoplasm and an eosinophilic nucleus (colours may
vary with stain used). It may be observed inside or outside the haemocytes. An observation time
of 10 minutes per slide is sufficient (under oil immersion, 8001000 magnification). The
organisms are enlarged by this method compared with those observed in histology. Heart smears
are inappropriate in stocks where the infection, if present, remains largely epitheliotropic.
2. PRESUMPTIVE DIAGNOSTIC METHODS
Cytological examination and histopathology, as described above, may be used.
3. CONFIRMATORY IDENTIFICATION OF THE PATHOGEN
3.1. Transmission electron microscopy examination
Transmission electron microscopy procedures are described in Chapter I.2. of this Aquatic
Manual.
Differences exist between Bonamia exitiosus forms and B. ostreae: there are fewer haplosporosomes,
mitochondrial profiles and lipoid bodies in dense forms of B. ostreae, smaller nucleus and
cytoplasm ratio (8, 17, 21).
Plasmodial forms of Bonamia exitiosus are distinguished by their size (4.04.5 m), irregular cellular
and nuclear outline, amorphous cytoplasmic inclusions (multi-vesicular bodies), and arrays of
Golgi-like smooth endoplasmic reticulum. Forms of intermediate cytoplasmic density are more
electron dense than the plasmodial forms and slightly smaller in diameter (3.03.5 m).
Haplosporosomes are formed from Golgi/nuclear material complexes and are similar in
construction and structure to some viruses.
Ultrastructural morphology differentiates M. mackini from Bonamia spp.; the nucleolus of
M. mackini is located towards the centre of the nucleus while that of B. ostreae has an eccentric
location and there is an apparent lack of mitochondria in M. mackini.
REFERENCES
1. BACHERE E., DURAND J.-L. & TIGE G. (1982). Bonamia ostreae (Pichot et al., 1979) parasite de
lhuitre plate : comparaison de deux mthodes de diagnostic. Cons. Int. Exploit. Mer, 28, 110.
2. BUCKE D., HEPPER B., KEY D. & BANNISTER C.A. (1984). A report on Bonamia ostreae in Ostrea
edulis in the UK. Cons. Inter. Explor. Mer, CM 1984/K, 9, 17.
3. CARNEGIE R., BARBER B.J., CULLOTY S.C., FIGUERAS A.J. & DISTEL D.L. (2000). Development of a
PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the
Haplosporidia. Dis. Aquat. Org., 42, 199206.
4. COCHENNEC N., LE ROUX F., BERTHE F. & GERARD A. (2000). Detection of Bonamia ostreae based
on small subunit ribosomal probe. J. Invertebr. Pathol., 76, 2632.
5. COCHENNEC N., REECE K.S., BERTHE F.C.J. & HINE P.M. (2003). Revisiting Mikrocytos roughleyi
taxonomic affiliation points to the genus Bonamia (Haplosporidia). Dis. Aquat. Org., (in press).
6. COCHENNEC N., RENAULT T., BOUDRY P., CHOLLET B. & GERARD A. (1998). Bonamia-like parasite
found in the Suminoe oyster Crassostrea rivularis reared in France. Dis. Aquat. Org., 34, 193197.
7. CULLOTY S.C. & MULCAHY M.F. (1996). Season-, age-, and sex-related variation in the prevalence of
bonamiosis in flat oysters (Ostrea edulis L.) on the south coast of Ireland. Aquaculture, 144, 5363.
8. DINAMANI P., HINE P.M. & JONES J.B. (1987). Occurrence and characteristics of the haemocyte
parasite Bonamia sp. in the New Zealand dredge oyster Tiostrea lutaria. Dis. Aquat. Org., 3, 3744.
9. DOONAN I.J., CRANFIELD H.J. & MICHAEL K.P. (1994). Catastrophic reduction of the oyster,
Tiostrea chilensis (Bivalvia: Ostreidae), in Foveaux strait, New Zealand, due to infestation by the
protistan Bonamia sp. NZ J. Mar. Freshwater Res., 28, 335344.
10. ELSTON R.A., FARLEY C.A. & KENT M.L. (1986). Occurrence and significance of bonamiasis in
European flat oysters Ostrea edulis in North America. Dis. Aquat. Org., 2, 4954.
11. FARLEY C.A., WOLF P.H. & ELSTON R.A. (1988). A long-term study of microcell disease with a
description of a new genus, Mikrocytos (g. n.), and two new species, Mikrocytos mackini (sp. n.) and
Mikrocytos roughleyi (sp. n.). Fishery Bull., 86, 581593.
12. GRIZEL H. (1985). Etudes des rcentes pizooties de lhuitre plate Ostrea edulis L. et de leur impact
sur lostreiculture bretonne. Thse de doctorat, Universit des Sciences et Techniques de Languedoc,
Montpellier, France.
13. GRIZEL H., COMPS M., RAGUENNES D., LEBORGNE Y., TIGE G. & MARTIN A.G. (1983). Bilan des
essais dacclimation dOstrea chilensis sur les ctes de Bretagne. Rev. Trav. Inst. Peches Marit., 46, 209
225.
14. HERVIO D., BACHERE E., BOULO V., COCHENNEC N., VUILLEMIN V., LE COGUIC Y.,
CAILLETAUX G., MAZURIE J. & MIALHE E. (1985). Establishment of an experimental infection
protocol for the flat oyster Ostrea edulis with the intrahaemocytic protozoan parasite Bonamia ostreae:
application in the selection of parasite-resistant oyster. Aquaculture, 132, 183194.
15. HINE P.M. (1991). The annual pattern of infection by Bonamia sp. in New Zealand flat oysters,
Tiostrea chilensis. Dis. Aquat. Org., 11, 163171.
16. HINE P.M., BOWER S.M., MEYER G.R., COCHENNEC-LAUREAU N. & BERTHE F. (2001).
Ultrastructure of Mykrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp.
and Ostrea spp. in British Columbia, Canada. Dis. Aquat. Org., 45, 215227.
17. HINE P.M., COCHENNEC-LAUREAU N. & BERTHE F.C.J. (2001). Bonamia exitiosus n. sp.
(Haplosporidia) infecting flat oysters Ostrea chilensis (Philippi) in New Zealand. Dis. Aquat. Org., 47,
6372.
18. MCARDLE J.F., MCKIERNAN F., FOLEY H. & JONES D.H. (1991). The current status of Bonamia
disease in Ireland. Aquaculture, 93, 273278.
19. MONTES J. & MELENDEZ I. (1987). Donnes sur la parasitose de Bonamia ostreae chez lhuitre plate de
Galice, cte Nord-Ouest de lEspagne. Aquaculture, 67, 195198.
20. PASCUAL M., MARTIN A.G., ZAMPATTI E., COATANEA D., DEFOSSEZ J. & ROBERT R. (1991).
Testing of the Argentina oyster, Ostrea puelchana, in several French oyster farming sites. Cons. Inter.
Explor. Mer, CM 1991/K30.
21. PICHOT Y., COMPS M., TIGE G., GRIZEL H. & RABOUIN M.A. (1979). Recherches sur Bonamia ostreae
gen. n., sp. n., parasite nouveau de lhuitre plate Ostrea edulis L. Rev. Trav. Inst. Peches Marit., 43, 131
140.
22. TIGE G., GRIZEL H., COCHENNEC N. & RABOUIN M.A. (1984). Evolution de la situation
pizootiologique en Bretagne en 1983 suite au dveloppement de Bonamia ostreae. Cons. Inter. Explor.
Mer, CM 1984/F, 14, 110.
23. VAN BANNING P. (1987). Further results of the Bonamia ostreae challenge tests in Dutch oyster
culture. Aquaculture, 67, 191194.
24. ZABALETA A. & BARBER B.J. (1996). Prevalence, intensity and detection of Bonamia ostreae in Ostrea
edulis L in the Damariscotta River area, Maine. J. Shellfish Res., 15, 395400.
*
* *
C HA P T E R 3 . 1 . 2 .

MS X DI S E AS E
( Ha p l o s p o r i d i u m n e l s o n i )

GENERAL INFORMATION
MSX disease is caused by the protistan Haplosporidium nelsoni (= Minchinia nelsoni) of the phylum
Haplosporidia (14). Haplosporidium nelsoni is commonly known as MSX (multinucleate sphere X).
The oysters Crassostrea virginica and Crassostrea gigas are infected by H. nelsoni; however, the
prevalence and virulence of this pathogen are much higher in C. virginica than in C. gigas (1, 5, 10, 11).
The geographical distribution of H. nelsoni in C. virginica is the east coast of North America from
Florida, USA, to Nova Scotia, Canada (9, 12). Enzootic areas are Delaware Bay and Chesapeake Bay,
with occasional epizootics in North Carolina estuaries, Long Island Sound, Cape Cod and Nova Scotia
(1, 8, 22). Haplosporidium nelsoni has been reported from Crassostrea gigas in California, USA
(10, 11), and in Korea, Japan, and France (5, 10, 15, 16, 18). Another Haplosporidium sp. also
occurs in C. gigas in France (6).
The plasmodium stage of H. nelsoni occurs intercellularly in connective tissue and epithelia. Spores of
H. nelsoni occur exclusively in the epithelium of the digestive tubules. Sporulation of H. nelsoni is rare in
infected adult oysters, but is frequently observed in infected juvenile oysters (2, 3). Infection by H. nelsoni
takes place between mid-May and the end of October. Mortalities from new infections occur throughout the
summer and peak in July/August. Mortalities may occur in the spring from over-wintering infections.
MSX disease is restricted to salinities over 15 ppt (parts per thousand); rapid and high mortalities can
occur at 20 ppt (1, 8, 13). Holding infected oysters for 2 weeks in water of 10 ppt salinity or less at 20C
kills H. nelsoni, but not C. virginica (7).
Mortality of highly susceptible oysters from H. nelsoni infections is rapid and occurs without loss of
condition. More tolerant oysters survive longer and show reduction in condition index correlated with
infection intensity (9). At death they are grossly emaciated and the storage cells appear shrunken and
disrupted. Sporulation causes disruption of digestive tubule epithelium.
It has not been possible to transmit H. nelsoni experimentally in the laboratory. No life cycle has been
elucidated for any member of the phylum Haplosporidia, but an intermediate host is suspected (12).
For diagnosis, the recommended guidelines for sampling are those stated in Chapter 1.1.4 and Chapter I.2.
of this Aquatic Manual.
1.1. Histological examination
General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. Cut a
transverse section through the visceral mass that includes mantle, gill and digestive gland, and
place the sample in a fixative, such as Davidsons or Carsons fluid (the latter enables the samples
to be re-used for electron microscopy, if necessary). The ratio must be no more than one volume
Chapter 3.1.2. - MSX disease (Haplosporidium nelsoni)
of tissue to ten volumes of fixative. The sections are subsequently treated by conventional
histological procedures (4). Haplosporidia are revealed by many nonspecific stains, such as
haematoxylin & eosin (H&E).
Multinucleate plasmodia of Haplosporidium nelsoni (usually 515 m in diameter, but can be up to
25 m) occur throughout the connective tissue and often in epithelia of the gill and gut.
Plasmodia are detectable throughout the year. Sporocysts containing spores are restricted to the
epithelium of the digestive tubules. Sporulation of H. nelsoni is rare in infected adult oysters, but is
frequently observed in infected juvenile oysters. Mature spores measure about 6 8 m.
Sporocysts can disrupt the digestive epithelium releasing developing and mature spores into the
lumen of the digestive tubules. Spores can be found from June through December in C. virginica.
Infection intensity has been rated (4) as follows: localised (LO) = any infection where plasmodia
are localised in one small area in one tissue type, usually the epithelium of gills or gut; rare (R) =
systemic infections with less than ten plasmodia in the entire section; light (L) = systemic
infections with less than two plasmodia per field at 400 magnification, but more than ten
plasmodia in the entire section; moderate (M) = systemic infections with 25 plasmodia per field
at 400 magnification; heavy (H) = more than five plasmodia per field at 400 magnification;
sporulation (S) = any infection where spores are present.
2.1. Histology
See Section 1.1. above for the technical procedure. When spores are present, H. nelsoni can be
presumptively diagnosed in oysters if the spores are the correct size and occur exclusively in the
epithelium of the digestive tubules. In areas along the east coast of the USA where salinity is
consistently less than 25 ppt., haplosporidian plasmodia in oysters can be presumed to be
H. nelsoni. In areas where salinity is consistently greater than 25 ppt., H. nelsoni and H. costale (the
causative agent of SSO disease) co-occur and plasmodia of the two species cannot be
distinguished by histology (see Chapter 3.1.6. SSO disease).
2.2. Polymerase chain reaction of DNA from oyster tissue
A positive polymerase chain reaction (PCR) amplification is only a presumptive diagnosis because
it detects DNA and not necessarily a viable pathogen. Other techniques, preferably in-situ
hybridisation, must be used to visualise the pathogen.
The PCR primers developed for H. nelsoni detection, MSX-A (5-CGA-CTT-TGG-CAT-TAG-
GT-TTC-AGA-CC-3) and MSX-B (5-ATG-TGT-TGG-TGA-CGC-TAA-CCG-3), target small
subunit ribosomal DNA and have been shown to be sensitive and specific for this pathogen (18,
21). A multiplex PCR (17) for H. nelsoni and H. costale (SSO) has been developed using these
primers, but it has not been validated. PCR reaction mixtures contain reaction buffer (10 mM
Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl
2
; 10 g/ml gelatin), 400 g/ml bovine serum albumin,
25 pmoles each of MSX-A and MSX-B, 200 M each of dATP, dCTP, dGTP, dTTP, 0.6 units
AmpliTaq DNA polymerase (Applied Biosystems), and template DNA, in a total volume of 25 l.
The reaction mixtures are cycled in a thermal cycler. The programme for the GeneAmp PCR
System 9600 thermal cycler (Applied Biosystems) is: initial denaturation at 94C for 4 minutes,
35 cycles at 94C for 30 seconds, 59C for 30 seconds, and 72C for 1.5 minutes, and a final
extension at 72C for 5 minutes. An aliquot (10% of reaction volume) of each PCR reaction is
checked for the 573 base pairs (bp) amplification product by agarose gel electrophoresis and
ethidium bromide staining.
3.1. In-situ hybridisation examination of Haplosporidium nelsoni
In-situ hybridisation is the method of choice for confirming identification because it allows
visualisation of a specific probe hybridised to the target organism. DNA probes must be
thoroughly tested for specificity and validated in comparative studies before they can be used for
confirmatory identification.
In-situ hybridisation has recently been developed to differentiate plasmodia of H. nelsoni from
those of H. costale in tissue sections (19, 20). Species-specific, labelled oligonucleotide probes
hybridise with the small subunit ribosomal RNA of the parasites. This hybridisation is detected by
an antibody conjugate that recognises the labelled probes. Substrate for the antibody conjugate is
added, causing a colorimetric reaction that enables visualisation of probeparasite RNA
hybridisations.
The procedure for in-situ hybridisation is conducted as follows. Positive and negative controls
must be included in the procedure.
i) A transverse section is cut through the visceral mass and placed in Davidsons AFA fixative
(glycerin [10%], formalin [20%], 95% ethanol [30%], dH
2
O [30%], glacial acetic acid [10%])
for 2448 hours, and then transferred to 70% ethanol until processed by histological
procedures (step ii). The ratio must be no more than one volume of tissue to ten volumes of
fixative.
ii) The samples are subsequently embedded in paraffin by conventional histological procedures.
Sections are cut at 56 m and placed on positively-charged slides or 3-aminopropyl-
triethoxylane-coated slides. Histological sections are then dried overnight in an oven at 40C.
iii) The sections are deparaffinised by immersion in xylene or another less toxic clearing agent
for 10 minutes. The solvent is eliminated by immersion in two successive absolute ethanol
baths for 10 minutes each and rehydrated by immersion in an ethanol series. The sections are
then washed twice for 5 minutes in phosphate buffered saline (PBS).
iv) The sections are treated with proteinase K, 50 g/ml in PBS, at 37C for 15 minutes. The
reaction is then stopped by washing the sections in PBS with 0.2% glycine for 5 minutes. The
sections are then placed in 2 SSC (standard saline citrate) for 10 minutes.
v) The sections are prehybridised for 1 hour at 42C in prehybridisation solution (4 SSC, 50%
formamide, 5 Denhardts solution, 0.5 mg/ml yeast tRNA, and 0.5 mg/ml heat-
denaturated herring sperm DNA).
vi) The prehybridisation solution is then replaced with prehybridisation buffer containing
2 ng/l of the digoxigenin-labelled oligonucleotide probe. The sequence of the probe
designated MSX1347 (19) is 5-ATG-TGT-TGG-TGA-CGC-TAA-CCG-3. The sections are
covered with in-situ hybridisation plastic cover-slips and placed on a heating block at 90C
for 12 minutes. The slides are then cooled on ice for 1 minute before hybridisation overnight
at 42C in a humid chamber.
vii) The sections are washed twice for 5 minutes each in 2 SSC at room temperature, twice for
5 minutes each in 1 SSC at room temperature, and twice for 10 minutes each in 0.5 SSC
at 42C. The sections are then placed in Buffer 1 (100 mM Tris, pH 7.5, 150 mM NaCl) for
12 minutes.
viii) The sections are placed in Buffer 1 (see step vii) supplemented with 0.3% Triton X-100 and
2% sheep serum for 30 minutes. Anti-digoxigenin alkaline phosphatase antibody conjugate is
diluted 1/500 (or according to the manufacturers recommendations) in Buffer 1
supplemented with 0.3% Triton X-100 and 1% sheep serum and applied to the tissue
sections. The sections are covered with in-situ hybridisation cover-slips and incubated for
3 hours at room temperature in the humid chamber.
ix) The slides are washed twice in Buffer 1 for 5 minutes each (see step vii) and twice in Buffer 2
(100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl
2
) for 5 minutes each. The slides are then
placed in colour development solution (337.5 g/ml nitroblue tetrazolium, 175 g/ml 5-
bromo-4-chloro-3-indolylphosphate p-toluidine salt, 240 g/ml levamisole in Buffer 2) for 2
hours in the dark. The colour reaction is stopped by washing in TE buffer (10 mM Tris, pH
8.0, 1 mM EDTA [ethylene diamine tetra-acetic acid]).
x) The slides are then rinsed in dH
2
O. The sections are counterstained with Bismarck Brown Y,
rinsed in dH
2
O, and cover-slips are applied using an aqueous mounting medium. The
presence of H. nelsoni is demonstrated by the purple-black labelling of the parasitic cells.
REFERENCES
1. ANDREWS J.D. & WOOD J.L. (1967). Oyster mortality studies in Virginia. VI. History and distribution
of Minchinia nelsoni, a pathogen of oysters in Virginia. Chesapeake Sci., 8, 113.
2. BARBER B.J., KANALEY S.A. & FORD S.E. (1991). Evidence for regular sporulation by Haplosporidium
nelsoni (MSX) (Acestospora: Haplosporidiidae) in spat of the American oyster, Crassostrea virginica. J.
Protozool., 38, 305306.
3. BURRESON E.M. (1994). Further evidence of regular sporulation by H. nelsoni in small oysters,
C. virginica. J. Parasitol., 80, 10361038.
4. BURRESON E.M., ROBINSON M.E. & VILLALBA A. (1988). A comparison of paraffin histology and
hemolymph analysis for the diagnosis of Haplosporidium nelsoni (MSX) in Crassostrea virginica (Gmelin).
J. Shellfish Res., 7, 1923.
5. BURRESON E.M., STOKES N.A. & FRIEDMAN C.S. (2000). Increased virulence in an introduced
pathogen: Haplosporidium nelsoni (MSX) in the eastern oyster Crassostrea virginica. J. Aquat. Anim.
Health, 12, 18.
6. COMPS M. & PICHOT Y. (1991). Fine spore structure of a haplosporidian parasitizing Crassostrea gigas:
taxonomic implications. Dis. Aquat. Org., 11, 7377.
7. FORD S.E. (1985). Effects of salinity on survival of the MSX parasite Haplosporidium nelsoni (Haskin,
Stauber, and Mackin) in oysters. J. Shellfish Res., 2, 8590.
8. FORD S.E. & HASKIN H.H. (1982). History and epizootiology of Haplosporidium nelsoni (MSX), an
oyster pathogen in Delaware Bay, 195780. J. Invertebr. Pathol., 40, 118141.
9. FORD S.E. & TRIPP M.R. (1996). Diseases and defense mechanisms. In: The Eastern Oyster
Crassostrea virginica, Kennedy V.S., Newell R.I.E. & Eble A.F., eds. Maryland Sea Grant College,
College Park, Maryland, USA, 581660.
10. FRIEDMAN C.S. (1996). Haplosporidian infections of the Pacific oyster, Crassostrea gigas (Thunberg),
in California and Japan. J. Shellfish Res., 15, 597600.
11. FRIEDMAN C.S., CLONEY D.F., MANZER D. & HEDRICK R.P. (1991). Haplosporidiosis of the Pacific
oyster, Crassostrea gigas. J. Invertebr. Pathol., 58, 367372.
12. HASKIN H.H. & ANDREWS J.D. (1988). Uncertainties and speculations about the life cycle of the
eastern oyster pathogen Haplosporidium nelsoni (MSX). In: Disease Processes in Marine Bivalve
Molluscs, Fisher W.S., ed. Amer. Fish. Soc. Spec. Publ. 18. Bethesda, Maryland, USA, 522.
13. HASKIN H.H. & FORD S.E. (1982). Haplosporidium nelsoni (MSX) on Delaware Bay seed oyster beds: a
host-parasite relationship along a salinity gradient. J. Invertebr. Pathol., 40, 388405.
14. HASKIN H.H., STAUBER L.A. & MACKIN J.A. (1966). Minchinia nelsoni sp. n. (Haplosporida,
Haplosporidiidae): causative agent of the Delaware Bay oyster epizootic. Science, 153, 14141416.
15. KAMAISHI T. & YOSHINAGA T. (2002). Detection of Haplosporidium nelsoni in Pacific oyster
Crassostrea gigas in Japan. Fish Pathol., 37, 193195.
16. KERN F.G. (1976). Sporulation of Minchinia sp. (Haplosporida, Haplosporidiidae) in the Pacific
oyster Crassostrea gigas (Thunberg) from the Republic of Korea. J. Protozool., 23, 498500.
17. PENNA M.-S., KHAN M. & FRENCH R.A. (2001). Development of a multiplex PCR for the detection
of Haplosporidium nelsoni, Haplosporidium costale and Perkinsus marinus in the eastern oyster (Crassostrea
virginica, Gmelin, 1791). Molec. Cell. Probes, 15, 385390.
18. RENAULT T., STOKES N.A., CHOLLET B., COCHENNEC N., BERTHE F. & BURRESON E.M. (2000).
Haplosporidiosis in the Pacific oyster, Crassostrea gigas, from the French Atlantic coast. Dis. Aquat.
Org., 42, 207214.
19. STOKES N.A. & BURRESON E.M. (1995). A sensitive and specific DNA probe for the oyster
pathogen Haplosporidium nelsoni. J. Euk. Microbiol., 42, 350357.
20. STOKES N.A. & BURRESON E.M. (2001). Differential diagnosis of mixed Haplosporidium costale and
Haplosporidium nelsoni infections in the eastern oyster, Crassostrea virginica, using DNA probes. J.
Shellfish Res., 20, 207213.
21. STOKES N.A., SIDDALL M.E. & BURRESON E.M. (1995). Detection of Haplosporidium nelsoni
(Haplosporidia: Haplosporidiidae) in oysters by PCR amplification. Dis. Aquat. Org., 23, 145152.
22. SUNILA I., KAROLUS J. & VOLK J. (1999). A new epizootic of Haplosporidium nelsoni (MSX), a
haplosporidian oyster parasite, in Long Island Sound, Connecticut. J. Shellfish Res., 18, 169174.
*
* *
C HA P T E R 3 . 1 . 3 .

MART E I L I OS I S
( Ma r t e i l i a r e f r i n g e n s a n d M. s y d n e y i )

GENERAL INFORMATION
Marteiliosis here refers only to the diseases caused by Marteilia refringens and M. sydneyi. Other
Marteilia spp. infect oysters and other bivalves. Until more is known about the identity and biology of these
other Marteilia spp., their presence in any bivalve should be regarded as potentially serious and the OIE
Reference Laboratory should be consulted.
Marteiliosis is primarily caused by two protistan parasites of the genus Marteilia: M. refringens and
M. sydneyi (phylum Paramyxea) (3, 11, 12, 26, 30). Marteiliosis is also known as Aber disease
(M. refringens), and QX disease (M. sydneyi).
The type species of the genus, M. refringens, is a lethal parasite of the European flat oyster, Ostrea
edulis (12). Marteilia sydneyi infects Saccostrea glomerata (= commercialis) and possibly also
Saccostrea echinata (30, 31).
In addition, M. maurini (9) in Mytilus galloprovincialis and Mytilus edulis has been described from
France, Italy and Spain (21). Because M. maurini is not easily distinguished from M. refringens,
detection of the parasite in mussels would require confirmatory diagnosis as described in this chapter.
Marteilia lengehi (7, 16) in Saccostrea cucullata from the Persian Gulf and Western Australia, and
M. christenseni (8) in Scrobicularia plana from France can apparently be differentiated from other
species by the characteristics of the cytoplasmic contents of sporangia and spore morphology. Marteilia spp.
has also been described in the following species: Tiostrea chilensis (13), Ostrea angasi (5),
O. puelchana (25), Cerastoderma (= Cardium) edule (10), Mytilus edulis (10, 22), Mytilus
galloprovincialis (29), Crassostrea gigas (6) and C. virginica (27). A protistan similar to
M. sydneyi was reported in giant clams Tridacna maxima (24). The scallop Argopecten gibbus is
infected by a nonspecified Marteilia sp. in Florida, United States of America (USA) that has not been
identified to species level (23).
The geographical distribution of M. refringens is: France, Greece, Italy, Morocco, Portugal, and Spain.
Marteilia sydneyi is found in New South Wales, Queensland and Western Australia.
Marteilia refringens and M. sydneyi sporulate in the epithelia of the digestive gland, where infection is
associated with poor condition index, emaciation of the oyster and exhaustion of its reserves of energy
(glycogen), discoloration of the digestive gland, cessation of growth, and mortalities. Mortality appears to be
related to the sporulation of the parasite (12). The initial site of infection of M. sydneyi is in the epithelia
of the palps and gills and presporulating stages are found throughout the connective tissue and in digestive
gland epithelia (18). Presporulating stages of M. refringens occur in the epithelia of the palps, stomach,
digestive ducts and possibly the gills. Oysters infected with M. sydneyi and M. refringens are in poor
condition with completely resorbed gonads (28). Massive invasion by M. sydneyi leads to complete
disorganisation of the digestive gland epithelia. Death results from starvation in under 60 days after initial
infection
The period of infection for M. refringens in Ostrea edulis is confined to spring and summer, when water
temperature is greater than 17C (1, 12, 14). Oysters may become infected with M. sydneyi in summer
Chapter 3.1.3. - Marteiliosis (Marteilia refringens, M. sydneyi)
and early autumn. However, the disease is not seasonal, heavy mortality occurs and spores may be found all
year round. High salinities limit the development of Marteilia spp.
The mode of infection and the life cycle outside the host are unknown. Because it has not been possible to
transmit the disease experimentally in the laboratory, possible intermediate hosts are suspected (2, 4, 14,
22, 28).
1.1. Histology
General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. For
histological examination, cut a section through the digestive gland and place the sample in a
fixative liquid, such as Davidsons or Carsons solutions or 10% formalin in filtered seawater
(these latter enables the sample to be reused for electron microscopy, if necessary). The ratio must
be no more than 1 volume of tissue to 10 volumes of fixative.
The samples are subsequently handled in accordance with classical histological methods (see
chapter I.2.). Several nonspecific stains allow M. refringens to be observed, e.g. haematoxylineosin
or Millots trichrome. These examples are not exhaustive, and modified staining techniques (15)
may enhance the detection of the parasite.
The young stages of Marteilia refringens are present in the epithelia of the stomach, intestine and
digestive ducts. Initial infective stages of Marteilia sydneyi are present in the palp and gill epithelia
and presporulating stages are found in the connective tissues and the epithelia of the digestive
tubules, digestive ducts, intestine and stomach. Sporulating stages of M. refringens and M. sydneyi
can be found in the epithelia of the digestive tubules. Free sporangia can also be observed in the
lumen of the intestine. The unique feature of internal cleavage to produce cells within cells during
sporulation differentiates Marteilia spp. from all other protista.
2.1. Histology
See Section 1.1. above.
In order to prepare the smears, cut a section through the digestive gland, remove excess water by
placing the sample on blotting paper, then imprint the sample corresponding to the section that
passes through the digestive tract on to a slide. The slides are air-dried and then fixed in methanol
(23 minutes).
The samples are stained using a commercially available staining kit for blood cells in accordance
with the manufacturers instructions. After staining, rinse in tap water, allow to dry completely and
mount with a cover-slip using an appropriate synthetic resin.
The parasite is 58 m in size in the early stages and may reach up to 40 m during sporulation.
The cytoplasm of the cells stains basophilic, the nucleus is eosinophilic. The secondary cells or
sporoblasts are surrounded by a bright halo (colour may vary slightly with the stain used). An
observation time of 10 minutes per slide is considered sufficient.
3.1. In-situ hybridisation examination of Marteilia spp.
An in-situ hybridisation assay may be performed to assess the taxonomic affiliation of the
observed organisms at the genus level (20). Cross reaction studies using this protocol
demonstrated that both M. refringens and M. sydneyi may be detected by the probe (17, 19, 20). An
M. sydneyi-specific DNA probe has been developed and determined to be specific to the pathogen
in in-situ hybridisation assays (17, 19).
The probe, Smart 2, is used to detect Marteilia spp. by in-situ hybridisation. For probe production,
the probe is obtained by polymerase chain reaction (PCR) using the primers SS2 (5-CCG-GTG-
CCA-GGT-ATA-TCT-CG-3) and SAS1 (5-TTC-GGG-TGG-TCT-TGA-AAG-GC-3). The
PCR reaction is performed as described in Section 3.2., from M. refringens purified from Ostrea
edulis DNA, except that 1 l of digoxigenin-dUTP (DIG-dUTP) 25 mM is added to the mix.
The probe, M. sydneyi the ITS (internal transcribed spacer) 1, is used to specifically detect
M. sydneyi among related species. For probe production, the probe is obtained by polymerase
chain reaction (PCR) using the primers PRO2 (5-TCA-AGG-GAC-ATC-CAA-CGG-TC-3) and
LEG1 (5-CGA-TCT-GTG-TAG-TCG-GAT-TCC-GA-3). The PCR reaction is performed as
described in Section 3.2., from M. sydneyi purified from Saccostrea glomerata DNA, except that 1 l
of digoxigenin-dUTP (DIG-dUTP) 25 mM is added to the mix.
The in-situ hybridisation is conducted as follows. Positive and negative controls must be included
in the procedure.
For the smart 2 probe, the visceral mass of molluscs is placed in Davidsons fixative AFA
(glycerine [10%], formalin [20%], 95 ethanol [30%], dH
2
O [30%], and glacial acetic acid [10%])
or 10% buffered formalin for approximately 24 hours and then embedded in paraffin. Sections are
cut 5 m thick and placed on aminoalkylsilane-coated slides, which are then baked overnight in an
oven at 40C. The sections are dewaxed by immersing in xylene for 10 minutes. This step is
repeated once and then the solvent is eliminated by immersion in two successive absolute ethanol
baths for 10 minutes each. The sections are then rehydrated by immersion in an ethanol series.
The sections are treated with proteinase K (100 g/ml) in TE buffer (Tris [50 mM], ethylene
diamine tetra-acetic acid [EDTA, 10 mM]), at 37C for 30 minutes. Slides are dehydrated by
immersion in an ethanol series and then air dried. Sections are incubated with 100 l of
hybridisation buffer (4 SSC [standard saline citrate], 50% formamide, 1 Denhardts solution,
250 g/ml yeast tRNA, 10% dextran sulfate) containing 10 ng (1 l of the PCR reaction) of the
digoxigenin-labelled probe. Sections are covered with in-situ plastic cover-slips and placed on a
heating block at 95C for 5 minutes. Slides are then cooled on ice for 1 minute before overnight
hybridisation at 42C in a humid chamber. Sections are washed twice for 5 minutes in 2 SSC at
room temperature, and once for 10 minutes in 0.4 SSC at 42C. The detection steps are
performed according to manufacturers instructions. The slides are then rinsed in dH
2
O. The
sections are counterstained with Bismarck Brown Y, rinsed in dH
2
O, and cover-slips are applied
using an aqueous mounting medium. The presence of Marteilia sp. is demonstrated by the purple-
black labelling of the parasitic cells.
3.2. Polymerase chain reaction-restriction fragment length polymorphism for Marteilia
refringens/M. sydneyi and M. refringens/M. maurini discrimination
A PCR-restriction fragment length polymorphism (RFLP) assay was developed to differentiate M.
refringens from M. maurini (21). A PCR assay was developed for the specific detection of M. sydneyi
(1719).
For DNA extraction, infected animals are frozen at 80C and the tissues are ground to powder.
Around 10 volumes of extraction buffer (NaCl [100 mM], EDTA [25 mM], pH 8, sodium dodecyl
sulfate [SDS, 0.5%]) are added with proteinase K (100 g/ml). Following an overnight incubation
at 50C, DNA is extracted using a standard phenol/chloroform protocol, and precipitated with
ethanol.
PCR is carried out in 50 l volume. After denaturation of DNA at 94C for 5 minutes, 30 cycles
are run as follows: denaturation at 94C for 1 minute, annealing at 55C for 1 minute, and
elongation at 72C for 1 minute per kilo-base pair. A final elongation step of 10 minutes at 72C is
performed.
For the detection of M. refringens, PCR is performed with primers (4 + 5) that target the ITS 1 (4:
5-CCG-CAC-ACG-TTC-TTC-ACT-CC-3 and 5: 5-CTC-GCG-AGT-TTC-GAC-AGA-CG-3).
For the detection of M. sydneyi, PCR is performed with primers LEG1 (5-CGA-TCT-GTG-
TAG-TCG-GAT-TCC-GA-3) and PRO2 (5-TCA-AGG-GAC-ATC-CAA-CGG-TC-3) that
target the ITS.
To differentiate M. refringens from M. maurini, polymorphism among the PCR products is
identified by testing for cleavage with restriction enzyme Hha1. The resulting restriction fragments
are analysed electrophoretically on 2% agarose gels. The profile corresponding to M. maurini gives
three bands of 156 + 157 and 68 bp, respectively, whereas the profile corresponding to M.
refringens gives two bands of 226 and 156 bp, respectively.
3.3. Electron microscopy examination
Transmission electron microscopy procedures are outlined in Chapter I.2. of this Aquatic Manual.
Marteilia sydneyi can be differentiated from M. refringens by a lack of striated inclusions in the
plasmodia, formation of eight to sixteen sporangial primordia in each plasmodium (instead of
eight for M. refringens), occurrence of two or three spores in each sporangium (rather than four in
M. refringens), and the presence of a heavy layer of concentric membranes surrounding mature
M. sydneyi spores.
REFERENCES
1. AUDEMARD C., BARNAUD A., COLLINS C.M., LE ROUX F., SAURIAU P.G., COUSTAU C., BLACHIER
P. & F. BERTHE (2001). Claire ponds as an experimental model for Marteilia refringens life-cycle
studies: new perspectives. J. Exp. Mar. Biol. Ecol., 257, 87108.
2. AUDEMARD C., LE ROUX F., BARNAUD A., COLLINS C.M., SAUTOUR B., SAURIAU P.G., DE
MONTAUDOIN X., COUSTAU C., COMBES C. & F. BERTHE (2002). Needle in a haystack: involvement
of the copepod Paracartia grani in the life cycle of the oyster pathogen Marteilia refringens. Parasitology,
124, 315323.
3. BERTHE F.C.J., LE ROUX F., PEYRETAILLADE E., PEYRET P., RODRIGUEZ D., GOUY M. & VIVARES
C.P. (2000). The existence of the phylum Paramyxea Desportes and Perkins, 1990 is validated by the
phylogenetic analysis of the Marteilia refringens small subunit ribosomal RNA. J. Euk. Microbiol., 47,
288293.
4. BERTHE F.C.J., PERNAS M., ZERABIB M., HAFFNER P., THEBAULT A. & FIGUERAS A.J. (1998).
Experimental transmission of Marteilia refringens with special considerations for its life cycle. Dis.
Aquat. Org., 34, 135144.
5. BOUGRIER S., TIGE G., BACHERE E. & GRIZEL H. (1986). Ostrea angasi acclimatization to French
coasts. Aquaculture, 58, 151154.
6. CAHOUR A. (1979). Marteilia refringens and Crassostrea gigas. Mar. Fish Rev., 41, 1920.
7. COMPS M. (1976). Marteilia lengehi n. sp. parasite de lhuitre Crassostrea cucullata Born. Rev. Trav. Inst.
Peches Marit., 40, 347349.
8. COMPS M. (1985). Etude morphologique de Marteilia christenseni sp. n. parasite du lavignon
Scrobicularia piperata P. (mollusque plcypode). Rev. Trav. Inst. Peches Marit., 47, 99104.
9. COMPS M., GRIZEL H. & PAPAYANNI Y. (1982). Infection parasitaire cause par Marteilia maurini sp.
n. chez la moule Mytilus galloprovincialis. Cons. Inter. Explor. Mer, CM, F: 24, 2 pp.
10. COMPS M., TIGE G., GRIZEL H. & DUTHOIT J.-L. (1975). Parasites nouveaux de la glande digestive
des mollusques marins Mytilus edulis L. et Cardium edule L. C.R. Acad., Paris, srie D., 281, 179181.
11. DESPORTES I. & PERKINS F.O. (1990). Phylum Paramyxea. In: Handbook of Protoctista, Margulis
L., Corliss J.O., Melkonian M. & Chapman D.J., eds. Jones and Bartlett Publishing Corporation,
Boston, USA, 3035.
12. GRIZEL H., COMPS M., BONAMI J.R., COUSSERANS F., DUTHOIT J.L. & LE PENNEC M.A. (1974).
Recherche sur lagent de la maladie de la glande digestive de Ostrea edulis L. Sci. Peche, 240, 730.
13. GRIZEL H., COMPS M., RAGUENES D., LEBORGNE Y., TIGE G. & MARTIN A.G. (1982). Bilan des
essais dacclimatation dOstrea chilensis sur les ctes de Bretagne. Rev. Trav. Inst. Peches Marit., 46, 209
225.
14. GRIZEL H. & TIGE G. (1979). Observations sur le cycle de Marteilia refringens. Haliotis, 8, 327330.
15. GUTIERREZ M. (1977). Tecnica de cloracion del agente de la enfermedad de la glandula digestiva de
la ostra plana, Ostrea edulis L. Investigacion Pesquera, 41, 643645.
16. HINE P.M. & THORNE T. (2000). A survey of some parasites and diseases of several species of bivalve
mollusc in northern Western Australia. Dis. Aquat Org., 40, 6778
17. KLEEMAN S. & ADLARD R. (2000). Molecular detection of Marteilia sydneyi, pathogen of Sydney rock
oysters. Dis. Aquat. Org., 40 (2), 137146.
18. KLEEMAN S., ADLARD R.. & LESTER R. (2002). Detection of the initial infective stages of the
protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to
sporogensis. Int. J. Parasitol., 32, 767784.
19. KLEEMAN S., LE ROUX F., ADLARD R., & BERTHE, F.C.J. (2002). Specificity of PCR and in situ
hybridisation assays designed for detection of Marteilia sydneyi and M. refringens: Parasitology, 125, 131
141.
20. LE ROUX F., AUDEMARD C., BARNAUD A. & BERTHE F.C.J. (1999). DNA probes as potential tools
for the detection of Marteilia refringens. Mar. Biotechnol., 1, 588597.
21. LE ROUX F., LORENZO G., PEYRET P., AUDEMARD C., FIGUERAS A. J., VIVARES C.P., GOUY M. &
BERTHE F. (2001). Molecular evidence for the existence of two species of Marteilia in Europe. J.
Euk. Microbiol., 48, 449454.
22. LESTER R.J.G. (1986). Field and laboratory observations on the oyster parasite Marteilia sidneyi. In:
Parasites Lives, Cremin M., Dobson C. & Moorhouse D.E., eds. University of Queenland Press,
Brisbane, Australia, 3340.
23. MOYER M.A., BLAKE N.J. & ARNOLD W.S. (1993). An ascetosporan disease causing mass mortality
in the Atlantic calico scallop, Argopecten gibbus (Linnaeus, 1758). J. Shellfish Res., 12, 305310.
24. NORTON J.H., PERKINS F.P. & LEDUA E. (1993). Marteilia-like infection in a giant clam, Tridacna
maxima, in Fiji. J. Invertebr. Pathol., 61, 328330.
25. PASCUAL M., MARTIN A.G., ZAMPATTI E., COATANEA D., DEFOSSEZ J. & ROBERT R. (1991).
Testing of the Argentina oyster, Ostrea puelchana, in several French oyster farming sites. Cons. Inter.
Explor. Mer, C.M. K:30, 17 pp.
26. PERKINS F.O. & WOLF P.H. (1976). Fine structure of Marteilia sydneyi sp. n. Haplosporidian
pathogen of Australian oysters. J. Parasitol., 62, 528538.
27. RENAULT T., COCHENNEC N. & CHOLLET B. (1995). Marteiliosis in American oysters Crassostrea
virginica reared in France. Dis. Aquat. Org., 23, 161164.
28. ROUBAL F.R., MASEL J. & LESTER R.J.G. (1989). Studies on Marteilia sydneyi, agent of QX disease in
the Sydney rock oyster, Saccostrea commercialis, with implications for its life cycle. Aust. J. Mar.
Freshwater Res., 40, 155167.
29. VILLALBA A., MOURELLE S.G., CARBALLAL M.J. & LOPEZ M.C. (1993). Effects of infection by the
protistan parasite Marteilia refringens on the reproduction of cultured mussels Mytilus galloprovincialis in
Galicia (NW Spain). Dis. Aquat. Org., 17, 205213.
30. WOLF P.H. (1972). Occurrence of a Haplosporidian in Sydney Rock oysters (Crassostrea commercialis)
from Morton Bay, Queensland, Australia. J. Invertebr. Pathol., 19, 416417.
31. WOLF P.H. (1979). Life cycle and ecology of Marteilia sydneyi in the Australian oyster, Crassostrea
commercialis. Mar. Fish Rev., 41, 7072.
*
* *
C HA P T E R 3 . 1 . 4 .

MI KROCYT OS I S
( Mi k r o c y t o s ma c k i n i )

GENERAL INFORMATION
Mikrocytosis here refers only to the disease in oysters caused by Mikrocytos mackini on the west coast of
Canada. If detected outside the known range of M. mackini, electron microscopy or molecular probes, if
available, must be used to identify and distinguish the detected organism from other microcell species (e.g.
Mikrocytos roughleyi, Bonamia ostreae, B. exitiosus). The presence of these pathogens in any bivalve
should be regarded as potentially serious and the OIE Reference Laboratory should be consulted.
Mikrocytos mackini has uncertain taxonomic affinities, but it is not closely related to other microcells (8,
10, 11). Mikrocytosis is also known as Denman Island disease (2, 13) and microcell disease of oysters.
Mikrocytos mackini infects Crassostrea gigas, Ostrea edulis and O. conchaphila (= O. lurida); and
infects experimentally Crassostrea virginica (1, 3, 4, 9). Crassostrea gigas seems to be more resistant to
the disease than the other species.
Mikrocytos mackini occurs on the south-west coast of Canada. It is probably ubiquitous throughout the
Strait of Georgia, including Henry Bay, Denman Island, and is confined to other localities around
Vancouver Island (British Columbia).
Mikrocytos mackini produces a focal intracellular infection of muscle and vesicular connective tissue cells,
which results in haemocyte infiltration and tissue necrosis (5). The parasite may induce yellow-greenish
pustules, abscesses and ulcers, mainly on the mantle, with corresponding brown scars on the shell.
Macroscopic lesions are not always present. Abscesses are composed of granular haemocytes and hyalinocytes,
and may contain small cells of 13 m in diameter. Severe infections appear to be restricted to oysters over
2 years of age.
The mortality rate has been recorded at around 40% for older oysters at low tide levels (13). The disease
occurs most often in April and May, after a 34-month period when temperatures are less than 10C.
Harvesting or moving large oysters to locations high in the intertidal zone prior to March and not planting
oysters at lower tide levels before June have been successfully implemented to control the disease (1).
1.1. Histology
Cut a section through the body of the oyster to include the mantle and digestive gland. Include
pustules, abscesses and ulcers, if they are present. Fix in the sample Davidsons or Carsons
fixatives (the latter enables the samples to be re-used for electron microscopy, if necessary). The
ratio must be no more than one volume of tissue to ten volumes of fixative.
Chapter 3.1.4. - Mikrocytosis (Mikrocytos mackini)
After processing for histology, stain sections with haematoxylin and eosin. It is recommended that
two sections per oyster be examined at 100 magnification.
Intracellular M. mackini, 13 m in diameter, may be detected by examination of vesicular
connective tissue cells immediately adjacent to abscess-like lesions. This parasite has also been
observed in muscle cells and occasionally in haemocytes or free within the lesions, but it is much
more difficult to detect in these situations.
Microcytos mackini differs from all other microcells by its occurrence within the cytoplasm of
vesicular connective tissue cells on the periphery of the lesions.
For detection of the parasite, tissue imprints are better than histology (9). Cut a section through
the abscesses or ulcers if present, blot on filter paper to remove excess water, then make imprints
on a slide of the sample surface corresponding to the section that passed through the lesion. Air-
dry the slides and fix in methanol (23 minutes).
Stain the slides using a commercially available staining kit for blood cells in accordance with the
manufacturers instructions. After drying, the imprint can be examined directly using oil
immersion at 100 magnification. However, if the slides are to be retained for future examination,
they should be mounted with a cover-slip using a synthetic resin prior to applying immersion oil.
The parasite, 13 m in diameter, appears free of the host cell or, rarely, in haemocytes, and has
blue (basophilic) cytoplasm and a small red (eosinophilic) nucleus (colours may vary with stain
used). An observation time of 10 minutes per slide is sufficient at 100 magnification.
3.1. Histology
Where infections have been observed within the known geographical range of M. mackini,
visualisation of intracellular microcells within vesicular connective tissue cells, by histological
examination, may be considered to be a confirmatory method (7).
Transmission electron microscopy procedures are described in Chapter I.2. of this Aquatic
Manual.
Ultrastructural morphology differentiates M. mackini from other microcells; M. mackini lacks
mitochondria and haplosporosomes (10), and the nucleolus of M. mackini is located towards the
centre of the nucleus while in Bonamia spp. it has an eccentric location.
REFERENCES
1. BOWER S.M. (1988). Circumvention of mortalities caused by Denman Island oyster disease during
mariculture of Pacific oysters. Am. Fish. Soc. (Special Publication), 18, 246248.
2. BOWER S.M. (2001). Synopsis of Infectious Diseases and Parasites of Commercially Exploited
Shellfish: Mikrocytos mackini (Denman Island Disease) of Oysters.
URL: http://www-sci.pac.dfo-mpo.gc.ca/sealane/aquac/pages/mikmacoy.htm
Chapter 3.1.4. - Mikrocytosis (Mikrocytos mackini)
3. BOWER S.M., HERVIO D. & MCGLADDERY S.E. (1994). Potential for the Pacific oyster, Crassostrea
gigas, to serve as a reservoir host and carrier of oyster pathogens. ICES Council Meeting Papers,
Copenhagen, Denmark, ICES-CM-1994/F:30.
4. BOWER S.M., HERVIO D. & MEYER G.R. (1997). Infectivity of Mikrocytos mackini, the causative agent
of Denman Island disease in Pacific oysters, Crassostrea gigas, to various species of oysters. Dis. Aquat.
Org., 29, 111116.
5. BOWER S.M., McGLADDERY S.E. & PRICE I.M. (1994). Synopsis of infectious diseases and parasites
of commercially exploited shellfish. Ann. Rev. Fish Dis., 4, 1199.
6. BOWER S.M. & MEYER G.R. (1999). Effects of cold water on limiting or exacerbating some oyster
diseases. J. Shellfish Res., 18, 296 (Abstract).
7. COCHENNEC N., LE ROUX F., BERTHE F. & A. GERARD (2000). Detection of Bonamia ostreae based
on small subunit ribosomal probe. J. Invertebr. Pathol., 76, 2632.
8. FARLEY C.A., WOLF P.H. & ELSTON R.A. (1988). A long-term study of microcell disease with a
description of a new genus, Mikrocytos (g. n.), and two new species, Mikrocytos mackini (sp. n.) and
Mikrocytos roughleyi (sp. n.). Fishery Bull., 86, 581593.
9. HERVIO D., BOWER S.M. & MEYER G.R. (1996). Detection, isolation and experimental transmission
of Mikrocytos mackini, a microcell parasite of Pacific oysters Crassostrea gigas (Thunberg). J. Invertebr.
Pathol., 67, 7279.
10. HINE P.M., BOWER S.M., MEYER G.R., COCHENNEC-LAUREAU N. & BERTHE F.C.J. (2001).
Ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp.
and Ostrea spp. in British Columbia, Canada. Dis. Aquat. Org., 45, 215227.
11. HINE P.M., COCHENNEC-LAUREAU N. & BERTHE F.C.J. (2001). Bonamia exitiosus n. sp.
(Haplosporidia) infecting flat oysters Ostrea chilensis (Philippi) in New Zealand. Dis. Aquat. Org., 47,
6372.
12. JOLY J.-P., BOWER S.M. & MEYER G.R. (2001). A simple technique to concentrate the protozoan
Mikrocytos mackini, causative agent of Denman Island disease in oysters. J. Parasitol., 87, 432434.
13. QUAYLE D.B. (1982). Denman Island oyster disease 19601980. British Columbia Shellfish Mariculture
Newsletter, 2, 15, (Victoria, Canada).
*
* *
C HA P T E R 3 . 1 . 5 .

P E RKI NS OS I S
( P e r k i n s u s ma r i n u s a n d P . o l s e n i / a t l a n t i c u s )

GENERAL INFORMATION
Perkinsosis here refers only to the diseases caused by Perkinsus marinus and P. olseni/atlanticus. Other
described species of Perkinsus include P. chesapeaki from Mya arenaria (26), P. andrewsi in Macoma
balthica (14), both from the east coast of the United States of America (USA), and P. qugwadi from
Patinopecten yessoensis from western Canada (5). These species are not considered at this time to cause
diseases notifiable to the OIE. Other unidentified Perkinsus spp. infect many species of bivalves in tropical
and subtropical waters (22). Until more is known about the identity, biology and pathology of these other
Perkinsus spp., their presence in any bivalve should be regarded as potentially serious and the OIE
Reference Laboratory should be consulted.
Perkinsosis is an infection of marine molluscs caused by protistan parasites of the genus Perkinsus. Recent
investigations using molecular sequence data (21, 39, 42) indicate that Perkinsus is not in the phylum
Apicomplexa as suggested by ultrastructural data (25), but is closely related to the Dinoflagellida. Some
authors have placed Perkinsus in the phylum Perkinsozoa (33), or the phylum Dinozoa subphylum
Protalveolata (12), but more molecular data are necessary on Perkinsus and genera related to Perkinsus
before phylogenetic relationships will be clear.
Perkinsus marinus causes disease of economic importance in Crassostrea virginica (2, 7). Although
infection of C. gigas and C. ariakensis is possible, these species seem to be more resistant to the disease (4,
9, 10). Perkinsus marinus was formerly named Dermocystidium marinum (29), and then
Labyrinthomyxa marina (30). Infection by P. marinus is commonly known as Dermo disease (19).
Perkinsus marinus is found on the east coast of the USA (2, 7) from Maine to Florida, and along the
Gulf of Mexico coast to the Yucutan Peninsula (6). The recent northward range extension of P. marinus
into Delaware Bay, New Jersey, Cape Cod and Maine, USA, is attributed to repeated introductions over
several years in conjunction with recent increases in winter sea-surface temperatures (13, 18). The effects of
P. marinus infection in C. virginica range from a pale appearance of the digestive gland and reduction in
condition index, to severe emaciation, gaping, retraction of the mantle, inhibition of gonadal development,
retarded growth, and death (27, 28).
Perkinsus olseni was originally described from Haliotis ruber in Australia (24) and P. atlanticus was
originally described from Ruditapes decussatus in Portugal (3). Similarities in the nucleotide sequences of
the internal transcribed spacers (ITS) of the ribosomal RNA gene cluster (20) have long suggested that P.
olseni is conspecific with P. atlanticus. Recently, the synonymy of the two species has been formally
established based also on sequence similarities in the rRNA nontranscribed spacer (NTS) region (32);
Perkinsus olseni has taxonomic priority. Other susceptible hosts for P. olseni/atlanticus include
Haliotis cyclobates, H. scalaris, H. laevigata, Anadara trapezia, Austrovenus stutchburyi and
Ruditapes philippinarum (22, 23, 32, 34). The geographical distribution of P. olseni/atlanticus is
eastern and southern Australia, New Zealand, Korea, Japan, Portugal, France, Italy and Spain (1, 3, 11,
22, 23, 34, 40).
Proliferation of P. olseni/atlanticus results in the disruption of connective tissue and epithelial cells leading
to weakening. Cysts are visible macroscopically on the gills of infected R. decussatus (3). Abscesses
occasionally can be noted in abalone (Haliotis spp.) (24). Pustules up to 8 mm in diameter in the foot and
mantle of infected Haliotis spp. reduce market value. Perkinsus olseni/atlanticus was directly associated
Chapter 3.1.5. - Perkinsosis (Perkinsus marinus, P. olseni/atlanticus)
with high losses of the abalone H. laevigata in Australia (22) and mortality in the clam R. decussatus in
Portugal (3) and in the clam R. philippinarum in Korea (34).
Morphology of life history stages is similar for all Perkinsus species. Trophozoites, characterised by a large
vacuole and a displaced nucleus, occur intercellularly in connective and epithelial tissue. Mature trophozoites
divide by successive binary fission resulting in the release of 832 immature trophozoites (36, 44). The
developmental cycle of P. marinus often occurs within phagocytes. Proliferation of all Perkinsus species is
correlated with warm summer water temperatures (higher than 20C) when pathogenicity and associated
mortalities are highest. All life history stages appear to be infective (45). Under certain, poorly understood,
conditions mature trophozoites enlarge and undergo zoosporulation. Although biflagellate zoospores are
infective, the role of the zoospore in transmission in nature is unclear.
Reference methods for the detection of Perkinsus spp. are histological sections and culture in fluid
thioglycollate medium (8, 17, 37, 38). For diagnosis, the recommended guidelines for sampling are those
stated in Chapter 1.1.4 and Chapter I.2. of this Aquatic Manual.
1.1. Diagnosis by culture in Rays fluid thioglycollate medium
Tissue samples measuring approximately 5 10 mm are excised giving preference to rectal, gill
and mantle tissue from oysters and clams, and adductor or foot muscles or mantle for abalone,
and placed in fluid thioglycollate medium (Difco) containing antibiotics. Recommended
antifungal/antibiotics are 200 units of mycostatin (Nystatin), 500 units penicillin G and 500 mg
dihydro-streptomycin per ml of media (37). Chloromycetin can be used in place of penicillin/
streptomycin (38). Incubation is at 2225C for between 4 and 7 days, in the dark.
Cultured parasites enlarge from 210 to 5070 m during incubation. After incubation, the
fragments of tissue are collected and macerated with a scalpel blade on a glass slide, a drop of
Lugols iodine 1/5 solution is added, and the preparation is covered with a cover-slip and allowed
to sit for 10 minutes. The preparations are examined in the fresh state. Perkinsus hypnospores are
spherical and the walls stain blue or bluish-black with Lugols iodine solution.
Infection intensity has been ranked (37) on a scale of 0 to 5 as follows: 0 = uninfected; 0.5 (very
light) = fewer than ten parasites found in entire tissue preparation; 1 (light) = 11100 cells present
in entire preparation, parasites may be scattered or occur in isolated clusters of 1015 cells; 2 (light
moderate) = some areas free of parasites, but other areas show localised concentrations of 24
50 cells, or cells uniformly distributed so that 23 cells occur in each field at 100 magnification;
3 (moderate) = parasites so numerous expect to find >3 cells in all fields at 100 magnification,
masses of >50 cells are still more or less localised; tissue does not show blue/black colour
macroscopically; 4 (moderate heavy) = parasite cells present in large numbers in all tissues, but
less than half of tissue shows a blue/black colour macroscopically; 5 (heavy) = parasite cells occur
in enormous numbers; major part of tissue appears blue/black macroscopically.
1.2. Histology
General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. Cut a
transverse section through the visceral mass that includes gill, mantle and digestive gland and
place the tissue sample in a fixative, such as Davidsons fluid. The ratio must be no more than
1 volume of tissue to 10 volumes of fixative.
Samples are processed by conventional histological procedures. Perkinsus spp. are revealed by
many nonspecific stains, such as haematoxylin & eosin. Histology is not as sensitive a technique as
fluid thioglycollate culture and misses many light infections (19).
Perkinsus marinus infection is usually systemic, although cells can be localised in the gut epithelium.
For P. olseni/atlanticus, infection is usually located in connective tissues. Thus, the connective
tissue of all organs may harbour immature trophozoites, mature trophozoites (signet-ring stages),
and tomont division stages containing 2, 4, 8, 16 or 32 developing trophozoites (19, 36, 44).
Mature trophozoites are characterised by the presence of a vacuole that displaces the nucleus
towards the periphery of the cell.
Morphology of all life history stages is similar for all species. The size of trophozoites in infected
hosts varies from 210 m for P. marinus up to 16 m for P. olseni/atlanticus. However,
morphological characters are similar for all Perkinsus species and size is highly variable, so
morphology alone cannot be used to distinguish species.
2.1. Diagnosis by culture in thioglycollate medium and histology
These methods, as described above (see Sections 1.1. and 1.2. of this Chapter), and used together,
may be used as a presumptive diagnostic method for the genus Perkinsus. The techniques cannot
be used to distinguish species.
2.2. Transmission electron microscopy
Transmission electron microscopy procedures are given in detail in Chapter I.2 of this Aquatic
Manual. The ultrastructure of the zoospore has been thought to be diagnostic for the genus
Perkinsus (3, 5, 36). However, recent evidence based on molecular data has shown that Colpodella,
a predatory flagellate that has identical ultrastructure to Perkinsus zoospores, is not closely related
to Perkinsus (43). Thus, ultrastructural morphology of zoospores does not appear to be a suitable
diagnostic tool even for the genus Perkinsus.
it detects DNA and not necessarily a viable pathogen. PCR primers have been developed for
P. marinus (31, 41, 46) and P. atlanticus (15, 40), but they have not been thoroughly tested for
inclusivity, specificity and sensitivity, especially in light of known sequence variability within a
single Perkinsus species in the internal transcribed spacers (ITS) region (11, 16). A multiplex PCR
(35) has been developed for P. marinus, Haplosporidium nelsoni (MSX) and Haplosporidium costale
(SSO), but it has not been validated. Development of species-specific and sensitive molecular
diagnostic tools is an area of active research; however, PCR is not recommended at this time as a
presumptive diagnostic method for any species of Perkinsus.
3.1. ITS region sequence analysis
At the present time, the only way to confirm the identification of Perkinsus species is to compare
ITS region nucleotide sequences with reference sequences deposited in the GenBank database
(http://www.ncbi.nlm.nih.gov/entrez/). PCR primers have been designed that amplify the ITS
region of any described Perkinsus spp. except P. qugwadi, from infected host tissue (11). The PCR
products can then be sequenced for species identification.
REFERENCES
1. ALMEIDA M., BERTHE F., THEBAULT A. & DINIS M.T. (1999). Whole clam culture as a quantitative
diagnostic procedure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams Ruditapes decussatus.
2. ANDREWS J.D. (1996). History of Perkinsus marinus, a pathogen of oysters in Chesapeake Bay 1950
1984. J. Shellfish Res., 15, 1316.
3. AZEVEDO C. (1989). Fine structure of Perkinsus atlanticus n. sp. (Apicomplexa, Perkinsea) parasite of
clams, Ruditapes decussatus, from Portugal. J. Parasitol., 75, 627635.
4. BARBER B.J. & MANN R. (1994). Growth and mortality of eastern oysters, Crassostrea virginica
(Gmelin, 1791), and Pacific oysters, Crassostrea gigas (Thunberg, 1793) under challenge from the
parasite, Perkinsus marinus. J. Shellfish Res., 13, 109114.
5. BLACKBURN J., BOWER, S.M. & MEYER G.R. (1998). Perkinsus qugwadi sp. nov. (incertae sedis), a
pathogenic protozoan parasite of Japanese scallops, Patinopectin yessoensis, cultured in British
Columbia, Canada. Can. J. Zool., 76, 627635.
6. BURRESON E.M., ALVAREZ R.S., MARTINEZ V.V. & MACEDO L.A. (1994). Perkinsus marinus
(Apicomplexa) as a potential source of oyster Crassostrea virginica mortality in coastal lagoons of
Tabasco, Mexico. Dis. Aquat. Org., 20, 7782.
7. BURRESON E.M. & RAGONE CALVO L.M. (1996). Epizootiology of Perkinsus marinus disease of
oysters in Chesapeake Bay, with emphasis on data since 1985. J. Shellfish Res., 15, 1734.
8. BUSHEK D., FORD S.E. & ALLEN S.K. (1994). Evaluation of methods using Ray's fluid thioglycollate
medium for diagnosis of Perkinsus marinus infection in the eastern oyster, Crassostrea virginica. Ann.
Rev. Fish Dis., 4, 201217.
9. CALVO G.W., LUCKENBACH M.W., ALLEN S.K. & BURRESON E.M. (1999). A comparative field
study of Crassostrea gigas (Thunberg 1793) and Crassostrea virginica (Gmelin 1791) in relation to salinity
in Virginia. J. Shellfish Res., 18, 465474.
10. CALVO G.W., LUCKENBACH M.W., ALLEN S.K. & BURRESON E.M. (2001). A comparative field
study of Crassostrea ariakensis (Fujita 1913) and Crassostrea virginica (Gmelin 1791) in relation to
salinity in Virginia. J. Shellfish Res., 20, 221229.
11 CASAS S.M., VILLALBA A. & REECE K.S. (2002). Study of the perkinsosis of the carpet shell clam
Tapes decussatus in Galicia (NW Spain). I. Identification of the etiological agent and in vitro
modulation of zoosporulation by temperature and salinity. Dis. Aquat. Org., 50, 5165.
12. CAVALIER-SMITH T. (1998). A revised six-kingdom system of life. Biol. Rev., 73, 203266.
13. COOK T., FOLLI M., KLINCK J., FORD S. & MILLER J. (1998). The relationship between increasing
sea-surface temperature and the northward spread of Perkinsus marinus (Dermo) disease epizootics in
oysters. Estuarine Coastal Shelf Sci., 46, 587597.
14. COSS C.A., ROBLEDO J.F., RUIZ G.M. & VASTA G.R. (2001). Description of Perkinsus andrewsi n. sp.
isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and
development of a species-specific PCR-based diagnostic assay. J. Eukaryot. Microbiol., 48, 5261.
15. DE LA HERRN R., GARRIDO-RAMOS M.A., NAVAS J.I., RUIZ REJN, C. & RUIZ REJN, M. (2000).
Molecular characterization of the ribosomal RNA gene region of Perkinsus atlanticus: its use in
phylogenetic analysis and as a target for a molecular diagnosis. Parasitology, 120, 345353.
16. DUNGAN C.F., HAMILTON R.M.., HUDSON K.L., MCCOLLOUGH C.B. & REECE K.S. (2002). Two
epizootic infectious diseases in Chesapaeke Bay commercial clams Mya arenaria and Tagelus plebius.
17. FISHER W.S. & OLIVER L.M. (1996). A whole-oyster procedure for diagnosis of Perkinsus marinus
disease using Rays fluid thioglycollate culture medium. J. Shellfish Res., 15, 109117.
18. FORD S.E. (1996). Range extension by the oyster parasite Perkinsus marinus into the northeastern
United States: response to climate change? J. Shellfish Res., 15, 4556.
20. GOGGIN C.L. (1994). Variation in the two internal transcribed spacers and 5.8S ribosomal RNA
from five isolates of the marine parasite Perkinsus (Protista, Apicomplexa). Molec. Biochem. Parasitol.,
65, 179182.
21. GOGGIN C.L. & BARKER S.C. (1993). Phylogenetic position of the genus Perkinsus (Protista,
Apicomplexa) based on small subunit ribosomal RNA. Molec. Biochem. Parasitol., 60, 6570.
22 GOGGIN C.L. & LESTER R.J.G. (1995). Perkinsus, a protistan parasite of abalone in Australia: a
review. Aust. J. Mar. Freshwater Res., 46, 639646.
23. HAMAGUCHI M., SUZUKI N., USUKI H. & ISHIOKA H. (1998). Perkinsus protozoan infection in short-
necked clam Tapes (=Ruditapes) philippinarum in Japan. Fish Pathol., 33, 473480.
24. LESTER R.J.G. & DAVIS G.H.G. (1981). A new Perkinsus species (Apicomplexa, Perkinsea) from the
abalone, Haliotis ruber. J. Invertebr. Pathol., 37, 181187.
25. LEVINE N.D. (1978). Perkinsus gen. n. and other new taxa in the protozoan phylum Apicomplexa. J.
Parastitol., 64, 549.
26. MCLAUGHLIN S.M., TALL B.D., SHAHEEN A., ELSAYED E.E. & FAISAL M. (2000). Zoosporulation
of a new Perkinsus species isolated from the gills of the softshell clam Mya arenaria. Parasite, 7, 115
122.
27. MACKIN J.G. (1951). Histopathology of infection of Crassostrea virginica Gmelin by Dermocystidium
marinum Mackin, Owen and Collier. Bull. Marine Sci. Gulf Caribb., 1, 7287.
28. MACKIN J.G. (1962). Oyster disease caused by Dermocystidium marinum and other microorganisms in
Louisiana. Publ. Inst. Mar. Sci. Univ. Texas, 7, 132299.
29. MACKIN J.G., OWEN H.M. & COLLIER A. (1950). Preliminary note on the occurrence of a new
protistan parasite, Dermocystidium marinum n. sp., in Crassostrea virginica (Gmelin). Science, 111, 328
329.
30. MACKIN J.G. & RAY S.M. (1966). The taxonomic relationship of Dermocystidiium marinum, Mackin,
Owen and Collier. J. Invert. Pathol., 8, 544545.
31. MARSH A.G., GAUTHIER J.D. & VASTA G.R. (1995). A semiquantitative PCR assay for assessing
Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. J. Parasitol., 81, 577583.
32. MURRELL A., KLEEMAN S.N., BARKER S.C. & LESTER R.J.G. (2002). Synonymy of Perkinsus olseni
Lester & Davis, 1981 and Perkinsus atlanticus Azevedo, 1989 and an update on the phylogenetic
position of the genus Perkinsus. Bull. Euro. Assoc. Fish Pathol., 22, 258265.
33. NOREN F., MOESTRUP O. & REHNSTAM-HOLM A. (1999). Parvilucifera infectans Norn et Moestrup
gen. et sp. nov. (Perkinsozoa phylum nov.): a parasitic flagellate capable of killing toxic microalgae.
Europ. J. Protistol., 35, 233254.
34. PARK K.-I. & CHOI K.-S. (2001). Spatial distribution of the protozoan parasite Perkinsus sp. found in
Manila clams, Ruditapes philippinarum, in Korea. Aquaculture, 203, 922.
36. PERKINS F.O. (1996). The structure of Perkinsus marinus (Mackin, Owen & Collier, 1950) Levine,
1978 with comments on taxonomy and phylogeny of Perkinsus spp. J. Shellfish Res., 15, 6787.
37. RAY S.M. (1954). Biological studies of Dermocystidium marinum, a fungus parasite of oysters. Rice
Institute Pamphlet, Houston, Texas, USA, 114 pp.
38. RAY S.M. (1966). A review of the culture method of detecting Dermocystidium marinum with suggested
modifications and precautions. Proc. Natl. Shellfish. Assoc., 54, 5569.
39. REECE K.S., SIDDALL M.E., BURRESON E.M. & GRAVES J.E. (1997). Phylogenetic analysis of
Perkinsus based upon actin gene sequences. J. Parasitol., 83, 417423.
40. ROBLEDO J.A.F., COSS C.A. & VASTA G.R. (2000). Characterization of the ribosomal RNA locus of
Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay. J.
Parastol., 86, 972978.
41. ROBLEDO J.A.F., GAUTHIER J.D., COSS, C.A., WRIGHT A.C. & VASTA G.R. (1998). Species-
specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern Oyster, Crassostrea
virginica: a comparison with the fluid thioglycollate assay. J. Parasitol., 84, 12371244.
42. SIDDALL M.E., REECE K.S., GRAVES J.E. & BURRESON E.M. (1997). Total evidence refutes the
inclusion of Perkinsus species in the phylum Apicomplexa. Parasitology, 115, 165176.
43. SIDDALL M.E., REECE K.S., NERAD T.A. & BURRESON E.M. (2001). Molecular determination of the
phylogenetic position of a species in the genus Colpodella (Alveolata). Am. Mus. Novitates, 3314, 110.
44. SUNILA I., HAMILTON R.M. & DUNGAN C.F. (2001). Ultrastructural characteristics of the in vitro cell
cycle of the protozoan pathogen of oysters, Perkinsus marinus. J. Eukaryot. Microbiol., 48, 348361.
45. VOLETY A.K. & CHU F.-L.E. (1994). Comparison of infectivity and pathogenicity of meront
(trophozoite) and prezoosporangiae stages of the oyster pathogen Perkinsus marinus in eastern
oysters, Crassostrea virginica (Gmelin, 1791). J. Shellfish. Res., 13, 521527.
46. YARNALL H.A, REECE K.S., STOKES N.A. & BURRESON E. M. (2000). A quantitative competitive
polymerase chain reaction assay for the oyster pathogen Perkinsus marinus. J. Parasitol., 86, 827837.
*
* *
C HA P T E R 3 . 1 . 6 .

S S O DI S E AS E
( Ha p l o s p o r i d i u m c o s t a l e )

GENERAL INFORMATION
SSO disease is caused by the protistan Haplosporidium costale (= Minchinia costalis) (13) of the phylum
Haplosporidia. Haplosporidium costale, commonly known as SSO (seaside organism), infects the oyster
Crassostrea virginica (3).
The geographical distribution of H. costale is in high salinity bays (>25 part per thousand) along the east
coast North America from Virginia, USA, to Nova Scotia, Canada (2, 7). Significant oyster mortalities
attributable to SSO disease have historically been restricted to coastal Virginia, Maryland and Delaware,
USA (2, 3), but significant mortality attributable to SSO recently occurred in Massachusetts, USA.
The plasmodial stage of H. costale occurs intercellularly in connective tissue of the digestive gland, mantle and
gonad (3, 11); spores occur throughout connective tissue in the same organs (2, 3, 10). Haplosporidium
costale has a limited seasonality. Oysters become infected in May and June at the time when spores are released
from previously infected moribund oysters (3, 6). Infections remain subclinical through the autumn and winter,
then plasmodia develop the following March/April, followed by synchronous sporulation and oyster mortalities
in May/June (2, 3, 6). This seasonal epizootiology may be more complicated than historically noted; H.
costale sporulation recently has been detected, and verified using molecular tools, in oysters collected in October
(11, 12).
The distribution of H. costale and H. nelsoni (the causative agent of MSX disease) overlaps in high salinity
areas from Virginia, USA, to Nova Scotia, Canada, and co-infections with both pathogens have been reported
(5, 6, 11). The pathogens can be readily differentiated during sporulation. Haplosporidium costale
sporulates throughout the connective tissue of most organs, whereas H. nelsoni sporulates only in the epithelium
of the digestive tubules (1, 3, 5). The plasmodial stages of these two pathogens, however, cannot be reliably
differentiated based on morphology (5), and definitive diagnosis in the absence of spores requires molecular tools
(11, 12).
Sporulation disrupts connective tissue and the high prevalence of spores in dead oysters suggests that sporulation
results in the death of the oyster. Sporulation is believed to be the end result of all H. costale infections (1) and
thus infections are invariably fatal (7).
It has not been possible to transmit H. costale experimentally in the laboratory. No life cycle has been
elucidated for any member of the phylum Haplosporidia, but an intermediate host is suspected (7).
For diagnosis, the recommended guidelines for sampling are those stated in Chapter 1.1.4 and Chapter I.2. of
this Aquatic Manual.
1.1. Histological examination
General histological procedures are detailed in Chapter I.2. of this Aquatic Manual. A transverse
section is cut through the visceral mass and placed in a fixative such as Davidsons or Carsons
Chapter 3.1.6. - SSO disease (Haplosporidium costale)
fluid (the latter enables the samples to be re-used for electron microscopy, if necessary). The ratio
must be no more than one volume of tissue to ten volumes of fixative. The sections are
subsequently treated by conventional histological procedures (4). Haplosporidia are revealed by
many nonspecific stains, such as haematoxylin & eosin (H&E).
Multinucleate plasmodia of Haplosporidium costale (usually 58 m in diameter) occur throughout
the connective tissue. Plasmodia are only easily detectable between March and June. Synchronous
sporulation of H. costale can be observed throughout the connective tissue of the digestive gland,
mantle and gonads; sporulation does not occur in the epithelia of the digestive tubules, as seen
with H. nelsoni spores (1, 10). Spores (3 5 m in size) are commonly found in gaping, moribund,
oysters. The parasite cannot readily be detected between July and March (1, 3); however, H. costale
infections, co-occurring with H. nelsoni, recently have been reported in October (11, 12).
Plasmodia of H. costale cannot reliably be distinguished from those of H. nelsoni on the basis of
morphology.
Infection intensity has been rated (4) as follows: localised (LO) = any infection where plasmodia
are localised in a small area of one tissue type; rare (R) = systemic infections with less than ten
plasmodia in the entire section; light (L) = systemic infections with less than two plasmodia per
field at 400 magnification, but more than ten plasmodia in the entire section; moderate (M) =
systemic infections with 25 plasmodia per field at 400 magnification; heavy (H) = more than
five plasmodia per field at 400 magnification; sporulation (S) = any infection where spores are
present.
2.1. Histology
When spores are present, H. costale can be presumptively diagnosed in the oyster Crassostrea
virginica if the spores are the correct size and occur throughout the connective tissue. For detailed
histological procedures see Section 1.1. of this Chapter and Chapter I.2. of this Aquatic Manual.
2.2. Polymerase chain reaction of DNA from oyster tissue
it detects DNA and not necessarily a viable pathogen. Other techniques, preferably in-situ
hybridisation, must be used to visualise the pathogen.
Two sets of PCR primers have been developed for H. costale detection: SSO1358F (5-TAC-TGC-
TAG-CGC-TTG-TTC-GCA-AGA-T-3) and SSO1507R (5-TCG-GGT-CGG-CCC-GCT-GAC-
TGG-GT-3) (8) and SSO-A (5-CAC-GAC-TTT-GGC-AGT-TAG-TTT-TG-3) and SSO-B (5-
CGA-ACA-AGC-GCT-AGC-AGT-ACA-T-3) (11). Both of these primer pairs target the small
subunit ribosomal DNA and have been shown to be sensitive and specific for this pathogen (8,
11). A multiplex PCR (9) for H. costale and H. nelsoni (MSX) has been developed using the former
SSO primers (8), but it has not been validated. PCR reaction mixtures contain reaction buffer (10
mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl
2
; 10 g/ml gelatin), 400 g/ml bovine serum
albumin, 25 pmoles each of SSO1358F and SSO1507R or SSO-A and SSO-B, 200 M each of
dATP, dCTP, dGTP, dTTP, 0.6 units AmpliTaq DNA polymerase (Applied Biosystems), and
template DNA, in a total volume of 25 l. The reaction mixtures are cycled in a thermal cycler.
The programme for the GeneAmp PCR System 9600 thermal cycler (Applied Biosystems) using
SSO-A and SSO-B is: initial denaturation at 94C for 4 minutes, 35 cycles of 94C for 30 seconds,
59C for 30 seconds, and 72C for 1.5 minutes, and a final extension at 72C for 5 minutes. The
cycle programme is identical using SSO1358F and SSO1507R except that the annealing
temperature is 55C. An aliquot (10% of reaction volume) of each PCR reaction is checked by
agarose gel electrophoresis and ethidium bromide staining for the 150 base pairs (bp)
amplification product of SSO1358F and SSO1507R or the 557 bp amplification product of SSO-
A and SSO-B.
Chapter 3.1.6. SSO disease (Haplosporidium costale)
3.1. In-situ hybridisation examination of Haplosporidium costale
In-situ hybridisation has recently been developed to differentiate plasmodia of H. costale from
those of H. nelsoni in tissue sections (11). Species-specific labelled oligonucleotide probes
hybridise with the small subunit ribosomal RNA of the parasites. This hybridisation is detected by
an antibody conjugate that recognises the labelled probes. Substrate for the antibody conjugate is
added, causing a colorimetric reaction that enables visualisation of probeparasite RNA
hybridisations.
The procedure for the in-situ hybridisation is conducted as follows. Positive and negative controls
must be included in the procedure.
i) Cut a transverse section through the visceral mass that includes mantle, gill and digestive
gland and place it in Davidsons AFA fixative (glycerin [10%], formalin [20%], 95% ethanol
[30%], dH
2
O [30%], glacial acetic acid [10%]) for approximately 24 hours, then transfer to
70% ethanol until processed by histological procedures (step ii). The ratio must be no more
than one volume of tissue to ten volumes of fixative.
Sections are cut at 56 m and placed on positively-charged slides or 3-aminopropyl-
iii) The sections are deparaffinised by immersing them in xylene or another less toxic clearing
agent for 10 minutes. The solvent is eliminated by immersion in two successive absolute
ethanol baths for 10 minutes each and rehydrated by immersion in an ethanol series. The
sections are then washed twice for 5 minutes in phosphate buffered saline (PBS).
v) The sections are prehybridised for 1 hour at 42C in prehybridisation solution (4 SSC, 50%
5 ng/l of the digoxigenin-labelled oligonucleotide probe. The sequence of the probe
designated SSO1318 (11) is 5-CGA-ACA-AGC-GCT-AGC-AGT-ACA-T-3. The sections
are covered with in-situ hybridisation plastic cover-slips and placed on a heating block at
90C for 12 minutes. The slides are then cooled on ice for 1 minute before hybridisation
overnight at 42C in a humid chamber.
vii) The sections are washed twice for 5 minutes each in 2 SSC at room temperature, twice for
5 minutes each in 1 SSC at room temperature, and twice for 10 minutes each in 0.5 SSC
at 42C. The sections are then placed in Buffer 1 (100 mM Tris, pH 7.5, 150 mM NaCl) for
12 minutes.
Chapter 3.1.6. - SSO disease (Haplosporidium costale)
sections. The sections are covered with in-situ hybridisation cover-slips and incubated for
2
hours in the dark. The colour reaction is stopped by washing in TE buffer (10 mM Tris, pH
8.0, 1 mM EDTA [ethylene diamine tetra-acetic acid]).
2
rinsed in dH
2
presence of H. costale is demonstrated by the purple-black labelling of the parasitic cells.
REFERENCES
1. ANDREWS J.D. (1982). Epizootiology of late summer and fall infections of oysters by Haplosporidium
nelsoni, and comparison to annual life cycle of Haplosporidium costalis, a typical haplosporidan. J.
2. ANDREWS J.D. (1988). Haplosporidium costale disease of oysters. In: Disease Diagnosis and Control in
North American Marine Aquaculture, Sindermann C.J. & Lightner D.V., eds. Elsevier, Amsterdam,
The Netherlands, 296299.
3. ANDREWS J.D. & CASTAGNA M. (1978). Epizootiology of Minchinia costalis in susceptible oysters in
seaside bays of Virginias eastern shore, 19591976. J. Invert. Pathol., 32, 124138.
4. BURRESON E.M., ROBINSON M.E. & VILLALBA A. (1988). A comparison of paraffin histology and
hemolymph analysis for the diagnosis of Haplosporidium nelsoni (MSX) in Crassostrea virginica (Gmelin).
J. Shellfish Res., 7, 1923.
5. COUCH J.A. (1967). Concurrent haplosporidian infections of the oyster, Crassostrea virginica (Gmelin).
J. Parasitol., 53, 248253.
6. COUCH J.A. & ROSENFIELD A. (1968). Epizootiology of Minchinia costalis and Minchinia nelsoni in
oysters introduced into Chincoteague Bay, Virginia. Proc. Natl. Shellfish. Assoc., 58, 5159.
8. KO Y.-T., FORD S.E. & FONG D. (1995). Characterization of the small subunit ribosomal RNA gene
of the oyster parasite Haplosporidium costale. Molec. Marine Biol. Biotechnol., 4, 236240.
10. PERKINS F.O. (1969). Electron microscope studies of sporulation in the oyster pathogen Minchinia
costalis (Sporozoa: Haplosporida). J. Parasitol., 55, 897920.
11. STOKES N.A. & BURRESON E.M. (2001). Differential diagnosis of mixed Haplosporidium costale and
Haplosporidium nelsoni infections in the eastern oyster, Crassostrea virginica, using DNA probes. J.
Chapter 3.1.6. SSO disease (Haplosporidium costale)
12. SUNILA I., STOKES N.A., SMOLOWITZ R., KARNEY R. C., & BURRESON E.M. (2002). Haplosporidium
costale (Seaside Organism), a parasite of the eastern oyster, is present in Long Island Sound. J. Shellfish
Res., 21, 113118.
13. WOOD J.L. & ANDREWS J.D. (1962). Haplosporidium costale (Sporozoa) associated with a disease of
Virginia oysters. Science, 136, 710711.
*
* *
C HA P T E R 3 . 1 . 7 .

WI T HE RI NG S YNDROME OF ABAL ONE
( Ca n d i d a t u s Xe n o h a l i o t i s c a l i f o r n i e n s i s )

GENERAL INFORMATION
Withering syndrome here refers only to the disease in abalone caused by Candidatus Xenohaliotis
californiensis on the west coast of California, United States of America (USA) and Baja California,
Mexico. However, as infected abalone have been transported to Chile, Japan, Israel and other countries, the
geographical range of the aetiological agent is suspected to be broad where abalone are cultured. If detected
outside the known range of Candidatus Xenohaliotis californiensis, light microscopy in combination with
molecular probes, if available, must be used to identify and distinguish the detected organism from other
rickettsial bacteria. The presence of these pathogens in any abalone should be regarded as potentially serious
and the OIE Reference Laboratory should be consulted.
Candidatus Xenohaliotis californiensis is an intracellular bacterium in the family Rickettsiaceae and is
closely related to members of the genera Ehrlichia, Anaplasma and Cowdria (4, 5, 6). The disease caused
by this bacterium is known as withering syndrome (5) and may be more appropriately termed abalone
rickettsiosis. Candidatus Xenohaliotis californiensis infects members of the genus Haliotis including black
abalone (H. cracherodii), red abalone (H. rufescens), pink abalone (H. corrugata), green abalone (H.
fulgens) and white abalone (H. sorenseni). Susceptibility varies with species and the bacterium is known
to cause disease in black, red, pink and green abalone.
Candidatus Xenohaliotis californiensis occurs along the south-west coast of North America in California,
USA and Baja California, Mexico. However, as infected abalone have been transported to Chile, Japan,
Israel and other countries, the geographical range of the aetiological agent is suspected to be broad where
California red abalone, Haliotis rufescens, are cultured.
Candidatus Xenohaliotis californiensis infects the gastrointestinal epithelial cells of the posterior
oesophagus, digestive gland and, to a lesser extent, intestine. The dimorphic rod-to-spherical shaped
bacterium measures an average of 332 x 1550 nm in the bacillus form and an average of 1405 nm in the
spherical morphotype. The bacteria reproduce within intracytoplasmic vacuoles 1456 m in diameter (6).
Severe infections result in withering syndrome, a disease that is characterised by morphological changes in the
digestive gland, which vary between species and may include degeneration (atrophy of tubules, increase in
connective tissues and inflammation) and/or metaplasia of the digestive tubules. The metaplasia involves the
replacement of terminal secretory/absorptive acini with absorptive/transport ducts similar in appearance to
the post-oesophagus. Some hyperplasia of the absorptive/transport ducts may also be involved. This
morphological change is accompanied by a decrease in feeding, depletion of glycogen reserves followed by use of
the foot muscle as an energy source and death. The foot of affected abalone contains fewer and less organised
muscle bundles, abundant connective tissue and may contain more cerous cells than unaffected individuals.
Disease (withering syndrome) occurs at elevated water temperatures (~18C and above) (9). The
incubation period of withering syndrome is prolonged and ranges between 3 and 7 months (10).
Cumulative mortality has been recorded at over 99% in black abalone and over 30% in red abalone (3, 5,
7). The pathogen and disease (withering syndrome) may occur year round, but losses due to the disease occur
most often in the summer and autumn, after a 34-month period when temperatures are elevated over
15C. Reducing densities and application of an oxytetracycline-medicated diet may reduce losses.
Chapter 3.1.7. Withering syndrome of abalone (Candidatus Xenohaliotis californiensis)
1.1. Histology
Histological procedure is detailed in chapter I.2 of this Aquatic Manual. Remove the shell and cut
several 35 mm cross sections that contain posterior oesophagus (post-oesophagus), digestive
gland, and foot muscle and placed in Davidsons or Carsons solutions (see Chapter I.2. of this
Aquatic Manual) for 24 hours and process for routine paraffin histology. Cross sections are most
easily handled when placed in cassettes prior to fixation. The ratio must be no more than one
volume of tissue to ten volumes of fixative.
Deparaffinised 35 m sections should be stained with haematoxylin and eosin and viewed by
light microscopy for RLP (Rickettsiales-like prokaryote) inclusions in the post-oesophagus and
digestive gland, and morphological changes in the digestive gland and foot. It is recommended
that sections should be examined at 200 or 400 magnification.
Candidatus Xenohaliotis californiensis may be morphologically similar to other marine rickettsial
bacteria. Definitive diagnosis of the bacterium may include molecular tools (in-situ hybridisation).
Definitive diagnosis of withering syndrome by histology must include the presence of the
bacterium and morphological changes to the digestive gland and may include those of the foot
muscle.
Where losses have been observed within the known geographical range of withering syndrome,
visualisation of intracellular bacterial foci within digestive epithelia, by histological examination,
may be considered to be a confirmatory method. However, confirmation by in-situ hybridisation is
recommended to verify the identity of the rickettsial bacteria in abalone species previously not
known to be susceptible to the bacterium or in a new geographical location.
2.1. Histology
See Section 1.1. above.
Tissue imprints may be used to detect moderate to high intensities of infection of Candidatus
Xenohaliotis californiensis. However, histology is more sensitive than tissues imprints.
Excise a section of the post-oesophagus, mince and lay on a slide, dry with a hair dryer for
~20 minutes. Stain the slides using a fluorescent stain for nucleic acid such as propidium
iodide or Hoechst. Incubate in the dark for 3 minutes and view by epifluorescence at
200 magnification. Bacterial inclusions are differentiated from host nuclei by size and frequency.
However, if the sample slides are to be retained for future examination, they should be thoroughly
dried and stored desiccated until staining.
Inclusions of the parasite, 1456 m in diameter, appear interspersed with the smaller host nuclei.
An observation time of 5 minutes per slide is sufficient at 200 magnification.
it detects DNA and not necessarily a viable pathogen. Other techniques, preferably histology and
in-situ hybridisation, must be used to visualise the pathogen.
The PCR primers developed for Candidatus Xenohaliotis californiensis detection specifically
amplify a 160 base-pair segment of the Rickettsia-like pathogen. Primers are currently designated
as: RA 5-1 (5-GTT-GAA-CGT-GCC-TTC-AGT-TTA-C-3) and RA 3-6 (5-ACT-TGG-ACT-
CAT-TCA-AAA-GCG-GA-3). They target small subunit ribosomal DNA and have been shown
to be sensitive and specific for this pathogen (1). PCR amplification is performed in a standard
50 l reaction volume containing 10 mM Tris, pH 8.3 (at 25C), 50 mM KCl, 1.5 mM MgCl
2
,
0.001% (w/v) gelatin, 400 M of dNTP, 5 M tetramethyl ammonium chloride, 40 pmoles of
each primers, 2 units of Taq polymerase, and template DNA. The reaction mixtures are cycled in a
thermal cycler. The programme for amplification reaction is: initial denaturation at 95C for
5 minutes, 40 cycles at 95C for 1 minute, 50C for 30 seconds, and 72C for 30 seconds, and a
final extension at 72C for 10 minutes. An aliquot of each PCR reaction is checked for the
160 base pair amplification product by agarose gel electrophoresis and ethidium bromide staining.
3.1. In-situ hybridisation
In-situ hybridisation has recently been developed to detect Rickettsiales-like prokaryotes in tissue
sections (2). Specific labelled oligonucleotide probes hybridise with the small subunit ribosomal
RNA of the bacterium. This hybridisation is detected by an antibody conjugate that recognises the
labelled probes. Substrate for the antibody conjugate is added, causing a colorimetric reaction that
enables visualisation of probeparasite RNA hybridisations.
The procedure of in-situ hybridisation is conducted as follows. Positive and negative controls must
be included in the procedure.
i) After removing the shell, a transverse section (35 mm) is cut that contains posterior
oesophagus (post-oesophagus), digestive gland, and foot muscle and placed in Davidsons
AFA fixative (glycerin [10%], formalin [20%], 95% ethanol [30%], dH
2
O [30%], glacial acetic
acid [10%]) for 2448 hours, then transferred to 70% ethanol until processed by histological
procedures (step ii). The ratio must be no more than 1 volume of tissue to 10 volumes of
fixative.
Sections are cut at 56 m and placed on positively charged slides or 3-aminopropyl-
iii) The sections are deparaffinised by immersion in xylene or other less toxic clearing agent for
10 minutes. The solvent is eliminated by immersion in two successive absolute ethanol baths
for 10 minutes each and rehydrated by immersion in an ethanol series. The sections are then
washed twice for 5 minutes in phosphate buffered saline (PBS).
v) The sections are prehybridised for 1 hour at 42C in prehybridisation buffer (4 SSC, 50%
2 ng/l of the digoxigenin-labelled oligonucleotide probes. The sequences of the probes
designated as RA 5-1, RA 3-6, RA 3-8 and RA 5-6 (2) are, respectively: 5-GTT-GAA-CGT-
GCC-TTC-AGT-TTA-C-3, 5-ACT-TGG-ACT-CAT-TCA-AAA-GCG-GA-3, 5-CCA-
CTG-TGA-GTG-GTT-ATC-TCC-TG-3, and 5-GAA-GCA-ATA-TTG-TGA-GAT-AAA-
GCA-3. The sections are covered with in-situ hybridisation plastic cover-slips and placed on
a heating block at 90C for 12 minutes. The slides are then cooled on ice for 1 minute before
hybridisation overnight at 40C in a humid chamber.
vii) The sections are washed twice for 5 minutes in 2 SSC at room temperature, twice for
5 minutes in 1 SSC at room temperature, and twice for 10 minutes in 0.5 SSC at 40C.
The sections are then placed in Buffer 1 (100 mM Tris, pH 7.5, 150 mM NaCl) for 1
2 minutes.
sections. The sections are covered with in-situ hybridisation cover slips and incubated for
2
hours in the dark. The colour reaction is stopped by washing in TE buffer (10 mM Tris,
pH 8.0, 1 mM EDTA [ethylene diamine tetra-acetic acid]).
2
rinsed in dH
2
presence of the pathogen is demonstrated by the purple-black labelling of the parasitic cells.
Transmission electron microscopy procedures are described in Chapter I.2. of this Manual. Rod-
shaped, ribosome-rich prokaryotes with trilaminar cell walls accumulated into intracellular
colonies within membrane-bound vacuoles in the cytoplasm of gastrointestinal epithelial cells are
observed (6).
REFERENCES
1. ANDREE K.B., FRIEDMAN C.S., MOORE J.D. & HEDRICK R.P. (2000). A polymerase chain reaction
for detection of genomic DNA of a Rickettsiales-like prokaryote associated with Withering Syndrome
in Black Abalone (Haliotis cracherodii). J. Shellfish Res., 19, 213218.
2. ANTONIO D.B., ANDREE K.B., MOORE J.D., FRIEDMAN C.S. & HEDRICK R.P. (2000). Detection of
Rickettsiales-like prokaryotes (RLPs) by In situ hybridization in black abalone Haliotis cracherodii with
Witheirng Syndrome. J. Invertebr. Pathol., 75, 180182.
3. FRIEDMAN C.S., THOMSON M., CHUN C., HAAKER P.L. & HEDRICK, R.P. (1997). Withering
syndrome of the black abalone, Haliotis cracherodii (Leach): Water temperature, food availability, and
parasites as possible causes. J. Shellfish Res., 16, 403411.
4. FRIEDMAN C.S., ANDREE K.B., BEAUCHAMP K.A., MOORE J.D., ROBBINS T.T., SHIELDS J.D. &
HEDRICK R.P. (2000). Candidatus Xenohaliotis californiensis a newly described pathogen of
abalone, Haliotis spp., along the west coast of North America. Int. J. Syst. Evol. Microbiol., 50, 847
855.
5. FRIEDMAN C.S., BIGGS W., SHIELDS J.D. & HEDRICK R.P. (2002). Transmission of Withering
Syndrome in black abalone, Haliotis cracherodii Leach. J. Shellfish Res., In press.
6. GARDNER G.R., HARSHBARGER J.C., LAKE J., SAWYER T.K., PRICE K.L., STEPHENSON M.D.,
HAAKER P.L. & TOGSTAD H.A. (1995). Association of prokaryotes with symptomatic appearance of
withering syndrome in black abalone Haliotis craherodii. J. Invertebr. Pathol., 66, 111120.
7. HAAKER P.L., PARKER D.O., TOGSTAD H., RICHARDS D.V., DAVIS G.E. & FRIEDMAN C.S. (1992).
Mass mortality and withering syndrome in black abalone Haliotis cracherodii, in California. In: Abalone
of the World, Shepard S.A., Tegner M.J. & Guzman del Proo S.A., eds, Blackwell Scientific, Oxford,
UK, 214224.
8. MOORE J.D., ROBBINS T.T. & FRIEDMAN C.S. (2000). Withering syndrome in farmed red abalone
Haliotis rufescens: Thermal induction and association with a gastrointestinal rickettsia-like prokaryote. J.
9. STEINBECK J.R., GROFF J.M., FRIEDMAN C.S., MCDOWELL T. & HEDRICK R.P. (1992).
Investigations into mortality among populations of the California black abalone, Haliotis cracherodii,
on the central coast of California, USA. In: Abalone of the World, Shepard S.A., Tegner M.J. &
Guzman del Proo S.A., eds. Blackwell Scientific, Oxford, UK, 203213.
*
* *
S E C T I ON 4 . 1 .


C HA P T E R 4 . 1 . 1 .

T AURA S YNDROME
SUMMARY
Taura syndrome (TS), caused by TS virus (TSV), has resulted in serious disease epizootics throughout the
shrimp-growing regions of the Western Hemisphere (1, 36, 1023, 27, 33). The virus has recently been
introduced into Asia with infected imported Pacific white shrimp, Penaeus vannamei, from Central and
South American sources (30, 32).
Although it is not listed in the most recent report of the International Committee on Nomenclature of
Viruses (31), TSV has been characterised and tentatively assigned to the family Picornaviridae (2, 12,
24, 29). TSV was recently placed in the genus Cricket paralysis-like viruses (25). The virus replicates in
the cytoplasm of host cells. TSV particles are 32 nm, nonenveloped icosahedrons with a buoyant density of
1.338 g/ml. The genome of TSV consists of a linear, positive-sense single-stranded RNA of 10,205
nucleotides, excluding the 3 poly-A tail, and it contains two large open reading frames (ORFs). ORF 1
contains the sequence motifs for nonstructural proteins, such as helicase, protease and RNA-dependent
RNA polymersae. ORF 2 contains the sequences for TSV structural proteins, including the three major
capsid proteins VP1, VP2 and VP3 (55, 40, and 24 kDa, respectively) (2, 24, 25).
The principal host for TSV is the Pacific white shrimp, P. vannamei, although other species can be
infected and present disease (1, 11, 17, 27, 29). Cumulative mortalities due to TSV epizootics have
ranged from 40 to >90% in cultured populations of postlarval (PL), juvenile, and subadult P. vannamei.
Survivors of TSV infections may carry the virus for life (4, 11, 15, 16, 22). TSV has been demonstrated
to remain infectious in the faeces of sea gulls that have ingested infected shrimp carcasses, which may
implicate birds as being an important route of transmission of the virus within affected farms or farming
regions (8, 17).
TSV can also infect other Western Hemisphere penaeid species (i.e. P. stylirostris, P. setiferus, and
P. schmitti), sometimes resulting in disease and mortalities in PL or yearly juvenile stages, but also in
asymptomatic persistent infections (4, 27). Other Western Hemisphere penaeids (P. aztecus and
P. duorarum) and Eastern Hemisphere penaeids (P. chinensis, P. monodon, and P. japonicus)
have been experimentally infected with TSV (4, 15, 16, 27).
Following its recognition in 1992 as a distinct disease of cultured P. vannamei in Ecuador (1, 3, 4, 13,
15, 16, 20), TS has spread rapidly throughout many of the shrimp-farming regions of the Americas
through shipments of infected PL and broodstock (36, 10, 15). Within the Western Hemisphere, TS and
TSV have been reported from virtually every penaeid shrimp-growing region in the Americas and Hawaii
(1, 16, 19, 29, 33). TSV is enzootic in cultured penaeid shrimp stocks on the Pacific coast of the
Americas from Peru to Mexico, and it is occasionally found in some wild stocks of P. vannamei from the
same region (1517, 19). TSV has also been reported in cultured penaeid stocks from the Atlantic,
Caribbean, and Gulf of Mexico coasts of the Americas, but it has not been reported in wild stocks from the
Chapter 4.1.1. - Taura syndrome
these regions (10, 16). Until the recent reports of an introduction of the disease to Taipei China with
imported P. vannamei from Central America, TSV had not been confirmed to occur in wild or cultured
penaeid shrimp stocks outside of the Western Hemisphere (30, 32).
During the 19992000 shrimp farming seasons, a new stain of TSV emerged in Mexico that caused high
mortalities (as high as 90%) in stocks of Pacific blue shrimp, P. stylirostris, that were previously resistant
to TS (17, 33). What may be a second newly emerged strain of TSV appeared in 2001 in Central
America where it caused similarly high mortalities in selected stocks of P. vannamei that had been selected
because of demonstrated resistance to TSV (7). The new Mexican strain of TSV may be distinguished
with antibody-based methods from the reference (Hawaiian 1994) isolate of TSV (2, 12, 25), and from
other (but otherwise indistinguishable) geographic and temporal isolates of the virus (7).
Standard histological and molecular methods are the surveillance methods for TSV in penaeid shrimp (13,
14, 19, 24). With standard histological methods, diagnostic lesions during the acute phase of the disease are
demonstrated in specific target tissues, especially the cuticular epithelium. In chronic-phase infections,
lymphoid organ spheroids are the only lesion apparent in shrimp with persistent TSV infections. In some
circumstances, molecular methods may be more effective for surveillance than routine histology. Specific
cDNA probes applied to in-situ hybridisation assays with paraffin sections provide the greatest diagnostic
certainty of this agent (912, 16, 17, 19). The use of specific primer sets in reverse-transcription
polymerase chain reaction assays for TSV provides the advantages of larger sample sizes and nonlethal
sampling of broodstock (19, 26). A variety of other diagnostic methods can be used to provide presumptive
and confirmatory diagnoses of TSV infections. Among these methods are live shrimp bioassay (3-5, 8,
12, 15) and antibody-based methods with monoclonal antibodies (28).
Eradication methods for TSV have been successfully applied to certain aquaculture situations. These
methods are dependent on the total depopulation of infected stocks, disinfection of the culture facility,
avoidance of re-introduction of the virus (from other nearby culture facilities, wild shrimp, etc.), and re-
stocking with TSV-free postlarvae that have been produced from TSV-free broodstock (6, 17, 18).
The available diagnostic methods for Taura syndrome (TS) and its agent TS virus (TSV) include traditional
methods that employ clinical signs, gross pathology(ies), histology, and bioassays. Antibody-based
methods that use monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assay (ELISA) formats
and molecular methods that use nonradioactively labelled gene probes, and reverse-transcription
polymerase chain reaction (RT-PCR) are additional methods available for diagnosis of infection by TSV.
The methods currently available for surveillance, detection, and diagnosis of TSV infections are listed in
Table 1. The designations used in the Table indicate: the method is presently unavailable or unsuitable;
? the method is available but untested; the method has application in some situations, but cost,
accuracy, or other factors severely limits its application; the method is a standard method with good
diagnostic sensitivity and specificity; and the method is the recommended method for reasons of
availability, utility, and diagnostic specificity and sensitivity.
Table 1. TSV surveillance, detection and diagnostic methods
Method Screening Presumptive Confirmatory
Larvae PLs Juveniles Adults
Gross signs

Direct BF/LM

Histopathology

Table 1 (cont.). TSV surveillance, detection and diagnostic methods
Bioassay

Transmission EM

Antibody-based methods

DNA probes in situ
RT-PCR

PLs = postlarvae; BF = bright field; LM = light microscopy; EM = electron microscopy;
RT-PCR = reverse-transcription polymerase chain reaction.
Sampling procedures: see Chapter I.3.
1. STANDARD SCREENING METHODS FOR TSV
1.1. Histological method
As in Section 2.2.
1.2. In-situ hybridisation with nonradioactive cDNA probes
Nonradioactive, DIG-labelled cDNA probes for TSV may be produced in the laboratory or
obtained from commercial sources. A ShrimProbe
TM
kit for TSV in an in-situ hybridisation format
is available from DiagXotics, (Wilton, Connecticut [CT], USA). This method provides greater
diagnostic sensitivity than do more traditional methods for TSV detection and diagnosis that
employ classic histological methods (1518, 24, 26).
The in-situ hybridisation assay of routine histological sections of acute- and transition-phase
lesions in the cuticular epithelium, other tissues, and of lymphoid organ spheroids (LOS) in
transition and chronic phase with a specific DIG-labelled cDNA probe to TSV, provides a
definitive diagnosis of TSV infection. Pathognomonic TSV-positive lesions display prominent
blue to blue-black areas in the cytoplasm of affected cells when reacted with the cDNA probes.
Not reacting to the probe are the prominent karyorrhectic nuclear fragments and pyknotic nuclei
that contribute to the pathognomonic buckshot riddled appearance of TS lesions (9, 15, 16, 24).
(See Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis virus [IHHNV] for details
of the in-situ hybridisation method. See Chapter I.3. Section 4.2. for detailed information on the
use of Davidsons AFA and RF-fixatives.)
False-negative in-situ hybridisation results may occur with Davidsons fixed tissues if tissues are
left in fixative for more than 2448 hours. The low pH of Davidsons fixative causes acid
hydrolysis of the TSV single-stranded RNA genome, resulting in false-negative probe results. This
artefact can be avoided through the use of neutral fixatives, including an RNA-friendly fixative
developed for shrimp, or by the proper use (avoiding fixation times over 24 hours) of Davidsons
fixative (9, 16, 19).
1.3. Reverse-transcription polymerase chain reaction method
Haemolymph samples may be assayed for TSV using RT-PCR (26). Primers designated as 9195
and 9992, amplify a 231 base pair (bp) sequence of the TSV genome.
Primer Sequence G:C ratio Temperature
9195 5-TCA-ATG-AGA-GCT-TGG-TCC-3 50 63C
9992 5-AAG-TAG-ACA-GCC-GCG-CTT-3 55 69C
i) The RT-PCR assay is done in solution, using 1.0 l of shrimp haemolymph as the RNA
template, or 10 l of total RNA extracted from tissue or haemolymph (concentration of
RNA = 1100 ng/ml).
Note: 10% sodium citrate is commonly used as an anticoagulant for shrimp haemolymph
samples and, without dilution, it may inhibit reverse transcription or PCR. Hence, diluting
crude haemolymph 1/10 in water is recommended if inhibition by sodium citrate is
suspected.
ii) The following controls should be included in every RT-PCR assay for TSV: a) known
TSV-negative tissue sample; b) a known TSV-positive sample (tissue or purified virus);
and C) a no-template control.
iii) The GeneAmp, EZ rTth RNA PCR kit (Applied Bioscience, Norwalk, CT) is used for
all amplification reactions described here.
iv) The optimised RT-PCR conditions (final concentrations in 50 l total volume) for
detection of TSV in Penaeus vannamei are: primers (0.46 M each), dNTPs (300 M each),
rTth DNA polymerase (2.5 U/50 l), manganese acetate (2.5 mM), in 5 EZ buffer
(25 mM Bicine, 57.5 mM potassium acetate, 40% [w/v] glycerol, pH 8.2).
v) The optimised RT-PCR conditions for detection of TSV from infected P. stylirostris are
the same as for P. vannamei, except that the amount of the rTth DNA polymerase should
be increased to 5.0 U/50 l.
vi) If the thermal cycler does not have a heated lid, then light mineral oil (50 l) is overlaid on
the top of the 50 l reaction mixtures to prevent condensation or evaporation during
thermal cycling.
vii) The RNA template and all the reagents are combined and reverse transcription is allowed
to proceed at 60C for 30 minutes, followed by 94C for 2 minutes.
Note: The reaction conditions described here were optimised using an automatic DNA
Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, CT). The conditions should be
optimised for each thermal cycler using known positive controls.
viii) At the completion of reverse transcription, the samples are amplified for 35 cycles under
the following conditions: denaturation at 94C for 45 seconds, and then annealing/
extension at 60C for 45 seconds. A final extension step for 7 minutes at 60C follows the
last cycle and the process is terminated in a 4C soak file.
ix) Following the termination of RT-PCR, the amplified cDNA solutions are drawn off from
beneath the mineral oil and placed into clean 0.5 ml microfuge tubes.
x) A 10 l sample of the amplified product can then be added to the well of a 2.0% agarose
gel, stained with ethidium bromide (0.5 g/ml), and electrophoresed in 0.5 TBE (Tris,
boric acid, ethylene diamine tetra-acetic acid [EDTA]).
xi) A 100 bp DNA ladder (Gibco/BRL, Gaithersburg, Maryland, USA) is used as a marker.
xii) Details of the composition of the reagents and buffers used here may be found in
Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis (IHHN).
2. DIAGNOSTIC METHODS FOR CONFIRMATORY TESTS
2.1. Gross signs
TS is best known as a disease of nursery- or growout-phase P. vannamei, which occurs within
~1440 days of stocking of postlarvae into growout ponds or tanks. Hence, shrimp with TS are
typically small juveniles of from ~0.05 g to <5 g. Larger shrimp may also be affected, especially if
they are not exposed to the virus until they are larger juveniles or adults (3, 16, 17, 22).
TS disease has three distinct phases, acute, transition, and chronic, which are grossly
distinguishable (11, 16). Gross signs presented by shrimp in the acute and especially the transition
phases of TS are unique and can provide a provisional diagnosis of the disease (9, 11, 15, 16).
Gross signs displayed by moribund P. vannamei with acute-phase TS include expansion of the red
chromatophores giving the affected shrimp a general, overall pale reddish coloration and making
the tail fan and pleopods distinctly red; hence red tail disease was one of the names given by
farmers when the disease first appeared in Ecuador (20). In such shrimp, close inspection of the
cuticular epithelium in thin appendages (such as the edges of the uropods or pleopods) with a 10
hand lens reveals signs of focal epithelial necrosis. Shrimp showing these gross signs of acute TS
typically have soft shells, an empty gut and are often in the late D stages of the molt cycle. Acutely
affected shrimp usually die during ecdysis. If the affected shrimp are larger than ~1 g, moribund
shrimp may be visible to sea birds at the pond edges and surface. Thus, during the peak of severe
epizootics, hundreds of sea birds (gulls, terns, cormorants, etc.) may be observed feeding on
affected shrimp (35, 8, 16, 20.).
Although only present for a few days during TS epizootics, the gross signs presented by shrimp in
the transition phase can provide a tentative diagnosis of TSV infection. During the transition
phase (which may be occurring while many shrimp in the affected populations are still in the acute
phase and daily mortalities are high), fair to moderate numbers of shrimp in affected ponds show
random, multifocal, irregularly shaped melanised cuticular lesions. These melanised spots are
haemocyte accumulations indicating resolving TS lesions in the cuticular epithelium. Such shrimp
may or may not have soft cuticles and red-chromatophore expansion, and may be behaving and
feeding normally. After successfully molting, shrimp in the transition phase move into the chronic
phase of TS in which the virus maintains a persistent infection of the lymphoid organ, perhaps for
life. Such TSV carriers may pass the virus horizontally to other susceptible shrimp or possibly
vertically to its progeny. Although vertical transmission is suspected, the actual mechanism of
transmission of TSV from parent broodstock to their progeny has not been demonstrated (11, 15,
16, 20).
TSV typically infects cells in tissues of ectodermal and mesodermal origin. TSV infections in
P. vannamei and P. stylirostris have three phases: acute, transition and chronic. The cuticular
epithelium is the most severely affected tissue in the acute phase, while only the lymphoid organ
seems to be infected by the virus during the chronic phase of the disease. In P. vannamei, acute-
phase TSV infection may result in high mortalities, while most strains of P. stylirostris can be
infected, but are resistant to the development of TS disease. Survivors of TSV acute infections
pass through a brief transition phase and enter a long-term chronic phase in which the affected
host may remain persistently infected for life. Such TSV carriers may pass the virus horizontally to
other susceptible shrimp (2, 3, 9, 12, 13, 16, 17).
Diagnosis of TS in the acute phase of the disease is dependent on the histological demonstration
(in haematoxylin-and-eosin-stained preparations), of multifocal areas of necrosis in the cuticular
epithelium of the general body surface, all appendages, gills, hindgut, oesophagus and stomach.
Occasionally affected are cells of the subcuticular connective tissues and adjacent striated muscle
fibres basal to affected cuticular epithelium. Rarely, the antennal gland tubule epithelium is
affected. Prominent in the multifocal cuticular lesions are conspicuous foci of affected cells that
display an increased eosinophilia of the cytoplasm and pyknotic or karyorrhectic nuclei.
Cytoplasmic remnants of necrotic cells are often extremely abundant in these TS acute-phase
lesions and these are generally spherical bodies (120 m diameter) that range in staining from
eosinophilic to pale basophilic. These structures, along with pyknotic and karyorrhectic nuclei,
give acute-phase TS lesions a characteristic peppered or buckshot-riddled appearance, which is
considered to be pathognomonic for the disease. Pyknotic or karyorrhectic nuclei give a positive
(for DNA) Feulgen reaction, which distinguishes them from the less basophilic to eosinophilic
cytoplasmic inclusions that do not contain DNA. The absence of haemocytic infiltration or other
signs of a significant host-inflammatory response distinguishes the acute phase of TS from the
transitional phase of the disease (35, 912, 15, 16 20).
In the transitional phase of TS, typical acute-phase cuticular lesions decline in abundance and
severity and are replaced by conspicuous infiltration and accumulations of haemocytes at the same
sites. The masses of haemocytes may become melanised giving rise to the black spots that
characterise the transition phase of the disease. Such lesions may show erosion of the cuticle,
surface colonisation and invasion of the affected cuticle and exposed surface haemocytes by
Vibrio spp. (11, 16).
Shrimp in the chronic phase of TS display no gross signs of infection, and histologically the only
sign of infection is the presence of prominent LOS, which are spherical accumulations of cells
that lack the central vessel of normal lymphoid organ tubules (11, 16, 17).
2.3. Bioassay method
Further confirmation of TSV infection may be accomplished by bioassay of TSV-suspect animals
with specific pathogen free (SPF) juvenile P. vannamei serving as the indicator for the virus (3, 5, 8,
11, 12, 16, 22, 27). Oral or injection protocols may be used. The oral method is relatively simple to
perform and is accomplished by feeding chopped carcasses of suspect shrimp to SPF juvenile
P. vannamei in small tanks. The use of a negative control tank of indicator shrimp, which receives
only a normal feed, is required. When the carcass feeding (per os) protocol is used to bioassay for
TSV, TS-positive indicator shrimp (by gross signs and histopathology) are typically apparent
within 34 days of initial exposure, and significant mortalities occur by 38 days after initial
exposure. The negative control shrimp must remain negative for gross or histological signs of TS
disease and unusual mortalities (11, 12, 16).
With the injection bioassay protocol, a variety of sample types may be tested for TSV. Whole
shrimp are used if they were collected during a TSV epizootic. Heads only are used if shrimp
display gross transition-phase lesions (multifocal melanisation spots on the cuticle) or no clinical
signs of infection (chronic phase) as the virus, if present, will be concentrated in the lymphoid
organ (11, 16). For nonlethal testing of broodstock, haemolymph samples may be taken and used
to expose the indicator shrimp (16).
To perform the bioassay:
i) Prepare a 1:2 or 1:3 ratio of TSV-suspect shrimp heads or whole shrimp with TN buffer (see
Chapter 4.1.6. IHHNV for the composition of this buffer) or sterile 2% saline prepared with
distilled water.
ii) Homogenise the mixture using a tissue grinder or blender. Do not permit the mixture to heat
up by excessive homogenisation or grinding. Tissues and resulting homogenate should be
kept cool during the entire protocol by maintaining on ice.
iii) Clarify the homogenate by centrifugation at 3000 g for 10 minutes. Decant and save the
supernatant fluid. Discard the pellet.
iv) Centrifuge the supernatant fluid at 27,000 g (15,000 rpm) for 2030 minutes at 4C. Decant
and save the supernatant fluid. Discard the pellet.
v) Dilute the supernatant fluid from step iv to 1/10 to 1/100 with sterile 2% saline. This
solution may now be used as the inoculum to inject indicator shrimp (or filtered sterilised as
described in step vi).
vi) Filter the diluted supernatant fluid from step v using a sterile syringe (size depends on final
volume of diluted supernatant) and a sterile 0.45 m syringe filter. Multiple filters may have
to be used as they clog easily. Filtrate should be collected in a sterile test tube or beaker. The
solution can now be stored frozen (recommend 20C for short-term [weeks] storage and
80C for long-term [months to years] storage) or used immediately to inject indicator
shrimp.
vii) Indicator shrimp should be from TSV susceptible stocks of SPF P. vannamei (such as the
Kona stock), which are commercially available from a number of sources in the Americas,
and not from selected lines of known TSV-resistant stocks.
viii) Inject 0.01 ml per gram of body weight using a 1 ml tuberculin syringe. Indicator shrimp
should be injected intramuscularly into the third tail segment. If the test shrimp begin to die
within minutes post-injection, the inoculum contains excessive amounts of proteinaceous
material and should be further diluted prior to injecting additional indicator shrimp. Sudden
death occurring post-injection is referred to as protein shock, and is the result of systemic
clotting of the shrimps haemolymph in response to the inoculum.
ix) Haemolymph samples may be diluted (1/10 or 1/20 in TN buffer), filter sterilised (if
necessary), and injected into the indicator shrimp without further preparation.
x) If TSV was present in the inoculum, the indicator shrimp should begin to die within 24
48 hours post-injection. Lower doses of virus may take longer to establish a lethal infection
and shrimp should be monitored for at least 7 days post-injection.
xi) The presence of TSV in the indicator shrimp should be confirmed by histological analysis
(and in-situ hybridisation by gene probe if available) of Davidsons fixed moribund shrimp.
As a variation to the bioassay technique, a sentinel shrimp system may be used. For example,
TSV-sensitive stocks of small juvenile SPF P. vannamei may be held in net-pens in tanks, or in the
same water system, with other shrimp of unknown TSV status to bioassay for the presence of
infectious agents such as TSV.
2.4. Antibody-based methods
MAb for detection of TSV may be used to assay samples of haemolymph, tissue homogenates, or
Davidsons AFA fixed tissue sections from shrimp (28). Currently, TSV-1A1-MAb (a mouse
immunoglobulin isotype IgG1) is available in kit form from DiagXotics (Wilton, CT, USA).
TSV-1A1-Mab may be used to distinguish an apparently new strain of TSV from other strains (7).
During the 19992000 shrimp farming seasons, a new stain of TSV emerged in Mexico that
caused high mortalities (as high as 90%) in stocks of Pacific blue shrimp, P. stylirostris, that were
previously resistant to TS (17, 33). What may be a second newly emerged strain of TSV appeared
in 2001 in Central America where it caused similarly high mortalities in selected stocks of
P. vannamei that had been selected because of demonstrated resistance to TSV (7). By not reacting
with TSV-1A1-MAb, while giving positive tests for TSV with available molecular tests (i.e. RT-
PCR and in-situ hybridisation), the new Mexican strain of TSV may be distinguished from the
reference (Hawaiian 1994) isolate of TSV (2, 12, 25), and from other (but otherwise
indistinguishable) geographical and temporal isolates of the virus that do react with TSV-1A1-
MAb (7).
Dot-blot immunoassay method
i) For the dot-blot immunoassay method, 1 l of test antigen (purified virus, infected shrimp
haemolymph or SPF shrimp haemolymph) is dotted on to the surface of MA-HA-N45 assay
plates (Millipore, South San Francisco, California [CA], USA).
ii) After air drying, the wells are blocked for 1 hour at room temperature with 200 l of a buffer
containing phosphate buffered saline and 0.05% Tween 20 (PBST) mixed with 10% normal
goat serum (Life Technologies, Gibco BRL) and 2% Hammersten casein (Amersham Life
Sciences, Arlington Heights, Illinois, USA).
iii) The wells are washed three times with PBST and then reacted with 100 l primary antibody
(MAb or mouse polyclonal antibodies) for 30 minutes at room temperature.
iv) Alkaline-phosphatase-labelled goat anti-mouse IgG, chain specific, secondary antibody
(Zymed, South San Francisco, CA) diluted 1/1000 in PBST plus 10% normal goat serum is
used for detection (30 minutes at room temperature).
v) After washing three times with PBST, once with PBS and once with distilled water, the
reactions are visualised by development for 15 minutes at room temperature with nitroblue
tetrazolium and bromo-chloro-indoyl phosphate (Roche Diagnostics, Corp.) in Tris-NaCl
(100 mM each) buffer containing 50 mM MgCl
2
, pH 9.5.
vi) Reactions are stopped with distilled water.
vii) The reactions are graded using a scale from 0 to 4, with the highest intensity reaction being
equivalent to the reaction generated using the MAb against the reference control consisting
of semipurified TSV. A negative reaction is one in which no coloured spot is visible in the
well.
Other antibody-based methods
The TSV-1A1-MAb may be applicable to other antibody-based test formats (i.e. indirect
fluorescent antibody or immunohistochemistry (IHC) tests with tissue smears, frozen sections,
or deparaffinised fixed tissues). TSV-1A1-MAb is applicable for use in an IHC format using
Davidsons AFA fixed tissue sections (7), and a kit for this procedure is available commercially
(DiagXotics, Wilton, CT, USA). Application to other antibody-based test formats (i.e. indirect
fluorescent antibody test with tissue smears, frozen sections, or deparaffinised fixed tissues) is
possible, but still experimental at this time.
Recent evidence indicates that there are serological differences among some geographical
isolates of TSV that may cause a false-negative reaction when using TSV-1A1-MAb, which is
the only TSV antibody currently available from commercial sources (DiagXotics, Wilton CT,
USA). Therefore, it is recommended that unexpected results from MAb-based tests for TSV
should be interpreted in the context of clinical signs, case history, and in conjunction with
other test results (e.g., histology or in-situ hybridisation with a TSV-specific DNA probe).
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* *
C HA P T E R 4 . 1 . 2 .

WHI T E S P OT DI S E AS E

SUMMARY
White spot disease (WSD) of penaeid shrimp is characterised by high and rapid mortality accompanied by
gross signs in moribund shrimp of white, initially circular, inclusions or spots in the cuticle, sometimes
accompanied by overall red body coloration. Disease progression is characterised by cessation of shrimp
feeding followed within a few days by the appearance of moribund shrimp swimming near the surface at the
edge of rearing ponds. The cuticular inclusions range from minute spots to discs several millimetres in
diameter, which may coalesce into larger plates. These are most easily observed by removing the cuticle over
the cephalothorax, scraping away any attached tissue with the thumbnail and holding the cuticle up to the
light. Rapid and severe pond mortality follows shortly after the first appearance of gross signs. However, the
appearance of white spots in the cuticle is unreliable even for preliminary diagnosis of WSD as similar
inclusions can be produced by high alkalinity and other environmental conditions and bacterial white spot
syndrome (61). Therefore, preliminary diagnosis must include histopathological examination.
The causative agent of WSD is white spot syndrome virus (WSSV) or white spot virus (WSV), a double-
stranded DNA (dsDNA) virus of the genus whispovirus within the family nimaviridea (39, 40).
WSSV has a wide host range among crustaceans (9, 29) and is potentially lethal to most of the
commercially cultivated penaeid shrimp species. Virions are rod-shaped to elliptical with a trilaminar
envelope and they are large (80120 250380 nm) (7, 15, 16, 52, 62, 66). Negatively stained virions
purified from shrimp haemolymph show unique, tail-like appendages (66). The virions are generated in
hypertrophied nuclei of infected cells without the production of occlusion bodies. In initial reports, WSSV
was described as a non-occluded baculovirus, but even while the molecular data were still limited, the
preliminary WSSV DNA sequence analysis (Maeda & Walker, pers. comm., 28), the morphological
characteristics and the general biological properties of the virus (29, 66) already highlighted its uniqueness.
Recent data, including the genome sequence and phylogenies based on DNA polymerase and protein kinase,
suggest that WSSV is a member of a new virus family (3, 27, 59, 68). The status of work on WSSV
has been reviewed by Lightner (26), Flegel (9), Flegel et al. (10), Loh et al. (35), Lo & Kou (31) and
Lo et al. (33).
The size of the WSSV genome has been differently reported for different isolates: 305107 bp (GenBank
Accession No. AF332093), 292967 bp (GenBank Accession No. AF369029) and 307287 bp
(GenBank Accession No. AF440570) for viruses isolated from the Peoples Republic of China, Thailand
and Taipei China, respectively. The sequences of these three isolates are almost identical, with the size
differences being due mostly to several small insertions and one large (~12 kbp) deletion (4). For two of
these isolates (from the Peoples Republic of China and Thailand), the analysis of the complete WSSV
genome has been published (59, 68). The following descriptions are based mostly on the WSSV isolate
from the Peoples Republic of China (68). The WSSV genome has a total G+C content of 41%. Three
per cent of the WSSV genome is made up of nine homologous regions containing 47 repeated mini-
fragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes, while the
remaining 97% of the sequence is unique. A total of 531 putative open reading frames (ORFs) were
identified by sequence analysis, among which 181 ORFs are likely to encode functional proteins. Thirty-six
of these 181 ORFs have been identified by screening and sequencing a WSSV cDNA library or else have
already been reported to encode functional proteins. Transcription of another 52 ORFs was confirmed by
reverse-transcription polymerase chain reaction (RT-PCR). For 80% of the putative 181 ORFs there is a
potential polyadenylation site (AAT-AAA) downstream of the ORF.
Chapter 4.1.2. - White spot disease
Among the 181 annotated ORFs, the proteins encoded by 18 ORFs show 4068% identity to known
proteins from other viruses or organisms or contain an identifiable functional domain. These proteins include
enzymes involved in nucleic acid metabolism and DNA replication, a collagen-like protein, and six
published WSSV structural proteins. Thirty ORFs have predicted proteins that show a partial homology
(2039% identity) to known proteins or contain one or two sequence motifs (as opposed to a functional
domain). The remaining ORFs encode proteins with no homology to any known proteins or motifs. The
uniqueness of this virus means that it is hard to directly apply other virus infection models to interpret the
infection strategy of WSSV; on the contrary, WSSVs infection strategy must be investigated ab initio.
The continuing thorough study of its functional genomics and molecular biology are therefore urgently needed
to better understand its nature, replication strategy and the molecular mechanisms of its pathogenesis, and
also to provide useful information for the development of virus control measures. To date, although much of
the sequence of the WSSV genome is already known, and the predicted sequences for many genes have
already been published, only a few WSSV genes have been studied beyond this sequence analysis. These
include genes encoding the DNA polymerase (3), ribonucleotide reductase small and large subunits (55),
structural proteins (58, 60, 69), a novel chimeric polypeptide of cellular-type thymidine kinase and
thymidylate kinase (56, 57), a protein kinase (27) and a nucleocapsid protein with nuclear targeting
behaviour (4). Many genes that are important for the completion of WSSVs infection cycle still remain to
be studied.
WSD outbreaks were first reported from farmed Penaeus japonicus in Japan in 1993 (15, 16, 42, 43,
45) and the causative agent was named penaeid rod-shaped DNA virus (PRDV) or rod-shaped nuclear
virus of P. japonicus (RV-PJ) (15, 16, 23, 42, 52). Later, outbreaks of viral disease with similar gross
signs and caused by similar rod-shaped viruses were reported from elsewhere in Asia and other names were
applied: hypodermal and haematopoietic necrosis baculovirus (HHNBV) in the Peoples Republic of China
(13, 14); white spot baculovirus (WSBV) and PmNOBIII in Taipei China (5, 28, 29, 62, 64); and
systemic ectodermal and mesodermal baculovirus (SEMBV) or PmNOBII in Thailand (66, 67). The
virus from the Peoples Republic of China has also been called Chinese baculovirus (CBV) (34, 37).
Shrimp exhibiting the gross signs and histopathology of WSD have also been reported from Korea (22, 48),
India (18, 19, 41), the Philippines (38) and the USA (26, 34). During 1999, WSD also had a severe
impact on the shrimp industries of both Central and South America (11, 12). Because of similar gross
signs, ultrastructure and molecular biology, Lightner (26) has included these viruses in a single white spot
syndrome baculovirus or white spot syndrome virus (WSSV) complex, which is referred to here simply as
white spot virus or WSSV.
The histopathology of WSD is distinctive and can be used for diagnosis with moribund shrimp during
outbreaks (26, 42, 43, 66). These moribund shrimp exhibit systemic destruction of tissues of ectodermal
and mesodermal origin with many infected cells showing hypertrophied nuclei with lightly to deeply basophilic
central inclusions (haematoxylin and eosin [H&E] staining) surrounded by marginated chromatin. These
begin as Cowdry type-A inclusions. The inclusions can be seen in rapidly stained squash mounts of gills or
subcuticular tissue (10) or in tissue sections. In tissue sections, the best tissues for examination are gill
tissue and subcuticular tissue of the stomach or cephalothorax.
Confirmation of WSD requires more detailed analysis by PCR (21, 22, 24, 33, 46, 53), antibody-based
assays (34, 44, 49, 50), in-situ DNA hybridisation (6, 46), or transmission electron microscopy (TEM)
(7, 52, 66). Using such techniques, it has been shown that WSSV from captured juvenile and broodstock
shrimp or other carriers are identical or closely related (29, 30, 34, 53, 62, 65, 67). These molecular
techniques have also been used to confirm infection of more than 40 penaeid and non-penaeid crustacean
carriers (9) and, tentatively, an aquatic insect larval carrier (29). Some of these carriers have been shown to
transmit WSSV to shrimp (17, 51) in laboratory tests. Detection of WSSV in carrier shrimp or other
crustaceans cannot be reliably accomplished by histological methods, and more sophisticated techniques are
required.
As WSSV readily infects species of shrimp such as P. vannamei that are geographically separated from
the WSSV endemic area (25, 26, 36, 37), it is possible that such shrimp, and especially specific pathogen
free (SPF) lines from controlled breeding programmes, could be used for WSSV bioassays (8, 47). Another
approach would be to use continuous cell lines, but unfortunately none is presently available for the study of
shrimp viruses, although WSSV has been cultivated in primary cell lines from the lymphoid organ of P.
vannamei (34, 54) and P. monodon (20, 63) where it was used to measure WSSV titres and to
observe virus replication, respectively. However, the need to continually prepare cultures from different
shrimp or from shrimp of undefined or uncertain history currently presents problems for test standardisation.
Because of these limitations, it is recommended that detection of WSSV in the absence of overt signs of
disease, as for surveillance or for certification of broodstock or fry for stocking, should be carried out by
PCR, Western blot analysis, or in-situ DNA hybridisation.
There are two main concerns regarding detection of white spot syndrome virus (WSSV). One is the
confirmation of disease outbreaks and the other is the certification of broodstock and post-larvae or fry
used to stock rearing ponds. For presumptive diagnosis of outbreaks in suspected ponds, it is sufficient to
carry out histological analysis by light microscopy of haematoxylin and eosin (H&E)-stained tissues from
moribund shrimp. For definitive diagnosis and certification of WSSV infection status of broodstock and
fry, polymerase chain reaction (PCR) technology is recommended. However, definitive diagnosis can also
be accomplished by in-situ DNA hybridisation, Western blot analysis and transmission electron
microscopy (TEM).
The various methods for surveillance, detection and diagnosis of WSSV are listed in Table 1. The
designations used in the Table indicate: the method is presently unavailable or unsuitable; ? the
method is available but untested; the method has application in some situations, but cost, accuracy, or
other factors severely limits its application; the method is a standard method with good diagnostic
sensitivity and specificity; and the method is the recommended method for reasons of availability,
utility, and diagnostic specificity and sensitivity. These are somewhat subjective as suitability involves
issues of reliability, sensitivity, and utility.
Table 1. WSV surveillance, detection and diagnostic methods
Gross signs +
Bioassay

Direct dark-field LM

Histopathology

Transmission EM

Antibody-based assays ? ?

DNA Probes in situ
? ?

PCR

PLs = postlarvae; LM = light microscopy; EM = electron microscopy; PCR = polymerase chain reaction.
Shrimp material suitable for virological examination:
Clinically affected shrimp: whole shrimp, haemolymph, gills or pleopods.
Asymptomatic shrimp (apparently healthy shrimp): whole larvae, postlarvae and juvenile shrimp;
haemolymph, gills or pleopods from juvenile to broodstock specimens.
1. STANDARD SCREENING METHODS FOR WSSV
1.1. Nested polymerase chain reaction of tissues and haemolymph
The protocol described here is according to Lo et al. (28, 32), although alternative assays have
been described (21, 22, 24, 46, 53). In brief, collect living shrimp specimens and, using a
disposable stick, homogenise approximately 100200 mg of tissue (or use 100 l of haemolymph)
from live juvenile to subadult shrimp, postlarvae 11 upwards (PL11 up) with removed heads, or
whole PL10 downwards in a 1.5 ml microfuge tube with 600 l of lysis solution (100 mM NaCl,
10 mM Tris/HCl, pH 8, 25 mM EDTA [ethylene diamine tetra-acetic acid], 0.5% SLS [sodium N-
laurylsarcosinate] or 2% SDS [sodium dodecyl sulfate], and 0.5 mg/ml proteinase K added just
before use). Except for PL10 or below, be careful to exclude shrimp eyes from samples, as these
are known to contain PCR inhibitor(s) (28, 32). After homogenisation, incubate at 65C for
1 hour. (If the virus load is very low, or if very high quality, less degraded genomic DNA is
required, start with 1000 l of lysis solution and insert this step: centrifuge at 13,000 g for
5 minutes and transfer 600 l of the supernatant to a fresh 1.5 ml tube.) Add 5 M NaCl to a final
concentration of 0.7 M. Next slowly add 1/10 volume of N-cetyl N,N,N-trimethylammonium
bromide (CTAB)/NaCl solution (10% CTAB in 0.7 M NaCl) and mix thoroughly. Incubate at
65C for 10 minutes, and then, at room temperature, add an equal volume of chlorofoum/
isoamyl alcohol (24/1) and mix gently. Centrifuge at 13,000 g for 5 minutes and then transfer the
aqueous solution (upper layer) to a fresh 1.5 ml tube and add an equal volume of phenol. Mix
gently and centrifuge at 13,000 g for 5 minutes. Collect the upper layer solution and repeat the
phenol extraction process 1 to 2 times. Transfer the final upper layer to a new tube, mix gently
with two volumes of chloroform/isoamyl alcohol (24/1) and centrifuge at 13,000 g for 5 minutes.
Transfer the upper layer to a new tube and precipitate DNA by adding two volumes of 95% or
absolute ethanol followed by standing at 20C for 30 minutes or 80C for 15 minutes.
Centrifuge at 13,000 g for 30 minutes and discard the ethanol. Wash the DNA pellet with 70%
ethanol, dry and resuspend in 100 l sterilised double-distilled water at 65C for 15 minutes. Use 1
l of this DNA solution for one PCR reaction.
For the first-step PCR reaction, add 1 l DNA template solution (containing about 0.10.3 g
DNA) to a PCR tube containing 100 l of reaction mixture (10 mM Tris/HCl, pH 8.8, 50 mM
KCl, 1.5 mM MgCl
2
, 0.1% Triton X-100, 200 M of each dNTP, 100 pmol of each primer, 2 units
of thermal stable DNA polymerase). The outer primer sequences are 146F1, 5-ACT-ACT-AAC-
TTC-AGC-CTA-TCT-AG-3 and 146R1, 5-TAA-TGC-GGG-TGT-AAT-GTT-CTT-ACG-A-3.
The PCR profile is one cycle of 94C for 4 minutes, 55C for 1 minute, and 72C for 2 minutes,
followed by 39 cycles of 94C for 1 minute, 55C for 1 minute, and 72C for 2 minutes and a final
5-minute extension at 72C. The WSSV-specific amplicon from this reaction is 1447 bp. The
sensitivity is approximately 20,000 copies of a plasmid template.
For the second-step of the (nested) PCR reaction, add 10 l of the first-step PCR reaction product
to 90 l of PCR cocktail of the same composition as above except that it contains the second
(inner) primer pair: 146F2 (5-GTA-ACT-GCC-CCT-TCC-ATC-TCC-A-3) and 146R2 (5-TAC-
GGC-AGC TGC-TGC-ACC-TTG-T-3). Use the same PCR amplification protocol as above.
The WSSV-specific amplicon from this reaction is 941 bp. The overall sensitivity of both steps is
approximately 20 copies of a WSSV plasmid template.
To visualise, electrophorese 10 l of PCR reaction products on 1% agarose gels containing
ethidium bromide at a concentration of 0.5 g/ml. Decapod-specific primers (143F 5-TGC-CTT-
ATC-AGC-TNT-CGA-TTG-TAG-3 and 145R 5-TTC-AGN-TTT-GCA-ACC-ATA-CTT CCC-
3 yielding an 848 bp amplicon; N represents G, A, T, or C) are available to verify the quality of
the extracted DNA (32) and the integrity of the PCR reaction. A positive control (WSSV DNA
template) and negative controls (no template and shrimp DNA template) should be included in
every assay.
1.2. PCR of postlarvae
From a nursery or hatchery tank containing 100,000 postlarvae (PL) or more, sample
approximately 1000 PL from each of five different points. Pool the samples in a basin, gently swirl
the water in the basin and then select an assay sample from living PL that collect at the centre of
the basin. Choose the sample number according to the assumed prevalence (see Chapter I.3.). For
PL11 and above be careful to exclude shrimp eyes from samples, as these are known to contain
PCR inhibitor(s) (28, 32). Homogenise the sample in an appropriate volume of lysis solution (see
Section 1.1.) and then follow the procedure for nested PCR with a 600 l fraction as described in
Section 1.1. Homogenates that have been stored in lysis solution at room temperature for more
than 1 year can still be used for PCR, but prolonged storage in lysis solution decreases the DNA
quality and reduces assay sensitivity.
1.3. Antibody-based assays
Both polyclonal (PAbs) and monoclonal antibodies (MAbs) to the virus have been used for the
detection of WSSV in clinical specimens.
The protocol described here for the production and application of polyclonal antibodies for
WSSV detection is from Nadala et al. (44) and Loh et al. (34). This includes purification of WSSV
virions from laboratory-infected shrimp, generation of immunoglobulins (Ig) in New Zealand
white rabbits, purification of the IgG using recombinant bacterial protein-G columns, and
removal of cross-reacting normal shrimp antigens by adsorption on to acetone-dried, ground
shrimp muscle tissue and haemolymph. For assay, remove 0.1 ml of haemolymph from live
shrimp specimens and dilute in an equal volume of citrate buffer (19.3 mM sodium citrate,
239.8 mM NaCl, 182.5 mM glucose, 6.2 mM EDTA, pH 7.0) for immediate use or storage at
80C until used. For Western blotting, use a 200 l sample, clarify at 8000 g for 5 minutes and
then pellet the supernatant at 140,000 g for 5 minutes. Resuspend pellets in 100 l 2 loading
buffer (2.5 ml 0.5 mM Tris/HCl, pH 6.8, 4 ml 10% SDS, 2 ml glycerol, 1 ml beta-
mercaptoethanol, and 0.5 ml deionised distilled water) and heat at 95C for 5 minutes. Load a
10 l sub-sample on to 10% SDSPAGE (polyacrylamide gel electrophoresis), and electrophorese
at 200 V. Blot the electrophoresed gel on to a nitrocellulose membrane (0.1 m pore size) in
blotting buffer (3.03 g Tris base, 14.4 g glycine, and 200 ml methanol per litre) at 100 V for
1 hour. Rinse the membrane with phosphate buffered saline (PBS), pH 7.4, soak in 5% skim milk
(in PBS) for 1 hour, and rinse with PBS for 5 minutes. Next, treat the membrane with a 1/1000
dilution of the primary antibody (IgG) for 1 hour, rinse three times with PBS for 5 minutes, and
then treat with a 1/2500 dilution of goat anti-rabbit IgG-horseradish peroxidase conjugate for
1 hour. Rinse again three times with PBS for 5 minutes and then treat with the substrate, 3,3, 5,5-
tetramethylbenzidine (TMB), until a bluish purple colour develops. Stop the reaction by soaking
the membrane in distilled water. All incubations should be carried out at 25C2C. Use a
purified viral preparation as a positive control and identify four major protein bands characteristic
of WSSV at 19, 23.5, 27.5 and 75 kDa. The sensitivity is 1 ng of WSSV protein.
The protocol described here for the production and application of MAbs for WSSV detection is
from Poulos et al. (49). Inject BALB/cByJ mice intraperitoneally with purified WSSV emulsified
in synthetic adjuvant (MPL+TDM [monophosphoryl lipid A and trehalose dimycolate]). Test sera
from the mice for antibody production after two booster immunisations. For the final
immunisation, use WSSV structural proteins in the range of 1535 kDa that have been eluted
from SDSPAGE gel slices and emulsified in synthetic adjuvant. Two days after immunisation,
remove spleen cells from one mouse and fuse them with SP2/0-Ag-14 myeloma cells using
polyethylene glycol. Use purified WSSV in a dot-immunoblot assay to screen the hybridoma for
the production of WSSV-specific MAbs. The highly specific MAbs can be adapted to various test
formats (49, 50). The following three formats are relatively rapid and sensitive enough and for the
detection of WSSV in clinical shrimp specimens (49).
For fluorescent antibody detection of WSSV in fresh tissue impression smears, remove the
stomachs of moribund shrimp and prepare impression smears on positively charged microscope
slides. Make a sagittal section and transfer the epithelial cells to the slide. Immediately spray-fix
using Surgipath cytology fixative and store at room temperature until used for reaction with the
antibodies. Just before antibody staining, fix the smears by immersion in 100% methanol for
10 minutes Rehydrate the smears by incubation in three changes of PBS for 2 minutes Apply
500 l of blocking reagent (PBS with 10% normal goat serum and 0.2% Hammersten casein) to
the smears for 15 minutes at room temperature. Apply the MAbs for 45 minutes at room
temperature. Wash the smears in three changes of PBS, and apply a fluorescein-conjugated goat
anti-mouse IgG secondary antibody for 45 minutes at room temperature. Wash three times in
PBS, and mount the smears with a cover-slip using a fluorescent mounting medium. Observe
under an epifluorescent microscope. Green reactions are fluorescing WSSV-infected cells.
For immunohistochemistry on fixed sections, fix moribund shrimp with Davidsons AFA fixative
for 2448 hours. Embed the tissues in paraffin and cut into 4 m sections. Place sections on to
Superfrost Plus positively charged microscope slides. Heat at 65C to melt the paraffin, rehydrate
the sections through a series of washes in Hemo-De (a xylene substitute), 95% alcohol, 80%
alcohol, 50% alcohol and distilled water. Incubate the sections for 5 minutes in PBS and then
block with PBS containing 10% normal goat serum and 2% Hammersten casein for 15 minutes at
room temperature. Apply the MAbs to the sections for 30 minutes at room temperature. Wash in
three changes of PBS, and react the sections with a goat anti-mouse IgG (H&L) F(ab)
2
antibody
conjugated to alkaline phosphatase and diluted 1/1000 in PBS for 30 minutes at room
temperature. Develop the reactions for 1530 minutes using nitroblue tetrazolium and
bromochloroindoyl phosphate in a pH 9.5 buffer. Counterstain the sections with Bismarck brown
and dehydrate through a series of alcohol washes ending with Hemo-De. Mount with a cover-slip
and permanent mounting medium and examine under a light microscope for the presence of a
blue-black precipitate.
For the whole mount assay, fix tissue from WSSV-infected shrimp in Davidsons AFA and
transfer to 70% alcohol after 2448 hours. Cut the tissue into 24 mm blocks using a razor blade
and place into a microfuge tube. All subsequent reactions are performed by adding 250500 l of
reagent to the tube, allowing to incubate and then removing the solution with a pipette. First
rehydrate the tissue by immersing in 50% ethanol for 15 minutes and wash six times with distilled
water over a 10-minute period. Homogenise the tissue blocks with a plastic pestle and treat with a
blocking reagent (PBS, 2% Hammersten casein, 10% normal goat serum) for 15 minutes at 37C.
React the tissues with 10 concentrated MAb tissue culture supernatant fluid for 30 minutes at
37C. Wash the tissues with several changes of PBS and add the secondary antibody (goat anti-
mouse IgG [H&L] F[ab]
2
conjugated to alkaline phosphatase and diluted 1/1000) for 30 minutes
at 37C. Wash the tissue again with PBS and add the detection reagent (nitroblue tetrazolium
[NBT] and bromochloro-indoyl phosphate in pH 9.5 buffer) for 3060 minutes at room
temperature. Wash the tissue with distilled water, counterstain with Bismarck brown and
dehydrate through a series of alcohol washes ending with Hemo-De. Place the crushed tissue on
to a microscope slide, mount with a cover-slip and permanent mounting medium and examine
under a light microscope for the presence of a blue-black precipitate within the nuclei of WSSV-
infected cells.
1.4. In-situ DNA hybridisation
The protocol described here was developed based on Nunan & Lightner (46), although an
alternative protocol has been described by Chang et al. (2). In brief, place 5 m paraffin sections
on silanised or positively charged slides, and heat the slide on a hot plate at 65C for 30 minutes.
Deparaffinise, rehydrate and then treat for 230 minutes (depending on tissue type) with
100 g/ml proteinase K in Tris/NaCl/EDTA (TNE) buffer at 37C. Post-fix slides by chilling in
pre-cooled 0.4% formaldehyde for 5 minutes at 4C and wash the slides in 2 standard saline
citrate (SSC; 1 SSC = 150 mM NaCl, 15 mM tri-sodium citrate, pH 7.0) at room temperature.
Prehybridise the slides with prehybridisation solution (50% formamide, 0.2% Ficoll 400, 0.2%
polyvinylpyrrolidone, 0.2% bovine serum albumin, 5 SSC, 1 mM EDTA, 50 mM Tris/HCl,
pH 8) for 30 minutes at 42C. Follow with hybridisation with the 1447 bp WSSV-specific PCR
amplicon (described in Section 1.1. above) that has been labelled with digoxigenin (DIG), and
then with visualisation using a light microscope. For hybridisation, boil the probe for 10 minutes
and immediately place on ice. Dilute the probe to 3050 ng/ml in prehybridisation solution and
apply 500 l to each slide. Put the slide on a hotplate at 8595C for 610 minutes (make sure that
it does not reach boiling point), quench slides on ice for 5 minutes and then transfer to a humid
chamber for 1620 hours at 42C. After hybridisation, wash the slides twice for 15 minutes each
time with 2 SSC at room temperature, twice for 5 minutes with 1 SSC at 37C and twice for
5 minutes with 0.5 SSC at 37C. For hybridisation detection, wash slides with maleic acid buffer
(100 mM maleic acid, 150 mM NaCl, pH 7.5) for 5 minutes at room temperature. Block the slides
with blocking solution (2% normal goat serum and 0.3% Triton X-100 in maleic acid buffer) for
30 minutes at 37C. Add 250 l anti-DIG alkaline phosphatase (AP)-conjugated antibody solution
(1 l/ml anti-DIG/AP-Fab fragment in maleic acid buffer containing 1% normal goat serum and
0.3% Triton X-100) to each slide, and incubate at 37C for 30 minutes Wash the slides twice with
maleic acid buffer for 10 minutes each and once with detection buffer (100 mM Tris/HCl,
100 mM NaCl, pH 9.5) at room temperature. Add 500 l of development solution (prepare
immediately before use by adding 45 l NBT salt solution [75 mg/ml in 70% dimethyformamide],
35 l 5-bromo-4-chloro-3-indoyl phosphate, toluidinum salt [X-phosphate] solution [50 mg/ml in
dimethylformamide] and 1 ml 10% PVA to 9 ml of detection buffer) to each slide and incubate in
the dark in a humid chamber for 13 hours. Stop the reaction by washing slides in TE buffer
(10 mM Tri-HCl, 1 mM EDTA, pH 8.0) for 15 minutes at room temperature. Wash slides in
distilled water for ten dips, counterstain the slides in 0.5% aqueous Bismarck Brown Y for 30
90 seconds and then rinse with water. Wet mount using aqueous mounting media for observation
immediately or dehydrate the slides and mount with Permount mounting media for long-term
preservation. Mount the slides with cover-slips and examine with a bright field microscope.
Positive hybridisation appears as a dark blue to black precipitate against the yellow to brown
counterstain.
2. PRESUMPTIVE DIAGNOSTIC METHODS FOR WSSV
Presumptive diagnosis of WSSV can be achieved by the following method.
2.1. Rapid staining of squash mount preparations
There are two general types of rapid squash mount preparations that can be used for presumptive
diagnosis of white spot disease (WSD). One employs fresh, unstained wet mounts fixed with
formalin and viewed by dark-field microscopy with a wet-type condenser. The other employs
fixed tissue stained with H&E.
The dark-field technique is described by Momoyama et al. (42). In brief: dissect out the stomach
as a source of subcuticular tissue or peel off thin layers of subcuticular tissue from the
cephalothorax and fix in a 10% formalin solution. Using fine forceps spread thin pieces of the
subcuticular tissue on a slide in a small volume of 10% formalin. Add a cover-slip and remove
excess solution by placing a filter paper at the edge of the cover-slip. Using dark-field optics, focus
the microscope on an area of the preparation where shrimp pigment cells are poorly distributed.
Specimens with WSD will show moderate to large numbers of refractile, hypertrophied nuclei.
For rapidly stained H&E preparations (10), collect moribund shrimp from a suspected outbreak
of WSD and fix the whole shrimp or gill filaments in Davidsons fixative (26) overnight. After
fixation, wash some gill filaments thoroughly with tap water to remove the fixative. Then stain
with Meyers H&E (26). After staining and dehydration, when the tissue is in xylene, place a gill
filament on a microscope slide in a drop of xylene. Using a fine pair of needles (a stereo
microscope is helpful), break off several secondary filaments and then replace the main filament in
xylene where it can be stored indefinitely as a permanent reference in a sealed vial. Being careful
not to let the xylene dry, tease apart the secondary filaments on the microscope slide and remove
any large fragments or particles that would thicken the mount unnecessarily. Finally, add a drop of
mounting fluid and a cover glass. Use light pressure to flatten the mount as much as possible. This
procedure may also be used with thin layers of subcuticular tissue. With WSD outbreaks,
examination with the 40 objective of the light microscope will reveal the presence of moderate
to large numbers of hypertrophied nuclei with basophilic central inclusions surrounded by
marginated chromatin (10). It is important also to detect some nuclei with Cowdry type-A
inclusions characteristic of the early stage of WSSV infection. Like the fixed tissues and the
filaments in xylene, these whole mount slides can be kept as a permanent record.
In the event that rapid results are required, the fixation step can be shortened to only 2 hours by
changing the acetic acid portion in the Davidsons fixative formula to 50% concentrated HCl (10).
For best results, this fixative should not be stored for more than a few days before use. After
fixation, wash thoroughly to remove the fixative and check that the pH has returned to near
neutral before staining. Do not fix for longer periods or above 25C as this may result in excessive
tissue damage that will make interpretation difficult or impossible.
2.2. Bioassay
Bioassay methods for WSSV are available, but their application is limited to labs with access to
susceptible WSSV-free reference shrimp stocks (8, 47). For bioassay, remove the pleopods from
shrimp suspected of WSSV infection and homogenise in TN buffer (0.02 M Tris/HCl, 0.4 M
NaCl, pH 7.4). Following centrifugation at 1000 g for 10 minutes, dilute the supernatant fluid
1/10 with 2% NaCl and filter (0.2 m filter). Inject 0.2 ml of inoculum into the dorso-lateral
aspect of the fourth abdominal segment of indicator shrimp (specific pathogen free shrimp at
juvenile stage), injecting between the tergal plates into the muscle of the third abdominal segment.
Examine moribund shrimp grossly orby using the methods described in Section 2.1.
3. CONFIRMATORY DIAGNOSTIC METHODS FOR WSSV
Confirmatory diagnosis of WSSV can be achieved by any of the following methods.
3.1. Histopathology of tissue sections
Fix moribund shrimp from a suspected WSD outbreak in Davidsons fixative and process for
preparation of standard H&E-stained tissue sections (1, 26). Examine the sections by light
microscopy for the presence of moderate to large numbers of hypertrophied nuclei with
basophilic central inclusions surrounded by marginated chromatin in tissues of ectodermal and
mesodermal origin (66). Some of the hypertrophied nuclei will have Cowdry type-A inclusions.
Particularly useful for viewing WSD histopathology are the subcuticular tissues of the stomach
and cephalothorax and the gills.
3.2. Nested PCR of tissues and haemolymph (see Section 1.1.)
3.3. Antibody-based assays (see Section 1.3.)
3.4. In-situ DNA hybridisation (see Section 1.4.)
For TEM, the most suitable tissues of moribund shrimp suspected of WSV infection are
subcuticular tissues, gills and pereiopods. For screening or surveillance of grossly normal shrimp,
the most suitable tissue is subcuticular tissue from the stomach. The reagents described below are
from ref. 24. In brief, stun living shrimp by immersion in ice water until just immobilised or kill by
injection of fixative. Quickly dissect and remove small portions of target tissue (no larger than a
few millimetres in diameter) and fix in at least 10 volumes of 6% glutaraldehyde held at 4C and
buffered with sodium cacodylate (Na[CH
3
]
2
AsO
2
.3H
2
O) solution (Na cacodylate 8.6 g, NaCl 10 g,
distilled water to make 100 ml adjusted to pH 7 with 0.2 N HCl) or phosphate solution
(NaH
2
PO
4
.H
2
O 0.6 g, Na
2
HPO
4
1.5g, NaCl 1 g, sucrose 0.5 g, distilled water to make 100 ml
adjusted to pH 7 with 0.2 N HCl). Fix for at least 24 hours prior to processing. For long-term
storage in fixative at 4C, reduce glutaraldehyde to 0.5~1.0%. Processing involves post-fixation
with 1% osmium tetroxide, dehydration, embedding, sectioning and staining with uranyl acetate
and lead citrate according to standard TEM methods.
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*
* *
C HA P T E R 4 . 1 . 3 .

YE L L OWHE AD DI S E AS E

SUMMARY
Yellowhead disease (YHD) of penaeid shrimp is characterised by high and rapid mortality, typically
accompanied by the gross signs of yellowing of the cephalothorax and general bleaching of body colour.
Histologically, moribund shrimp show systemic necrosis of cells of ectodermal and mesodermal origin, with
the formation of intensely basophilic cytoplasmic inclusions (by haematoxylin and eosin staining) that arise
from phagocytosed nuclei and viral inclusions. However, gross signs of infection and histopathology are not
entirely reliable indicators of YHD and the use of molecular methods for confirmatory diagnosis required.
In initial Thai reports, gross signs of disease and mortality occurred within 24 days following an interval
of exceptionally high feeding activity that ended in abrupt cessation of feeding (4, 17). Many moribund
shrimp congregated near the surface at pond edges. Moribund shrimp exhibited a bleached overall
appearance and a yellowish discoloration of the cephalothorax caused by the underlying yellow
hepatopancreas (HP), which was exceptionally soft when compared with the brown HP of normal shrimp.
In many cases, total crop loss was experienced within a few days of the appearance of shrimp showing gross
signs of YHD. Yellowhead virus (YHV) can infect cultured shrimp from late postlarval stages onwards,
but massive mortality is usually encountered from early to late juvenile stages in rearing ponds. Several
carrier crustaceans are known or suspected (8, 39). While cessation of feeding, congregation at pond edges
and a generally bleached appearance are always seen in YHD outbreaks, these disease features are not
particularly distinctive for YHD. The other more pathognomonic gross signs are not always seen and thus
they are not reliable, even for preliminary diagnosis of YHD.
YHV is a positive-sense, single-stranded RNA (ssRNA) virus (27, 36, 43), originally thought to be
related to rhabdoviruses but now known to be related to coronaviruses and arteriviruses (32, 39). YHV
has now been formally classified in the new genus Okavirus and new family Roniviridae within the viral
order Nidovirales (24, 25). The status of work on YHV has been reviewed (8, 10, 12, 14, 39).
Transmission electron microscopy (TEM) of YHV-infected ectodermal and mesodermal tissues shows
enveloped bacilliform virions. They range from approximately 150 nm to 200 nm in length and from
40 nm to 50 nm in diameter and are located within vesicles in the cytoplasm of infected cells and in
intercellular spaces (3, 4, 27). The virions arise from long, filamentous nucleocapsids (approximately
15 nm 130800 nm), which accumulate in the cytoplasm and obtain an envelope by budding at the
endoplasmic reticulum into intracellular vesicles. Negatively stained YHV virions show regular arrays of
short spikes on the viral envelope. YHV is potentially lethal to most of the commercially cultivated penaeid
shrimp species.
A virus with a similar ultrastructure to that of YHV has been reported from Australia (33, 35). Gill-
associated virus (GAV) was first described in moribund Penaeus monodon during an outbreak of a
yellowhead-like disease (33). Previously, a yellowhead-related virus called lymphoid organ virus (LOV)
was described in healthy Australian P. monodon. However, it is now evident that these are reports of the
same virus, which may occur as a chronic, asymptomatic infection or an acute, fatal infection. Chronic
GAV infection occurs in wild healthy P. monodon with high prevalence at some sites in eastern
Australia (39) and is transmitted vertically during spawning (6). Acute GAV infection causes high
mortality, but the gross signs and patterns of tissue tropism may differ from those of YHV (33).
Comparison of DNA sequences of reverse-transcription polymerase chain reaction (RT-PCR) products from
YHV and GAV indicates that they are closely related viral genotypes (5, 32, 39). Recently, GAV was
classified as the type species of the genus Okavirus (24, 25). A third yellowhead genotype also occurs
Chapter 4.1.3. - Yellowhead disease
commonly in healthy P. monodon in Thailand and Vietnam, but this virus has not yet been recovered
from shrimp displaying signs of YHD. The third genotype is more closely related in nucleotide sequence to
GAV than to YHV. A fourth, more distantly related genotype has also recently been detected in healthy
P. monodon from India.
YHD was first described as an epizootic from Thai shrimp farms (17), and subsequent outbreaks have
been reported mostly from cultivated shrimp in Asia (1, 3, 4, 26, 39, 41). YHV has also been reported in
farmed shrimp from the Americas (16, 18). However, these cases were not confirmed and presumptive
histological diagnoses appear to have been due to severe infections with white spot virus, which can cause
similar histopathology in the lymphoid organ (30). Natural YHV infections have been reported in
P. monodon, Marsupenaeus japonicus, Fenneropenaeus merguiensis, Litopenaeus setiferus,
Metapenaeus ensis, Palaemon styliferus and Euphasia superba (8). Laboratory trials have shown
that YHV can cause high mortality in other penaeid shrimp species including Litopenaeus vannamei, L.
stylirostrus, Farfantepenaeus aztecus and F. duorarum (15, 19, 20). GAV has been reported only
from Australia where P. monodon appears to be the only natural host (31, 33, 35, 38). Experimental
GAV infection and mortalities have also been reported in Penaeus esculentus, M. japonnicus, F.
merguiensis and L. vannamei, but there is evidence of species- and age-related variation in susceptibility
to disease (34, 37).
Histopathology of YHD can be used for presumptive diagnosis during outbreaks (4, 14). In moribund
shrimp collected near the surface at pond edges, extensive necrosis can be found in tissues of ectodermal and
mesodermal origin. The areas of necrosis are characterised by the presence of large numbers of intensely
basophilic, mostly spherical, cytoplasmic inclusions ranging from approximately 2 m in diameter or
smaller. These inclusions can be seen in rapidly stained squash mounts of gills or subcuticular tissue (10),
and in tissue sections. In the latter, the lymphoid organ is usually enlarged and extensively necrotised;
however the subcuticular tissue of the stomach and gill tissue are the best tissues to visualise the
characteristic inclusions. Grossly normal shrimp should also be collected from the same ponds where
moribund shrimp are collected, for the preparation of haemolymph smears. In YHD outbreaks some of these
smears will show moderate to large numbers of haemocytes with pycnotic and karyorrhectic nuclei, in the
absence of bacteraemia (28). This is an early sign of impending morbidity, but it is usually absent or
difficult to see in moribund shrimp because of the loss of haemocytes. Together, the results from tissues of
moribund shrimp and from haemolymph smears can be used for presumptive diagnosis of a YHD outbreak.
However, as severe infection with white spot virus can cause necrosis and similar histopathology in the
lymphoid organ (30), confirmatory diagnosis using molecular tests is recommended. Presumptive diagnosis of
GAV disease is based on gross signs including swimming near the surface and at the pond edges, cessation
of feeding, a reddening of body and appendages and pink to yellow coloration of the gills (33). Lymphoid
organ spheroids are commonly observed in healthy P. monodon chronically infected with GAV and
lymphoid organ necrosis often accompanies GAV disease (33, 35). However, spheroid formation and
degeneration of lymphoid organ tissue also occur during infection with other shrimp viruses (14, 30).
Confirmation of YHD requires more detailed analysis by RT-PCR (36, 43), Western-blot analysis (18,
20), in-situ nucleic acid hybridisation (37) and TEM (3, 4, 27). The only test currently available that
reliably distinguishes YHV from GAV and other closely related viruses is RT-PCR. However, as chronic
infections with YHV-related viruses appear to be common in healthy shrimp in parts of Asia, reliable
disease diagnosis requires consideration of all available evidence. Dual and multiple infections with several
shrimp viruses are not uncommon (9, 40) so detection of a virus in a diseased shrimp does not, in itself,
establish a causal relationship. For both YHV and GAV, the use of in-situ hybridisation probes to
demonstrate invasive necrotic infection of the lymphoid organ is the most reliable method of confirmatory
disease diagnosis presently available (37).
Detection of YHV in carrier shrimp or other crustaceans cannot be reliably accomplished by histological
methods, and more sophisticated techniques are required. As YHV readily infects species of shrimp such as
L. vannamei, which are geographically separated from the YHV endemic area (15, 19, 20), bioassay in
uninfected or specific pathogen free shrimp from controlled breeding programmes can be used (7, 29, 37).
No continuous cell lines are presently available for the study of shrimp viruses, although YHV has been
cultivated in primary cell lines from the lymphoid organ of L. vannamei (23). These cultures can be used to
measure YHV titres and tissue tropism (22). However, the need to continually prepare cultures from
different shrimp or from shrimp of undefined or uncertain history currently presents problems for test
standardisation. Because of these limitations, it is recommended that detection of YHV for surveillance or
certification of broodstock or fry for stocking, should be carried out by RT-PCR.
Methods for detection of yellowhead virus (YHV) and gill-associated virus (GAV) may be applied for
confirmation of disease outbreaks, certification of broodstock and postlarvae used to stock rearing ponds,
and for disease surveillance. For presumptive diagnosis of disease outbreaks, it is sufficient to carry out
histological analysis by light microscopy of haematoxylin and eosin (H&E)-stained tissues from moribund
shrimp, combined with light microscopy of stained haemolymph smears from grossly normal shrimp in
the same pond. However, for definitive disease diagnosis the reverse-transcription polymerase chain
reaction (RT-PCR) assay and in-situ nucleic acid hybridisation are recommended. Definitive diagnosis can
also be accomplished by Western-blot analysis and transmission electron microscopy (TEM), but methods
other than in-situ hybridisation and RT-PCR using suitable discriminatory primers should be used
cautiously due to the high prevalence of yellow head-related viruses in healthy wild and cultured shrimp in
some regions of Asia and Australia. Detection of YHV, GAV or a related virus in diseased shrimp is not,
in itself, sufficient to establish a causal association. Certification of YHV or GAV infection status of
broodstock and fry should be conducted using the RT-PCR assay that is sufficiently sensitive and specific
to detect low-level chronic infections.
The various methods for surveillance, detection and diagnosis of YHV are listed in Table 1. The
designations used in the Table indicate: the method is presently unavailable or unsuitable; ? the
method is available but untested; + the method has application in some situations, but cost, accuracy, or
other factors severely limits its application; ++ the method is a standard method with good diagnostic
sensitivity and specificity; and +++ the method is the recommended method for reasons of availability,
utility, and diagnostic specificity and sensitivity. These are somewhat subjective as suitability involves
issues of reliability, sensitivity, and utility.
Table 1. YHV surveillance, detection and diagnostic methods
Gross signs +
Histopathology ++
Transmission EM + + +++ +
Western blot ? ? + + +++ +
In-situ hybridisation ? ? + + +++ +++
RT-PCR + +++ +++ +++ +++ +++
PLs = postlarvae; EM = electron microscopy; RT-PCR = reverse-transcription polymerase chain reaction.
Note that EM, Western blot or in-situ hybridisation cannot distinguish among YHV, GAV and yellowhead-related
viruses. The viruses can be distinguished by RT-PCR using suitable discriminatory primers.
Shrimp material suitable for virological examination is:
Clinically affected shrimp: whole shrimp.
Asymptomatic shrimp (apparently healthy shrimp): whole larvae, postlarvae and juvenile shrimp;
lymphoid organs, haemolymph or gills from juvenile to broodstock specimens.
1. STANDARD SCREENING METHODS FOR YHV AND GAV
1.1. Reverse-transcription polymerase chain reaction assay of haemolymph
The protocol described here is adapted from Wongteerasupaya et al. (42). An alternate assay for
YHV is reported by Tang & Lightner (36). A nested RT-PCR method, which is more sensitive
and will detect and discriminate both YHV and GAV is described below in Section 1.2.
Collect fresh shrimp haemolymph (50 l), mix immediately with 500 l of Trizol reagent
(Bethesda Research Laboratories, Gaithersberg, Maryland, United States of America [USA]) and
extract RNA according to the product manual. Resuspend RNA (2 l) in 20 l of PCR buffer
(10 mM Tris/HCl, pH 8.3, 50 mM KCl) containing 2.5 U of M-MLV (Moloney murine leukaemia
virus) reverse transcriptase, 1.0 U of ribonuclease inhibitor, 0.75 M of antisense primer (144R,
below), 1 mM each of dATP, dTTP, dCTP, and dGTP, and 5 mM of MgCl
2
, and incubate at 42C
for 15 minutes to synthesise cDNA. Incubate the mixture at 100C for 5 minutes to inactivate the
reverse transcriptase and allow the mixture to cool to 5C. Add the PCR mixture
(10 mM Tris/HCl, pH 8.3, 50 mM KCl) containing 2.5 U of Taq DNA polymerase (Perkin Elmer
Cetus), 2 mM MgCl
2
and 0.75 M of sense primer (10F, below) to give a final volume of 100 l.
Overlay the tubes with 100 l of mineral oil and carry out PCR amplification for 40 cycles at 94C
for 30 seconds, 58C for 30 seconds, 72C for 30 seconds, and finishing at 72C for 10 minutes.
Include a negative control containing diethyl pyrocarbonate (DEPC)-treated distilled water instead
of RNA extract, and a positive control containing YHV RNA. Detect the characteristic YHV
amplicon of 135 bp by electrophoresis of 20 l aliquots through 2% agarose gels. The sensitivity
of the assay is approximately 0.01 pg of purified YHV RNA ( 10
3
genomes). The sequences of
the PCR primers are:
10F: 5-CCG-CTA-ATT-TCA-AAA-ACT-ACG-3
144R: 5-AAG-GTG-TTA-TGT-CGA-GGA-AGT-3
1.2. Reverse-transcription polymerase chain reaction assay for differential detection of YHV
and GAV
The RT-PCR method of Wongteerasupaya et al. (42) will not detect GAV due to sequence
differences from YHV at the PCR primer sites (38). The following method (provided by J.A.
Cowley & P.J. Walker) is a nested RT-PCR procedure that allows detection of both YHV and
GAV in the first amplification and discrimination of the genotypes in the nested amplification.
The third yellowhead genotype, which occurs commonly in healthy P. monodon in some parts of
Asia, will usually react as GAV in this test. The fourth genotype, which has been detected in
healthy P. monodon from India, does not react at all in this test or the test described by
Wongteerasupaya et al. (42).
Fresh lymphoid organ, gill tissue or haemolymph provide a good source of RNA. Lymphoid
organ and gill tissue preserved in 95% analytical-grade ethanol or stored frozen at 70C are also
suitable for total RNA preparation. Approximately 1020 mg lymphoid organ or gill tissue or
50 l haemolymph is disrupted in 500 l TRIzol reagent (Gibco-BRL) and total RNA is
extracted according to the product manual. RNA is resuspended in 25 l DEPC-treated water,
heated at 56C for 15 minutes, kept on ice and used immediately or stored at 70C until
required. Ideally, a 1/200 dilution (i.e. 2.5 l in 500 l DEPC-treated water) should be prepared,
and absorbances A
260
nm and A
280
nm (UV spectrophotometer required) should be determined to
quantify and check the quality of the RNA. For each set of RNA samples to be tested, DEPC-
treated water and extracts known to contain either GAV or YHV RNA should be included as
negative and positive controls, respectively. For cDNA synthesis, 0.11.0 g RNA (1.0 g for
optimum sensitivity) and 0.7 l of 50 pmol/l primer GY5 are adjusted to 6 l with DEPC-
treated water, incubated at 70C for 10 minutes and chilled on ice. cDNA is synthesised by adding
2 l Superscript II buffer 5 (250 mM Tris/HCl, pH 8.3, 375 mM KCl, 15 mM MgCl
2
), 1 l 100
mM DTT and 0.5 l 10 mM dNTP stock mixture (i.e. 10 mM dATP, 10 mM dTTP, 10 mM
dCTP, 10 mM dGTP), preheating to 42C for 2 minutes prior to adding 0.5 l of 200 U/l
Superscript II reverse transcriptase (Gibco-BRL) and incubating at 42C for 1 hour. The reaction
is then heated at 70C for 10 minutes, placed on ice and spun briefly in a microcentrifuge to
collect the contents of the tube. In the first PCR step, 1 l cDNA is amplified in a 50 l reaction
containing 1 Taq buffer (10 mM Tris/HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 1.5 mM
MgCl
2
, 35 pmol each primer GY1 and GY4, 200 M each of dATP, dTTP, dCTP and dGTP and
2.5 U Taq polymerase (Promega) using a 0.5 ml thin-walled tube. The reaction mixture is overlaid
with 50 l liquid paraffin and heated at 85C for 23 minutes hot start prior to adding cDNA.
DNA is amplified using 35 cycles of 95C for 30 seconds, 66C for 30 seconds, and 72C for 45
seconds, followed by final extension at 72C for 7 minutes. In the second step, multiplex
differential PCR, 0.55.0 l (5.0 l for optimum sensitivity) of the first PCR product is amplified
using the reaction conditions above except that 35 pmol of each primer GY2, Y3 and G6 are used
in place of GY1 and GY4, and DNA is amplified using 35 cycles of 95C for 30 seconds, 66C
for 30 seconds, and 72C for 30 seconds, followed by final extension at 72C for 7 minutes. The
PCR products (10 l) are resolved in 2% agarose/TAE (Tris-acetate-EDTA [ethylene diamine
tetra-acetic acid]) gels containing 0.5 g/ml ethidium bromide alongside a suitable DNA ladder,
detected using a UV transilluminator, and the gel is photographed as a record.
A 794 bp DNA is amplified from either GAV or YHV in the first PCR step and can be visualised
by agarose gel electrophoresis if virus RNA levels are sufficiently high. In the differential second
PCR step, either a 406 bp DNA is amplified from GAV or a 277 bp DNA is amplified from
YHV. The presence of both 406 bp and 277 bp DNA products will indicate a dual infection with
GAV and YHV. The detection sensitivity of the second-step PCR is ~1000-fold greater than the
first-step PCR and, depending on the level of infection, GAV or YHV RNA can be detected to a
limit of 10 fg lymphoid organ total RNA.
The sequences of RT-PCR primers generic for GAV and YHV (GY) or specific for GAV (G) or
YHV (Y) are as follows:
GY1 5-GAC-ATC-ACT-CCA-GAC-AAC-ATC-TG-3
GY2 5-CAT-CTG-TCC-AGA-AGG-CGT-CTA-TGA-3
GY4 5-GTG-AAG-TCC-ATG-TGT-GTG-AGA-CG-3
GY5 5-GAG-CTG-GAA-TTC-AGT-GAG-AGA-ACA-3
Y3 5-ACG-CTC-TGT-GAC-AAG-CAT-GAA-GTT-3
G6 5-GTA-GTA-GAG-ACG-AGT-GAC-ACC-TAT-3
1.3. Reverse-transcription polymerase chain reaction assay of postlarvae
From a nursery or hatchery tank containing 100,000 postlarvae or more, sample approximately
1000 postlarvae from each of five different points. Pool the samples in a basin, gently swirl the
water in the basin and then select an assay sample from living postlarvae that collect at the centre
of the basin. Choose the sample number according to the assumed or target prevalence (see
Chapter I.3. Section 3.). Homogenise the sample in an appropriate volume of Trizol reagent
and extract RNA according to the product manual. For subsequent steps, follow the RT-PCR
protocol for haemolymph given in Sections 1.1. or 1.2 above, using this extract as the template.
1.4. Western-blot assay
Prepare reagents and carry out assays according to the protocols of Lu et al. (21) and Loh et al.
(18). This includes purification of YHV virions from laboratory-infected shrimp, generation of
immunoglobulins (Igs) in New Zealand white rabbits, purification of the IgG using recombinant
bacterial protein-G columns and removal of cross-reacting normal shrimp antigens by adsorption
on to acetone-dried, ground shrimp muscle tissue and haemolymph. For assay, remove 0.1 ml of
haemolymph from live shrimp specimens and dilute in an equal volume of citrate buffer for
immediate use or storage at 80C until used. For Western blotting, use 200 l sample, clarify at
8000 g for 5 minutes and then pellet the supernatant at 140,000 g for 5 minutes. Resuspend
pellets in 100 l 2 loading buffer (2.5 ml 0.5 mM Tris/HCl, pH 6.8, 4 ml 10% sodium dodecyl
sulfate [SDS], 2 ml glycerol, 1 l beta-mercaptoethanol, and 0.5 ml deionised distilled water) and
heat at 95C for 5 minutes. Load a 10 l subsample on to 5% SDS/polyacrylamide gel, and
conduct electrophoresis at 200 V. Blot the gel on to a nitrocellulose membrane (pore size,
0.1 mm) in blotting buffer (3.03 g Tris base, 14.4 g glycine, and 200 ml methanol per litre) at
100 V for 1 hour. Rinse the membrane with phosphate buffered saline (PBS), pH 7.4, soak in 5%
skim milk (in PBS) for 1 hour, and rinse with PBS for 5 minutes. Next, treat the membrane with a
1/1000 dilution of the primary antibody (IgG) for 1 hour, rinse three times with PBS for
5 minutes, and then treat with a 1/2500 dilution of goat anti-rabbit IgG-horseradish-peroxidase
conjugate for 1 hour. Rinse again three times with PBS for 5 minutes and then treat with the
substrate, 3,3,5,5-tetramethylbenzidine, until a bluish/purple colour develops. Stop the reaction
by soaking the membrane in distilled water. All incubations should be carried out at 25C 2C.
Use a purified viral preparation as a positive control and identify 24 major protein bands
characteristic of YHV at 116, 64 and 20 kDa. The sensitivity is 0.4 ng of YHV protein (
10
6
YHV virions).
2. PRESUMPTIVE DIAGNOSTIC METHODS FOR YHV AND GAV
Presumptive diagnosis of YHV can be achieved by the following three methods.
2.1. Rapid staining of squash mount preparations
Collect moribund shrimp from a suspected outbreak of YHD and fix the whole shrimp or gill
filaments in Davidsons fixative (14) overnight. After fixation, wash some gill filaments thoroughly
with tap water to remove the fixative, and stain with Meyers H&E (14). After staining and
dehydration, when the tissue is in xylene, place a gill filament on a microscope slide in a drop of
xylene and, using a fine pair of needles (a stereo microscope is helpful), break off several
secondary filaments and then replace the main filament in xylene where it can be stored
indefinitely as a permanent reference in a sealed vial. Being careful not to let the xylene dry, tease
apart the secondary filaments on a microscope slide and remove any large fragments or particles
that would thicken the mount unnecessarily. Finally, add a drop of mounting fluid and a cover-
slip. Use light pressure to flatten the mount as much as possible. This procedure may also be used
with thin layers of subcuticular tissue. With YHD outbreaks, examination with a 40 objective of
the light microscope will reveal the presence of moderate to large numbers of deeply basophilic,
evenly stained, spherical, cytoplasmic inclusions approximately 2 m in diameter or smaller (10).
This evidence should be used together with the results from haemolymph smears (see below) in
making a presumptive diagnosis of a YHD outbreak. As for the fixed tissues and the filaments in
xylene, these whole-mount slides can be kept as a permanent record.
In the event that rapid results are required, the fixation step can be shortened to only 2 hours by
changing the acetic acid portion of the Davidsons fixative formula to 50% concentrated HCl. For
best results, this fixative should not be stored for more than a few days before use. After fixation,
wash thoroughly to remove the fixative and check that the pH has returned to near neutral before
staining. Do not fix for longer periods or above 25C as this may result in excessive tissue damage
that will make interpretation difficult or impossible.
2.2. Staining of haemolymph smears
Haemolymph smears from moribund shrimp infected with YHV are not useful because
haemocytes are usually depleted in advanced stages of YHD. Sample haemolymph from grossly
normal shrimp from a suspect pond where moribund shrimp have also been collected. Draw the
haemolymph into a syringe containing twice the haemolymph amount of 25% formalin or of
Davidsons fixative in which the acetic acid of the formula has been replaced by either water or
formalin. Mix thoroughly, ignore clots in the syringe, place a drop on a microscope slide, smear
and then air-dry before staining with H&E or other standard blood smear stains. Dehydrate, add
mounting fluid and add a cover-slip. Examine with the 40 lens of a light microscope. In YHD
outbreaks, some of the smears will show moderate to large numbers of haemocytes with
karyorhectic or pycnotic nuclei (28). It is important that the slides with these nuclei show no
evidence of concomitant bacterial infection, as bacterial infections may cause similar changes in
haemocytes. The results from haemolymph smears should be used together with the results from
rapidly stained whole mounts (see above) or stained tissue sections when making a presumptive
diagnosis of a YHD outbreak.
2.3. Histopathology of tissue sections
Fix moribund shrimp from a suspected YHV outbreak in Davidsons fixative and process for
preparation of standard H&E-stained tissue sections (2, 14). Examine the sections by light
microscopy for the presence of moderate to large numbers of deeply basophilic, evenly stained,
spherical, cytoplasmic inclusions approximately 2 m in diameter or smaller in tissues of
ectodermal and mesodermal origin (4). Tissues of the lymphoid organ, stomach subcuticulum and
gills are particularly useful.
3. CONFIRMATORY DIAGNOSTIC METHODS FOR YHV AND GAV
Confirmatory diagnosis of YHV can be achieved by a combination of the following methods. Note
that in-situ nucleic acid hybridisation, Western-blot assay or TEM may not distinguish YHV from GAV
or other related viruses. As low-level chronic infections are common in some regions, detection of the
presence of virus is not, in itself, evidence of aetiology. A combination in-situ nucleic acid hybridisation
and discriminatory RT-PCR assay is required for definitive diagnosis.
3.1. Reverse-transcription polymerase chain reaction
See Sections 1.1 and 1.2.
3.2. Western-blot assay
See Section 1.4.
3.3. In-situ nucleic acid hybridisation
Follow the protocol according to Tang & Lightner (36) as briefly described here. The method is
suitable for detection of YHV or GAV (37). To preserve the viral RNA, fix live shrimp with
neutral-buffered, modified Davidsons fixative without acetic acid (RF-fixative; 13). Process the
fixed shrimp using standard histological methods and prepare 4 m thick sections on Superfrost
Plus slides (Fisher Scientific, Pennsylvania, USA). Prior to hybridisation, incubate sections at 65C
for 45 minutes, remove paraffin with Hemo de (Fisher Scientific, Pennsylvania, USA), and
rehydrate through an ethanol series to water. Digest sections with proteinase K (100 g/ml, in
50 mM Tris/HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA) for 15 minutes at 37C, followed by post-
fixation in formaldehyde (0.4%) for 5 minutes. Rinse in 2 SSC (standard saline citrate), then
prehybridise with 500 l prehybridisation solution (4 SSC, 50% formamide, 1 Denhardts,
0.25 mg/ml yeast RNA, 0.5 mg/ml sheared salmon sperm DNA, 5% dextran sulfate) at 42C for
30 minutes. For hybridisation, overlay the sections with 250 l hybridisation solution containing a
digoxigenin-labelled probe (2040 ng/ml) at 42C overnight. The next day, wash the sections as
follows: 2 SSC once for 30 minutes at room temperature; 1 SSC twice for 5 minutes at 37C;
0.5 SSC twice for 5 minutes at 37C. Incubate the sections with sheep anti-digoxigenin-alkaline
phosphatase conjugate (Boehringer Mannheim, Germany) at 37C for 30 minutes. Wash with
0.1 M Tris/HCl, pH 7.5, 0.15 M NaCl twice for 10 minutes at room temperature and rinse with
0.1 M Tris/HCl, pH 9.5, 0.1 M NaCl. Incubate with nitroblue tetrazolium and 5-bromo-4-chloro-
3-indoyl phosphate in the dark for 12 hours for colour development. Counterstain with
Bismarck Brown Y (0.5%), dehydrate through a series of ethanol and Hemo de, add Permount
(Fisher Scientific, Pennsylvania, USA) and cover with a cover-slip. YHV-infected cells give a blue
to purple-black colour against the brown counterstain. Include positive controls of YHV-infected
tissue and negative controls of uninfected shrimp tissue. The diagnostic probe can be prepared by
PCR labelling using the following primers:
YHV1051F: 5-ACA-TCT-GTC-CAG-AAG-GCG-TC-3
YHV1051R: 5-GGG-GGT-GTA-GAG-GGA-GAG-AG-3
For TEM, the most suitable tissues of moribund shrimp suspected of YHV infection are those of
the lymphoid organ and gills. For screening or surveillance of grossly normal shrimp, the most
suitable tissue is from the lymphoid organ. The reagents described are from ref. 14. In brief: stun
live shrimp by immersion in iced water until just immobilised or kill by injection of fixative.
Quickly dissect and remove small portions of target tissue (no larger than a few mm in diameter)
and fix in at least 10 volumes of 6% glutaraldehyde held at 4C and buffered with sodium
cacodylate (Na[CH
3
]
2
AsO
2
.
3H
2
O) solution (8.6 g Na cacodylate, 10 g NaCl, distilled water to
make 100 ml, adjusted to pH 7 with 0.2 N HCl) or phosphate solution (0.6 g NaH
2
PO
4
.
H
2
O,
1.5 g Na
2
HPO
4
, 1 g NaCl, 0.5 g sucrose, distilled water to make 100 ml, adjusted to pH 7 with
0.2 N HCl). Fix for at least 24 hours prior to processing. For long-term storage in fixative at 4C,
reduce glutaraldehyde to 0.51.0%. Processing involves post-fixation with 1% osmium tetroxide,
dehydration, embedding, sectioning and staining with uranyl acetate and lead citrate according to
standard TEM methods.
3.5. Bioassay
The bioassay procedure is based on that described by Spann et al. (33) but similar procedures have
been described by several other authors (19, 29, 41). Bioassay should be conducted in susceptible
shrimp (see Summary) that have been certified as specific pathogen free and have been obtained
from a biosecure breeding facility. Alternatively, susceptible wild or farmed shrimp to be used for
bioassay should be screened by nested RT-PCR on haemolymph samples to confirm the absence
of pre-existing chronic infections with YHV, GAV or related viruses. Shrimp should be
maintained throughout the procedure under optimal conditions for survival of the species in
laboratory culture.
Collect moribund shrimp from a disease outbreak or shrimp suspected of being carriers of
infection and maintain at 4C or on ice. Remove and discard the tail and appendages. If necessary,
the whole shrimp or the retained cephalothorax may be snap-frozen and stored at
80C or in liquid nitrogen until required. Thaw stored samples rapidly in a 37C water bath
within two snap-seal plastic bags and then maintain at 4C or on ice during all procedures.
Remove the carapace and calciferous mouth-parts. Suspend the remaining tissues in six volumes
of TN buffer (0.02 M Tris/HCl, pH 7.4, 0.4 M NaCl) and homogenise in a tissue grinder to form
a smooth suspension. Clarify the homogenate at 1300 g for 20 minutes at 4C. Remove the
supernatant fluid below the lipid layer and pass through a 0.45 m filter. Maintain the filtrate at
4C for immediate use or snap-freeze and store in aliquots at 80C or in liquid nitrogen. Thaw
the filtrate rapidly at 37C and maintain on ice prior to use.
Inject at least twelve juvenile (15 g) shrimp of a known susceptible species (P. monodon,
P. esculentus, M. japonicus, F. merguiensis, L. vannamei, L. stylirostris), with 5 l of filtrate per gram
body weight into the second abdominal segment using a 26-gauge needle. Inject two equivalent
groups of at least twelve shrimp with TN buffer and a filtered tissue extract prepared from
uninfected shrimp. One additional group of at least twelve shrimp should be injected last with a
known and calibrated positive control inoculum from shrimp infected with YHV or GAV (as
required). Maintain each group of shrimp in a separate covered tank with a separate water supply
for the duration of the bioassay. Ensure no inadvertent transfer of water between tanks by good
laboratory practice. Observe the shrimp and record mortalities for at least 21 days or until the test
and positive control groups reach 100% mortality. Collect at least one moribund shrimp from
each of the four groups for examination by histology, TEM, in-situ nucleic acid hybridisation, and
PCR or Western-blot analysis to confirm the presence of YHV or GAV (as required) in the
sample (refer Sections above for test procedures).
Note that shrimp to be tested that are suspected of being carriers of low level chronic infections
may produce an inoculum containing a very low dose of virus. In bioassay, such an inoculum may
not necessarily cause mortalities, gross signs of disease or histology characteristic of a lethal
infection. In this event, molecular tests or TEM must be applied to the bioassay shrimp.
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WITHYCHUMNARNKUL B. (2002). Yellow head-complex viruses occur commonly in healthy
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April 2002. Abstract.
41. WANG Y.-C. & CHANG P.-S. (2000). Yellow head virus infection in the giant tiger prawn Penaeus
monodon cultured in Taiwan. Fish Pathol., 35, 110.
42. WONGTEERASUPAYA C., BOONSAENG V., PANYIM S., TASSANAKAJON A., WITHYACHUMNARNKUL B.
& FLEGEL T.W. (1997). Detection of yellow-head virus (YHV) of Penaeus monodon by RT-PCR
amplification. Dis. Aquat. Org., 31, 181186.
43. WONGTEERASUPAYA C., SRIURAIRATANA S., VICKERS J.E., ANUTARA A., BOONSAENG V., PANYIM
S., TASSANAKAJON A., WITHYACHUMNARNKUL B. & FLEGEL T.W. (1995). Yellow-head virus of
Penaeus monodon is an RNA virus. Dis. Aquat. Org., 22, 4550.
*
* *
C HA P T E R 4 . 1 . 4 .

T E T RAHE DRAL BACUL OVI ROS I S
( Ba c u l o v i r u s p e n a e i )

SUMMARY
Baculovirus penaei (BP) is an occluded baculo-like virus that contains double-stranded DNA as its
nucleic acid type (2, 3, 11, 12, 16, 25, 32). Although BP was not listed with the baculoviruses in the
Seventh Report of International Committee on Taxomony of Viruses (ICTV) (34), it had been cited and
recognised as a possible member of the nuclear polyhedrosis genus of the Baculoviridae in the Fifth Report of
the ICTV (14). Furthermore, Penaeus monodon-type baculovirus (MBV), a related nuclear
polyhedrosis virus of Eastern Hemisphere penaeid shrimp, was listed as a possible member of this group in
the Seventh Report of the ICTV (34). According to ICTV guidelines (14, 26, 34), BP was designated
PvSNPV (for singly enveloped nuclear polyhedrosis virus from P. vannamei, the most characterised
geographical strain of BP from P. vannamei) (2). Although PvSNPV may be the most correct name for
BP, the term BP will be used to designate this virus (and its closely strains) in this Manual.
BP is considered to be a potentially serious pathogen in the larval, postlarval, and early juvenile stages of
host shrimps. BP possesses a wide geographical distribution and diverse host range, and multiple strains of
the virus have been documented (6, 10, 13, 1719, 21, 2931). Infections by BP are characterised by the
presence of prominent, tetrahedral intranuclear occlusion bodies, which are referred to as polyhedral occlusion
bodies or polyhedral inclusion bodies, in affected epithelial cells of the hepatopancreas and midgut, or free
within lysed cell debris in the faeces (35, 21, 22). Crowding, chemical stress, or environmental stress may
enhance the pathogenicity and increase the prevalence of BP in its hosts (11). Infection by BP is exclusively
by the oral route in which cannibalism and faecaloral contamination are the principal mechanisms of
transmission (15, 16, 21, 22, 27, 29).
BP has a widespread distribution in cultured and wild penaeid shrimps in North and South America, but
its geographical distribution is limited to the Western Hemisphere (25, 10, 2024, 27, 35). However,
within the Americas and Hawaii, multiple geographical strains of BP exist, and some of these may be
distinguished by the size of the virion or by molecular methods (9, 13, 21, 24).
Several surveillance methods are available for use in certification of the BP infection status of shrimp stocks.
The simplest method is based on the microscopic demonstration of the characteristic occlusion bodies
produced by the virus. With direct microscopy, characteristic tetrahedral occlusion bodies are demonstrated in
wet-mounts of whole larvae, of excised portions of the hepatopancreas from postlarvae or older shrimp, or of
faeces from large juvenile to adult shrimp. Wet-mount examination of the faeces of adult broodstock for
characteristic occlusion bodies may be used as a simple nonlethal method to detect carriers with moderate or
heavier infections (35, 20, 21). However, the wet-mount method may not detect developing, low-grade, or
low prevalence BP infections. Histology of fixed specimens may also be used for surveillance (1, 21).
Routine histology provides a positive diagnosis of BP infection when characteristic tetrahedral occlusion
bodies are demonstrated in hypertrophied nuclei of mucosal epithelial cells of the hepatopancreas or midgut
(35, 20, 21). Molecular detection methods for BP, (gene probes applicable to in-situ hybridisation assays
and polymerase chain reaction methods) are also available and provide a satisfactory method for surveillance
applications (3, 79, 13, 21, 22, 35).
Chapter 4.1.4. - Tetrahedral baculovirosis (Baculovirus penaei)
Because BP is transmitted from adults to their offspring by faecal contamination of the spawned eggs,
prevention of infection in hatcheries may be achieved by taking steps to eliminate faecal contamination of
spawned eggs and larvae thorough the use of disinfectants, drying, and other routine sanitation methods with
tanks and equipment (18), and by direct decontamination of spawns by washing nauplii or eggs with
formalin, iodophores, and clean sea water (28).
Infection of the hepatopancreas by the nuclear polyhedrosis virus Baculovirus penaei (BP) is among the
most readily diagnosable disease of the penaeid shrimps. The occlusion bodies formed by the virus are
very conspicuous and easily demonstrated by direct light microscopy with fresh specimens or by routine
histological methods with fixed specimens. Direct microscopic methods are most suitable for the
postlarvae stages, which are commonly moved in regional and international commerce (35, 2022).
Highly sensitive molecular methods for BP are also available and provide alternative methods for
surveillance applications, especially for nonlethal testing of broodstock (21, 22, 35). Hence, there are
several options to choose from as surveillance methods for certification of the BP-infection status of
shrimp stocks.
The methods currently available for surveillance, detection, and diagnosis of BP are listed in Table 1. The
designations used in the Table indicate: = the method is presently unavailable or unsuitable; ? = the
method is available but untested; + = the method has application in some situations, but cost, accuracy, or
other factors severely limit its application; ++ = the method is a standard method with good diagnostic
sensitivity and specificity; and +++ = the method is the recommended method for reasons of availability,
utility, and diagnostic specificity and sensitivity.
Table 1. Surveillance, detection and diagnostic methods for the nuclear polyhedrosis virus BP.
Gross signs +
Direct BF/LM +++ +++ + ++ +++ +++
Histopathology +++ +++ ++ ++ +++ +++
Bioassay + +
Transmission EM + +
DNA probes dot-blot ? ?
DNA probes in situ +++ +++
PCR +++ +++ +++ +++ +++ +++
PCR = polymerase chain reaction.
1. STANDARD SCREENING METHODS FOR BP
1.1. Direct microscopic methods
1.1.1. Wet-mount of fresh tissue
Diagnosis of BP infections is made by the demonstration of single or multiple tetrahedral
occlusion bodies in epithelial cell nuclei in squash preparations of hepatopancreas or midgut
examined by phase-contrast or bright-field microscopy. Occlusion bodies are tetrahedral or
pyramidal in three-dimensional form, and range in size from less than 0.1 m to nearly 20 m
from pyramidal base to peak, with a modal, vertical length of 8 m. In some publications, the
occlusion bodies of BP are referred to as PIBs (polyhedral inclusion bodies) (35, 11, 12, 16,
2022).
1.1.2. Faecal examination technique
This nonlethal method may be used to screen for carriers of BP. The method can be applied to
juvenile or older shrimp, and it is perhaps most useful as a nonlethal method for screening
valuable broodstock. Faecal samples from shrimp to be tested may be obtained by placing the
shrimp in an aquarium, spawning tank, or other suitable tanks for a few hours until faecal
strands are present on the tank bottom. The faecal strands are best collected using a clear
plastic siphon hose (an air line fitted with a section of plastic pipette as a tip is ideal) and placed
in a beaker, cup, or suitable container. The faecal strands may be made into wet-mounts and
examined directly for occlusion bodies. BP occlusion bodies are prominent, refractive
tetrahedrons that range from just resolvable to nearly 20 m in height (35, 2022).
Collected faeces may also be used as the sample for nonlethal testing for BP by polymerase
chain reaction (PCR). PCR will provide greater diagnostic sensitivity for low-grade infections
than will direct microscopic examination (3, 21).
1.2. Histological methods
See Section 2.2.
1.3. Polymerase chain reaction procedure
PCR methods have been developed for BP. Primer sets designed from certain segments of the BP
genome are unique and provide positive test results only in samples infected with particular stains
of this virus (3, 35).
Substances in the hepatopancreas and faeces of shrimp have been found to inhibit the DNA
polymerase used in the PCR assay. Therefore, DNA extraction is required before PCR can be
successfully used to detect BP (35).
1.3.1. DNA extraction
DNA extraction kits are convenient and commercially available. Otherwise, a suitable DNA
extraction procedure is as follows:
i) A sample of faeces or hepatopancreas is added to digestion buffer (~1:10 ratio of
sample:buffer in up to 400 l of buffer) containing 50 mM KCl, 10 mM Tris/HCl,
pH 8.3, 0.1 mg/ml gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 80 g/ml
proteinase K, crushed, and dispersed with a wooden toothpick or a pipette tip.
ii) After dispersing the sample in the digestion buffer, heat to 60C for 1 hour and then to
95C for 10 minutes.
iii) Centrifuge at 12,000 g for 2 minutes, transfer the supernatant fluid to a new tube, and
store on ice.
iv) Remove 50 l of the digested sample and dilute with 150 l of dilution buffer (10 mM
Tris/HCl, pH 8.0, 0.1 mM ethylene diamine tetra-acetic acid [EDTA]) and extract with
200 l of phenol/isoamyl alcohol/chloroform (PIC) (25/1/24).
v) After vortexing the sample for 5 seconds, leave the tube for 5 minutes and then
centrifuge at 12,000 g for 2 minutes.
vi) Remove 160 l of the aqueous (upper) phase and transfer to a new microcentrifuge tube.
vii) The extraction step may be repeated if necessary.
viii) Precipitate the DNA by adding 20 g of glycogen (1 l of a 20 mg/ml stock), 65 l of a
7.5 M ammonium acetate and 390 l of ethanol, store at 20C for >1 hour, and then
pellet the DNA by centrifugation at 12,000 g for 5 minutes.
ix) Rinse the DNA pellet with 200 l of 70% ethanol to remove residual ammonium acetate,
dry, and then dissolve the DNA pellet in 30 l of dilution buffer or distilled water prior to
adding the sample (template) to the PCR reaction mixture and beginning the PCR.
1.3.2. BP PCR method
Three forward and three reverse primers selected from an ~1430 base pair (bp) segment of the
BP polyhedrin gene have been reported by Wang et al. (35).
The sequences for these primers are:
BPA 5-GAT-CTG-CAA-GAG-GAC-AAA-CC-3 Temperature 61C
BPB 5-ATC-GCT-AAG-CTC-TGG-CAT-CC-3 Temperature 64C
BPD 5-TGT-TCT-CAG-CCA-ATA-CAT-CG-3 Temperature 62C
BPE 5-TAC-ATC-TTG-GAT-GCC-TCT-GC-3 Temperature 63C
BPF 5-TAC-CCT-GCA-TTC-CTT-GTC-GC-3 Temperature 68C
BPG 5-ATC-CTG-TTT-CCA-AGC-TCT-GC-3 Temperature 64C
The combinations of these primers amplify segments from BP template DNA of: BPA/BPF
196 bp; BPA/BPB 560 bp; BPA/BPG 933 bp; BPD/BPB 207 bp; BPD/BPG 580 bp;
and BPE/BPG 221 bp.
The following PCR procedure was adapted from Wang et al. (35):
i) For BP PCR, the DNA in each extracted sample (Section 1.3.1.) is denatured by heating
in a boiling water bath for 3 minutes followed by quick chilling in ice-water.
ii) 25 l reaction mixture containing 5 mM of each primer, 1.5 mM MgCl
2
, and 0.51 unit of
DNA polymerase is added.
iii) After heating the reaction mixture for 3 minutes at 95C, 30 PCR cycles (a DNA melting
step at 94C, a primer annealing step at 60C, and an elongation step at 72C) are
performed followed by an elongation step of 5 minutes at 72C.
iv) The resultant PCR products may be compared with molecular standards by 2% agarose
gel electrophoresis or assayed for with a specific DNA probe for the fragment following a
Southern transfer.
v) The following controls should be included in every PCR assay for BP: a known negative
tissue or faecal sample; a known positive tissue or faecal sample (this can be the DNA
clone from which a specific set of primers was designed); and a no- template control.
Confirmation of infection by BP may be accomplished with any of the methods listed in Section 1 (i.e.
wet-mounts of hepatopancreas tissue squashes or of faecal strands, or by PCR). The other methods
available for confirmatory diagnosis of BP include: autofluorescence with phloxine, and routine
histological methods (1, 35, 2022, 33).
2.1. Autofluorescence method with phloxine stain
Another method for detecting BP occlusion bodies is based on the fluorescence of phloxine-
stained occlusion bodies (3, 21, 33). Aqueous 0.001% phloxine may be added to tissue squash
preparations to make wet-mounts of hepatopancreas or faeces for direct examination. Histological
sections stained with routine haematoxylin and eosin (H&E) containing 0.005% phloxine, are also
suitable for this procedure. BP occlusions in wet-mounts of tissue squashes, in faeces, or in
histological sections fluoresce bright yellow-green against a pale green background under epi-
fluorescence (barrier filter of 0515 nm and a 490 nm exciter filter). Other objects in the tissues
and insect baculovirus occlusion bodies do not fluoresce with this method. Hence, the method
can provide a rapid and specific diagnosis.
Histology may be used to provide a definitive diagnosis of BP infection. Because 10% buffered
formalin and other fixatives provide, at best, only fair fixation of the shrimp hepatopancreas (the
principal target organ for BP and other baculo-like virus infections of penaeid shrimp), the use of
Davidsons fixative (containing 33% ethyl alcohol [95%], 20% formalin [approximately 37%
formaldehyde], 11.5% glacial acetic acid, and 33.5% distilled or tap water) is highly recommended
for all routine histological studies of shrimp (1, 3, 21). To obtain the best results, dead shrimp
should not be used. Only live, moribund, or compromised shrimp should be selected for fixation
and histological examination. Selected shrimp are killed by injection of fixative directly into the
hepatopancreas; the cuticle over the cephalothorax and abdomen just lateral to the dorsal midline
is opened with fine-pointed surgical scissors to enhance fixative penetration (the abdomen may be
removed and discarded), the whole shrimp (or cephalothorax less the abdomen) is immersed in
fixative for 2448 hours, and then transferred to 70% ethyl alcohol for storage. After transfer to
70% ethyl alcohol, fixed specimens may be transported by wrapping in cloth or a paper towel
saturated with 70% ethyl alcohol and packed in leak-proof plastic bags. To begin histological
processing, fixed shrimp are cut-in (see ref. 1 for a photographic guide to this procedure) to
facilitate eventual sectioning of the hepatopancreas and midgut. After dehydration, the specimens
are embedded in paraffin and sections of 57 m thickness are cut. Routine histological stains
such as MayerBennetts or Harris H&E, Giemsa stains, and Gram tissue-staining methods may
be used for the demonstration of pathognomonic (for BP) tetrahedral occlusion bodies in
hepatopancreatocytes, gut epithelial cells, or gut lumen (3, 4, 6, 21). Typically, BP-infected
hepatopancreatic (or occasionally midgut) cells will present markedly hypertrophied nuclei with
single or, more often, multiple occlusion bodies, chromatin diminution and margination.
Occlusion bodies may be stained bright red with H&E stains, and intensely, but variably, with
Grams tissue stains. Brown and Brenns histological Gram stain, although not specific for
baculovirus occlusion bodies, tends to stain occlusions more intensely (either red or purple,
depending on section thickness, time of decolouration, etc.) than the surrounding tissue, aiding in
demonstrating their presence in low-grade infections (35, 2022).
2.3. Molecular methods
Nonradioactive DIG-labelled gene probes to BP have been developed and some are commercially
available (3, 79, 21, 22, 25). Gene probe and PCR methods may provide greater diagnostic
sensitivity in detecting low-grade infections than do more traditional wet-mount or histological
techniques. Furthermore, the PCR method (see Section 1.3.) has the added advantage of being
applicable to nonlethal testing of faecal samples collected from valuable broodstock shrimp.
DIG-labelled DNA probes for representative strains of BP are commercially available as
ShrimProbe
TM
kits from DiagXotics (Wilton, Connecticut, USA). The probes are labelled with a
nonradioactive label, digoxigenin-11-dUTP (DIG). These probes only work well with the in-situ
hybridisation method with histological sections because there are substances present in the
hepatopancreas and faeces of shrimp that provide both false-positive and false-negative results
with samples that are blotted directly and not extracted prior to probing.
2.3.1. Dot-blot hybridisation procedure for BP
While specific DNA probes for BP are available, their use in dot-blot hybridisation procedures
is not recommended for most routine diagnostic applications. Pigments present in the
hepatopancreas leave a coloured spot on the hybridisation membrane that can result in the
masking of a positive test or in the false interpretation of a negative test. Likewise, bits of
chitin (which nonspecifically bind DNA probes), pigments, and other materials present in the
faecal sample may also result in false-positive or false-negative dot-blot hybridisation tests.
Extraction of DNA from the hepatopancreas or faeces prior to blotting or the use of
chemiluminescent or radioactively labelled probes may circumvent these problems, but the
adequacy of other test methods (i.e. direct wet-mounts, histology, or PCR) has not indicated a
need for the further refinement and application of the dot-blot method (21).
2.3.2. In-situ hybridisation procedure
The in-situ hybridisation protocol given in detail in Section 1.2. of Chapter 4.1.6. Infectious
hypodermal and haematopoietic necrosis virus (IHHNV), uses the Genius
TM
System
developed by Boehringer Mannheim Biochemicals (now Roche Diagnostics) and was adapted
from the Boehringer Mannheims Nonradioactive In Situ Hybridization Application Manual. An
additional step is required for the in-situ hybridisation test for BP. Follow steps ivi of Section
1.2., in-situ hybridisation procedure for IHHNV, in Chapter 4.1.6. For BP substitute the
following modifications to step vii before proceeding with steps viiixvii as given for IHHNV:
i) (Modified step vii): Boil the DIG-labelled probe for 10 minutes and quench on ice; spin
briefly in the cold (~410C) using a refrigerated centrifuge or a chilled microcentrifuge
to bring all the liquid down to the base of the microcentrifuge tube; keep on ice. Dilute
the probe to 50 ng/ml in prehybridisation solution and cover the tissue with 500 l of the
solution. Denature the double-stranded viral DNA in the tissue of the histological section
by placing the slides on a 85C heat block (or on aluminium foil that is placed over a
boiling water bath) for 610 minutes. Quench the slides on ice for 5 minutes. Incubate
the slides overnight at 42C in a humid chamber. Drain the fluid on to blotting paper.
During this incubation step, keep the wash buffers at 37C to prewarm them.
ii) Proceed with steps viiixvii as given for IHHNV.
With DIG-labelled probes, accumulations of BP viral DNA within infected cell nuclei, in
cytoplasmic phagosomes, or in necrotic tissue debris are stained blue to a dark blue-black.
Although they contain virus, occlusion bodies do not normally react with DIG-labelled DNA
probes because the occlusion body protein crystalline matrix does not permit penetration of
the probe (21).
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Aquat. Org., 23, 5966.
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8. BRUCE L.D., REDMAN R.M. & LIGHTNER D.V. (1994). Application of gene probes to determine
target organs of a penaeid shrimp baculovirus using in situ hybridization. Aquaculture, 120, 4551.
9. BRUCE L.D., REDMAN R.M., LIGHTNER D.V. & BONAMI J.R. (1993). Application of gene probes to
detect a penaeid shrimp baculovirus in fixed tissue using in situ hybridization. Dis. Aquat. Org., 17,
215221.
10. BUENO S.L.S., NASCIMENTO R.M. & NASCIMENTO I. (1990). Baculovirus penaei infection in Penaeus
subtilis: A new host and a new geographic range of the disease. J. World Aquaculture Soc., 21, 235237.
11. COUCH J.A. (1974). An enzootic nuclear polyhedrosis virus of pink shrimp: ultrastructure, prevalence,
and enhancement. J. Invertebr. Pathol., 24, 311331.
12. COUCH J.A. (1974). Free and occluded virus similar to Baculovirus in hepatopancreas of pink shrimp.
Nature, 247 (5438), 229231.
13. DURAND S., LIGHTNER D.V. & BONAMI J.R. (1998). Differentiation of BP-type baculovirus strains
using in situ hybridization. Dis. Aquat. Org., 32, 237239.
14. FRANCKI R.I.B., FAUQUET C.M., KNUDSON D.L. & BROWN F. (1991). Classification and
Nomenclature of Viruses. Fifth Report of the International Committee on Taxonomy of Viruses.
Arch. Virol. (Suppl. 2). Springer-Verlag, Vienna, Austria and New York, USA, 450 pp.
15. HAMMER H.S., STUCK K.C. & OVERSTREET R.M. (1998). Infectivity and pathogenicity of Baculorirus
penaei (BP) in cultured larval and postlarval Pacific white shrimp, Penaeus vannamei, related to the stage
of viral development. J. Invertebr. Pathol., 72, 3843.
16. JOHNSON P.T. & LIGHTNER D.V. (1988). Rod-shaped nuclear viruses of crustaceans: gut-infecting
species. Dis. Aquat. Org., 5, 123141.
17. LE BLANC B.D. & OVERSTREET R.M. (1990). Prevalence of Baculovirus penaei in experimentally
infected white shrimp (Penaeus vannamei) relative to age. Aquaculture, 87, 237242.
18. LE BLANC B.D. & OVERSTREET R.M. (1991). Effect of dessication, pH, heat and ultraviolet irridation
on viability of Baculovirus penaei. J. Invertebr. Pathol., 57, 277286.
19. LE BLANC B.D., OVERSTREET R.M. & LOTZ J.M. (1991). Relative susceptibility of Penaeus aztecus to
Baculovirus penaei. J. World Aquaculture Soc., 22, 173177.
20. LIGHTNER D.V. (1988). Diseases of cultured penaeid shrimp and prawns. In: Disease Diagnosis and
Control in North American Marine Aquaculture, Sindermann C.J. & Lightner D.V., eds. Elsevier,
Amsterdam, The Netherlands, 8127.
pp.
22. LIGHTNER D.V & REDMAN R.M. (1998). Shrimp diseases and current diagnostic methods.
23. LIGHTNER D.V., REDMAN R.M. & ALMADA RUIZ E.A. (1989). Baculovirus penaei in Penaeus stylirostris
(Crustacea: Decapoda) cultured in Mexico: unique cytopathology and new geographic record. J.
Invertebr. Pathol., 53, 137139.
24. LIGHTNER D.V., REDMAN R.M., WILLIAMS R.R., MOHNEY L.L., CLERX J.P.M., BELL T.A. &
BROCK J.A. (1985). Recent advances in penaeid virus disease investigations. J. World Mariculture Soc.,
16, 267274.
25. MACHADO C.R., BUENO S.L. DE S. & MENCK C.F.M. (1995). Cloning shrimp Baculovirus penaei DNA
and hybridization comparison with Autographa californica nuclear polyhedrosis virus. Rev. Brasil. Genet.
(Brazil. J. Genetics), 18, 16.
26. MURPHY F.A., FAUQUET C.M., MAYO M.A., JARVIS A.W., GHABRIAL S.A., SUMMERS M.D.,
MARTELLI G.P. & BISHOP D.H.L. (1995). Virus Taxonomy. Sixth Report of the International
Committee on Taxonomy of Viruses. Arch. Virol. (Suppl. 10). Springer-Verlag, Vienna, Austria and
New York, USA. 586 pp.
27. OVERSTREET R.M., STUCK K.C., KROL R.A. & HAWKINS W.E. (1988). Experimental infections with
Baculovirus penaei in the white shrimp Penaeus vannamei as a bioassay. J. World Aquaculture Soc., 19,
175187.
28. SANO T. & MOMOYAMA K. (1992). Baculovirus infection of penaeid shrimp in Japan. In: Diseases of
Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K., eds. The Oceanic
Institute, Makapuu Point, Honolulu, Hawaii, USA, 169174.
29. STUCK K.C. & OVERSTREET R.M. (1994). Effect of Baculovirus penaei on growth and survival of
experimentally infected postlarvae of the Pacific white shrimp, Penaeus vannamei. J. Invertebr. Pathol.,
64, 1825.
30. STUCK K.C., STUCK L.M., OVERSTREET R.M. & WANG S.Y. (1996). Relationship between BP
(Baculovirus penaei) and energy reserves in larval and postlarval Pacific white shrimp Penaeus vannamei.
Dis. Aquat. Org., 24, 191198.
31. STUCK K.C. & WANG S.Y. (1996). Establishment and persistence of Baculovirus penaei infections in
cultured Pacific white shrimp Penaeus vannamei. J. Invertebr. Pathol., 68, 5964.
32. SUMMERS M.D. (1977). Characterization of Shrimp Baculovirus. U.S. Environmental Protection
Agency Report, EPA-600/3-77-130, US E.P.A., Gulf Breeze, FL, USA, 35 pp.
33. THURMAN R.B., LIGHTNER D.V., BELL T.A. & HAZANOW S. (1990). Unique physicochemical
properties of the occluded penaeid shrimp baculoviruses and their use in diagnosis of infections. J.
LEMON S.M., MANILOFF J., MAYO M.A., MCGEOCH, D.J., PRINGLE C.R. & WICKNER R.B. (2000).
Academic press, San Diego, USA, 1162 pp.
35. WANG S.Y., HONG C. & LOTZ J.M. (1996). Development of a PCR procedure for the detection of
Baculovirus penaei in shrimp. Dis. Aquat. Org., 25, 123131.
*
* *
C HA P T E R 4 . 1 . 5 .

S P HE RI CAL BACUL OVI ROS I S
( P e n a e u s mo n o d o n - t y p e b a c u l o v i r u s )

SUMMARY
Penaeus monodon-type baculovirus (MBV) is an occluded baculo-like virus that contains double-
stranded DNA as its nucleic acid type (20, 22, 24). MBV from P. monodon was designated
PmSNPV (for singly enveloped nuclear polyhedrosis virus from P. monodon) in accordance with the
guidelines for virus nomenclature published by the International Committee on Taxomony of Viruses
(ICTV) (28), but it appears as Penaeus monodon NPV, or PemoNPV, in the Seventh Report of the
ICTV (37). Although PemoNPV may be the most correct name for the virus, the term MBV will be used
to designate this virus (and its closely strains) in this Aquatic Manual.
MBV is considered to be a potentially serious pathogen in the larval, postlarval, and early juvenile stages of
its host penaeid shrimps and prawns. MBV has a wide geographical distribution and diverse host range,
and multiple strains of the virus are likely to exist (1, 2, 5, 6, 821, 25, 29, 30, 34, 40). MBV
infections are characterised by the presence of prominent, spherical intranuclear occlusion bodies in affected
epithelial cells of the hepatopancreas and midgut, or free within lysed cell debris in the faeces (5, 20).
Crowding or environmental stress may enhance the pathogenicity and increase the prevalence of MBV in its
hosts (2, 13, 20, 24, 31). Transmission and infection by MBV is exclusively by the oral route in which
cannibalism and faecaloral contamination are the principal mechanisms of transmission (17, 20, 31, 32,
35).
MBV-type baculoviruses are widely distributed in cultured and wild penaeid shrimp and prawns in the
Eastern Hemisphere and have been observed or reported from Australia, East Asia, South-East Asia,
India, East Africa, the Middle East, from many of the Indo-Pacific countries (1, 2, 5, 6, 815, 1824,
26, 27, 29, 30, 34, 40, 41). MBV-type baculoviruses have been found in cultured (but not in wild)
shrimp and prawns only at sites in the Mediterranean and in West Africa (20). In most of these examples,
the affected stocks were either introduced P. monodon or the facility had a recent history of P. monodon
introduction. MBV has been found in introduced stocks of P. monodon in the Western Hemisphere.
MBV has been observed in introduced P. monodon in the Pacific in Tahiti and Hawaii, and in the
Americas in a number of shrimp-farming sites in North America, South America, and the Caribbean.
Despite the simultaneous culture in a number of farms, and the consequent direct exposure of certain
Western Hemisphere penaeids to MBV-infected P. monodon, MBV did not produce significant infections
in native species, nor has it become established in shrimp farms or in wild stocks (20, 23).
Several surveillance methods are available for use in certification of the MBV-infection status of shrimp and
prawn stocks. The simplest method is based on the microscopic demonstration of the characteristic occlusion
bodies produced by the virus. With direct microscopy, characteristic occlusion bodies (clusters of refractive
spheres for MBV) are demonstrated in wet-mounts of whole larvae, of excised portions of the
hepatopancreas from postlarvae or older shrimp, or of faeces from large juvenile to adult shrimp. Wet-mount
examination of the faeces of adult broodstock for characteristic occlusion bodies may be used as a nonlethal
method to detect carriers. Histology of fixed specimens may also be used for surveillance. Routine histology
provides a positive diagnosis of MBV infection when characteristic occlusion bodies are demonstrated in
hypertrophied nuclei of mucosal epithelial cells of the hepatopancreas or midgut. Molecular methods for
MBV are also available and provide a satisfactory method for surveillance applications. An antibody-based
enzyme-linked immunosorbent assay for diagnosis of MBV has been reported (16), but the method used
polyclonal antibodies and the antibodies are not commercially available. Gene probes applicable to in-situ
Chapter 4.1.5. - Spherical baculovirosis (Penaeus monodon-type baculovirus)
hybridisation assays and polymerase chain reaction methods are also available for MBV (3, 7, 16, 20, 26,
28, 38, 39). A nested PCR method for MBV may provide the most sensitive method available for
screening and surveillance applications (3).
Because MBV is transmitted from adults to their offspring by faecal contamination of the spawned eggs,
prevention of infection in hatcheries may be achieved by taking steps to eliminate faecal contamination of
spawned eggs and larvae by thoroughly washing nauplii or eggs with formalin, iodophores, and clean sea
water (9).
Infection of the hepatopancreas by Penaeus monodon-type baculovirus (MBV) is among the most readily
diagnosable diseases of the penaeid shrimps and prawns. The occlusion bodies formed by the virus are
very conspicuous and easily demonstrated by direct light microscopy with fresh specimens or by routine
histological methods with fixed specimens. Direct microscopic methods are most suitable for the
postlarvae stages, which are commonly moved in regional and international commerce (5, 6, 19, 20).
Highly sensitive molecular methods for MBV are also available and provide alternative methods for
surveillance applications, especially for nonlethal testing of broodstock (3, 7 20, 23). Hence, there are
several options to choose from as surveillance methods for certification of the MBV-infection status of
shrimp and prawn stocks.
The methods currently available for surveillance, detection, and diagnosis of MBV are listed in Table 1.
The designations used in the Table indicate: = the method is presently unavailable or unsuitable; ? = the
method is available but untested; + = the method has application in some situations, but cost, accuracy, or
other factors severely limits its application; ++ = the method is a standard method with good diagnostic
sensitivity and specificity; and +++ = the method is the recommended method for reasons of availability,
utility, and diagnostic specificity and sensitivity.
Table 1. Surveillance, detection and diagnostic methods for the nuclear polyhedrosis virus MBV
Gross signs +
Direct BF/LM +++ +++ + ++ +++ +++
Histopathology +++ +++ ++ ++ +++ +++
Bioassay + +
Transmission EM + +
Antibody-based methods + +
DNA probes dot-blot
DNA probes in situ +++ +++
PCR +++ +++ +++ +++ +++ +++
1. STANDARD SCREENING METHODS FOR MBV
1.1. Direct microscopic methods
1.1.1. Wet-mount of fresh tissue
Diagnosis of MBV infections is made by the demonstration of single or multiple generally
spherical occlusion bodies in wet-mounts of squash preparations of hepatopancreas or midgut
examined by phase-contrast or bright-field microscopy. In carefully prepared preparations,
MBV occlusion bodies are shown to be intranuclear and to range in diameter from less than
0.1 m to nearly 20 m. Staining the tissue squash with a stain such as 0.05% aqueous
malachite green aids in demonstration of the occlusion bodies by staining them more intensely
than other similar sized spherical objects, such as normal host cell nuclei, nucleoli, secretory
granules, phagolysosomes, and lipid droplets (5, 6, 19, 20, 23).
1.1.2. Faecal examination technique
This method may be used as a nonlethal method to screen for carriers of MBV. The method
can be applied to juvenile or older shrimp, and it is perhaps most useful as a nonlethal method
for screening valuable broodstock. Faecal samples from shrimp to be tested may be obtained
by placing the shrimp in an aquarium, spawning tank, or other suitable tanks for a few hours
until faecal strands are present on the tank bottom. The faecal strands are best collected using
a clear plastic siphon hose (an air line fitted with a section of plastic pipette as a tip is ideal) and
placed in a beaker, cup, or other suitable container. The faecal strands may be made into wet-
mounts and examined directly for occlusion bodies. MBV occlusion bodies are roughly
spherical, refractive bodies (similar in size to Baculovirus penaei [BP] occlusion bodies). The
addition of a drop of 0.05% aqueous malachite green to the wet-mount preparation aids in
demonstrating the MBV occlusion bodies by staining them more intensely green than other
round objects in faeces. In fresh faeces, MBV occlusions often remain in clusters, held
together by the nuclear membrane (5, 9, 19, 20 36).
Collected faeces may also be used as the sample for nonlethal testing for MBV by polymerase
chain reaction (PCR). PCR will provide greater diagnostic sensitivity for low-grade infections
than will direct microscopic examination (3, 7, 16, 20, 26, 38).
See Section 2.2.
1.3. Polymerase chain reaction procedure
PCR methods have been developed for MBV. Primer sets designed from certain segments of the
MBV genome are unique and provide positive test results only in samples infected with particular
stains of this virus (11, 19, 27, 29, 33, 38, 39).
Substances in the hepatopancreas and faeces of shrimp have been found to inhibit the DNA
polymerase used in the PCR assay. Therefore, DNA extraction is required before PCR can be
successfully applied to the detection of this virus (3, 7, 16, 33).
The following controls should be included in every PCR assay for MBV: a known negative tissue
or faecal sample; a known positive tissue or faecal sample (this can be the DNA clone from which
a specific set of primers was designed); and a no-template control.
1.3.1. DNA extraction
DNA extraction kits are convenient and commercially available. Otherwise, refer to Section
1.3.1. in Chapter 4.1.4. Tetrahedral baculovirosis (BP) for a suitable DNA extraction
procedure.
1.3.2. MBV PCR method segment of the polyhedrin gene
A PCR method with primer sequences was published by Lu et al. (25). Two primers, p35 and
p36, were designed using insect baculovirus polyhedrin gene sequences as a reference, and they
amplify a 674 base pairs (bp) segment of the MBV polyhedrin gene. The primer sequences are:
p35: 5-AC(CT)-TA(CT)-GTG-TCA-GAC-AAC-AAA-TA(CT)-TAC-AAA-3
p36: 5-GG(CT)-GCG-TC(GT)-GG(CT)-GCA-AA(CT)-TT(CT)-TT(AT)-AC(CT)-TT(AG)-
AA-3
1

i) The reaction is carried out in a final volume of 100 l of a reaction mixture containing
67 mM Tris/HCl, pH 8.0, 2 mM MgCl
2
, 0.2 mM of each dNTP, 15 pmol of each primer,
and 1 unit of Taq DNA polymerase.
ii) A drop of mineral oil is added to cover the reaction mixture (if the thermal cycler used
does not have a heated lid), which is then amplified through 35 cycles (95C for 1 minute,
45C for 1 minute, and 72C for 2 minutes) in a thermal cycler.
iii) The 674 bp PCR product may be visualised by electrophoresis in a 1% agarose gel or by
Southern transfer and hybridisation with a labelled MBV probe prepared from the same
DNA segment.
1.3.3. MBV PCR method segment of the polymerase gene
A PCR method with primer sequences was published by Hsu et al. (16). Two primers, pA1 and
pA2, were designed using a conserved sequence from the insect baculovirus polymerase gene
as a reference, and they amplify a 511 bp segment of the MBV polymerase gene. The primer
sequences are:
pA1: 5-GTG-CTG-CAA-CGA-CTG-GAC-TGT-CC-3
pA2: 5-TGA-TCC-TAG-GCG-TGC-CTG-TAT-TGG G-3
i) The reaction is carried out in a final volume of 100 l of a reaction mixture containing
10 ng of viral DNA, 1.6 M each of the two primers, 10 l of 10 Dynazyme buffer and
five units of Dynazyme (Finnzymes, Espoo, Finland), and 20 mM mixture of all four
dNTP.
ii) The reaction mixes were overlaid with mineral oil (if required), and then heated to 94C
for 10 minutes before starting the PCR in a thermal cycler.
iii) The first five cycling parameters are: denaturing for 2 minutes at 94C, annealing for
1 minute at 42C, extension for 1 minute at 72C.
iv) This is followed by 30 cycles using the following parameters: denaturing at 94C,
annealing for 1 minute at 59C, and extension for 1 minute at 72C. During the last cycle,
extension is 10 minutes. PCR reactions are stopped at 4C.
v) The 511 bp PCR product may be visualised by electrophoresis in a 2% agarose gel.
1.3.4. MBV nested PCR method
A nested PCR method capable of detecting low concentrations of MBV (down to eight viral
genome equivalents) was published by Belcher & Young (3). Two external and two internal
primers were designed using a DNA sequence derived from the plasmid p4Ec196, which was

1 Bases in parentheses indicate that either base can be used in this position and that the primers synthesised
should be a mixture containing both possible base combinations at the sites indicated by the parentheses.
constructed from a 7.4 kb EcoRI fragment of an Australian isolate of MBV (3). The primer
sequences are:
Primer Sequence Temperature
MBV1.4F 5-CGA- TTC-CAT-ATC-GGC-CGA-ATA-3 62C (68.9C)
MBV1.4r 5-TTG-GCA-TGC-ACT-CCC-TGA-GAT-3 64C (70.8C)
MBV1.4NF 5-TCC-AAT-CGC-GTC-TGC-GAT-ACT-3 64C (70.8C)
MBV1.4NR 5-CGC-TAA-TGG-GGC-ACA-AGT-CTC-3 66C (72.8C)
The melting temperatures of the primers are according to the formula 2(A+T) + 4(G+C), or
according to the %GC method (values in parentheses).
DNA extraction
i) PCR inhibitors were noted by the authors of this method (3) to be present in DNA
samples prepared from whole MBV-infected postlartval Penaeus monodon when using the
extraction method recommended by Wang et al. (42) for BP, which incorporates
proteinase K. However, when hot phenol was used to extract the DNA, this inhibitory
effect was removed.
ii) With the hot phenol method, the sample to be tested (postlarvae, shrimp hepatopancreas,
faeces) are freeze-dried and ground to a powder in liquid nitrogen with a motor and
pestle.
iii) Approximately 300 mg of the resulting material is added immediately to 400 l of
preheated (65C) lysis buffer (100 mM Tris/HCl, 100 mM ethylene diamine tetra-acetic
acid [EDTA], 1% sodium dodecyl sulfate, pH 8.0) and incubated at 65C for 5
10 minutes.
iv) The resulting suspensison is coarsely homogenised by spot centrifugation and
homogenisation with a microfuge tube pestle. Tris/HCl-buffered phenol, pH 8.0 (600 l)
is added and the mixture is incubated for 2 hours at 65C with occasional inversion.
v) Following centrifugation at 12,000 g for 10 minutes at room temperature, the aqueous
layer is transferred to a fresh microfuge tube and extracted twice with an equal volume of
phenol/chloroform (1/1). Then, a total of 50 l of the aqueous layer is transferred to a
fresh microfuge tube containing 150 l dilution buffer and extracted once more with an
equal volume of phenol/chloroform (1/1) followed by a straight chloroform extraction.
vi) Ammonium acetate is added to the aqueous layer to a final concentration of 2.5 M, mixed
briefly, and two volumes of 20C ethanol are added with 1 l of 20 mg/litre glycogen to
precipate the DNA.
vii) DNA is precipitated by incubation at 20C overnight or by incubation at 70C for
1 hour.
viii) DNA is pelleted at 12,000 g for 15 minutes at 4C. The resulting DNA pellet is rinsed
twice, first with 500 l 80% cold ethanol and centrifuged at 12000 g for 10 minutes at
4C, followed by an identical rinse and centrifugation at room temperature.
ix) The final DNA pellet is dried in vacuo, resuspended in 100 l dilution buffer (10 mM
Tris/HCl, pH 8.0, 0.1 mM EDTA, pH 8.0) at room temperature overnight or at 37C for
2 hours. Following spectrophotometric analysis, and prior to PCR, the DNA is diluted to
50 ng/l in dilution buffer.
Nested PCR method
i) Prior to PCR, the extracted total DNA is denatured in boiling water for 3 minutes
followed by followed by quick chilling in ice-water.
ii) A total of 100 ng of extracted DNA is used as template.
iii) Each reaction tube contains 50 mM KCl, 10 mM Tris/HCl, pH 9, 0.1% Triton X-100, 0.2
mM of each dNTP, 1.5 mM MgCl
2

, 0.25 M of each MBV1.4F and MBV1.4R, 2.5 U of
Taq, and made up to a final volume of 50 l.
iv) The reaction mixes are overlaid with mineral oil (as necessary).
v) The conditions for the first round of amplification are: one cycle of 96C for 5 minutes;
40 cycles of 94C for 30 seconds, 65C for 30 seconds, 72C for 60 seconds; and one
cycle of 72C for 7 minutes.
vi) The second step of the nested PCR is accomplished with 0.5 l of the primary PCR
reaction used as template with the internal primers.
vii) The second round of amplification reaction contains 50 mM KCl, 10 mM Tris/HCl, pH
9.0, 0.1% Triton X-100, 1.5 mM MgCl
2
, 0.2 mM of each dNTP, 0.25 M of each of the
primers MBV1.4NF and MBV1.4NR, and 2.5 U of Taq, and made up to a final volume of
50 l.
viii) The reaction mixes are overlaid with mineral oil (as necessary).
ix) The conditions for the second round of amplification are: one cycle of 96C for
5 minutes; 35 cycles of 94C for 30 seconds, 60C for 30 seconds, 72C for 60 seconds;
and one cycle of 72C for 7 minutes.
x) Demonstration of the PCR products (533 bp first step and 361 bp second step) is
accomplished by adding 1 l of gel-loading buffer (0.25% [w/v] bromophenol blue, 15%
[w/v] Ficoll-type 400, 100 mM EDTA, pH 8.0) to 10 l of each reaction mixture and
electrophoresis through a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM
EDTA, pH 8.0) containing 0.5 g/litre ethidium bromide.
Confirmation of infection by MBV may be accomplished with any of the methods listed in Section 1
(i.e. wet-mounts of hepatopancreas tissue squashes or of faecal strands, or by PCR). The other
methods available for confirmatory diagnosis of MBV include: autofluorescence with phloxine, and
routine histological methods (46, 19, 20, 23, 36).
2.1. Autofluorescence method with phloxine stain
Another method for detecting MBV occlusion bodies is based on the fluorescence of phloxine-
stained occlusion bodies (5, 20, 36). Aqueous 0.001% phloxine may be added to tissue squash
preparations to make wet-mounts of hepatopancreas or faeces for direct examination. Histological
sections stained with routine haematoxylin and eosin (H&E) containing 0.005% phloxine, are also
suitable for this procedure. MBV occlusions in wet-mounts of tissue squashes, in faeces, or in
histological sections fluoresce bright yellow-green against a pale green background under epi-
fluorescence (barrier filter of 0515 nm and a 490 nm exciter filter). Other objects in the tissues
and insect baculovirus occlusion bodies do not fluoresce with this method. Hence, the method
can provide a rapid and specific diagnosis.
Histology may be used to provide a definitive diagnosis of MBV infection. Because 10% buffered
formalin and other fixatives provide, at best, only fair fixation of the shrimp hepatopancreas (the
principal target organ for MBV), the use of Davidsons fixative (containing 33% ethyl alcohol
[95%], 20% formalin [approximately 37% formaldehyde], 11.5% glacial acetic acid and 33.5%
distilled or tap water) is highly recommended for all routine histological studies of shrimp (4, 20).
To obtain the best results, dead shrimp should not be used. Only live, moribund, or compromised
shrimp should be selected for fixation and histological examination. Selected shrimp are killed by
injection of fixative directly into the hepatopancreas; the cuticle over the cephalothorax and
abdomen just lateral to the dorsal midline is opened with fine-pointed surgical scissors to enhance
fixative penetration (the abdomen may be removed and discarded), the whole shrimp (or
cephalothorax less the abdomen) is immersed in fixative for 2448 hours, and then transferred to
70% ethyl alcohol for storage. After transfer to 70% ethyl alcohol, fixed specimens may be
transported by wrapping in cloth or a paper towel saturated with 70% ethyl alcohol and packed in
leak-proof plastic bags. To begin histological processing, fixed shrimp are cut-in (see ref. 4 for a
photographic guide to this procedure) to facilitate eventual sectioning of the hepatopancreas and
midgut. After dehydration, the specimens are embedded in paraffin and sections of 57 m
thickness are cut. Routine histological stains such as MayerBennetts or Harris H&E, Giemsa
stains, and Gram tissue-staining methods may be used for the demonstration of MBV diagnostic
spherical occlusion bodies in hepatopancreatocytes, gut epithelial cells, or gut lumen (5, 6, 10, 20,
24). Typically, MBV-infected hepatopancreatic (or occasionally midgut) cells will present markedly
hypertrophied nuclei with single or, more often, multiple occlusion bodies, chromatin diminution
and margination. Occlusion bodies may be stained bright red with H&E stains, and intensely, but
variably, with Grams tissue stains. Brown and Brenns histological Gram stain, although not
specific for baculovirus occlusion bodies, tends to stain occlusions more intensely (either red or
purple, depending on section thickness, time of decolouration, etc.) than the surrounding tissue,
aiding in demonstrating their presence in low-grade infections (19, 20, 23).
Nonradioactive DIG-labelled gene probes to MBV have been developed (20, 23, 2628, 33).
Gene probe and PCR methods may provide greater diagnostic sensitivity in detecting low-grade
infections than do more traditional wet-mount or histological techniques. Furthermore, PCR
methods (see Section 1.3.) have the added advantage of being applicable to nonlethal testing of
faecal samples collected from valuable broodstock shrimp.
DIG-labelled DNA probes for representative strains of BP and MBV are commercially available
as ShrimProbe
TM
kits from DiagXotics (Wilton, Connecticut, USA). The probes are labelled with
a nonradioactive label, digoxigenin-11-dUTP (DIG). These probes only work well with the in-situ
hybridisation method with histological sections because there are substances present in the
hepatopancreas and faeces of shrimp that provide both false-positive and false-negative results
with samples that are blotted directly and not extracted prior to probing.
2.3.1. Dot-blot hybridisation procedure for MBV
While specific DNA probes for MBV are available, their application to dot-blot hybridisation
procedures is not recommended for most routine diagnostic applications. Pigments present in
the hepatopancreas leave a coloured spot on the hybridisation membrane that can result in the
masking of a positive test or in the false interpretation of a negative test. Likewise, bits of
chitin (which nonspecifically bind DNA probes), pigments, and other materials present in the
faecal sample may also result in false-positive or false-negative dot-blot hybridisation tests.
Extraction of DNA from the hepatopancreas or faeces prior to blotting or the use of
chemiluminescent or radioactively labelled probes may circumvent these problems, but the
adequacy of other test methods (i.e. direct wet-mounts, histology, or PCR) has not indicated a
need for the further refinement and application of the dot-blot method (20).
2.3.2. In-situ hybridisation procedure
The in-situ hybridisation protocol given in detail in Section 1.2. of Chapter 4.1.6. Infectious
hypodermal and haematopoietic necrosis virus (IHHNV) uses the Genius
TM
System developed
by Boehringer Mannheim Biochemicals (now Roche Molecular Biochemicals) and was adapted
from the Boehringer Mannheims Nonradioactive In Situ Hybridization Application Manual. An
additional step is required for the in-situ hybridisation test for MBV. Follow steps ivi of
Section 1.2., in-situ hybridisation procedure for IHHNV, in Chapter 4.1.6. For MBV substitute
the following modifications to step vii before proceeding with steps viiixvii as given for
IHHNV:
i) (Modified step vii): Boil the DIG-labelled probe for 10 minutes and quench on ice; spin
briefly in the cold (~410C) using a refrigerated centrifuge or a chilled microcentrifuge)
to bring all the liquid down to the base of the microcentrifuge tube; keep on ice. Dilute
the probe to 50 ng/ml in prehybridisation solution and cover the tissue with 500 l of the
solution. Denature the double-stranded viral DNA in the tissue of the histological section
by placing the slides on a 85C heat block (or on aluminium foil that is placed over a
boiling water bath) for 610 minutes. Quench the slides on ice for 5 minutes. Incubate
the slides overnight at 42C in a humid chamber. Drain the fluid on to blotting paper.
During this incubation step, keep the wash buffers at 37C to prewarm them.
ii) Proceed with steps viiixvii as given for IHHNV.
With DIG-labelled probes, accumulations of MBV viral DNA within infected cell nuclei, in
cytoplasmic phagosomes, or in necrotic tissue debris are stained blue to a dark blue-black.
Although they contain virus, occlusion bodies do not normally react with DIG-labelled DNA
probes because the occlusion body protein crystalline matrix does not permit penetration of
the probe (20).
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Observations on the ultrastructure of baculovirus in Australian Penaeus monodon and Penaeus
merguiensis. Aust. J. Mar. Freshwater Res., 39, 743749.
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development and histopathology of monodon baculovirus in Southern Thailand. Aquaculture, 96,
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CHANRATCHAKOOL P. (1999). Presence of the two viral pathogens WSSV and MBV in three wild
shrimp species (Penaeus indicus, Metapenaeus ensis and Metapenaeus lysianassa) cultured in the mangrove
forest of Ca Mau Province. Asian Fish. Sci., 12, 309325.
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Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Dis.
Aquat. Org., 40, 9399.
17. JOHNSON P.T. & LIGHTNER D.V. (1988). Rod-shaped nuclear viruses of crustaceans: gut-infecting
species. Dis. Aquat. Org., 5, 123141.
18. LESTER R.J.G., DOUBROVSKY A., PAYNTER J.L., SAMBHI S.K. & ATHERTON J.G. (1987). Light and
electron microscope evidence of baculovirus infection in the prawn Penaeus plebejus. Dis. Aquat. Org.,
3, 217219.
20. LIGHTNER, D.V. (ED.) (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for
Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA, 304
pp.
21. LIGHTNER D.V., HEDRICK R.P., FRYER J.L., CHEN S.N., LIAO I.C. & KOU G.H. (1987). A survey of
cultured penaeid shrimp in Taiwan for viral and other important diseases. Fish Pathol., 22, 127140.
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Penaeus monodon. J. Invertebr. Pathol., 38, 299302.
23. LIGHTNER D.V & REDMAN R.M. (1998). Shrimp diseases and current diagnostic methods.
24. LIGHTNER D.V., REDMAN R.M. & BELL T.A. (1983). Observations on the geographic distribution,
pathogenesis and morphology of the baculovirus from Penaeus monodon Fabricius. Aquaculture, 32,
209233.
25. LU C.C., TANG F.J.K. & CHEN S.N. (1996). Morphogenesis of the membranous labyrinth in penaeid
shrimp cells infected with Penaeus monodon baculovirus (MBV). J. Fish Dis., 19, 357364.
26. LU C.C., TANG F.J.K., KOU G.H. & CHEN S.N. (1993). Development of a Penaeus monodon-type
baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis. J. Fish Dis., 16,
551559.
27. LU C.C., TANG F.J.K., KOU G.H. & CHEN S.N. (1995). Detection of Penaeus monodon-type
baculovirus (MBV) infection in Penaeus monodon Fabricius by in situ hybridization. J. Fish Dis., 18,
337345.
28. MARI J., BONAMI J.R., POULOS B. & LIGHTNER D.V. (1993). Preliminary characterization and partial
cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. Aquat. Org.,
16, 207215.
29. NASH G., POERNOMO A. & NASH M.B. (1988). Baculovirus infection in brackishwater pond cultured
Penaeus monodon Fabricius in Indonesia. Aquaculture, 73, 16.
30. NATIVIDAD J.M. & LIGHTNER D.V. (1992). Prevalence and geographic distribution of MBV and
other diseases in cultured giant tiger prawns (Penaeus monodon) in the Philippines. In: Diseases of
Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K., eds. The Oceanic
Institute, Makapuu Point, Honolulu, Hawaii, USA, 139160.
31. NATIVIDAD J.M. & LIGHTNER D.V. (1992). Susceptibility of the different larval and postlarval stages
of black tiger prawn, Penaeus monodon Fabricius, to monodon baculovirus (MBV). In: Diseases in
Asian Aquaculture I, Shariff M., Subasinghe R. & Arthur J.R., eds. Fish Health Section, Asian
Fisheries Society, Manila, Philippines, 111125.
32. PAYNTER J.L., VICKERS J.E. & LESTER R.J.G. (1992). Experimental transmission of Penaeus monodon-
type baculovirus (MBV). In: Diseases in Asian Aquaculture I, Shariff M., Subasinghe R. & Arthur J.R.,
eds. Fish Health Section, Asian Fisheries Society, Manila, Philippines, 97110.
33. POULOS B.T., MARI J., BONAMI J.R., REDMAN R. & LIGHTNER D.V. (1994). Use of non-radioactively
labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization
on fixed tissue. J. Virol. Methods, 49, 187194.
34. SPANN K.M. & LESTER R.J.G. (1996). Baculovirus of Metapenaeus bennettae from the Moreton Bay
region of Australia. Dis. Aquat. Org., 27, 5358.
35. SPANN K.M., LESTER R.J.G. & PAYNTER J.L. (1993). Efficiency of chlorine as a disinfectant against
monodon baculovirus (MBV). Asian Fish. Sci., 6, 295301.
36. THURMAN R.B., LIGHTNER D.V., BELL T.A. & HAZANOW S. (1990). Unique physicochemical
properties of the occluded penaeid shrimp baculoviruses and their use in diagnosis of infections. J.
LEMON S.M., MANILOFF J., MAYO M.A., MCGEOCH D.J., PRINGLE C.R. & WICKNER R.B. (2000).
Academic Press, San Diego, USA, 1162 pp.
38. VICKERS J.E., LESTER R.J.G., SPRADBROW P.B. & PEMBERTON J.M. (1992). Detection of Penaeus
monodon-type baculovirus (MBV) in digestive glands of postlarval prawns using polymerase chain
reaction. In: Diseases in Asian Aquaculture I. Shariff M., Subasinghe R.P., & Arthur J.R., eds. Fish
Health Section, Asian Fisheries Society, Manila, Philippines, 127133.
39. VICKERS J.E., LESTER R.J.G., SPRADBROW P.B. & PEMBERTON J.M. (1994). Evidence for homology
between polyhedrin genes of baculoviruses of a prawn and an insect. J. Invertebr. Pathol., 63, 207208.
40. VIJAYAN K.K., ALAVANDI S.V., RAJENDRAN K.V. & ALAGARSWAMI K. (1995). Prevalence and
histopathology of monodon baculovirus (MBV) infection in Penaeus monodon and P. indicus in shrimp
farms in the south-east coast of India. Asian Fish. Sci., 8, 267272.
41. VOGT G. (1992). Transformation of anterior midgut and heaptopancreas by monodon baculovirus
(MBV) in Penaeus monodon postlarvae. Aquaculture, 107, 239248.
42. WANG S.Y., HONG C. & LOTZ J.M. (1996). Development of a PCR procedure for the detection of
Baculovirus penaei in shrimp. Dis. Aquat. Org., 25, 123131.
*
* *
C HA P T E R 4 . 1 . 6 .

I NF E CT I OUS HYP ODE RMAL AND
HAE MAT OP OI E T I C NE CROS I S

SUMMARY
Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is the smallest of the known penaeid
shrimp viruses. The IHHN virion is a 22 nm, nonenveloped icosahedron, with a density of 1.40 g/ml in
CsCl, contains linear single-stranded DNA with an estimated size of 4.1 kb, and has a capsid with four
polypeptides of molecular weight 74, 47, 39, and 37.5 kD. Because of these characteristics, IHHNV has
been classified as a member of the family Parvoviridae (5, 6, 14, 16, 24).
Following its discovery, infection by IHHNV was demonstrated to cause acute epizootics and mass
mortality (90%) only in Penaeus stylirostris, with the juvenile and subadult life stages being the most
severely affected. In marked contrast, IHHNV causes the chronic disease runt-deformity syndrome in
P. vannamei in which reduced, irregular growth and cuticular deformities, rather than mortalities, are the
principal effects (1, 2, 7, 9, 10, 12, 14, 20). Selected shrimp stocks that are not only resistant to IHHN
disease, but are also refractory to infection have been developed (14, 35). Some members of populations of
P. stylirostris and P. vannamei that survive IHHNV infections and/or epizootics, may carry the virus
for life and pass the virus on to their progeny and other populations by vertical and horizontal transmission
(1, 20, 28).
IHHNV has a world-wide distribution among cultured penaeid shrimp. IHHNV is commonly found in
wild penaeid shrimp in the eastern Pacific from Peru to Mexico. Although IHHNV has been reported
from cultured P. vannamei and P. stylirostris in most of the shrimp-culturing regions of the Western
Hemisphere and in wild penaeids throughout their range along the Pacific coast of the Americas (Peru to
northern Mexico), it has not been found in wild penaeid shrimp on the Atlantic coast of the Americas (1,
2, 431, 33, 34). IHHNV has been also reported in cultured penaeid shrimp from Pacific islands
including the Hawaiian Islands, French Polynesia, Guam, and New Caledonia. In the Indo-Pacific region,
the virus has been reported from cultured and wild penaeid shrimp in East Asia, South-East Asia, and
the Middle East (20). An IHHN-like virus has been reported from Australia (32). However, because
this agent does not react with the DNA probe BS4.5 to IHHNV, it is not closely related to the strain(s)
of IHHNV that do react with the probe and that are widely distributed in East Asia and in the
Americas (20). Although demonstrated in wild shrimp from the Indo-Pacific, there is a paucity of data
available on the distribution of this virus in wild shrimp from this region. The geographical distribution of
IHHNV in shrimp has increased significantly since its discovery in the early 1980s. Recent work suggests
that there is sufficient sequence variation among geographical isolates from diverse locations in Asia to
suggest that multiple geographical strains of the virus exist (37). IHHNV causes serious disease in two
species of Western Hemisphere penaeid shrimp and it is considered to be a potential pathogen in several
other Western and Eastern Hemisphere species of penaeid shrimp. While infection by IHHNV has been
demonstrated to cause disease in P. stylirostris, P. vannamei and P. monodon, natural or experimental
infections in other penaeid species have been observed without the occurrence of disease (8, 10, 1720, 22).
IHHNV typically infects cells in tissues of ectodermal and mesodermal origin, and only rarely is it detected
in endoderm-derived tissues, such as the epithelial mucosal cells of the midgut, midgut caeca and
hepatopancreas. In P. stylirostris, IHHNV infection may result in high mortalities, while in some strains
of P. stylirostris, in P. vannamei, and possibly in P. monodon, reduced growth and cuticular deformities
may be the only outcome of infection (15, 19, 20, 22).
Chapter 4.1.6. - Infectious hypodermal and haematopoietic necrosis
Molecular diagnostic tests are the surveillance methods for IHHNV in penaeid shrimp. With molecular
methods unique portions of the IHHNV genome are detected by dot-blot hybridisation using specific DNA
probes or by polymerase chain reaction (12, 14, 20, 24, 27, 28, 30, 32, 3437). While PCR methods
provide the greatest sensitivity for detection of IHHNV (14, 28, 30, 3537), the use of IHHNV-specific
DNA probes with in-situ hybridisation applied to paraffin sections provides the greatest diagnostic
certainty available for this agent (20). A variety of other diagnostic methods can be used to provide
presumptive and confirmed diagnoses of IHHNV infection. Among these are routine histology, bioassays,
and methods that use monoclonal antibodies against IHHNV with indirect fluorescent, enzyme-linked
immunosorbent, and Western blot assays. With standard histological methods, diagnostic intranuclear
eosinophilic inclusion bodies are demonstrated in specific target tissues (8, 20).
Eradication methods for IHHNV can be applied to certain aquaculture situations. These methods are
dependent on eradication of infected stocks, disinfection of the culture facility, the avoidance of re-
introduction of the virus (from other nearby culture facilities, wild shrimp, etc.), and re-stocking with
IHHNV-free postlarvae that have been produced from IHHNV-free broodstock (10, 20, 21).
The methods currently available for surveillance, detection, and diagnosis of infectious hypodermal and
haematopoietic necrosis virus (IHHNV) infections are listed in Table 1. The designations used in the
Table indicate: the method is presently unavailable or unsuitable; ? the method is available but
untested; the method has application in some situations, but cost, accuracy, or other factors severely
limits its application; the method is a standard method with good diagnostic sensitivity and
specificity; the method is the recommended method for reasons of availability, utility, and
diagnostic specificity and sensitivity; and R&D = the method is in development but is not yet available
though commercial sources.
Table 1. IHHNV surveillance, detection and diagnostic methods
Gross signs

Direct BF/LM

Histopathology

Bioassay

Transmission EM


R&D R&D
DNA probes dot-blot
DNA probes in situ
PCR

1. STANDARD SCREENING METHODS FOR IHHNV
Surveillance methods for IHHNV use nonradioactive DIG-labelled gene probes and polymerase chain
reaction (PCR) methods. Nonradioactive DIG-labelled DNA probes for IHHNV are now
commercially available as ShrimProbe
TM
kits from DiagXotics (Wilton, Connecticut [CT], USA) in dot-
blot and in-situ hybridisation formats. The probe is labelled with a nonradioactive label, digoxigenin-11-
dUTP (DIG-11-dUTP). The protocols given below use the Genius
TM
System developed by Boehringer
Mannheim Biochemicals (this company now owned by Roche Diagnostic Corporation) and was
adapted from the GeniusTM System Users Guide for Membrane Hybridization and from Boehringer
Mannheims Nonradioactive In Situ Hybridization Application Manual (12, 20, 27, 34).
Gene probe and PCR methods provide greater diagnostic sensitivity than do more traditional methods
for IHHN diagnosis that employ classic histological methods. Furthermore, these methods have the
added advantage of being applicable to nonlethal testing of valuable broodstock shrimp. A
haemolymph sample may be taken with a tuberculin syringe, or an appendage (a pleopod for example)
may be biopsied, and used as the sample for a direct dot-blot test or PCR assay (4, 12, 20, 30, 32).
Several PCR methods are available for IHHNV detection (28, 30, 3537), and several companies
market PCR test kits. PCR kits are available for IHHNV in the USA from DaigXotics (Wilton, CT,
USA) or from Farming Intelligene Technology (Taipei China). However, very recent information on
IHHNV suggests that there are multiple geographical strains and that some primer sets will not react
with some geographical strains of the virus isolates (37). Hence, confirmation of unexpected positive
and/or negative PCR results with a second primer set using another diagnostic method is advisable.
1.1. Dot-blot hybridisation procedure
The dot-blot hybridisation method using a DIG-labelled DNA probe for IHHNV follows
generally the methods outlined in Lightner (16) that are given below. Formulas for the required
reagents are given in Section 1.1.1. below.
i) Prepare a positively charged nylon membrane (Roche Diagnostics Cat. No. 1-209-299): cut
pieces to fit samples and controls and mark with soft-lead pencil making 1 cm squares for
each sample. Include a positive and a negative control on each filter. Lay out on to a piece of
filter paper (Whatman 3MM).
ii) If necessary, dilute samples to be assayed in TE plus 50 g/ml salmon sperm DNA, using
1 l sample in 9 l buffer in 1.5 ml microcentrifuge tubes. Samples for dot-blots can be
haemolymph or tissues homogenised in TN buffer.
iii) Boil samples for 10 minutes and quench on ice for 5 minutes. Briefly microfuge samples in
the cold to bring down all liquid and to pellet any coagulated protein. Keep on ice until
samples are dotted on to the membrane.
iv) Dot 13 l of each sample on to an appropriate place on the filters. Allow to air-dry and then
fix samples on to the membrane by baking at 80C for 30 minutes or by UV cross-linking
using a DNA transilluminator for 3 minutes.
v) Turn on a water bath to 68C and prepare prehybridisation solution. For a 10 15 cm
membrane, prepare 8 ml per membrane. Switch on a stirring hot plate set to low and stir
and warm for 30 minutes until the blocking agent has dissolved and the solution is cloudy.
Also prepare some heat-seal bags that are slightly larger in size than the membrane. Five to
six bags will be needed per membrane.
vi) Remove membranes from the oven or transilluminator and put into a heat-seal bag with 4 ml
per membrane of prehybridisation solution. Seal the bags and put into a 68C water bath for
0.51 hour.
vii) Boil the DIG-labelled probe for 10 minutes, quench on ice and then microfuge in the cold to
bring all the liquid down. Keep on ice. Remove the prehybridisation solution from the bags.
Add 2 ml of fresh prehybridisation solution to each bag and then add the correct,
predetermined amount of DIG-labelled probe to each, mixing well as it is being added. Seal
the bags, place back in the 68C water bath and incubate for 812 hours.
viii) Wash membranes well with:
2 standard saline citrate
(SSC)/0.1% sodium dodecyl
sulfate (SDS)
2
5 minutes at room temperature
0.1 SSC/0.1% SDS 3
15 minutes at 68C
(use 4 ml/filter and seal in bags)
Buffer I
1
Buffer II
1
Buffer I
1
(Buffers are prepared ahead of time).
ix) React the membranes in bags with anti-DIG AP conjugate (Roche Diagnostics 1-093-274)
diluted 1/5000 in Buffer I. Use 3 ml per membrane, incubate for 3045 minutes at room
temperature on a shaker platform.
x) Wash membrane well with:
Buffer I
2
Buffer III
1
xi) Develop the membranes in bags using 3 ml per membrane of development solution
(nitroblue tetrazolium salt [NBT]/X-phosphate in Buffer III) made up just prior to use.
React in the dark at room temperature for 12 hours. Stop the reactions in Buffer IV and dry
the membranes on 3MM filter paper.
xii) Photograph the results. (Colour fades over time.)
xiii) Store dry membranes in heat-seal bags.
1.1.1. Reagent formulas for dot-blot method
i) TN buffer
20 mM Tris/HCl 3.15 g Tris/HCl
0.4 M NaCl 23.38 g NaCl
DD H
2
O 1000 ml
pH to 7.4; autoclave to sterilise; store at 4C.
ii) Sample dilution buffer: Buffer IV plus 50 g/ml salmon sperm DNA. Prepare salmon (or
herring) sperm DNA as follows:
Dissolve 2.5 mg salmon sperm DNA in 5 ml DD H
2
O (500 g/ml).
Shear the DNA by passing the solution through an 1820-gauge needle several times.
Boil the DNA for 1015 minutes to denature it.
Filter 45 ml Buffer IV through a 0.45 m filter.
Mix 5 ml of sheared, denatured DNA with the 45 ml of filtered Buffer IV.
Store at 20C (can keep usable amount at 4C).
iii) 20 SSC buffer:
3 M NaCl 175.32 g NaCl
0.3 M Na citrate 88.23 g Na citrate.2H
2
O
DD H
2
O 1000 ml
pH to 7.0; filter through 0.45 m filter; autoclave to sterilise; store at 4C.

iv) 5 SSC buffer
5 SSC 250 ml 20 SSC stock
DD H
2
O 750 ml
Filter through 0.45 m filter; store at 4C.
v) Prehybridisation solution (prepare 30 minutes prior to use)
1% blocking agent 0.1 g blocking reagent (Roche Diagnostics 1-096-176)
0.1% Sarkosyl 50 l 20% Sarkosyl
0.02% SDS 20 l 10% sodium dodecyl sulfate
5 SSC buffer 10 ml 5 SSC buffer
Heat on a low setting with stirring for 30 minutes to dissolve blocking agent.
vi) Wash buffers
2 SSC, 0.1% SDS:
Mix 100 ml 20 SSC with 895 ml DD H
2
O; filter through 0.45 m filter; add 5 ml 20% SDS
and mix; store at room temperature.
0.1 SSC, 0.1% SDS:
Mix 5 ml 20 SSC with 990 ml DD H
2
O; filter through 0.45 m filter; add 5 ml 20% SDS
and mix; store at room temperature.
vii) Buffer I
100 mM Tris/HCl 12.11 g Tris Base
150 mM NaCl 8.77 g NaCl
DD H
2
O 1000 ml
pH to 7.5 with HCl; filter through 0.45 m filter; store at 4C (can make a 10 stock, autoclave;
filter the 1 buffer).
viii) Buffer II
0.5% blocking agent 0.05g blocking reagent (Roche Diagnostics 1-096-176)
Buffer I 10 ml
Heat on a low setting with stirring for 30 minutes to dissolve blocking agent.
ix) Buffer III
100 mM Tris/HCl 12.11 g Tris Base
DD H
2
O 1000 ml
pH to 9.5 with HCl
Then add:
50 mM MgCl
2
10.16 g MgCl
2
.6H
2
O
x) Buffer IV (TE)
10 mM Tris/HCl 1.21 g Tris base
1 mM EDTA 0.37 g ethylene diamine tetra-acetic acid.2 H
2
O (disodium salt)
DD H
2
O 1000 ml
pH to 8.0 with HCl; filter through 0.45 m filter; store at 4C.
xi) Development solution (prepare just prior to use)
Nitroblue tetrazolium salt 45 l NBT (Roche Diagnostics 1-383-213)
(75 mg/ml in 70% dimethylformamide)
5-bromo-4-chloro-3-indoyl
phosphate, toluidine salt
(50 mg/ml in dimethyl-
formamide)
35 l X-phosphate
(Roche Diagnostics 1-383-221)
Buffer III 10 ml
1.2. In-situ hybridisation procedure
The in-situ hybridisation method using a DIG-labelled DNA probe for IHHNV follows generally
the methods outlined in Lightner (20) and given below. Formulas for the required reagents are
given in Section 1.2.1.
i) Embed tissue in paraffin and cut sections at ~4 m thickness. Put sections on to positively
charged microscope slides (do not put gelatin in water to float sections; use just water).
ii) Put slides in a slide rack, such as a Tissue-Tek rack. Heat the slides in an oven for 45 minutes
at 60C. In the staining centre, rehydrate the tissue as follows:
Citri Solve*
3
5 minutes each
Absolute alcohol
2
1 minutes each
95% alcohol
2
10 dips each
80% alcohol
2
10 dips each
50% alcohol
1
10 dips
Distilled water six rinses (do not let slides dry out)
* Citri Solve is a xylene substitute.
iii) Wash the slides for 5 minutes in phosphate buffered saline (PBS or TNE). Prepare fresh
proteinase K at 100 g/ml in PBS (or TNE). Place slides flat in a humid chamber, pipette on
500 l of the proteinase K solution and incubate for 1015 minutes at 37C. Drain fluid on
to blotting paper.
iv) Return slides to slide rack. Fix sections in 0.4% cold formaldehyde for 5 minutes at room
temperature.
v) Incubate slides in 2 SSC for 5 minutes at room temperature.
vi) With slides flat, add 0.51 ml prehybridisation buffer and incubate in a humid chamber for
1530 minutes at 37C.
vii) Boil the DIG-labelled probe for 10 minutes and quench on ice; spin briefly in the cold and
keep on ice. Dilute the probe to 25 ng/ml in prehybridisation solution and cover the tissue
with 250 l of the solution. Incubate the slides for 24 hours at 42C or overnight at 37C in
a humid chamber. Drain fluid on to blotting paper. During this incubation, put the wash
buffers at 37C to prewarm them.
viii) Place slides in slide rack. Wash the slides as follows:
2 SSC 2
530 minutes at 37C
1 SSC 2
5 minutes at 37C
0.5 SSC 2
5 minutes at 37C
ix) Wash the slides for 5 minutes in Buffer I at room temperature. Put the slides flat in a humid
chamber and block with 0.5 ml per slide of Buffer II. Incubate for 15 minutes at 37C. Drain
the fluid on to blotting paper.
x) Dilute the anti-DIG AP conjugate (Boehringer Mannheim 1-093-274) 1/1000 in Buffer II
(1 l anti-DIG AP per 1 ml buffer). Cover tissue with 500 l of diluted conjugate and
incubate in a humid chamber for 30 minutes at 37C.
xi) Place the slides in a slide rack. Wash in Buffer I twice for 510 minutes each time at room
temperature. Wash one time with Buffer III for 510 minutes.
xii) Prepare the development solution by first adding 4.5 l NBT per 1 ml buffer III. Mix well.
Then add 3.5 l X-phosphate per ml of solution and mix well. Pipette on 500 l per slide and
incubate in a humid chamber in the dark for 23 hours at room temperature.
xiii) Stop the reaction by returning the slides to a slide rack and washing in Buffer IV for
15 minutes at room temperature.
xiv) Counterstain the slides by dipping for 5 minutes in 0.5% aqueous Bismarck brown Y.
xv) Dehydrate the slides in the staining centre as follows:
95% alcohol
3
10 dips each
Absolute alcohol
3
10 dips each
Citri Solve (or xylene)
4
10 dips each
Do not allow the slides to dry out leave them in the last Citri Solve (or xylene)
container until ready for cover-slips.
xvi) Mount with cover-slips and mounting medium (Permount).
xvii) Examine the slides under bright-field for dark-blue or black precipitate that marks sites
where IHHNV DNA is present. Pathodiagnostic intranuclear Cowdry type A inclusions are
well marked with probe. Also often marked are host cell nuclei without obvious inclusions,
cytoplasmic inclusions, and accumulation of free virus in the tissue spaces and haemolymph.
NOTE: Always run a known positive and negative control.
1.2.1. Reagent formulas for in-situ hybridisation method
i) 10 phosphate buffered saline
NaCl 160 g
KH
2
PO
4
4 g
Na
2
HPO
4
23 g
KCl 4 g
DD H
2
O 1950 ml (qs to 2 litres)
pH to 8.2 with NaOH; autoclave to sterilise; store at room temperature. To make 1 PBS,
dilute 100 ml 10 PBS in 900 ml DD H
2
O; Filter 1 solution through 0.45 m filter; store
at 4C.
ii) 10 Tris/NaCl/EDTA (TNE) buffer
Tris base 60.57 g
NaCl 5.84 g
EDTA 3.72 g
DD H
2
O 900 ml (qs to 1 litre)
pH to 7.4 with concentrated or 5 M HCl. To make 1 TNE, dilute 100 ml 10 TNE in
900 ml DD H
2
O; Filter 1 solution through 0.45 m filter; store at 4C.
iii) Proteinase K, 100 g/ml (prepare just prior to use)
PBS
10 ml 1 PBS
Proteinase K 1 mg
iv) 0.4% formaldehyde
37% formaldehyde 5.4 ml
DD H
2
O 500 ml
Store at 4C; can be reused up to four times before discarding.
v) Prehybridisation buffer (50 ml final volume)
4 SSC 10 ml 20 SSC
50% formamide 25 ml 100% formamide
1 Denhardts 2.5 ml 20 Denhardts
5% dextran sulfate 10 ml 25% dextran sulfate
Warm to 60C
Boil 2.5 ml of 10 mg/ml salmon sperm DNA and add to buffer for final concentration of 0.5
mg/ml salmon sperm DNA; store at 4C.
vi) 20 SSC buffer
3M NaCl 175.32 g NaCl
0.3 M Na
3
C
6
H
5
O
7
.2H
2
O 88.23 g Na citrate.2H
2
O
DD H
2
O 1000 ml (qs)
pH to 7.0; autoclave; store at 4C.
To make 2 SSC, dilute 100 ml 20 SSC in 900 ml DD H
2
O; To make 1 SSC, dilute
50 ml 20 SSC in 950 ml DD H
2
O; To make 0.5 SSC, dilute 50 ml 20 SSC in 1950 ml
DD H
2
O. Filter solutions through 0.45 m filter; store at 4C.
vii) 20 Denhardts solution
BSA (Fraction V) 0.4 g bovine serum albumin
Ficoll 400 0.4 g Ficoll
PVP 360 0.4 g polyvinylpyrollidine
DD H
2
O 100 ml
Filter solutions through 0.45 m filter; store at 4C. Aliquot 2.5 ml into small tubes and
store frozen.
viii) 25% dextran sulfate
Dextran sulfate 25 g
DD H
2
O 100 ml
Mix to dissolve; store frozen in 10 ml aliquots.
ix) Salmon sperm DNA (10 mg/ml)
Salmon sperm DNA 0.25 g
DD H
2
O 25 ml
To prepare, warm the water and slowly add the DNA with stirring until completely dissolved;
boil for 10 minutes; shear the DNA by pushing through an 18-gauge needle several times;
aliquot 2.5 ml into small tubes and store frozen; boil for 10 minutes just before using to
facilitate mixing in the buffer.
x) 10 Buffer I
1 M Tris/HCl 121.1 g Tris base
1.5 M NaCl 87.7 g NaCl
DD H
2
O 1000 ml (qs)
pH to 7.5 with HCl. Autoclave; store at 4C.
To make 1 Buffer I, dilute 100 ml of 10 stock in 900 ml DD H
2
O. Filter through
0.45 m filter; store at 4C.
xi) Buffer II (blocking buffer)
Blocking reagent 0.25 g Blocking reagent (Roche Diagnostics 1-096-176)
Buffer I
50 ml 1 Buffer I
Store at 4C for up to 2 weeks.
xii) Buffer III
DD H
2
O 100 ml (qs)
pH to 9.5 with HCl
Then add:
50 mM MgCl
2
1.02 g MgCl
2
.6H
2
O

xiii) 10% polyvinyl alcohol (PVA)
Polyvinyl alcohol 10 g
DD H
2
O 100 ml
To prepare, slowly add PVA to water while stirring on low heat. (It takes 23 hours for PVA
to go into solution.) Dispense 10 ml per tube and store frozen at 20C.
xiv) Development solution
Mix 90 ml Buffer III with 10 ml of 10% PVA. Store at 4C. Just prior to use, for each 1 ml
of Buffer III with PVA add:
4.5 l NBT 75 mg NBT/ml in 70% dimethylformamide
3.5 l X-phosphate 5-bromo-4-chloro-3-indoyl phosphate, toluidine salt
(50 mg/ml in dimethylformamide)
xv) Buffer IV
1 mM EDTA 0.37 g EDTA.2H
2
O (disodium salt)
DD H
2
O 1000 ml
pH to 8.0 with HCl. Filter through 0.45 m filter; store at 4C.
xvi) 0.5% Bismarck Brown Y
Bismarck Brown Y 2.5 g
DD H
2
O 500 ml
Dissolve the stain in water. Filter through a Whatman No. 1 filter; store at room temperature.
The PCR method for IHHNV follows generally the methods outlined in Lightner (20) and is
given below. Cumulative experience with the technique has led to modifications with respect to
template (DNA extraction of clinical specimens), choice of primers, and volume of reaction. The
primers described below were derived from the GenBank sequence AF218266 (30). The protocols
for thermal cycling and the PCR reaction mix are described in Nunan et al. (31).
i) Use as template, the DNA extracted from ground tissue homogenate (TN buffer, 0.4 M
NaCl, 20 mM Tris, pH 7.4) or haemolymph (collected with a small amount of 10% sodium
citrate) or from tissue or haemolymph that were fixed in 95% ethanol and then dried. A
control consisting of tissue or haemolymph from known negative animals should be included
during the DNA extraction step. The DNA can be extracted by a variety of methods but
excellent results have been obtained using kits from Roche Diagnostics (Cat. No. 1-796-828)
or Qiagen (Cat. No. 51304), or reagents from Gibco Life Sciences (DNazol Cat. No. 10503-
027). Spectrophotometric readings of the final DNA will indicate the purity of the DNA and
the amount of total DNA extracted from the sample. Use 15 l of extracted DNA per 50 l
reaction volume.
Note: Homogenised tissue material or haemolymph that has not had the DNA extracted
may also be used directly as template, but the concentration of virus will be lower and there
may be substances present that will inhibit the PCR.
ii) The following controls should be included in every PCR assay for IHHNV: a) DNA from a
known negative tissue sample; b) DNA from a known positive sample (either from tissue or
haemolymph or from a plasmid clone that contains the fragment that the specific set of
primers amplifies; and c) a no template control.
iii) Use as primers, primers 389F and 389R, which elicit a band 389 bp in size from IHHNV-
infected material, or primers 77012F and 77353R, which elicit a band 356 bp in size from
IHHNV-infected material. Prepare primers at 100 ng/l in distilled water. Keep frozen at
70C.
Primer Sequence G:C ratio Temperature
389F 5-CGG-AAC-ACA-ACC-CGA-CTT-TA-3 50 72C
389R 5-GGC-CAA-GAC-CAA-AAT-ACG-AA-3 45 71C
77012F 5-ATC-GGT-GCA-CTA-CTC-GGA-3 50 68C
77353R 5-TCG-TAC-TGG-CTG-TTC-ATC-3 55 63C
Note: The 77012/77353 primers are from the region in the genome that codes for the
structural proteins, whereas the 389F/R primers are from the region that codes for
nonstructural proteins. If a test result is ambiguous with one set of primers, the second set
can be used for confirmation of the result from a different region of the viral genome.
iv) Use a hot start method for the polymerase: if you use Applied Biosystems AmpliTaq Gold,
this involves a 5-minute step at 95C to denature DNA prior to the primers binding and
activation of the enzyme. This programme is then linked to the cycling programme
(35 cycles) and an extension programme. The programme is set as follows:
Hot start Programme 1 5 minutes 95C
Linked to Programme 2 30 seconds 95C
30 seconds 55C 35 cycles
1 minutes 72C
Linked to Programme 3 7 minutes 72C
Linked to Programme 4 4C until off
v) Prepare a master mix consisting of water, 10 PCR buffer, the four dNTPs, the two
primers, MgCl
2
, AmpliTaq Gold and water (assume use of 1 l of template; if using more,
adjust water accordingly). Add mix to each tube. Use thin-walled tubes designed for PCR.
Always run a positive and a negative control.
Master Mix:
DD H
2
O
32.5 l number of samples
10 PCR buffer 5 l number of samples
10 mM dTTP
1 l number of samples
10 mM dATP
10 mM dCTP
10 mM dGTP
25 mM MgCl
2

Forward primer (100 ng/l)
Reverse primer (100 ng/l)
AmpliTaq Gold
Vortex this solution to mix all reagents well; keep on ice.
Note: The volume of the PCR reaction may be modified. Previously, the PCR reactions for
IHHNV were run in 100 l volumes, but it is not necessary to use that amount of reagents,
therefore 50 l volumes are described in this procedure. Likewise, the PCR reactions can also
be run in volumes as small as 25 l. To do this, increase or decrease the volume of the
reagents accordingly.
vi) Add 49 l Master Mix to each tube and then add 1 l of the sample to be tested.
vii) Vortex each tube, spin quickly to bring down all liquid. If your thermal cycler does not have
a heated lid to prevent condensation, then carefully overlay the top of each sample with
2550 l mineral oil and re-cap the tubes. Insert tubes into thermal cycler and start
programme 1 (hot start), which is linked to cycling, extension and soak cycles.
viii) If mineral oil was used, recover samples from under the mineral oil using a pipette set at
50 l and transfer to a fresh tube. Using the long-tipped pipette tips (designed for loading
gels) results in less oil being carried over with the sample.
ix) Run 10 l of the sample in a 1.5% agarose gel (containing 0.5 g/ml ethidium bromide to
stain the DNA). Look for the 389 bp band (if using primers 389F and 389R) or for the
356 bp band (if using primers 77012F and 77353R). Bands are not always seen, as it is
necessary to have at least 10 ng DNA/l to see DNA in a gel. A Southern transfer of the gel
or a dot-blot can be run for more sensitive detection. The DNA can also be precipitated
(0.3 M sodium acetate and 2.5 volumes 100% ethanol, 70C, for 13 hours, centrifuge for
20 minutes) and resuspended in 1/10th volume (i.e. 4 l) TE (10mM Tris, 1 mM EDTA,
pH7.5) or water and either re-run in the gel or tested in a dot-blot.
2.1. Gross signs
2.1.1. IHHN disease in Penaeus stylirostris
IHHNV often causes an acute disease with very high mortalities in juveniles of the species.
Vertically infected larvae and early postlarvae do not become diseased, but in approximately
35-day old or older juveniles, gross signs of the disease may be observed, followed by mass
mortalities (1, 2, 8, 9, 1720, 22, 25, 26). In horizontally infected juveniles, the incubation
period and severity of the disease is somewhat size and/or age dependent, with young
juveniles always being the most severely affected. Infected adults seldom show signs of the
disease or mortalities (1, 2).
Gross signs are not IHHN specific, but juvenile P. stylirostris with acute IHHN show a marked
reduction in food consumption, followed by changes in behaviour and appearance. Shrimp of
this species with acute IHHN have been observed to rise slowly in culture tanks to the water
surface, there to become motionless and then to roll-over and slowly sink (ventral side up) to
the tank bottom. Shrimp exhibiting this behaviour may repeat the process for several hours
until they become too weak to continue, or until they are attacked and cannibalised by their
healthier siblings. Penaeus stylirostris at this stage of infection often have white or buff-coloured
spots (which differ in appearance and location from the white spots that sometimes occur in
shrimp with white spot syndrome virus infections) in the cuticular epidermis, especially at the
junction of the tergal plates of the abdomen, giving such shrimp a mottled appearance. This
mottling later fades in P. stylirostris. In P. stylirostris and in P. monodon with IHHN, moribund
shrimp are often distinctly bluish in colour, with opaque abdominal musculature (1720, 22).
2.1.2. IHHN disease in Penaeus vannamei
The chronic disease, runt deformity syndrome (RDS), occurs in this species as a result of
IHHNV infection. The severity and prevalence of RDS in infected populations of juvenile or
older P. vannamei may be related to infection during the larval or early postlarval stages (7, 8
13, 1620, 22). RDS has also been reported in cultured stocks of P. stylirostris. Juvenile shrimp
with RDS display bent or deformed rostrums, wrinkled antennal flagella, cuticular roughness,
and other cuticular deformities. Populations of juvenile shrimp with RDS display a relatively
wide distribution of sizes with many smaller than expected (runted) shrimp. The coefficient
of variation (CV = the standard deviation divided by the mean of different size groups within a
population) for populations with RDS is typically greater than 30% and may approach 90%,
while IHHNV-free (and thus RDS-free) populations of juvenile P. vannamei and P. stylirostris
usually show CVs of 1030% (7, 10, 11, 13, 16, 20, 22).
Histological demonstration of prominent intranuclear, Cowdry type A inclusion bodies provides a
provisional diagnosis of IHHNV infection. These characteristic IHHN inclusion bodies are
eosinophilic and often haloed (with haematoxylin and eosin stains of tissues preserved with
fixatives that contain acetic acid, such as Davidsons AFA and Bouins solution), intranuclear
inclusion bodies within chromatin-marginated, hypertrophied nuclei of cells in tissues of
ectodermal (epidermis, hypodermal epithelium of fore- and hindgut, nerve cord and nerve ganglia)
and mesodermal origin (haematopoietic organs, antennal gland, gonads, lymphoid organ, and
connective tissue). Intranuclear inclusion bodies due to IHHNV may be easily confused with
developing intranuclear inclusion bodies due to WSSV infection. In-situ hybridisation assay of
such sections with a specific DNA probe to IHHNV provides a definitive diagnosis of IHHNV
infection (see Section 1.2.) (3, 15, 20).
2.3. Enhancement of infection
The prevalence and severity of IHHNV infections may be enhanced in a quarantined population
by rearing shrimps in relatively crowded or stressful conditions. The crowding stress factors may
include high stocking densities and marginal water quality (i.e. low dissolved oxygen, elevated
water temperature, or elevated ammonia or nitrite) in the holding tank water. These conditions
may encourage expression of low-grade IHHNV infections and the transmission of the agent
from carriers to previously uninfected hosts in the population resulting in increased prevalence
and severity of infections that can be more easily detected using the available diagnostic and
detection methods for IHHNV (20).
See Sections 1.1.1.3.
Real-time PCR methods have been developed for the detection of IHHNV. These methods offer
extraordinary sensitivity that can detect a single copy of the target sequence from the IHHNV
genome (14, 35). While providing extraordinary sensitivity and the promise of being the most
appropriate PCR technology for pathogen detection, widespread adoption of these methods by
diagnostic facilities that service the international shrimp culture and related industries is not likely
to occur in the near future. The methods, which are evolving rapidly, require highly trained
technicians, costly reagents, and very expensive equipment. Some very costly real-time thermal
cyclers placed on the market only a few years ago are deemed to be obsolete by their
manufactures and, therefore, the sale and service of these instruments have been discontinued.
These instruments have been replaced by the next generation of instruments that employ the
newest technology.
REFERENCES
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stylirostris and Penaeus vannamei. Aquaculture, 38, 185194.
2. BELL T.A. & LIGHTNER D.V. (1987). IHHN disease of Penaeus stylirostris: effects of shrimp size on
disease expression. J. Fish Dis., 10, 165170.
No. 1, World Aquaculture Society, Baton Rouge, Louisiana, USA. 114 pp.
4. BELL T.A., LIGHTNER D.V. & BROCK J.A. (1990). A biopsy procedure for the non-destructive
determination of IHHN virus infection in Penaeus vannamei. J. Aquat. Anim. Health, 2, 151153.
5. BONAMI J.R., BREHELIN M., MARI J., TRUMPER B. & LIGHTNER D.V. (1990). Purification and
characterization of IHHN virus of penaeid shrimps. J. Gen. Virol., 71, 26572664.
6. BONAMI J.R. & LIGHTNER D.V. (1991). Chapter 24. Unclassified Viruses of Crustacea. In: Atlas of
Invertebrate Viruses, Adams J.R. & Bonami J.R., eds. CRC Press, Boca Raton, Florida, USA, 597
622.
7. BRAY W.A., LAWRENCE A.L. & LEUNG-TRUJILLO J.R. (1994). The effect of salinity on growth and
survival of Penaeus vannamei, with observations on the interaction of IHHN virus and salinity.
Germany, 245349.
9. BROCK J.A., LIGHTNER D.V. & BELL T.A. (1983). A review of four virus (BP, MBV, BMN, and
IHHNV) diseases of penaeid shrimp with particular reference to clinical significance, diagnosis and
control in shrimp aquaculture. Proceedings of the 71st International. Council for the Exploration of
the Sea, C.M. 1983/Gen:10/118.
vannamei. Oceanic Institute, Makapuu Point, P.O. Box 25280, Honolulu, Hawaii, USA, 241 pp.
11. BROWDY C.L., HOLLOWAY J.D., KING C.O., STOKES A.D., HOPKINS J.S. & SANDIFER P.A. (1993).
IHHN virus and intensive culture of Penaeus vannamei: effects of stocking density and water exchange
rates. J. Crustacean Biol., 13, 8794.
12. CARR W.H., SWEENEY J.N., NUNAN L., LIGHTNER D.V., HIRSCH H.H. & REDDINGTON J.J. (1996).
The use of an infectious hypodermal and hematopoietic necrosis virus gene probe serodiagnostic
field kit for the screening of candidate specific pathogen-free Penaeus vannamei broodstock.
13. CASTILLE F.L., SAMOCHA T.M., LAWRENCE A.L., HE H., FRELIER P. & JAENIKE F. (1993).
Variability in growth and survival of early postlarval shrimp (Penaeus vannamei Boone 1931).
14. DHAR A.K., ROUX M.M. & KLIMPEL K.R. (2001). Detection and quantification of infectious
hypodermal and hematopoeitic necrosis virus and white spot virus in shrimp using real-time
quantitative PCR and SYBR green chemistry. J. Clin. Microbiol., 39, 28352845.
15. KALAGAYAN G., GODIN D., KANNA R., HAGINO G., SWEENEY J., WYBAN J. & BROCK J. (1991).
IHHN virus as an etiological factor in runt-deformity syndrome of juvenile Penaeus vannamei cultured
in Hawaii. J. World Aquaculture Soc., 22, 235243.
16. LIGHTNER D.V. (1983). Diseases of Cultured Penaeid Shrimp. pp. In: CRC Handbook of
Mariculture. Vol. 1. Crustacean Aquaculture, McVey J.P., ed. CRC Press, Boca Raton, Florida, USA,
289320.
18. LIGHTNER D.V. (1993). Diseases of penaeid shrimp. In: CRC Handbook of Mariculture: Crustacean
Aquaculture, McVey J.P., ed. CRC Press, Boca Raton, Florida, USA.
pp.
20. LIGHTNER D.V. (1996). The penaeid shrimp viruses IHHNV and TSV: epizootiology, production
impacts and role of international trade in their distribution in the Americas. Rev. sci. tech. Off. int.
Epiz., 15, 579601.
21. LIGHTNER D.V., BELL T.A., REDMAN R.M. & PEREZ L.A. (1992). A collection of case histories
documenting the introduction and spread of the virus disease IHHN in penaeid shrimp culture
facilities in Northwestern Mexico. ICES Marine Science Symposia, 194, 97105.
22. LIGHTNER D.V., MOHNEY L.L., WILLIAMS R.R. & REDMAN R.M. (1987). Glycerol tolerance of
IHHN virus of penaeid shrimp. J. World Aquaculture. Soc., 18, 196197.
23. LIGHTNER D.V., POULOS B.T., BRUCE L., REDMAN R.M., MARI J. & BONAMI J.R. (1992). New
developments in penaeid virology: application of biotechnology in research and disease diagnosis for
shrimp viruses of concern in the Americas. In: Diseases of Cultured Penaeid Shrimp in Asia and the
United States, Fulks W. & Main K., eds. The Oceanic Institute, Makapuu Point, Honolulu, Hawaii,
USA, 233253.
24. LIGHTNER D.V., REDMAN R.M. & BELL T.A. (1983). Infectious hypodermal and hematopoietic
necrosis a newly recognized virus disease of penaeid shrimp. J. Invertebr. Pathol., 42, 6270.
25. LIGHTNER D.V., REDMAN R.M., BELL T.A. & BROCK J.A. (1983). Detection of IHHN virus in
Penaeus stylirostris and P. vannamei imported into Hawaii. J. World Mariculture Soc., 14, 212225.
26. MARI J., BONAMI J.R. & LIGHTNER D.V. (1993). Partial cloning of the genome of infectious
hypodermal and hematopoietic necrosis virus, an unusual parvovirus pathogenic for penaeid shrimps;
diagnosis of the disease using a specific probe. J. Gen. Virol., 74, 26372643.
27. MARTINEZ-CORDOVA L.R. (1992). Cultured blue shrimp (Penaeus stylirostris) infected with infectious
hypodermal and hematopoietic necrosis virus in northwestern Mexico. The Progressive Fish Culturist,
54, 265266.
28. MORALES-COVARRUBIAS M.S. & CHAVEZ-SANCHEZ M.C. (1999). Histopathological studies on wild
broodstock of white shrimp Penaeus vannamei in the Platanitos area, adjacent to San Blas, Nayarit,
Mexico. J. World Aquaculture Soc., 30, 192200.
29. MORALES-COVARRUBIAS M.S., NUNAN L.M., LIGHTNER D.V., MOTA-URBINA J.C., GARZA-
AGUIRRE M.C. & CHAVEZ-SANCHEZ M.C. (1999). Prevalence of IHHNV in wild broodstock of
Penaeus stylirostris from the upper Gulf of California, Mexico. J. Aquat. Anim. Health, 11, 296301.
30. NUNAN L.M., ARCE S.M., STAHA R.J. & LIGHTNER D.V. (2001). Prevalence of infectious
hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) in
Litopenaeus vannamei in the Pacific Ocean off the coast of Panama. J. World Aquaculture Soc., 32, 330
334.
31. NUNAN L.M., POULOS B.T. & LIGHTNER D.V. (2000). Use of polymerase chain reaction (PCR) for
the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid
shrimp. Mar. Biotechnol., 2, 319328.
32. OWENS L., ANDERSON I.G., KENWAY M., TROTT L. & BENZIE J.A.H. (1992). Infectious hypodermal
and hematopoietic necrosis virus (IHHNV) in a hybrid penaeid prawn from tropical Australia. Dis.
Aquat. Org., 14, 219228.
33. PANTOJA C.R., LIGHTNER D.V. & HOLTSCHMIT K.H. (1999). Prevalence and geographic distribution
of IHHN parvovirus in wild penaeid shrimp (Crustacea: Decapoda) from the Gulf of California,
Mexico. J. Aquat. Anim. Health, 11, 2334.
34. TANG K.F.J., DURAND S.V., WHITE B.L., REDMAN R.M., PANTOJA C.R. & LIGHTNER D.V. (2000).
Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal
and hematopoietic necrosis virus infection. Aquaculture, 190, 203210.
35. TANG K.F.J. & LIGHTNER D.V. (2001). Detection and quantification of infectious hypodermal and
hematopoietic necrosis virus in penaeid shrimp by real-time PCR. Dis. Aquat. Org., 44, 7985.
36. TANG K.F.J. & LIGHTNER D.V. (In press). Low sequence variation among isolates of infectious
hypodermal and hematopoietic necrosis virus (IHHNV) originating from Hawaii and the Americas.
Dis. Aquat. Org.,
37. TANG K.F.J. & LIGHTNER D.V. (2002). High genetic variation among isolates of infectious
hypodermal and hematopoietic necrosis virus (IHHNV) collected from southeast Asia, Madagascar
and east Africa. Book of Abstracts, Aquaculture America 2002. World Aquaculture Society, Baton
Rouge, LA, USA. p. 328.
*
* *
C HA P T E R 4 . 1 . 7 .

CRAYF I S H P L AGUE
( Ap h a n o my c e s a s t a c i )

SUMMARY
Crayfish plague is a highly infectious disease of all crayfish (Decapoda: Astacidae, Cambaridae) of non-
North American origin. The aetiological agent is an Oomycete fungus, Aphanomyces astaci, which is
now widespread in Europe as well as in North America. The European crayfish species, the Noble crayfish
Astacus astacus of north-west Europe, the stone crayfish Austropotamobius pallipes of south-west and
west Europe, the related Austropotamobius torrentium (mountain streams of south-west Europe) and
the slender clawed or Turkish crayfish Astacus leptodactylus of eastern Europe and Asia Minor are all
highly susceptible. The only other crustacean known to be capable of infection by A. astaci is the Chinese
mitten crab (Eriocheir sinensis) and this only under laboratory conditions.
The disease first occurred in Europe in the third quarter of the 19th century in the Franco-German border
region. From there region a steady spread of infection occurred, principally in two directions down the
Danube into the Balkans and towards the Black Sea, and across the North German plain into Russia and
from there south to the Black Sea and north-west to Finland and finally, in 1907, to Sweden. In the
1960s the first outbreaks in Spain were reported and in the 1980s further spread of infection to the British
Isles, Turkey, Greece and Norway was reported (1).
The reservoir for the original infections in the 19th century was never established, but the post-1960s
extensions are largely linked to movements of North American crayfish introduced more recently for
purposes of crayfish farming. These species (Pacifastacus leniusculus [the Signal crayfish] and
Procambarus clarki [the Louisiana swamp crayfish]) can act as largely or completely asymptomatic
carriers, but can be killed by A. astaci under adverse conditions. Transmission has also resulted from
contaminated crayfish traps and other contaminated equipment.
Clinically, infected crayfish may present a wide range of gross signs of infection or none at all. Focal
whitening of local areas of musculature beneath transparent areas of thin cuticle, especially of the ventral
abdomen and in the periopod (limb) joints, often accompanied by even more localised brown melanisation, is
the most consistent sign. In the terminal stages of infection, animals show a limited range of behavioural
signs, principally a loss of the normal aversion to bright light (they are seen in open water in daylight) later
accompanied by a loss of limb co-ordination, which produces an effect that has been described as walking on
stilts. Eventually, moribund animals lose their balance and fall onto their backs before dying.
Diagnosis requires isolation and identification of the pathogen by microscopic morphology; no molecular,
biochemical or serological methods that have been adequately validated exist.
Control of the spread of infection once a watershed is infected is in practical terms impossible. Prevention of
all introductions of crayfish to natural waters and into enclosed waters from which they may escape to
natural waters can be effective, although movement of fish can result in the movement of infected water
between watersheds and can transmit infection, as can contaminated equipment such as boots and fishing
gear. Sodium hypochlorite and iodophores are effective for disinfection of contaminated equipment. Thorough
drying of equipment (>24 hours) is also effective since the Oomycetes are not resistant to desiccation.
Chapter 4.1.7. - Crayfish plague (Aphanomyces astaci)
Diagnosis of crayfish plague strictly requires the isolation and characterisation of the pathogen, A. astaci,
using simple mycological media fortified with antibiotics to control bacterial contamination (3). Isolation is
only likely to be successful before or within 12 hours of the death of infected crayfish. However, there is
no other disease or pollution effect that can cause such total mortality of crayfish while leaving all other
animals in the same water unharmed, so that isolation of the pathogen is desirable but not essential,
particularly in regions where further spread of infection is known to be a potential hazard. Clinical signs of
crayfish plague include behavioural changes and a range of visible external lesions. The range of these
lesions is so large that, except for the experienced eye, such clinical signs are of limited diagnostic value.
1. STANDARD SCREENING METHODS FOR CRAYFISH PLAGUE
1.1. Isolation of Aphanomyces astaci
Isolation methods are as described by Alderman & Polglase (3). An agar medium (isolation
medium) is used that contains yeast extract and glucose in river water with antimicrobial agents
(penicillin G and oxolinic acid) to prevent the growth of most bacteria and enable easy and rapid
isolation of the pathogen.
Isolation medium (IM): 12.0 g agar; 1.0 g yeast extract; 5.0 g glucose; 10 mg oxolinic acid; 1000 ml
river water; and 1.0 g penicillin G (sterile) added after autoclaving and cooling to 40C. River
water = any natural river or lake water as opposed to demineralised water.
Simple aseptic excision of infected tissues, which are then placed as small pieces (12mm
2
) on the
surface of isolation medium plates, will normally result in successful isolation of A. astaci from
moribund or recently dead (<24 hours) animals. Depending on a range of factors, foci of
infection in crayfish may be easily seen by the naked eye or may not be discernable despite careful
examination. Such foci can best be seen under a low power stereo microscope and are most
commonly recognisable by localised whitening of the muscle beneath the cuticle. In some cases a
brown colouration of cuticle and muscle may occur and in others, hyphae are visible in infected
cuticle in the form of fine brown (melanised) tracks in the cuticle itself. Sites for particular
examination include the intersternal soft ventral cuticle of the abdomen and tail, the cuticle of the
perianal region, the cuticle between the carapace and tail, the joints of the periopods (walking
legs), particularly the proximal joint and finally the gills.
Provided that care is taken in excising infected tissues for isolation, contaminants need not present
significant problems. Small pieces of cuticle and muscle may be transferred to a Petri dish of
sterile distilled water and there further cut into small pieces with sterile instruments for transfer to
IM isolation medium. Suitable instruments for such work are cataract knives and fine electron
microscope or instrument grade forceps and scissors.
1.2. Identification of A. astaci
On IM agar, growth of new isolates of A. astaci is almost entirely within the agar except at
temperatures below 7C, when some superficial growth occurs. Colonies are colourless.
Dimensions and appearance of hyphae are much the same in crayfish tissue and in agar culture.
Vegetative hyphae are aseptate and (5)79(10) m in width (i.e. normal range 79 m, but
observations have ranged between 5 and 10 m). Young, actively growing hyphae are densely
packed with coarsely granular cytoplasm with numerous highly refractile globules. Older hyphae
are largely vacuolate with the cytoplasm largely restricted to the periphery leaving only thin strands
of protoplasm bridging the large central vacuole. The oldest hyphae are apparently devoid of
contents. Hyphae branch profusely, with vegetative branches often tending to be somewhat
narrower than the main hyphae for the first 2030 m of growth.
When actively growing thalli or portions of thalli from broth or agar culture are transferred to
river water (natural water with available cations encourages sporulation better than does distilled
water), sporangia form readily in 2030 hours at 16C and 1215 hours at 20C. Thalli transferred
from broth culture may be washed with sterile river water in a sterile stainless steel sieve, before
transfer into fresh sterile river water for induction of sporulation. Thalli in agar should be
transferred by cutting out a thin surface sliver of agar containing the fungus so that a minimum
amount of nutrient containing agar is transferred. Always use a large volume of sterile river water
relative to the amount of fungus being transferred (100:1). Sporangia are myceloid, terminal or
intercalary, developing from undifferentiated vegetative hyphae. Sporangial form is variable:
terminal sporangia are simple, developing from new extramatrical hyphae, while intercalary
sporangia can be quite complex in form. Intercalary sporangia develop by the growth of a new
lateral extramatrical branch, which forms the discharge tube of the sporangium. The cytoplasm of
such developing discharge tubes is noticeably dense, and these branches are slightly wider (10
12 m) than ordinary vegetative hyphae. Sporangia are delimited by a single basal septum in the
case of terminal sporangia and by septa at either end of the sporangial segment in intercalary
sporangia. Such septa are markedly thicker than the hyphal wall and have a high refractive index.
Successive sections of vegetative hypha may develop into sporangia, and most of the vegetative
thallus is capable of developing into sporangia.
Within developing sporangia the cytoplasm cleaves into a series of elongate units (1025 8 m)
that are initially linked by strands of protoplasm. Although the ends of these cytoplasmic units
become rounded, they remain elongate until and during discharge. Spore discharge is achlyoid,
that is, the first spore stage is an aplanospore which encysts at the sporangial orifice and probably
represents the suppressed saprolegniaceous primary zoospore. No evidence has been observed for
the existence of a flagellated primary spore, thus, in this description, the terms sporangium not
zoosporangium and primary spore not primary zoospore have been used. Discharge is fairly
rapid (<5 minutes) and the individual primary spores (=cytoplasmic units) pass through the tip of
the sporangium and accumulate around the sporangial orifice. The speed of cytoplasmic cleavage
and discharge is temperature dependent. At release, each primary spore retains its elongate
irregularly amoeboid shape briefly before encystment occurs.
Encystment is marked by a gradual rounding up followed by the development of a cyst wall,
which is evidenced by a change in the refractive index of the cell. The duration from release to
encystment is 25 minutes. Some spores may drift away from the spore mass at the sporangial tip
and encyst separately. Formation of the primary cyst wall is rapid, and once encystment has taken
place the spores remain together as a coherent group and adhere well to the sporangial tip so that
marked physical disturbance is required to break up the spore mass.
Encysted primary spores are spherical, (8)911(15) m in diameter, and are relatively few in
number, (8)1530(40) m per sporangium in comparison with other Aphanomyces spp. Spores
remain encysted for 812 hours. Optimum temperatures for sporangial formation and discharge
for the majority of European isolates of A. astaci are between 16 and 24C. For some isolates,
particularly from Spanish waters slightly higher temperature optima may prevail. The discharge of
secondary zoospores from the primary cysts peaks at 20C and does not occur at 24C. In new
isolates of A. astaci, it is normal for the majority of primary spore cysts to discharge as secondary
zoospores, although this varies with staling in long-term laboratory culture. Sporangial formation
and discharge occurs down to 4C. Aphanomyces astaci does not survive at 5C and below for
more than 24 hours either in culture or in crayfish tissues, nor does it remain viable in crayfish
tissues that have been subject to normal cooking procedures.
In many cases, some of the primary spores are not discharged from the sporangium and many
sporangia do not discharge at all. Instead, the primary spores appear to encyst in situ within the
sporangium, often develop a spherical rather than elongate form and certainly undergo the same
changes in refractive index that mark the encystment of spores outside the sporangium. This
within-sporangial encystment has been observed on crayfish. Spores encysted in this situation
appear to be capable of germinating to produce further hyphal growth.
Release of secondary zoospores is papillate, the papilla developing shortly before discharge. The
spore cytoplasm emerges slowly in an amoeboid fashion through a narrow pore at the tip of a
papilla, rounds up and begins a gentle rocking motion as a flagellar extrusion begins and spore
shape changes gradually from spherical to reniform. Flagellar attachment is lateral; zoospores are
typical saprolegniaceous secondary zoospores measuring 8 12 m. Active motility takes some 5
20 minutes to develop (dependent on temperature) and, at first, zoospores are slow and
uncoordinated. At temperatures between 16 and 20C, zoospores may continue to swim for at
least 48 hours.
2. PRESUMPTIVE DIAGNOSTIC METHODS FOR CRAYFISH PLAGUE
The first sign of a crayfish plague mortality may the presence of numbers of crayfish at large during
daylight (crayfish are normally nocturnal), some of which may show evident loss of co-ordination in
their movements, and easily fall over on their backs and are unable to right themselves. Often,
however, unless waters are carefully observed, the first recognition that there is a problem will be the
presence of large numbers of dead crayfish in a river or lake (3).
In susceptible species where sufficient numbers of crayfish are present to allow infection to spread
rapidly, particularly at summer water temperatures, infection will spread quickly and stretches of over
50 km may loose all their crayfish in under 21 days from first observed mortality. Crayfish plague has
unparalleled severity of effect, infected susceptible crayfish do not survive 100% mortality is the
norm. Resistant North American species survive infection in many cases and then act as largely
asymptomatic carriers, although under adverse conditions (stress, concurrent infections), mortality may
occur.
It must be emphasised, however, that presence of large numbers of dead crayfish, even in crayfish
plague affected watersheds is not on its own sufficient. The general condition of other aquatic fauna
must be assessed. Mortality or disappearance of other aquatic crustaceans as well as crayfish, even
though fish survive, may indicate pollution (e.g. insecticides).
3. CONFIRMATORY DIAGNOSTIC METHODS FOR CRAYFISH PLAGUE
Strictly, the identification of Oomycetes to genus depends on sporangial morphology and to species on
the morphology of the sexual reproductive stages (oogonia and antheridia). Such sexual stages are
absent in A. astaci so that identification is based on general morphology of isolates from crayfish
involved in an outbreak of crayfish plague. As no other crayfish disease produces such swift and drastic
mortalities this normally presents no practical diagnostic problem.
Exposing susceptible crayfish (e.g. A. leptodactylus or A. pallipes) to zoospores produced by suspect
isolates (see above) will result in characteristic rapid mortality (3) and with subsequent re-isolation of
the fungus, give firm confirmation of crayfish plague. However, susceptible crayfish species should
only be used for confirmation of diagnosis in exceptional circumstances since some are endangered
species (Berne Convention) and populations may be protected under conservation legislation.
No biochemical or molecular biological test methods yet exist that have been adequately validated for
cross reaction with other species of Aphanomyces such as Aphanomyces invadens, aetiological agent of
epizootic ulcerative syndrome (Chapter 2.1.10). Oidtmann et al. (4) have exploited the sequence
differences between the A. astaci isolates, other Aphanomyces and non-related fungi at the 28s rRNA
gene level to develop a PCR-based to detect the crayfish plague fungus. This work needs further
validation and requires the pathogen to be isolated into culture since the method currently lacks the
sensitivity required to be usefully applied to clinical samples. The areas that need attention are 1) The
specificity and sensitivity of the primer set in the presence of crayfish tissue 2) and the confirmed
ability to differentiate A. astaci in the presence of other Oomycetes in clinical samples.
These results therefore represent a major development towards a practical molecular biological
diagnostic method for A. astaci, but have not yet reached the stage at which they can be applied to
clinical samples.
REFERENCES
1. ALDERMAN D.J. (1996). Geographical spread of bacterial and fungal diseases of crustaceans. (Paper
given at the Office International des Epizooties [OIE] International Conference on Preventing the
Spread of Aquatic Animal Diseases, 79 June 1995, OIE Headquarters, Paris, France.) Rev sci. tech.
Off. int. Epiz., 15 (2), 603632.
2. ALDERMAN D.J. & POLGLASE J.L. (1986). Aphanomyces astaci: isolation and culture. J. Fish Dis., 9,
367379.
3. ALDERMAN D.J., POLGLASE J.L. & FRAYLING M. (1987). Aphanomyces astaci pathogenicity under
laboratory and field conditions. J. Fish Dis., 10, 385393.
4. OIDTMANN B., BAUSEWEIN S., HOTZLE L., HOFFMANN R. & WITTENBRINK M. (2002).
Identification of the crayfish plague fungus Aphanomyces astaci by polymerase chain reaction and
restriction enzyme analysis. Vet. Microbiol., 85, 183194.
*
* *
C HA P T E R 4 . 1 . 8 .

S P AWNE R- I S OL AT E D MORT AL I T Y VI RUS DI S E AS E

SUMMARY
Disease due to infection by spawner-isolated mortality virus (SMV) was first recognised in captive spawners
of Penaeus monodon at a research station in northern Queensland, Australia.
SMV is one of several viruses associated with mid-crop mortality syndrome (MCMS), which caused
significant mortalities among cultured juveniles and subadults of P. monodon cultured in Australia from
1994 to 1996. In the Philippines, P. monodon infected with luminous vibriosis were also found to be
infected with SMV (1). Infection and disease due to SMV has only been reported from cultured or captive
wild adult P. monodon and cultured Cherax quadricarinatus (4).
SMV has been tentatively classified as a parvovirus (2). Transmission electron microscopy of infected
P. monodon showed virus particles that were 20 nm in diameter, hexagonally shaped, and suggestive of an
icosahedral symmetry. Accumulations of these 20 nm particles in massive arrays were noted in the cytoplasm
of infected gut cells, and the virions appeared to be issuing through pores in the nuclear membrane (2).
Partial characterisation of the virus was accomplished by treatment of infected prawn tissue extracts with
DNase and RNase. These tests further indicated that SMV is a DNA virus, probably a parvovirus (2).
There are no practical surveillance methods presently available for SMV in penaeid prawns. Confirmatory
diagnosis of SMV can be accomplished by transmission electron microscopy of gut tissues.
There are neither pathognomic clinical signs nor pathognomic histopathological lesions associated with
spawner-isolated mortality virus disease (SMVD). The diagnosis of SMV is based on electron microscopy.
Molecular methods that use nonradioactively labelled gene probes are under development for diagnosis of
infection by SMV.
The various methods for surveillance, detection, and diagnosis of infections due to SMV are listed in Table
1. The designations used in the Table indicate: = the method is presently unavailable or unsuitable; + =
the method is least suitable; ++ = the method is moderately suitable; +++ = the method is most suitable;
and R&D = the method is in development but is not yet available though commercial sources. These are
somewhat subjective as suitability involves issues of reliability, sensitivity; and utility.
Table 1. SMV surveillance, detection and diagnostic methods
Gross signs

Direct BF/LM

Dark-field LM

Table 1 continued
Chapter 4.1.8. - Spawner-isolated mortality virus disease
Histopathology

Bioassay

Transmission EM

Scanning EM

Fluorescent antibody

ELISA with PAb/MAb

DNA probes dot-blot
DNA probes in situ
R&D R&D R&D R&D R&D R&D
PCR/RT-PCR R&D R&D R&D R&D R&D R&D
PLs = postlarvae; BF = bright field; LM = light microscopy; EM = electron microscopy; ELISA = enzyme-linked
immunosorbent assay; PAb/MAb = poly/monoclonal antibodies; RT-PCR = reverse-transcription polymerase chain reaction.
1. STANDARD SCREENING METHODS FOR SMV
The histopathological changes associated with SMVD are nonspecific, i.e. SMVD cannot be
diagnosed by histopathology. Nonspecific histopathological changes can occur in the
hepatopancreas, midgut, and anterior midgut caecum. Juvenile P. monodon infected experimentally
with tissue extracts from clinically diseased prawns with SMVD displayed haemocytic infiltration,
necrosis, and sloughing of cells into the lumens of the midgut and hepatopancreas (2). This,
however, is not always seen (figure 2 of ref. 3).
1.2. Molecular method
A nonradioactively labelled DNA probe and PCR test (in kit form) have been developed (3, 4).
Commercial availability of the kits is currently being negotiated.
2. PRESUMPTIVE DIAGNOSTIC METHODS FOR SMV
2.1. Clinical signs
There are no specific clinical signs for SMVD. Juvenile prawns in grow-out ponds with clinical
viral infections may exhibit signs such as discoloration, lethargy, fouling and anorexia.
See Section 1.2.
2.3. Bioassay method
This bioassay will confirm the presence of a pathogenic virus, but does not identify the specific
virus. The presence of any pathogenic virus may be detected using a relatively simple bioassay in
which healthy (specific pathogen free [SPF] if available) juvenile P. monodon are exposed to suspect
prawns by either feeding them with suspect prawn tissues or by injecting them with cell-free tissue
extracts prepared from suspect prawns. Fraser & Owens (2) reported that SMV could be
transmitted per os by feeding infected carcasses or by injection of cell-free extracts prepared from
infected carcasses. With intramuscular injection of cell-free extracts, mortalities began at 14 days
post-inoculation and approached 100% by 30 days post-inoculation. Disease development in the
bioassay indicator prawns following per os exposure required more time, with the first mortalities
occurring at ~30 days post-inoculation and reaching 76% mortality by 50 days post-inoculation
(2).
To perform the bioassay, use the generalised protocol as follows:
i) Prepare a 1:2 or 1:3 ratio (w:v) of SMV-suspect prawn heads or whole prawns with TN
buffer (see Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis virus for the
composition of this buffer) or sterile 2% saline prepared with distilled water.
ii) Homogenise the mixture using a tissue grinder or blender. Do not permit the mixture to heat
up by excessive homogenisation or grinding. Tissues and resulting homogenate should be
kept cool during the entire protocol by maintaining on ice.
iii) Clarify the homogenate by centrifugation at 3000 g for 10 minutes. Decant and save the
supernatant. Discard the pellet.
iv) Centrifuge the supernatant fluid at 27,000 g (15,000 rpm) for 2030 minutes at 4C. Decant
and save the supernatant fluid. Discard the pellet.
v) Dilute the supernatant fluid from step iv to from 1/10 to 1/100 with sterile 2% saline. This
solution may now be used as the inoculum to inject indicator prawns (or filter sterilised as
described in step vi).
vi) Filter the diluted supernatant from step v using a sterile syringe (size depends on final
volume of diluted supernatant) and a sterile 0.45 m syringe filter. Multiple filters may have
to be used as they clog easily. The filtrate should be collected in a sterile test tube or beaker.
The solution can now be stored frozen (20C for short-term [weeks] storage and 80C for
long-term [months to years] storage) or used immediately to inject indicator prawns.
vii) Indicator prawns should be from stocks of healthy (SPF if available) P. monodon.
viii) Inject 0.01 ml per gram of body weight using a 1-ml tuberculin syringe. Indicator prawns
should be injected intramuscularly into the third tail segment. If the test prawns begin to die
within minutes post-injection, the inoculum contains excessive amounts of proteinaceous
material and should be further diluted prior to injecting additional indicator prawns. Sudden
death occurring post-injection is referred to as protein shock, and is the result of systemic
clotting of the prawns haemolymph in response to the inoculum.
ix) Cell-free extract samples may be diluted (1/10 or 1/20 in TN buffer), filter sterilised (if
warranted), and injected into the indicator prawns without further preparation.
x) If a pathogenic virus is present in the inoculum, the indicator prawns should begin to die.
xi) The presence of SMV in the indicator prawns should be confirmed by transmission electron
microscopy (and in situ hybridisation by gene probe if available).
3. CONFIRMATORY DIAGNOSTIC METHODS FOR SMVD
Confirmation of SMV infection in prawns can be achieved by the following method.
The method is essentially that of Fraser and Owens (2). Moribund prawns are sedated in cold
water and then the organs of interest are removed into Petri dishes containing 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1. The tissues are cut into 1 mm cubes, fixed for
30 minutes at room temperature (24C) and washed twice for 10 minutes in the 0.1 M cacodylate
buffer. The samples are stored in the cacodylate buffer at 4C. Before embedding, the samples are
post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at 24C, and then
washed twice for 10 minutes with the same buffer. The post-fixed samples are dehydrated and
embedded in Spurrs resin. The sections are stained with 2% uranyl acetate in 50% ethanol
acidified with hydrochloric acid for 7 minutes, rinsed four times in distilled water and blotted dry.
The sections are then stained with lead acetate for 2 minutes and the rinsing step is repeated.
Aggregations of virus-like particles are found associated with the nuclear membrane in the
cytoplasm of the gut cells. The virions are approximately 2025 nm in diameter (3) and
hexagonally shaped suggesting icosahedral symmetry. SMV virions are not found in tissues other
than the gut.
See Section 1.2.
REFERENCES
1. ALBALADEJO J.D., TAPAY L.M., MIGO V.P., ALFAFARA C.G., SOMGA J.R., MAYO S.L., MIRANDA
R.C., NATIVIDAD K., MAGBANUA F.O., ITAMI T., MATSUMURA M., NADALA E.C.B., JR. & LOH P.C.
(1998). Screening for shrimp viruses in the Philippines. In: Advances in Shrimp Biotechnology, Flegel
T.W., ed. National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand, 251254.
2. FRASER C.A. & OWENS L. (1996). Spawner-isolated mortality virus from Australian Penaeus monodon.
Dis. Aquat. Org., 27, 141148.
3. OWENS L., HAQSHENAS G., MCELNEA C., & COELEN R. (1998). Putative spawner-isolated mortality
virus associated with mid-crop mortality syndrome in farmed Penaeus monodon from northern
Australia. Dis. Aquat. Org., 34, 177185.
4. OWENS L. & MCELNEA C. (2000). Natural infection of the redclaw crayfish cherax quadricarinatus
with presumptive spawner-isolated mortality virus. Dis. Aquat. Org., 40, 219223.
*
* *
L I S T OF OI E RE F E RE NCE L ABORAT ORI E S AND
COL L ABORAT I NG CE NT RE F OR DI S E AS E S OF F I S H,
MOL L US CS AND CRUS T ACE ANS
Disease Expert/Laboratory
Epizootic haematopoietic necrosis Dr A. Hyatt
Australian Animal Health Laboratory , CSIRO, Private Bag 24,
Geelong, Victoria 3220, 6135 AUSTRALIA
Tel.: (61.3) 52.27.54.19, Fax: (61.3) 52.27.55.55
E-mail: alex.hyatt@csiro.au
Dr R. Whittington
Chair, Farm Animal Health, Faculty of Veterinary Science,
University of Sydney, Private Bag 3, Camden NSW 2570,
AUSTRALIA
Tel.: (61.2) 93.51.16.19., Fax: (61.2) 93.51.16.18
E-mail: richardw@camden.usyd.edu.au
Infectious haematopoietic necrosis Dr J.-A. Leong
Director and Professor, Hawaii Institute of Marine Biology,
School of Ocean and Earth Sciences Technology, University of
Hawaii, P.O. Box 1346, Kaneohe, Hawaii 96744,
UNITED STATES OF AMERICA
Tel.: (1.808) 236.74.01, Fax: (1.808) 236.74.43
E-mail: joannleo@hawaii.edu
Dr J. Winton
Western Fisheries Research Center, 6505 N.E. 65
th
Street,
Seattle, Washington 98115,
Tel.: (1.206) 526.65.87, Fax: (1.206) 526.66.54,
E-mail: jim_winton@usgs.gov
Oncorhynchus masou virus disease Dr M. Yoshimizu
Laboratory of Microbiology, Graduate School of Fisheries
Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate,
Hokkaido 041-8611, JAPAN
Tel./Fax: (81.138) 40.88.10
E-mail: yosimizu@fish.hokudai.ac.jp
Spring viraemia of carp Prof. B.J. Hill
The Centre for Environment, Fisheries & Aquaculture Science
(CEFAS), Barrack Road, The Nothe, Weymouth, Dorset
DT4 8UB, UNITED KINGDOM
Tel.: (44.1305) 20.66.25, Fax: (44.1305) 20.66.27
E-mail: b.j.hill@cefas.co.uk
OIE Reference Laboratories and Collaborating Centre for diseases of fish, molluscs and crustaceans
Viral haemorrhagic septicaemia Dr N.J. Olesen
Danish Veterinary Laboratory, Hangvej 2,
DK-8200 arhus N, DENMARK
Tel.: (45) 89.37.24.31, Fax: (45) 89.37.24.70
E-mail: njo@vetinst.dk
Channel catfish virus disease Dr L.A. Hanson
Fish Diagnostic Laboratory, College of Veterinary Medicine,
Mississippi State University, P.O. Box 6100, Mississippi 39762,
Tel.: (1-662) 325.1202, Fax: (1-662) 325.1031
E-mail: hanson@cvm.msstate.edu
Viral encephalopathy and
retinopathy
Dr G. Bovo
Istituto Zooprofilattico Sperimentale delle Venezie, Dipartimento
di Ittiopatologia, Viale dellUniversit 10, 35020 Legnaro PD,
ITALY
Tel.: (39.049) 808.42.48, Fax: (39.049) 808.43.92
E-mail: gbovo@izsvenezie.it
Dr T. Nakai
Laboratory of Fish Pathobiology, Graduate School of Biosphere
Science, Hiroshima University, Higashihiroshima 739-8528,
JAPAN
Tel.: (81-824) 24.79.47, Fax: (81-824) 22.70.59
E-mail: nakaitt@hiroshima-u.ac.jp
Infectious pancreatic necrosis Prof. B.J. Hill
Tel.: (44.1305) 20.66.25, Fax: (44.1305) 20.66.27
E-mail: b.j.hill@cefas.co.uk
Infectious salmon anaemia Dr B. Dannevig
National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156
Dep., 0033 Oslo, NORWAY
Tel.: (47.23) 21.64.04, Fax: (47.23) 21.63.01
E-mail: birgit.dannevig@vetinst.no
Epizootic ulcerative syndrome Dr S. Kanchanakhan
Aquatic Animal Health Research Institute (AAHRI), Department
of Fisheries, Kasetsart University Campus, Paholyothin Road,
Jatuchak, Bangkok 10900, THAILAND
Tel.: (66.2) 579.41.22, Fax: (66.2) 561.39.93
E-mail: somkiatkc@fisheries.go.th
Bacterial kidney disease
(Renibacterium salmoninarum)
Dr R.J. Pascho
Western Fisheries Research Center, 6505 N.E. 65
th
Street,
Seattle, Washington 98115
Tel.: (1.206) 526.65.88, Fax: (1.206) 526.66.54,
E-mail: ron_pascho@usgs.gov
Enteric septicaemia of catfish
(Edwardsiella ictaluri)
Dr L.A. Hanson
Fish Diagnostic Laboratory, College of Veterinary Medicine,
Mississippi State University, P.O. Box 6100, Mississippi 39762,
Tel.: (1-662) 325.1202, Fax: (1-662) 325.1031
E-mail: hanson@cvm.msstate.edu
Piscirickettsiosis
(Piscirickettsia salmonis)
Dr M. Kent
Center for Fish Disease Research, Department of Microbiology,
220 Nash Hall, Oregon State University, Corvallis, Oregon 97331-
3804, UNITED STATES OF AMERICA
Tel.: (1.541) 737.44.41, Fax: (1.541) 737.04.96
E-mail: michael.kent@orst.edu
Gyrodactylosis (Gyrodactylus salaris) Dr T.A. Mo
National Veterinary Institute, Ullevlsveien 68, P.O. Box 8156
Dep., 0033 Oslo, NORWAY
Tel.: (47.23) 21.61.10, Fax: (47.23) 21.61.01
E-mail: tor-atle.mo@vetinst.no
Red sea bream iridoviral disease Dr K. Nakajima
Fisheries Research Agency, Research Promotion and Development
Division, 2-12-4 Fukuura, Kanagawa-ku, Yokohama-shi,
Kanagawa 236-8648, JAPAN
Tel.: (81.45) 788.75.12, Fax: (81.45) 788.50.90
E-mail: kazuhiro@fra.affrc.go.jp
Infection with Bonamia exitiosus,
Infection with Bonamia ostreae,
Infection with Mikrocytos roughleyi,
Infection with Marteilia refringens
and Infection with Marteilia sydneyi
Dr F. Berthe
IFREMER, Laboratoire de Gntique Aquaculture et Pathologie,
BP 133, 17390 La Tremblade, FRANCE
Tel.: 33 (0)5 46.36.98.36, Fax: 33 (0)5 46.36.37.51
E-mail: fberthe@ifremer.fr
Infection with Haplosporidium
nelsoni, Infection with
Haplosporidium costale, Infection
with Perkinsus olseni/atlanticus and
Infection with Perkinsus marinus
Dr E.M. Burreson
Director for Research and Advisory Services, Virginia Institute of
Marine Science, P.O. Box 1346, College of William and Mary,
Gloucester Point, VA 23062
Tel.: (1.804) 684.71.08, Fax: (1.804) 684.70.97
E-mail: gene@vims.edu
Infection with Mikrocytos mackini Dr S. Bower
Department of Fisheries and Oceans, Pacific Biological Station,
3190 Hammond Bay Road, Nanaimo, British Columbia V9R 6N7,
CANADA
Tel.: (1.250) 756.70.77, Fax: (1.250) 756.70.53
E-mail: bowers@dfo-mpo.gc.ca
Crustacean pathogens Prof. D. Lightner
Aquaculture Pathology Section, Department of Veterinary Science,
University of Arizona, Building 90, Room 202, Tucson AZ 85721,
Tel.: (1.520) 621.84.14, Fax: (1.520) 621.48.99
E-mail: dvl@u.arizona.edu
Crayfish plague
(Aphanomyces astaci)
Dr D.J. Alderman
Tel.: (44.1305) 20.66.41, Fax: (44.1305) 20.66.38
E-mail: d.j.alderman@cefas.co.uk
OI E C O L L A B O R A T I N G C E N T R E F O R
I N F O R M A T I O N O N A Q U A T I C A N I M A L DI S E A S E S
(CEFAS), Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB,
UNITED KINGDOM
Tel.: (44.1305) 20.66.25, Fax: (44.1305) 20.66.27
E-mail: b.j.hill@cefas.co.uk; http://www.collabcen.net

Manual of Diagnostic Tests For Aquatic Animals: Fourth Edition, 2003

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