Bacteriology Textbook 2009 PDF

Textbook for
Veterinary Microbiology (VETS3040) and

Animal Disease (VETS3038)
Bacteriology
(2009)
Jennie Hodgson, Jacqui Norris, Katrina Bosward,

Gary Muscatello,Denise Wigney
COPYRIGHT
This book is copyright of the various source textbooks and the

contributing staff member of the Faculty of Veterinary Science,
University of Sydney.
The book is distributed to enrolled students on a non-profit cost

recovery basis. Where illustrative material is taken form
standard textbooks, it is on the assumption of fair usage for
teaching and is for non-commercial purposes.
Commercial use of any material in the book is an infringement

of copyright.
TABLE OF CONTENTS
SECTION 1: Gram Positive Cocci 1
Genus Staphylococcus 3
Genus Streptococcus 18
Genus Enterococcus 38
SECTION 2: Gram Negative Rods 41
FAMILY Enterobacteriaceae 43
Species E. coli 53
Genus Salmonella 65
Genus Yersinia 71
Genus Klebsiella 72
Genus Proteus 72
Genus Pseudomonas 77
Genus Burkholderia 84
Genus Campylobacter 89
Genus Lawsonia 98
Genus Helicobacter 104
FAMILY Pasteurellaceae 111
Genus Pasteurella 113
Genus Mannheimia 126
Genus Actinobacillus 129
Genus Haemophilus 136
Genus Brucella 142
Genus Bordetella 152
Genus Moraxella 162
Genus Taylorella 168
SECTION 3: Gram Positive Rods 173
FAMILY Actinomycetes 175

Genus Actinomyces 177
Genus Arcanobacterium 185
Genus Nocardia 191
Genus Dermatophilus 198
“Diptheroids” 205
Genus Corynebacterium 207
Genus Rhodococcus 214
Genus Listeria 221
Genus Erysipelothrix 229
Genus Bacillus 238
Genus Clostridium 253
Genus Mycobacterium 277
SECTION 1
Gram Positive Cocci
1
2
STAPHYLOCOCCI
Staphylococci are gram positive cocci which occur in small clumps or clusters. They are one of the most
common pathogens of domestic species (and humans!). However, not all species of Staphylococci are
pathogenic, and it is important to be able to distinguish between pathogens and non-pathogens when
interpreting results of culture and sensitivity testing. The diseases caused by Staphylococci are
characterised by purulent inflammation, and may be acute or chronic. Chronic infections result from their
ability to survive intracellularly, which is a major virulence factor of these bacteria. Staphylococcal
infections may be difficult to treat due to their intracellular position and their widespread antimicrobial
resistance.
Summary Table: Species of Staphylococcus that have veterinary importance, their animal hosts, their
coagulase test result, and the diseases they cause
SPECIES HOSTS Coagulase Disease
S. aureus++++ humans, horses, common pathogen (pyogenic) of

ruminants, birds, humans and animals
positive
pigs, cats,
(dogs)
S. intermedius++++ dogs (cats, leading pus forming bacteria in dogs
positive
cattle)
S. epidermidis+ humans, cattle, part of normal flora of skin + some
dogs, horses mucous membranes; common
negative
(cats) contaminant of cultures; rarely
pathogenic
S. hyicus+ pigs some are exudative dermatis and arthritis in pigs
positive and rarely bovine mastitis
S. schleiferi ssp. dogs otitis externa in dogs
positive
coagulans++
NOTE: the number of + denotes the relative importance of these veterinary species within THIS genus (NOT
for all bacterial genera)
3
What are they?
Staphylococci are gram positive cocci
they are facultatively anaerobic, but grow best
aerobically
occur in pairs, short chains or clusters
all species of Staphylococcus are catalase positive
Staphylococci divide in several planes to form irregular CLUSTERS.

KEY POINT This can help you differentiate Staphylococci from Streptococci (which are
in chains) in gram stained smears.
there are ~35 different species, but only 5 are of veterinary importance and 2 are of MAJOR
IMPORTANCE (S. aureus and S. intermedius)
coagulase (and clumping factor) tests are biochemical tests used to differentiate between the pathogenic
species (which are coagulase positive e.g. S. aureus and S. intermedius)) and those species that are
rarely or non pathogenic (which are coagulase negative - e.g. S. epidermidis, S. saprophyticus)
organisms that are positive or negative for this test are generally referred to as coagulase positive and
coagulase negative staphylococci respectively
The distinction between coagulase positive and coagulase negative isolates

KEY POINT is IMPORTANT for determination of CLINICAL SIGNIFICANCE.
Where are they found?
Staphylococci are found world-wide

they are probably present in the upper respiratory tract and other epithelial surfaces of all warm-
blooded animals and there is good evidence that species of Staphylococcus have evolved together with
their host (host-adapted species)
studies in humans suggest widespread staphylococcal colonisation begins within hours of birth; the
same probably occurs in animals
KEY POINT Staphylococci are part of the NORMAL FLORA (commensals).
coagulase-positive species (e.g. S. aureus and S intermedius) inhabit the distal nasal passages, external
nares, and skin, especially near mucocutaneous borders such as the perineum, external genitalia, and
bovine udder (moist areas)
they also occur as transients in the GIT
humans are the natural hosts for S. aureus, though variants adapted to horses, ruminants and pigs
occur; this species is rarely found on dogs or cats
the predominant staphylococcal species of dogs is S. intermedius and of cats are S. felis and S. xylosus
(these 2 species are coagulase negative)
S. hyicus is found on the skin of pigs, on the skin and nares of healthy poultry, and more rarely on the
skin and in the milk of cattle
4
coagulase-negative staphylococci (especially S. epidermidis) are predominant among the resident skin
flora, but they also colonise the upper respiratory tract
staphylococci may also be found in animal products (meat, cheese, milk)
staphylococci can be found in the environment (soil, dust, air, water) as a result of contamination from
the skin and are comparatively stable
How do they enter the host?
KEY POINT
Many staphylococal infections are ENDOGENOUS
most infections are caused by the resident strain of bacteria on the animal host (ENDOGENOUS)
alternatively, staphylococcal infections may be EXOGENOUS, where the organism is transmitted from
one animal to another either by direct contact or indirectly via fomites, soil, air and water
for example, organisms causing mastitis may be transferred from one cow to another on the hands of
the milker, or in the teat dip at the time of milking and therefore management practices and milking
hygiene significantly influence the prevalence of staphylococcal mastitis
staphylococci survive well in the environment as they are relatively resistant to drying and disinfection
prolonged environmental survival of staphylococci assists their transmission between animals
interspecies spread (e.g. humans-to-cows, dogs-to-humans, humans-to-horses) appears to be limited,
but has been recorded – for the most part host adapted species are transiently present on another host
species
How do they cause disease?
I) Virulence Factors

coagulase positive staphylococci in general, and S. aureus specifically, possess a wide variety of
factors that are believed to contribute to their ability to cause disease
however, the specific contribution to the disease process of many of the described virulence factors is
not well understood
in addition, all virulence factors are not found in all strains of staphylococci
Staphylococci have many virulence factors and therefore are GOOD

KEY POINT PATHOGENS! However, their specific role in disease is often poorly
understood.
i) Intracellular Survival
this mechanism allows some strains of coagulase positive staphylococci to survive within cells (e.g.
macrophages, endothelial cells, or epithelial cells) and contributes to the chronicity of disease seen in
some staphylococcal infections
this ability is thought to be linked to the emergence of small colony variants which are non-haemolytic
(decreased production of alpha toxin) and therefore do not lyse the host cell; other mechanisms for
intracellular survival may also exist
this intracellular position shields the staphylococci from host defences (particularly antibody and
complement) and decreases exposure to antibiotics
5
thus the staphylococci can hide within the host cell, then once the host immune response has abated
and antibiotic therapy is completed, the staphylococci can revert to the virulent form, lyse the host cell
and resume causing inflammation and disease
Intracellular survival by some strains of Staphylococci allows development of

KEY POINT chronic, recurrent infections due to diffuculty in resolution of these
infections.
ii) Coagulase
this enzyme converts prothrombin to thrombin which in turn converts fibrinogen to fibrin - thus
causing plasma to clot (coagulate) in vitro
it is used to identify the pathogenic species as the strains which do not have coagulase activity tend to
be less pathogenic
coagulase positive species include S. aureus, S. intermedius, S. schleiferi ssp coagulans, S. delphini
and some strains of S. hyicus
however, the actual role of coagulase in virulence is questionable
iii) Capsule and Pseudocapsule

a number of cell wall constituents have antiphagocytic properties and include the capsule,
pseudocapsule and peptidoglycan
C3b is a component of complement cascade (formed through the alternative pathway) which can bind
to peptidoglycan on all gram positive bacteria, including Staphylococcus
part of the antiphagocytic effect of these molecules is to shield bound C3b from attaching to
complement receptors on phagocytic cells, and therefore preventing the phagocyte from being able to
attach to and internalise the bacteria
as only pathogenic Staphylococci have a capsule/pseudocapsule, only these species can prevent being
phagocytosed – while other non pathogenic Staphylococci are rapidly phagocytosed and cleared from
the animal
iv) Peptidoglycan and Teichoic Acids

teichoic acids function in the specific adherence of gram positive bacteria to mucosal surfaces
in addition, peptidoglycan and teichoic acids have
several biologic activities that are thought to contribute to virulence and include the ability to stimulate
antibody production, help trigger an inflammatory response (through the ability to activate
complement) and immobilise leucocytes at the focus of infection (hence suppuration)
they may also have an antiphagocytic role (interfere with opsonisation and therefore phagocytosis)
v) Urease
Urease activity is implicated in urolithiasis in dogs due to its ability to breakdown urea and cause
ammonia production which alters the pH of the urine
iii) Exotoxins
a large variety of exotoxins are produced by strains of S. aureus and S. intermedius and are thought to
enhance virulence through an increased ability to invade tissues (tissue destruction) and protect the
bacteria from body defence mechanisms (lyse inflammatory cells to avoid be killed)
these toxins and their specific actions are listed in Table 1.
6
TABLE: Exotoxins produced by Staphylococcus spp. The important toxins are in bold.
Toxin Action
Leucocidin Destroys PMNs and macrophages of some species
Haemolysin – alpha1 Produces clear zone of haemolysis around colonies due to
cytotoxic action on cells causing complete cell lysis. It is
necrotising and potentially lethal. Mutant strains without alpha
toxin are < virulent. Major toxin in gangrenous mastitis.
Haemolysin – beta1 Produces partial zone of haemolysis due to partial cell lysis.
Mutants without beta toxin are < virulent.
Haemolysin – delta Small zone of complete haemolysis. May have a role in
causing diarrhoea.
Haemolysin – gamma No lysis observed on blood plates.
Enterotoxins A, B, C1, C2, C3, D, E Mostly of human medical interest, as they are involved in food
poisoning. Clinical signs (nausea, vomiting diarrhoea) occur
within 1-6 hours (versus Salmonella food poisoning takes 24-
48 hours).
Exfoliatins (staphylococcal exfoliative Produced by some strains of S. aureus (sET) and S. hyicus
toxins – sET and shET) (shET). These toxins cause cleavage of desmosomes in the
stratum granulosum of the epidermis. sET is thought to be
responsible for staphylococcal scalded-skin syndrome (SSSS)
in infants and dogs, and shETfor porcine exudative dermatitis.
Toxic Shock Syndrome Toxin – 1 (TSST- Implicated in Toxic Shock Syndrome in humans. ~15% of S.
1) aureus produce this toxin.
Protein A Is a surface component of most strains of virulent S. aureus.
Has the unique ability to bind to Fc region of IgG which
interferes with opsonisation and therefore phagocytosis. It
also activates complement.
Extracellular Slime Substance – ESS Produced by S. epidermidis – important role in implant-
(glycocalyx) related infections. Helps adhesion of bacteria to implants
(e.g. bone plate) and impairs antibiotic access and killing by
host defence mechanisms.
Others Lipase, esterase, deoxyribonuclease, staphylokinase (a
plasminogen activator), hyaluronidase, phospholipase
1
Double haemolysis – animal strains of S. aureus and S. intermedius often produce both alpha and beta
haemolysins resulting in two zones of haemolysis around bacterial colonies. The inner zone is a small
zone of complete haemolysis (alpha haemolysin) and the outer zone a larger zone of partial haemolysis
(beta haemolysin).
II) The Pathogenesis of Staphylococcal Infections

Staphylococci are not inherently invasive, rather they usually colonise the intact epithelium of healthy
animals without causing disease
clinical infections appear to be determined by host factors – that is infections are opportunistic – they
require some disturbance in the host-parasite balance in order for disease to occur
for example, in cases of pyoderma, staphylococci can only invade epithelium damaged by
environmental insult (e.g. maceration or prolonged wetting) traumatic insult (e.g incisions, wounds),
other infections (e.g. demodicosis, dermatophytosis), clinical conditions (e.g. seborrhoea, allergies,
thyroid dysfunction), or immunodeficiencies and immunosuppression (glucocorticoid administration)
A BREAKDOWN in HOST DEFENCE MECHANISMS is required for staphylococcal infections

to establish (= OPPORTUNISTIC INFECTIONS). It is therefore VITAL if you diagnose an
KEY POINT infection with Staphylococcal spp that you look for UNDERLYING PROCCESSES that have
allowed the infection to develop. However, once Staphylococci invade host tissues, they
are GOOD PATHOGENS, and can difficult for the host to clear!!!
7
the predominant pattern of staphylococcal infections is
suppuration and abscess formation
components of the cell wall of Staphylococci attract many
neutrophils to the site of infection, which leads to a pyogenic
response (purulent inflammation) and suppuration
in addition, the leucocidins, haemolysins and other toxins
damage RBCs, macrophages, epithelial cells and other tissue
cells, which leads to further pus formation and inflammation
these processes result in abscess formation, frequently
followed by rupture of the skin and drainage of pus at the
surface
the immune system can also plays a role in the development
of lesions:
a. type IV hypersensitivity (delayed or cell mediated)

intensifies the inflammatory responses in
staphylococcal infection while spatially confining
them
b. type III hypersensitivity (antibody/antigen complexes)
thought to play a role in development of lesions in
some forms of canine pyoderma (juvenile pyoderma, FIGURE: “Butch” who has severe deep
folliculitis), and dogs with recurrent pyoderma or pyoderma on his face. This case was
secondary to generalised demodicosis secondary to infection with Sarcoptes mites
and therefore is consistent with requiring
an underlying predisposing factor for
infection to occur.
What do you see?
KEY POINT The typical lesion produced in staphylococcal infections is a pus filled abscess.
the typical lesion is the abscess

this is an inflammatory lesion in which participating cells have been destroyed by the combined effects
of bacterial and inflammatory cell activity
this confrontation between leucocytes and micro-organisms produces pus, a mixture of host cell debris
and bacteria (living and dead)
in an abscess, pus is surrounded by intact leucocytes and fibrin strands
unless the pus is drained, a fibrous capsule will gradually be formed
in chronic, ulcerative staphylococcal wound infections (“botryomycosis”), fibrous tissue predominates,
interspersed with pockets of suppuration
FIGURE: Pus-filled Abscess in vertebral

space.
Vertebral column from “Muffy” the dog.
There is a pus filled abscess in one of
the intervertebral spaces (=
discospondylitis). “Muffy” had evidence
of back pain and was reluctant to walk.
Despite long term therapy the lesion did
not resolve and “Muffy” was euthanised.
8
What Diseases can Staphylococci Cause?
I) Coagulase Positive Staphylococci

all warm-blooded animals can be clinically affected by coagulase-positive staphylococci, however, the
prevalence and form of such interactions vary among host species
S. aureus is the most common coagulase positive staphylococci isolated, and causes a wide range of
diseases in a range of species (including humans)
S. intermedius is primarily isolated in infections of dogs and occasionally from bovine mastitis and
feline infections (10% of staphylococcal isolates); but has been recovered from other species (horse,
pigeons) in which its significance is not always clear
S. aureus and S. inermedius can cause a wide range of different diseases in

KEY POINT a variety of animals.
Although, coagulase positive staphylococci are common causes of disease in domestic species, few
specific disease processes can be attributed to these bacteria (ie most of the diseases staphylococci
cause may be caused by other bacteria)
Exudative dermatitis and tick pyaemia are the only clinical conditions of
KEY POINT domestic animals specifically attributable to pathogenic staphylococci.
1. Pyogenic Infections ×Øb Õ

coagulase-positive staphylococci can affect every organ system independently or concurrently
S. aureus produces a wide range of diseases in a variety of species (including humans) and includes
mastitis, septicaemia, osteomyelitis, arthritis, sinusitis, endocarditis, pyometra, pneumonia, wound
infections, pyoderma, impetigo etc
S. intermedius is the chief pyogenic agent of dogs – with disease reported most commonly in the skin,
eyes, ears, respiratory tract, genitourinary tract, skeleton and joints
in cats, the infections sites are similar with S. felis being the most common isolate (45%), then S.
aureus (13%) and S. intermedius (10%)
2. Arthritis and Osteomyelitis

S aureus is a relatively common cause of septic arthritis in adult horses (and other species)
infections are commonly iatrogenic, occurring in performance horses that have been given intra-
articular injections
the source of the infection is either the horse’s skin, the human’s skin or the environment.
9
FIGURE: A swollen hock joint
in a standardbred racehorse
called “Used to Run Fast” (THE
CASE USED IN LECTURES).
The joint had been injected 3
days previously with a
corticosteroid. The fluid obtained
from this joint had many white
blood cells (predominantly
neutrophils) and occasional gram
positive cocci were observed. A
pure culture of a coagulase
positive S. aureus was isolated
from the joint. This is called septic
arthritis and is BAD if you want to
be a racehorse!
Swollen
hock joint
3. Bovine Mastitis
FIGURE: A case of gangrenous

mastitis in a cow caused by S. aureus.
Peracute and gangrenous forms of the
disease are associated with severe
systemic reactions and can be life-
threatening (as you can see in this cow
which is DEAD!!!). In gangrenous
mastitis, the affected quarter becomes
cold and blue-black, and eventually
sloughs. Tissue necrosis is attributed to
the alpha toxin which causes
contraction and necrosis of smooth
muscle in blood vessel walls, impeding
blood flow in the affected quarter (hence
the tissue necrosis as if there is no blood,
there is no oxygen and the tissues die!!!).
S. aureus is one of the major causes of bovine mastitis worldwide (along with Streptococci and coliforms)
and results in significant economic losses
infections occur via the teat canal
the course of infection may be subclinical, acute suppurative, gangrenous (due to alpha haemolysin) or
chronic depending on infecting strain, infecting dose, and host resistance
chronic and subclinical infections are most common
this organism may also cause mastitis in the ewe, goat and sow
4. Urolithiasis and Cystitis ×

phosphate (struvite or apatite) urolithiasis of dogs and mink is commonly associated with S.
intermedius infections and is associated with urease production by the bacteria (urease breaks down
urea to produce ammonia, which increases the pH of urine and leads to precipitation of struvite
crystals)
cystitis is a common sequelae to urolithiasis
10
5. Canine Pyoderma ×
the term “canine pyoderma” covers many clinical pictures, all of which include some degree of
pyogenic skin inflammation associated with bacterial infection
S. intermedius is the chief bacterium implicated (occasionally S. aureus), however, the contribution of
S. intermedius to the disease process, and the degree of suppuration, is variable
in chronic and recurrent forms, type III and IV hypersensitivity reactions are also thought to be
involved
host aspects (concurrent infections, genetic, endocrine, immunological factors) also play an important
role
pyoderma is also a problem in horses, and wild and domestic rabbits
6. Otitits Externa ×
S. intermedius together with S. schleiferi ssp coagulans are common causes of otitis externa in dogs
(see your notes from last years practical classes)
7. Bumble Foot Õ
Bumble Foot is a disease of gallinaceous birds
it is seen as chronic pyogranulomatous process in the subcutaneous tissues of the foot resulting in
thick-walled swelling of one or more joints
8. Botryomycosis
is characterised by chronic, suppurative, granulomatous lesions, especially of the udder of sows, mares
and cows and in the equine spermatic cord (scirrhous cord) after castration
9. Turkeys
In turkeys staphylocococcosis usually presents as a bacteremia localising in joints and tendon sheaths
Diseases ONLY caused by Staphylococcus spp
10. Exudative Epidermitis = Greasy Pig Disease

caused by S. hyicus and occurs worldwide in sucklers and weaned pigs
usually affects young pigs (<7 weeks), but is also found in weaned pigs up to 3 months of age
S. hyicus is found on vaginal mucosa and skin of sows and is transmitted to piglets; organism enters
skin through minor abrasions such as bite wounds
disease is characterised by skin lesions which appear as a thick, greyish-brown exudate, especially
around face and ears; lesions may occur in the lungs, LN, kidneys and/or brain
when infections are systemic they may be rapidly fatal (24 – 48 hours), but varies in severity from one
group of pigs to another
S. hyicus has also been implicated in septic arthritis of pigs, abortion in sows, seborrheic dermatitis of
pygmy goats, dermatitis of donkeys and horses and skin and udder infections in cattle
11
FIGURE: “Babe III”with exudative dermatitis or “Greasy Pig Disease”
(this is not a very attractive disease and so Babe III wasn’t included in the
movie!!!). Predisposing factors include agalactia in the sow, intercurrent
infections and weaning. The disease is characterised by widespread
excessive sebaceous secretions, exfoliation and exudation on the skin
surface. This results in a non-pruritic dermatitis with a greasy exudate
(which you can see on Babe III). Affected piglets are also not very well
(anorexic, depressed and febrile) and the mortality rate for this disease can
be high. Unfortunately, this disease is highly contagious!
11. Tick Pyemia
a disease of lambs due to inoculation of S. aureus (part of normal flora of skin) by tick bites
occurs in Britain and Ireland as this is where there are suitable habitats for the tick Ixodes ricinus
(which bites the lambs and transmits the bacteria).
it is often linked with tick-borne fever (caused by the rickettsial agent Ehrlichia phagocytophila) as this
organism can cause immunosuppression which predisposes to S. aureus infection
infections may be acute with toxaemic death, or chronic with disseminated abscess formation
II) Coagulase Negative Staphylococci

in the past, coagulase negative staphylococci were generally considered to be contaminants, with little
clinical significance
however, over the past 2 decades, these organisms have become increasingly recognised as important
agents of human disease; in particular in immunocompromised humans or compromised sites and as
nosocomial infections.
BUT, care must be taken in interpretation of their isolation due to their ubiquitous nature
Coagulase negative staphylococci rarely cause disease in

KEY POINT immunocompetent animals.
12
i) Infections of indwelling devices ×Øb
e.g. intravenous catheters, bone plates and screws
S. epidermidis is the main species involved, and these infections are being increasingly recognised as
important in human medicine
production of a glycocalyx slime layer is crucial to the pathogenesis of this infection
ii) Bovine Mastititis
the role of coagulase-negative infections in bovine mastitis is contentious – mostly S. epidermidis, S.

xylosus, and S. sciuri
How would you Diagnose an Infection?
If suppurative infections are observed, the likelihood of staphylococcal

KEY POINT infections must be considered and appropriate specimens collected for
laboratory confirmation.
Sample Collection
fine needle aspirates from unopened lesions (e.g pustule or abscess) collected into sterile syringes or
sterile containers are preferred (after surface has been gently cleaned with alcohol)
swabs may also be used to collect samples; for example from the ear where outer parts of the ear
should first be cleaned and then using a clean otoscope cone to protect the swab it can be inserted to
the level of the horizontal canal and a sample obtained
however, care must be taken if swabs are collected to ensure that contamination with normal flora does
not occur
this is especially the case if there is a delay in processing and samples are left in transport media; this
will result in overgrow of normal flora (which may contain staphylococci) and these bacteria will
predominate although they were not present in the original site of infection
punch biopsies are suitable for collection of samples from superficial and deep pyoderma
milk must be collected into sterile containers using sterile techniques
blood (venipuncture) and urine (cystocentesis) cultures are appropriate for isolation of staphylococci
Direct Examination
gram and/or Diff Quik stains will enable visualisation of single, pairs (diplococci), or more rarely
clusters of (gram positive) cocci
inflammatory cells (primarily PMNs) MUST also be present for interpretation of the significance of an
isolate
Inflammatory cells MUST be present in smears of collected samples to

ascribe any significance to the bacteria you subsequently isolate. If there
KEY POINT is no inflammation present – the bacteria that you have isolated must be
contaminants and you have not collected a sample from the site of
infection/inflammation.
13
Culture
cultivation on blood agar is preferred for primary isolation , though Staphylococci will also grow on
most common laboratory media – cultures should be incubated aerobically
enumeration of colonies (cfu’s) is helpful in interpretation of the significance of isolates
pure cultures and heavy growth (many colonies) are likely to be significant, especially if isolated from
a sterile site
Staphylococci are NOT difficult to ISOLATE, but their presence can be

KEY POINT difficult to INTERPRET unless appropriate collection techniques are used!!!
Identification
the catalase test is useful for differentiation between Staphylococci (positive) and Streptococci
(negative)
coagulase (clumping factor) test is useful to help interpretation of the significance of the isolate
phage typing has been used to identify strains of S. aureus, and is mostly used for investigating
outbreaks of disease in humans (epidemiological studies)
animal phage types are usually different to humans
Degenerating Neutrophils
Clumps of gram positive cocci
FIGURE: Smear of URINE from a 4 year old female Westie called “Maddie”. She was
presented because of pollakiuria (pain on urination). This smear demonstrates MANY cocci
(mostly in clusters, but there also appears to be some chains - ? mixed infection with
Streptococcus spp). There are also some inflammatory cells (neutrophils). These cells
MUST be present to verify that a bacterial INFECTION is occurring. Note: the neutrophils
are partially lysed because of the environment in the urine – that is why they do not look like
normal neutrophils found in the blood.
14
Immunity and Resistance
clearance of staphylococci depends chiefly on phagocytosis by macrophages and neutrophils - therefore

opsonisation of the bacteria by complement and/or specific antibodies is important
however, some strains (mutants – see earlier) are resistant to intracellular killing and can survive
inside the phagocytes (intracellular survival)
humoral factors (antibodies) are important opsonins as studies have shown that agammoglobulinaemic
individuals suffer frequent infections
the antibodies can be directed at either the bacteria (anti-bacterial) or their toxins (anti-toxic)
cell mediated factors contribute to localisation and resolution of lesions
recovery from staphylococcal infection confers no lasting resistance
How would you Treat an Infection?
General Approach to Therapy

removal of the underlying cause(s) or predisposing factors is essential for resolution of Staphylococcal
infections
Staphylococci cause opportunistic infections, therefore ALWAYS look for

KEY POINT underlying disease states or predisposing factors to infection and take
these into account when implementing a treatment plan.
in addition, abscesses, empyemas and infected sites (e.g. joints) should be drained of pus
In general, antibiotics penetrate poorly into abscess. Therefore drainage of

KEY POINT any pus-filled abscesses or infected sites (e.g. joints) is also required for
resolution of staphylococcal infections.
topical administration of mild antiseptics (e.g. bathing in antibacterial shampoos such as

4%.chlorhexidine (= Pyohex® ) is also important in all cases and may be adequate for resolution of
surface and/or superficial infections (e.g. hot spots and puppy impetigo), without requiring antibiotic
therapy (especially surface infections)
Specific Treatments – Antibiotics
antibiotics may be given either topically or systemically

extensive, inaccessible and disseminated processes require systemic antibiotic therapy
S. aureus had an unpredictable sensitivity pattern and strains are commonly resistant to a variety of
antibiotics including penicillin (equine isolates less so), streptomycin, tetracyclines etc
Antibiotic Sensitivity Testing should be performed on isolated of S. areus

KEY POINT and S. intermedius, especially if infection is in a compromised site (e.g.
bone or joints) or is refractory to therapy (e.g. deep pyoderma).
15
FIGURE: This is an agar plate used to
determine the sensitivity of bacteria to
chosen antibiotics. It is HIGHLY
RECOMMENDED that all isolates of
pathogenic Staphylococcus spp have a
sensitivity test performed as they do NOT
have a predictable sensitivity pattern.
Conversely, sensitivity testing should NOT
be performed on coagulase negative
Staphylococcus spp as these bacteria are
NOT of clinical significance in the majority of
cases.
antibiotics that may be effective in the treatment of S. aureus infections include penicillinase-resistance
penicillins, cephalosporins (first generation), gentamicin, fluoroquinolones, chloramphenicol,
erythromycin, vancomycin, lincomycin, and trimethoprim-sulphonamides, but resistance to most of
these antibiotics has been recorded
most resistance is conferred by genes encoded on plasmid (e.g. beta-lactam antibiotics resistance is
most often due to plasmid-encoded beta-lactamases, but other mechanisms for resistance to beta-lactam
antibiotics do occur that are often chromosomally encoded)
clavulanic acid inactivates the beta-lactamases produced by S. aureus and S. intermedius, therefore
antibiotics (e.g. clavulox) containing this substance are useful IF resistance is conferred via this
method
the antibiotic sensitivity patterns of clinically important isolates of staphylococci from dogs were
investigated and most strains were found to be sensitive to first generation cephalosporins,
penicillinase-resistance penicillins, fluoroquinolones, and gentamicin; but resistant to penicillin,
ampicillin, amoxicillin, and tetracyclines
HOWEVER, these sensitivity patterns have not been investigated for over 20 years, AND variability
in these patterns may also exist (therefore it is PREFERRABLE to always conduct sensitivity testing
for Staphylococcus isolates)
for exudative dermatitis in pigs, cloxacillin can be used systemically and topically
intramammary preparations (commonly cloxacillin) has been used with variable success in cases of
mastitis
in these cases dry cow preparations (ie preparations administered after drying the cow off) are more
effective as they provide antibiotics over a longer period of time
Antimicrobial therapy may be ineffective as Staphylococci can survive in

KEY POINT phagocytes.
16
Duration of Therapy
for acute infections short term therapy (e.g. ~ 7-10 days) may be sufficient
however, for chronic infections, long term therapy (weeks to months) may be required due to the ability of
the bacteria to survive inside cells (intracellular survival)
How would you control disease?
Vaccines to Staphylococci have been developed and marketed, but the benefits of vaccination are
dubious
commercial or autogenous whole culture preparations and toxoids plus bacterins have been used
prophylactically on dairy cattle and sometimes in small animal dermatology to treat persistent infection
although success has been reported, controlled evaluations are lacking
use of staphylococcal phage lysates, and non-specific stimulants of cell-mediated immunity in cases of
non-responsive pyoderma awaits support by adequate clinical or experimental evidence
Staphylococci withstand drying, especially in exudates for weeks; this may be of significance for
transmission, especially in cases of bovine mastitis
Staphylococci are inhibited by bacteriostatic dyes (e.g crystal violet), bile salts, disinfectants like
chlorhexidine, and many antimicrobial drugs
What are the Public Health Considerations?
humans may become infected with S. aureus, S. hyicus and possibly other staphylococci of animal
origin
This is particularly important if the S. aureus or S. intermedius isolate is methicillin resistant (MRSA
= methicillin resistant Staphylococcus aureus) which have been reported in dogs (including those in
Australia) and horses
however, inter-species transmission is generally thought to be rare
17
STREPTOCOCCI
Streptocococci are common pathogens in domestic species, especially horses and cattle. They are gram
positive cocci that like to form chains (which may be short or long). This can help to distinguish them
from Staphylococci when examining smears from exudates of lesions. Like Staphylococci, the diseases
they cause are characterised by purulent inflammation, and are commonly acute or peracute. One of the
most important diseases associated with this genus is “Strangles”, caused by S. equi ss equi. It is a most
commonly observed in young horses as a submandibular or retropharyngeal lymphadenitis. These
swellings can get very large (even to the point of “strangling” the horse – hence the name!). Furthermore,
this is a contagious disease which has important control implications. Mastitis is another important disease
caused by Streptococci and has significant financial costs to the dairy industries.
Summary Table: Species of Streptococcus that have veterinary importance, their Lancefield group (1),
their haemolytic pattern (2), their animal hosts, and the diseases they cause
SPECIES 1 2 HOSTS DISEASES
tonsillitis, pharyngitis, otitis, pyoderma,

humans, (dairy cattle, bacteraemia, toxic shock, necrotising fasciitis,
β
+
Str. pyogenes A
horses) erysipelas, puerperal fever, rheumatic fever,
glomerulonephritis (mastitis, pneumonia)
pneumonia, otitis, meningitis, bacteraemia,
Str. humans, primates
A α polyarthritis, (mastitis, calf septicaemia,
pneumoniae+ (cattle, horses, cat)
meningitis, pneumonia)
Mastitis (chronic)
dairy cattle (sheep,
Str. agalactiae+++ B β goats) neonatal sepsis, metritis, arthritis, pharyngitis,
humans (cats, dogs) skin and wound infections, peritonitis,
placentitis, etc
Str. equi ss strangles
C β horses
equi++++
Str. dysgalactiae pigs, horses miscellaneous suppurative conditions,
C β
ss equisimilis+ (humans, dogs) endometritis, mastitis
Str. equi ss pneumonia, miscellaneous suppurative
horses (fowl, dogs,
zooepidemicus++ C β conditions, neonatal infections, mastitis
++ ruminants, humans)
Enterococcus α many opportunistic conditions, genital and
D all species
spp++ (β,γ) neonatal conditions
Str. porcinus+ E β pigs, cattle jowl abscesses
feline lymphadenitis, toxic shock, necrotising
dog, cattle, cats
Str. canis+++ G β fasciitis, miscellaneous pyogenic conditions
(humans)
of dogs, cats and humans
Str. dysgalactiae acute mastitis
dairy cattle
ss C α pharyngitis, tonsillitis, wound infections,
(humans)
dysgalactiae+++ neonatal sepsis, endocarditis
neonatal infections, septicaemias,
Str. suis++ D α (β) pigs, (humans ), pneumonia, meningitis, arthritis (humans –
septicaemia, arthritis, meningitis)
Str. uberis+++ - α (γ) cattle, soil Mastitis
NOTE: the number of + denotes the relative veterinary importance of these species within THIS genus (NOT for all
bacterial genera)
18
What are they?
Streptococci are gram positive cocci

however, they frequently stain gram negatively, especially in exudates and older cultures
Streptococci are gram positive cocci but may stain negatively in exudates –
KEY POINT which is important to remember when examining smears of exudates.
their morphology also can vary from cocci to short rods

Streptococci divide in one plane to form pairs, short or
long CHAINS when grown in liquid medium (including
pus!)
the length of chains varies between species and media
KEY POINT The presence of gram positive cocci in chains is useful for DIAGNOSIS.
Streptococci are facultatively anaerobic; but many strains grow better in CO2
all species of Streptococci are catalase negative
there are more than 37 species; 12 species have medical or veterinary importance (see summary table)
several classification systems exist for streptococci, but the one most frequently used is the
“Lancefield” grouping as this tends to correlate to pathogencity
this test divides the Streptococci into groups based on their C-substance which is a polysaccharide in
their cell wall (= group antigen)
the groups are designated by capital letters A to V (see summary table)
Lancefield Groups are helpful for diagnosis and determination of the clinical
KEY POINT significance of the different Streptococci (See Summary Table for Lancefield
groups).
As a general rule, the haemolytic streptococci that belong to particular Lancefield group are usually
associated with disease in a particular species:
Group A human
Group B bovine mastitis
Group C horse
Groups D,E pigs (D also in other species)
Group G dog
19
streptococci are found worldwide, where they are widely distributed in nature, but are most commonly
found as commensals on humans and animals
potentially pathogenic and non pathogenic species can be present on the skin or on the mucous
membranes of the lower genital, upper respiratory and most of the digestive tract (enterococci)
many of these species are part of the normal flora of the animal (e.g. S. equi ss zooepidemicus), whilst
others species are not considered part of the normal flora rather survive living on a smaller group of
carrier animals (e.g. S. equi ss equi)
in general, Streptococci are fragile bacteria, and are susceptible to dessication (usually surviving for
only short periods off their host species)
Streptococci are frequently part of the NORMAL FLORA but some may be
KEY POINT maintained on carrier animals.
some species are obligate parasites (cannot live off the animal host) e.g. Str. agalactiae
others species survive for variable periods off the host (e.g. Str. uberis) and infections are acquired
from the environment (but these Streps are still susceptible to dessication)
most of the pathogenic streptococci can be carried by healthy individuals
KEY POINT Infections with Streptococci may be ENDOGENOUS or EXOGENOUS
many infections are caused by the resident strain of bacteria on the animal host (endogenous)
other infections originate from bacteria present in the environment (exogenous)
streptococci may be TRANSMITTED between animals by inhalation or ingestion, sexually, or
congenitally
they may also be transmitted indirectly via hands and fomites
neonatal infections are commonly maternal in origin
strangles and porcine lymphadenitis are contagious diseases, usually affecting young animals (past
infancy)
Some streptococcal infections are CONTAGIOUS. This has important

KEY POINT implications for CONTROL of these infections.
the 2 most important streptococcal diseases that are caused by Streptococci and that are contagious
involve Str. equi and Str. agalactiae
Str. equi is spread by contaminated food, drinking water, or utensils, or recovered animals (carriers)
which may remain shedders for months
Str. agalactiae is spread among dairy cows, often by milking equipment, unskilled attempts at
mammary medication, and unsanitary milking practices
20
I Virulence Factors
the primary way that streptococci avoid host defence mechanisms is to avoid (inhibit) phagocytosis (M
protein and capsule) and to kill phagocytes (haemolysins)
a number of other virulence factors have been described, but most of them are poorly understood
a) Haemolysins
the effect of the streptococcal haemolysins on sheep or bovine blood agar is used to divide streptococci
into three types (alpha, beta and gamma) depending on the type of haemolysis that is caused:
1. Alpha haemolysis
9 alpha haemolysis is an incomplete haemolysis and is seen as a zone of green discolouration or
haziness around colonies
9 the mechanism of partial cell lysis is poorly understood
9 most commensal streptococci (Streptococci that live as part of the normal flora) of animals are
alpha haemolytic
9 in general, alpha haemolytic Streptococci cannot cause disease as well as beta haemolytic
Streptococci (ie. they are less pathogenic), but when they do cause disease they may have
widespread antimicrobial resistance and therefore can be more difficult to treat
9 however, care must be taken when interpreting the isolation of alpha haemolytic Streptococci
from clinical samples to make sure that it is really causing the disease process and is not just
normal flora present as a contaminant
Alpha haemolytic Streptococci are commonly found as commensals and are

less virulent than beta haemolytic Streptococcrai. However, when they do
KEY POINT cause disease they may be difficult to treat as they may have broad
antibiotic resistance.
2. Beta haemolyis
9 beta haemolysis is a complete haemolysis and is a result of the action of STREPTOLYSINS
which destroy RBCs and produce a zone of transparent clarity around the streptococcal
colonies
9 beta haemolytic streptococci tend to be most pathogenic
9 to date all strain of beta haemolytic streptococci that have been isolated from clinical case have
been predictably sensitive to penicillin
Beta haemolytic Streptococci are more virulent than other types of

Streptococci and are common causes of disease in domestic species,
KEY POINT particularly in horses. In addition, they are predictably sensitive to
penicillin.
21
Streptococccal colonies with a
clear zone of haemolysis around
each individual colony
FIGURE: A blood agar plate with a pure culture of β-haemolytic Streptococci. It is

very helpful clinically to determine if the Streptocococcus spp involved in a disease
process is β-haemolytic as this group of organisms are predictably sensitive to
penicillin (at this stage!@!)
there are 2 main types of streptolysins: O and S (this is for your interest ONLY!)
Streptolysin O
• Streptolysin O is O2 sensitive, therefore only works in areas of low O2
• it is produced by most pathogenic streptococci that are beta haemolytic
• produced by Lancefield groups A, many C and G
• in addition to lysing RBC, this molecule is cytotoxic for WBC and platelets
• antibodies are formed against it
Streptoylysin S
• this Streptolysin is oxygen stable and cell bound
• it lyses RBCs, WBCs and platelets; it can stimulates the release of lysosomal contents after
cell engulfment with subsequent death of the phagocyte and results in irreversible tissue
damage
• produced by Lancefield groups A, C and G
• no antibodies formed against it
3. Gamma haemolysis (non haemolytic)

9 little or no clearing around the colony
9 most gamma haemolytic streptococci are non pathogenic
22
b) M Protein
M protein is a cell wall protein )
it is present on the surface of some streptococci e.g. Str. pyogenes, Str. equi ss equi, Str. porcinus
studies have shown that it is the most important virulence factor of these Streptococci
M protein allows streptococci to inhibit phagocytosis, reduce chemotaxis, and adhere to epithelial cells
its antiphagocytic activity is through interference of effective deposition of complement components
necessary for opsonisation
loss of M protein in some strains results in loss of virulence (change in colony type from rough to
smooth)
in addition, M protein stimulates antibody production that bind to it and neutralises the antiphagocytic
function
thus anti-M protein antibodies are associated with protection and are the basis of many streptococcal
vaccines (e.g. S. equi ss equi)
however, the M protein is antigenically variable and the antibodies produced type specific immunity
where limited cross protection is observed between serotypes
70 serotypes have been identified for S. pyogenes
M protein is a major virulence factor as it helps Streptococci to avoid

KEY POINT phagocytosis (anti-phagoyctic). Antibodies to the M protein confer type
specific protection
c) Capsules
these are only present on some species or strains
if they are present they can be polysaccharides (carbohydrates) or hyaluronic acid
they are antiphagocytic as they make it difficult for the neutrophil (phagocyte) to interact with their
surface
the polysaccharide capsules also provide the basis for serotyping in some species (e.g. S. pneumoniae
and S. agalactiae) as they are antigenic (stimulate antibody production)
this anti-capsular antibody is important as a host defence mechanism as it means the bacteria lose their
antiphagocytic properties
KEY POINT The Strepotococci with capsules can avoid phagocytosis.
d) Surface Protein F
responsible for adhesion of several streptococci species to extracellular matrix proteins
e) Peptidoglycan
can trigger inflammation, fever (pyrogen) and lymphocyte proliferation
it is dermonecrotic, and cytotoxic in vitro
f) Other Exotoxins (S. pyogenes)

the pathogenic significance of these further extracellular protein products is often poorly defined
they have been best described for S. pyogenes, but comparable toxins occur in animal streptococci
they include haemolysins (streptolysin O and S– see earlier), hyaluronidase, nucleases, NADasem
Proteinase, Streptokinase (a fibrinolysin) and Pyrogenic toxin (phage encoded) – responsible for the
rash in scarlet fever
II) The Pathogenesis of Streptococcal Infections

similar to staphylococci, many of the streptococci that cause disease are present as commensals on the
animal host
23
therefore some change in host-parasite relationship is required to produce disease (OPPORTUNISTIC
INFECTIONS)
development of disease also depends on other factors such as portal of entry, animal species and
streptococcal species
streptococci frequently induce pyogenic infections, as they trigger inflammatory processes that lead to
suppuration and abscess formation
The bacteria that most frequently result in the production of large amounts
KEY POINT of PUS are Staphylococci, Streptococci and Corynebacteria (= PYOGENIC
BACTERIA).
when pyogenic bacteria invade tissues they invoke an inflammatory response characterised by vascular
dilation and a marked exudation of plasma and neutrophils
within the tissues, the neutrophils then move towards the bacteria (through chemotaxis) and may
engulf them
however, pathogenic streptococci are antiphagocytic and produce toxins that kill phagocytic cells
furthermore, the enzymes liberated from the dead neutrophils bring about additional tissue destruction,
with partial liquefaction of the dead tissue and phagocytic cells
the liquefied mass becomes visible as thick, usually yellow, pus
the pus is viscous due to the large amount of DNA from the nuclei of dead cells
in response to streptococcal infections, the host produces type-specific antibodies against the anti-
phagocytic compounds (M protein or capsule) within 7 to 14 days
these antibodies allow rapid clearance of the streptococci
unlike staphylococci, no intracellular survival of streptococci occurs once ingested by phagocytes
thus, diseases produced by streptococci tend to be acute and of short duration
As Streptococci do not survive inside cells, streptococcal diseases tend to

KEY POINT be acute, of short duration, and involve pus formation and abscessation.
as with staphylococci, the immune system also plays a role in the development of lesions, especially as
sequelae to infections
type III hypersensitivity (antibody/antigen complexes) is thought to play a role in development of
lesions in human post-streptococcal diseases (rheumatic fever, acute glomerulonephritis)
similarly, equine purpura haemorrhagica following “strangles”(caused by Str. equi equi) is probably
immune-complex mediated (type III hypersensitivity)
24
Typical Pathogenesis of a streptococcal infection
Normal Horse in its Environment
Change in Host/Parasite Interaction creating OPPORTUNITY for invasion

(may be due to environmental influences e.g transportation, or host factors e.g. pre-existing viral infection
such as Equine Influenza)
Invasion by potential pathogens that are part of the normal flora and which can avoid host defences (e.g.
phagocytosis) and cause tissue damage (e.g. toxin/haemolysins)
Disease (e.g. Pneumonia) = Sick Animal
Lung abscess in a horse that

developed Streptococcal
pneumonia subsequent to long
distance transportation
25
What do you see?
the basic pathological process resembles that of Staphylococcal infections

the typical lesion is the pus-filled abscess (see Staph notes).
most of the streptococci within abscesses will be gram negative if a gram stain is performed
Streptococci typically cause pus filled abscesses. Smears of samples

obtained from these abscesses will frequently demonstrate gram negative
KEY POINT cocci in chains as the walls of the Strepotococci have been damaged by lytic
enzymes released by phagocytes.
Clinical lesions caused by Streptococci and Staphylococci can look the same
(ie the lesions are NOT pathognomonic for one genus or the other).
KEY POINT However, the antimicrobial sensitivity pattern for these two genera differs.
Therefore, it is important to isolate and dentify the causative bacteria in
clinical cases in order to be able to implement appropriate therapy.
What Diseases do Streptococci Cause?
a number of very important and common diseases are caused by streptococci

these organisms are a particularly common cause of a VARIETY of diseases in humans, horses, pigs,
dogs and cats, and to a lesser extent cattle (predominantly mastitis)
the sites in which they cause disease are related to the sites where they are commonly found as normal
flora ie near mucous membranes
in horses they may be found causing infections in most body tissues (eg. pneumonia, lymphadenitis, sc
abscesses, endometritis, osteomyelitis, septic arthritis etc) and S. equi ss zooepidemicus is one of the
most common pathogens isolated from clinical samples in horses
Most bacteria (including Streptococci) that are part of the NORMAL FLORA
tend to cause disease in sites that are NEAR to where they are found as
KEY POINT normal flora. They need some COMPROMISE to the host to be able to cause
disease in these sites.
1. CERVICAL LYMPHADENITIS
invasion of the mucous membranes of the upper respiratory tract by the organisms commensal in this
site will frequently lead to a local pharyngitis and cervical lymphadenitis (infection of lymph nodes of
the neck)
a) STRANGLES in HORSES
9 this is a highly contagious equine disease caused by S. equi ss equi
9 the disease occurs in equine populations world-wide and results in significant economic and
production losses
STRANGLES is a MAJOR disease of horses worldwide and is a particular

KEY POINT problem when horses gather together (e.g. at races, sales, horse shows etc).
26
9 the source of infection for horses is nasal discharge or purulent exudates from infected or carrier
animals, transmitted either directly or indirectly through contamination of the environment
(fomites)
9 the incubation period is 3 – 6 days, then infection is marked by inflammation of the upper
respiratory tract and abscessation of adjacent lymph nodes
9 strangles is most commonly observed in young horses (6 months to 5 years) , especially in foals
and weanlings
9 clinically, there is a serous or purulent nasal discharge, diphasic fever, local pain, cough, anorexia
and abscess formation in the regional lymph nodes (primarily submandibular and retropharyngeal)
9 the abscesses typically rupture and drain within 2 weeks (this pus is infectious)
9 clinical recovery usually follows, though most horses remain as carriers for variable periods after
cessation of clinical signs (usually up to 6 weeks, but a low % of horses may remain long term
carriers
9 in carrier animals the organism usually localises in guttural pouches
9 in naïve groups of horses, morbidity if usually high (40-80%) but mortality rate is low (2-10%)
FIGURE: Large submandibular and periocular swellings on the face of “Nelly” a 4 year old stock
horse mare. Both of these swellings were infected lymph nodes (submandibular and parotid lymph
nodes respectively). They were infected with the organism S. equi ss equi. This horse has
STRANGLES (cervical lymphadenitis). The other clinical signs that this horse had included fever, a
serous nasal discharge, inappetence, and lethargy. The course of this disease in uncomplicated
cases is 5 – 10 days. At this time, usually the lymph nodes will rupture, discharging highly purulent,
highly infectious material into the environment.
9 COMPLICATIONS can occur and include:
1. dissemination to distant lymph nodes, meninges, lungs, pericardium and abdominal viscera (=
“bastard” strangles or metastatic strangles)
2. extension to guttural pouches (gutteral pouch empyema +/- chondroid formation)
27
3. purpura haemorrhagica – a type III hypersensitivity reaction (Ab-Ag complex disease) manifested
by subcutaneous swellings, mucosal haemorrhage and petechiae, and fever – follows acute disease
by about 1 - 3 weeks
4. death due to asphyxia, especially if retropharyngeal LN are involved (hence the term “strangles”)
Pus that has been drained surgically from the Plastic tube positioned in the guttural pouch to
guttural pouch facilitate drainage
FIGURE: A showjumper called “Major”, who was presented because of a swelling behind
the jaw and a persistent muopurulent nasal discharge. Endoscopy revealed that he had
guttural pouch empyema which was drained surgically. Note the caseous pus on the left
of the picture (which may contain numerous viable organisms). This was drained from
the affected guttural pouch. The guttural pouch is the site where these bacteria can
persist in carriers.
9 re-infection has been reported in some horses after strangles, but is rare
9 Strangles is a notifiable disease of horses in NSW (but NOT elsewhere in Australia!)
b) JOWL ABSCESS of PIGS

9 this is cervical lymphadenitis caused by S. porcinus
9 it occurs in 8-10 week old pigs
9 it is analogous to strangles in horses, but clinically less dramatic
9 and often not detected until slaughter
9 its most damaging aspect is carcass condemnation
c) Cervical Lymphadenitis in young cats and guinea pigs

9 Caused by S. canis and S. equi ss zooepidemicus respectively
9 In young cats, juvenile streptococcosis occurs in the post-weaning period (2-4 months) and is due
to invasion of the tonsil and local lymphatics of he head/neck
9 This can lead to purulent lymphadentitis of the mandibular LN, or further dissemination of
infection with pneumonia, discospondylitis, osteomyeolitis and arthritis being recorded
28
2. NEONATAL SEPTICAEMIA
these diseases can often be traced to infections of the mucous membranes of the maternal genital tract
a) Foals
9 S.equi ss zooepidemicus is part of the normal flora of the genital tract and as a consequence is a
common cause of cervicitis endometritis in mares (opportunistic infection), and post natal
infections in foals
9 in foals, it may cause umbilical infections (navel ill) and be disseminated via the blood stream
(septicaemia), frequently localising in joints (joint ill = polyarthritis) and the renal cortices
(pyelonephritis)
b) Puppies
9 In newborn puppies, S. canis is associated with septicaemia
9 Sequelae include pneumonia, arthritis, and abscess formation in various locations
c) Kittens
9 kittens are infected with S. canis from vaginal secretions or from mouth of the queen when she
bites the umbilical cord close to the abdominal wall
9 the organisms enter the umbilical vein, are carried into the blood stream and seed distant organs
including liver, spleen, lungs, kidney, and muscles
9 gross and microscopic abscesses are formed and death may occur approximately one week after
birth
d) Piglets
9 in pigs, S. suis is a common and important cause of disease in piglets and is associated with
neonatal septicaemia, tonsilitis, skin abscesses, pneumonia, arthritis, meningitis, endocarditis and
abortion and mastitis (in sows)
9 S. suis is found in the pharynx and tonsillar tissues of carrier sows, and is transferred to pharynx
of piglets
9 if predisposing factors (e.g. overcrowding, poor ventilation, weaning) occur, then the bacteria can
invade the mucosa and move to regional lymph nodes (cervical lymphadenitis) from where they
may escape into the blood stream
9 there are 2 biotypes of S. suis (I and II), both types causing disease but in different age groups of
pig:
1. Type I piglets 2-4 weeks age
2. Type II post weaning piglets
9 it is important to remember that Str. suis can be a serious zoonotic infection (see later)
FIGURE: “Babe IV” who unfortunately

has S. suis meningitis. Notice the bare
areas of straw around her front and back
legs due to paddling associated with
convulsions! She also was displaying
tremors, inco-ordination and
opisthotonos and was febrile.
Unfortunately, Babe IV died and didn’t
make it to the second movie!
Streptococcus suis serotype 2 are
most commonly associated with these
infections in both pigs and also humans
(and has important public health
implications).
29
3. LOWER RESPIRATORY TRACT INFECTIONS
in addition to upper respiratory tract conditions, streptococci are a common cause of lower respiratory
tract disease (pneumonia) in a variety of animal species including humans
predisposing factors are important for the development of disease and include recent shipment
(travelling), viral infections, and poor ventilation or housing
Predisposing Factors are required for development of infections by

streptococcal species that are part of the normal flora. In the case of lower
KEY POINT respiratory tract disease predisposing factors may include transportation,
viral infections and poor housing conditions.
in horses, S. zooepidemicus is one of the most common causes of uncomplicated bacterial pneumonia
(along with Pasteurellaceae)
similar disease is produced in pigs (S. equisimilis), dogs and cats, (S. canis) and alpacas (S.
zooepidemicus)
in humans (and primates) S. pneumoniae is a major cause of disease including pneumonia,
bacteraemia, meningitis (pneumococcal meningitis), otitis media, sinusitis and endocarditis
pneumonia (and other diseases) disease have been reported in calves, horses, dogs, cats, goats, rabbits
and rats associated with Str. pneumoniae infection
4. Bovine mastitis
Streptococci are one of the leading agents of bovine mastitis, where the Streptococcal species most
commonly involved include S. agalactiae, S. dysgalactiae, and S. uberis
other species which may be isolated (but less frequently) include Str. pyogenes, S. zooepidemicus, S.
pneumoniae, S. bovis and Enterococcus faecalis
Bovine Mastitis may be divided into:

1. Contagious Mastitis S. agalactiae, Staphylococcus aureus
KEY POINT 2. Environmental mastitis S. dygalactiae, S. uberis, coliforms
3. Normal teat flora Staphyolococcus epidermidis, Staph hyicus.
This distinction is important for appropriate control of this disease.
Str. agalactiae
9 is a primary pathogen of the udder of cows and a major cause of mastitis world-wide
9 this organism is specifically associated with the mammary gland, colonising the milk ducts
S. agalactiae is an obligate parasite of the mammary gland of cows.

KEY POINT Infected cows (clinical or carriers) are the RESERVOIR for infections.
Mastitis caused by S. agalactiae is CONTAGIOUS.
9 therefore infections with S. agalactiae are contagious in that cows can only acquire infections from
another cow and NOT from organisms present continually in the environment
9 transmission from carriers or clinical cases usually occurs at milking either on the hands of
milking personnel or through milking equipment
9 therefore morbidity (infection rates) with Str. agalactiae is high on dairy farms where hygiene is
poor and efficient treatment regimens are not used
9 infection is through the teat canal with bacterial proliferation in lactiferous ducts and alveoli
resulting in an acute inflammation followed by some fibrosis
9 successive flare-ups cause replacement of secretory tissue by fibrous connective tissue and
eventually the entire mammary gland may be lost
30
9 during active phases of the chronic infection, milk yield is reduced, the milk may be abnormal and
contain clots
9 in acute mastitis, the gland will be swollen and hot, and the cow may be febrile and anorexic
Milk from affected quarter from

which S. agalactiae was isolated
FIGURE: Milk collected from the 4 quarters of a “Bessie” the cow. Note that the third vial
from the left contains milk that is more watery than normal, and contains multiple large
clots. This quarter has mastitis caused by S. agalactiae. This milk is NOT nice to drink!!!
S. dysgalactiae
9 causes sporadic acute mastitis and infection is sometimes severe
9 this organism is usually found in the mouth (buccal cavity), genital tract, or skin of the udder
S. uberis
9 present in soil, faeces, and also a common inhabitant of the skin, lips, tonsil, and mucous
membranes of cattle
9 therefore is an opportunistic pathogen that invades the udder under conditions of poor hygiene and
may be secondary to infection of the skin
9 infections are generally mild, though it is the most common cause of mastitis during the dry cow
period (intercalving period) especially in the month before parturition
9 cases may also occur in the first part of lactation
Other Streptococci
9 infections are variable (severe to mild)
9 less frequent causes of mastitis
5. MISCELLANEOUS PYOGENIC INFECTIONS (non respiratory tract disease)
a) CANINE CYSTITIS
9 streptococci (usually Enterococci) account for about 10% of canine urinary tract infections
31
b) SEPTICAEMIA
9 is observed in fresh water aquarium fish
9 a beta-haemolytic streptococcus spp is implicated
c) TOXIC SHOCK and NECROTISING FASICULITIS (“flesh eating bacteria”)

9 caused by a Group G streptococcus (S. canis) in dogs and cats
9 no virulence traits have been defined as they have for similar conditions seen in humans affected
with S. pyogenes (see CAVMOLs for more details on this disease)
d) ENDOCARDITIS
9 Streptococci may cause endocarditis in dogs and other species (e.g. horses)
9 beta-haemolytic belonging to Group G (dog) and C (horses) are usually implicated
9 in chickens, Enterococcus faecalis can cause endocarditis and septicaemia along with S.
zooepidemicus
FIGURE: Lesions on the heart valves of “Jessie” the border collie. Jessie
was presented for lethargy, coughing and weight loss. Upon auscultation
of her heart it was noted that she had a murmur. An ultrasound scan of her
heart revealed proliferative valvular lesions. Despite therapy, Jessie died
and she was subsequently diagnosed with bacterial endocarditis. Note that
the valvular lesion is caseating (pus-like) which is typical of Streptococcus as
well as other pyogenic bacteria. A group G streptococcus (S. canis) was
isolated from this lesion.
How do you Diagnose Infections?
Sample Collection
same procedures as for Staphylococci
Direct Examination
gram stain and Diff Quik stains of exudates or fluids should be made
32
streptococci appear as gram positive (or gram negative) cocci (or very short rods if they are in the
process of dividing) in pairs, short chains or long chains (long chains particularly prominent in
samples involving S. equi ss equi and S. agalactiae)
FIGURE: A sample of Str. equi ss equi grown in liquid culture media.

The organism is displaying the long chains that are characteristic of this
species. Other streptococcal species only have short chains and this can be
used to help differentiate streptococci.
Streptococcus equi ss equi forming long

chains in a broth culture (they will also do
this in pus in the live animal)
Culture
streptococci have fairly exacting growth needs and need blood, serum or a fermentable carbohydrate to
grow (e.g. glucose)
Streptococci are more difficult to grow than some of the other pathogenic
KEY POINT bacteria. They MUST have SERUM in the media to survive. Therefore
primary isolation of these bacteria should always be on blood agar plates.
Streptococci will also frequently grow better in 5% CO2

colonies are small; about 1mm after 48 hours, which helps differentiation from staphylococci
Identification
the catalase test is useful for differentiation between Staphylococci (positive) and Streptococci
(negative)
further biochemical tests may be required to identify the species of Streptococci
Identification of species of Streptococci is NECESSARY in some cases for

KEY POINT implementation of appropriate treatment or control measures.
33
an example of when identification of the streptococcal species is required is if you are presented with
a horse with lymphadenitis
it is important to differentiate S. equi ss equi from other beta haemolytic streptococci that may cause
lymphadenitis e.g. S. equi ss zooepidemicus, or S. dysgalactiae ss equisimilis, as S. equi ss equi is
contagious (therefore control measures would differ), and strangles is notifiable
similarly, S. agalactiae should be differentiated from other streptococci causing bovine mastitis as the
methods of control for these organisms differs
see the recommended texts for biochemical tests required for differentiation (sugar fermentations tests)
commercial kits are available for Lancefield grouping which may be of help in determining the
significance of the Streptococcal isolate (ie differentiating pathogenic from commensal species)
the main defence against streptococcal infection is phagocytosis, therefore antibodies to the anti-
phagocytic molecules produced by streptococci are protective
for example animals recovered from strangles and cervical lymphadenitis are at least temporarily
immune to re-infection due to the production of antibodies to M protein (immunity is often, but not
always, long term)
similarly, the anti-phagocytic polysaccharide capsules of S. agalactiae and Str. pneumoniae evoke the
formation of opsonising antibody and in cases of streptococcal pneumonia, the appearance of these
antibodies determines recovery from infection
in bovine mastitis – no useful immunity develops and cows, unless treated, remain infected
however, experimental evidence exists to suggest that anti-capsular IgG2 antibody is protective
all immunity is serotype specific
General Therapy
localised suppurative conditions are treated as for staphylococcal infections
abscesses must be drained of pus if possible
maintenance of adequate drainage during healing is important
extensive, inaccessible and disseminated processes require systemic antibiotic treatment
Antibiotics
if systemic therapy is required - beta-haemolytic streptococci are predictably sensitive to penicillin,
which is the drug of choice in most instances
KEY POINT Beta haemolytic Streptococci are PREDICTABLY susceptible to PENICILLIN.
other antibiotics to which they are commonly (but not always) sensitive include ampicillin,
cephalosporins, erythromycin, chloramphenicol, and trimethoprim-sulphonamides
they are frequently resistant to aminoglycosides, fluoroquinolones, and tetracyclines
enterococci and alpha haemolytic streptococci are more resistant to all of these drugs (vancomycin
resistant enterococci – VREs – are a big problem in hospitals; and occasionally in veterinary medicine)
34
FIGURE: A bottle of Procaine Penicillin – the drug
of choice to treat beta haemolytic Streptococci
Mastitis
intramammary (topical) therapy is effective for treatment of mastitis due to Str. agalactiae and most
other streptococci
unlike staphylococcal infections, intra-lactational therapy is frequently successful for mastitis due to
streptococci due to the acute, and short lived nature of these inflammatory processes
however, important aspects of mastitis control lie in the area of sanitation and herd management
Strangles
inappropriate or inadequate therapy of strangles may prolong the illness; however suggestions in the
literature that antibiotic therapy causes “bastard strangles”, has no current justification
there is considerable controversy regarding the optimal treatment of horses with strangles, but
treatment of horses with strangles can be put into into 4 categories:
1. recently infected horses that are febrile but have no obvious abscessation of their lymph nodes
should be treated with a 3-5 day course of penicillin
2. horses with evident abscess of their lymph nodes, should have the abscesses drained (if possible –
if not the abscess should be “encouraged to mature” and then drained). These hoses should NOT
be given penicillin as it prolongs the disease process
3. horses with evident abscesses that cannot be drained but are causing problems (eg retropharyngeal
abscesses that are occluding the trachea; or large internal abscesses) will need antibiotic for
prolonged periods of time (ie 6 – 8 weeks) and nursing therapy
4. horses that have been in contact with cases of strangles should be monitored for development of
fever (twice daily) and started on penicillin if fever develops
35
How would I Control/Prevent the Disease?
beta-haemolytic streptococci can survive in dried pus for weeks and for months in dry, dusty
conditions, which is important for control of the contagious streptococci (e.g. strangles and S.
agalactiae mastitis)
they are readily killed by most disinfectants and by 55-60°C for 30 minutes
Control of Strangles – Isolation and Quarantine Measures

horses suspected as having strangles MUST BE ISOLATD from other horses to prevent transmission
of the disease
horses should be isolated as far as possible/practical from other susceptible horses, but minimally a
distance of 10 m (and definitely not in nose-to-nose contact!)
it is also very important to stop transferring the organism on fomites (hands, clothes, shoes, halters
etc) so separate equipment should be used for isolated horses
buildings and equipment in contact with affected horses be disinfected (eg with bleach or
chlorhexidine) before re-use on susceptible individuals
horses may “carry” the organism in the URT and guttural pouches for prolonged periods (up to 6
weeks is common) after cessation of clinical signs; therefore it is recommended that quarantine be
maintained for at least this period of time (this is NOT liked by the horse owners!!!)
vaccines – a whole-cell bacterin and an M protein vaccine are available for vaccination against
strangles - neither is uniformly effective
an intranasal avirulent live vaccine that stimulates local antibody production is available in the US (but
not Australia), and appears promising, however, abscess production at other sites has been reported
with the use of this vaccine in conjunction with other vaccines
populations at risk may warrant vaccination to decrease morbidity and severity of the disease
to prevent outbreaks from occurring it is recommended that all new horses introduced to a property by
isolated for 10 days and monitored for signs of infection
Jowl Abscesses
immunity to porcine jowl abscesses has been produced by feeding live avirulent strains, but this is not
commercially produced
a preventative regimen for this condition in pigs includes tetracycline administration in feed (220mg/kg
feed)
animal streptococci have limited public health significance as there is rarely transmission of
streptococci directly from animals to humans (or vica versa)
the group A streptococci are significant causes of disease in humans, but there is little evidence that
dogs or cats (or other animal species) are long term reservoirs for infection of humans
nevertheless, it is generally recommended that household pets are treated at the same time as all
members of the household in cases of recurrent infection of children
the Group B streptococci that cause disease in human infants are apparently distinct from bovine
strains, but infections with S. zooepidemicus (group C) have been traced to infected milk
similarly, the group G streptococci affecting dogs (S. canis) are apparently different from group G
streptococci affecting human patients
however, S. suis (serotype 2) has caused serious infections in swine handlers, abattoir workers and
women including meningitis, arthritis, septicaemia, diarrhoea and ear infections
therefore it is important if dealing with suspected cases of this disease that precautions are taken to
prevent infection
36
Proposed Role of Pets in transmission of Group
A Streptococci in humans
Exposure to Group A Streptococci

at School or Day Care Centre
Pet remains infected Exposure of family

and pets who become infected
Child treated by Doctor
Diagram reproduced from “Infectious Diseases of the Dog and Cat” C.E. Greene ed.
37
ENTEROCOCCI
Summary Table: Species of Enterococci that have veterinary importance, their animal hosts, and the
diseases they cause
SPECIES HOSTS Disease

E. faecium humans, dogs, urinary tract infections, bacteraemia, endocarditis,
cats, horses, intra-abdominal and pelvic infections, wound and
ruminants, birds soft tissue infections, neonatal sepsis and rarely
etc meningitis
E. faecalis many animal spp as above
E. hirae many animal spp as above
38
Enterococci
What are they and what Diseases do they Cause?
Enterococci are gram positive cocci that are morphologically identical to Streptococci
this genus includes members that were previously classified as group D streptococci, but now have
their own genus which includes:
9 E. faecalis
9 E. faecium
9 E. hirae
these bacteria are normal residents of the GIT and biliary tracts and in lower numbers in the vagina
and male urethra in humans and animals
enterococci are not as virulent as other streptococci, but they are becoming increasing important agents
of human disease, largely because of their resistance to antibiotics to which other streptococci are
generally susceptible
therefore enterococci are important causes of nosocomial and community-acquired infections, and are
now the second most common cause of nosocomial infections in the USA
vancomycin resistant enterococci (VRE) are of particular concern due to their resistance to this
important group of antibiotics, and which is usually associated with widespread resistance to other
antibiotics
VRE have been isolated from dog food in the US!!!
unlike the streptococcal species, the factors that determine pathogenicity of the enterococci are not well
understood
in humans, enterococci have been associated with complicated urinary tract infections, bacteraemia,
endocarditis, intra-abdominal and pelvic infections, wound and soft tissue infections, neonatal sepsis
and rarely meningitis
the same syndromes have been reported in domestic species, though less frequently than in humans
(infections in dogs are the species that they are most commonly isolated, particularly complicated
urinary tract infections; increasingly they are being isolated from cases of bovine mastitis)
in addition, E. faecalis has been associated with urinary tract infections and endocarditis in chickens
39
40
SECTION 2
Gram Negative Rods
41
42
ENTEROBACTERIACEAE
Bacteria in the family Enterobacteriaceae are gram negative, oxidase negative rods. They are very
commonly causes of disease in all domestic species (E. coli is the most common pathogen isolated from
most domestic species AND humans!). Many of these bacteria are part of the normal flora, particularly of
the gastro-intestinal tract. In addition, they are frequently found in the environment due to faecal
contamination and their ability to survive in the environment for long periods of time. Therefore they are
in an ideal position to be able to invade and cause disease. They have a multitude of virulence factors that
aid disease production, in particular some specific exotoxins that act in the GIT, as well as endotoxin. As
a result, they can cause a WIDE variety of diseases in a wide variety of organs, particularly E. coli.
Diarrhoea is one of the most common diseases these organisms cause, and in production animals they are a
significant cause of neonatal losses. Treatment of systemic diseases can be hampered by the fact that they
do not have a predictable antimicrobial sensitivity pattern and may be resistant to a number of commonly
used antimicrobials.
Summary Table : Genera within the family Enterobacteriaceae that have veterinary importance, their
animal hosts, and the diseases they cause.
GENUS +/- SPECIES HOSTS DISEASE
Escherichia coli++++ All animals, birds, fish, etc Major diseases include enteritis (see table p22 + p27 for
details), septicaemia, cystitis, pyometra, mastitis,
osteomyelitis, etc, etc
Salmonella+++ All animals, birds, insects etc Enteritis, septicaemia (and localisation), abortion,
Klebsiella++ Dogs, cows, horses, mink Pneumonia, mastitis, endometritis, wound infections etc,
Proteus++ Dogs Cystitis, otitis externa, wound infections, diarrhoea (?)
Enterobacter+ A variety of animals Septicaemia, occasional infections in a range of sites
Serratia+ A variety of animals Septicaemia, occasional infections in a range of sites
Yersinia+ Ruminants, (humans) Dysentery (Y. pestis causes the plague in humans ++++!!!)
NOTE: the number of + denotes the relative importance of these veterinary species within THIS genus (NOT for all
bacterial genera). Other members of the family Enterobacteriaceae rarely cause disease and therefore have
not been included in this table.
43
KEY POINT The Enterobacteriaceae is a FAMILY not a GENUS.
The genera within this family have a number of common features, which will be discussed before
different genera are discussed individually.
What are they?
all members of the Enterobacteriaceae are large, gram negative rods

as they are gram negative they all have endotoxin as an integral part of their cell wall
As Enterobacteriaceae are gram negative they have ENDOTOXIN as an

KEY POINT integral component of their cell wall. This molecule always plays a role in
the diseases they cause.
they are all facultatively anaerobic

they are all oxidase negative, which is an important biochemical test for differentiating the
Enterobacteriaceae from other gram negative rods
they produce acid plus/minus gas from glucose
there are currently 28 genera and over 80 species within the family Enterobacteriaceae, some of which
have been only recently described (see table later)
HOWEVER, only the genera that commonly cause disease in domestic species will be covered in these
notes
Where are they Found?
many of the Enterobacteriaceae are PARASITES (commensals) of HUMANS and ANIMALS and live
as part of the NORMAL FLORA within the GIT (e.g. E. coli)
these bacteria are used to indicate faecal contamination of the environment (especially the coliforms)
others are members of the Enterobacteriaceae are FREE LIVING within the ENVIRONMENT (e.g.
Klebsiella, Citrobacter and Enterobacter)
in addition, most can survive for long periods in the environment regardless of whether they are
normally found on animal hosts or not, contaminating vegetation, soil and water
The Enterobacteriaceae are ubiquitous (they are found in MANY places).

KEY POINT Many of them are also part of the NORMAL FLORA of the GIT.
How do they Enter the Host?
the most common route of infection is INGESTION of the organism that is present in the environment
because of faecal contamination (oro-faecal route)
consequently, FOMITES are very important with regard to transmission of these infections
Oro-faecal transmission is the PRIMARY route of infection for this group of

KEY POINT organisms.
44
other infections arise when the organism directly enters the host e.g. wound infections, especially if
there is faecal contamination
some infections are ENDOGENOUS – the infection arises from bacteria already in the host e.g.
salmonellosis – especially when recurrent
but most infections are EXOGENOUS – the infections arises from bacteria present in the environment
How do they Cause Disease?
some background revision on the structures (antigens) found on the surface of Enterobacteriaceae will
help a discussion of virulence factors (see diagram below)
these structures have an added importance as they are used in serological tests to identify different
“strains” of bacteria within the genera of the Enterobacteriaceae
most of the Enterobacteriaceae have variation in these antigens and therefore we can use them to
identify strains of the different organisms, some of which may cause disease and others not
strains of Enterobacteriaceae are named by the different cell wall structures including:
1. Cell wall = O antigens

2. Flagella = H antigens
3. Capsule = K antigens
4. Fimbrae = F antigens (used to be called P for pilus)
Cell Wall Structures (Antigens) of Enterobacteriaceae
Capsule = K antigens
Flagella = H antigens
Cell Wall = O antigens =

Somatic Antigens
Pilus = F or fimbrial antigens
e.g. K99 (F5), 987P
For Example:
9 E. coli O141:K85:H3 is an isolate with antigens of the 141 serogroup, capsular antigen 85 and flagella
antigen 3
9 examples of “important” strains (serotypes) include E. coli O157:H7, E. coli K99 (which is now
called F5), Klebsiella pneumoniae K5 etc
45
E. coli
the virulence factors of E. coli have been the best described and will be used as the basis for a
discussion of the basic mechanisms for production of disease in the whole family
the basic mechanisms of action for the various virulence factors are:
1. attachment
a. fimbrae (pili)
2. avoidance of phagocytosis
a. capsule
b. O somatic antigens
3. cellular toxicity
a. haemolysins
b. enterotoxins
i. cytotonic
1. Heat Labile
2. Heat Stable
ii. cytotoxic
4. systemic effects
a. endotoxin
1. ATTACHMENT
a) Adhesins (e.g. Fimbriae)

fimbriae (the best characterised of the adhesin molecules and which used to be called PILI) are very
important virulence factors of Enterobacteriaceae as they assist in the attachment of the bacteria to the
surface of mucosal cells in the SI and lower urinary tract
firm attachment facilitates colonisation by diminishing the expulsive effects of peristalsis and the
flushing effect of urination
without attachment disease is usually not produced
Unless the Enterobacteriaceae can attach to the host’s epithelial cells

KEY POINT they usually cannot cause disease!
many members of the Enterobacteriaceae can produce fimbriae/pili (or other types of adhesins)
the fimbriae of E. coli which produce enteric disease are encoded on conjugative plasmids
in contrast, E. coli associated with extra-intestinal disease (particularly of the urogenital tract) have
their pili encoded by DNA located on their chromosome
in general, pili are specific to the disease situation; ie pili required to produce enteric disease do not
help to produce urogenital tract disease
this is because PILI are SPECIFIC for certain CELL SURFACE MOLECULES (Receptors)
E. coli that produce GIT disease have fimbriae [usually with the prefix “K” or “F” (e.g. K99 = F5; or
K88 = F4))] that interact with very specific receptors elaborated on the surface of enterocytes at specific
sites (e.g. ileum) in the small and large intestine, whereas uropathogenic E. coli (“p” or “s” prefix)
have fimbriae that attach to specific receptors on uroepithelial cells
46
E. coli attached to the surface of the
enterocytes through their fimbrae
Enterocytes within the ileum
Fimbrae
FIGURE: An ileum of a calf with colibacillosis (diarrhoea due to E. coli). The E. coli are attached
through their fimbrae to specific receptors on the surface of the enterocytes and can thus adhere
to the surface of the ileum. This process is NECESSARY for disease to occur.
2. AVOIDANCE of PHAGOCYTOSIS
The term “serum resistance” is applied to strains of Enterobacteriaceae (particularly E. coli) that avoid
phagocytosis and are thought to help in the establishment of these organisms within the blood stream
Capsular antigens and O side chains (somatic antigens) play a key role in
KEY POINT “SERUM RESISTANCE” by the Enterobacteriaceae.
a) Capsule
many Enterobacteriaceae produce a polysaccharide capsule ( = K antigen)
there are a large number of different types of capsules within the Enterobacteriaceae (e.g. K1, K5, and
K12 of E. coli)
capsules enhance virulence by:
47
9 preventing activation of complement (they cover the sites required for complement binding and
therefore activation)
9 preventing deposition of Membrane Attack Complexes (MAC) (so cannot get complement
mediated lysis)
9 preventing phagocytosis by neutrophils and macrophages
these features of capsule are particularly useful in the pre-antibody phase of disease
9 specific antibodies to the capsules will bind to the capsule and prevent these mechanisms of
evasion
however, not all capsules of Enterobacteriaceae are immunogenic
9 some are poorly immunogenic due to natural “mimicry” by the capsules of common antigens (e.g.
food substances)
9 animal doesn’t want to make antibodies to these antigens, so little antibody is produced to these
capsules
9 this allows recurrent infections with these organisms to occur (e.g. some strains of E. coli can
cause recurrent septicaemia or bacteraemia)
Diagramatic representation of the capsule on a gram negative bacteria and its relationship with endotoxin
(= Lipid A + core sugars + O side chains)
b) Somatic (O) Antigens

“O” side chains are composed of polysaccharides (sugars) and are an integral part of the LPS
(endotoxin) molecule
they are thought to function in the same way as capsular antigens in their interaction with complement
some of the newer antimicrobial agents are designed to interfere with polysaccharide production –
therefore decrease the effectiveness of the somatic antigens in avoiding the immune system
3. CELLULAR TOXICITY
a) Haemolysins
haemolysins act to lyse RBC and thus increases the availability of Fe++ for growth of the bacteria
these molecules are frequently found in E. coli associated with extra-intestinal disease e.g. meningitis
and oedema disease in pig, but some strains of E. coli, which can cause septicaemia, do not have
haemolysins (they have other mechanisms for scavenging of iron)
b) Exotoxins ( = ENTEROTOXINS)
these are the most studied of the virulence factors of E. coli
the vast majority of enteric disease (diarrhoea) caused by E. coli revolves around production of
enterotoxins
there are a number of different GIT syndromes produced by E. coli depending on the type of
enterotoxin produced (and other factors such as pili, haemolysins etc) – see later in notes
Although most of the gastrointestinal disease manifestation of

Enbacteriaceae are due to the production of enterotoxins, these toxins can
KEY POINT do NOTHING unless the bacteria “finds the right place” to manifest disease
and can attach at this site (ie have pili as well!!!).
there are a two important types enterotoxins associated with disease production in the
Enterobacteriaceae; the cytotonic enterotoxins and the cytotoxic enterotoxins:
48
i) CYTOTONIC ENTEROTOXINS
these toxins do NOT damage the integrity of the cell upon which they act (cell is still physically
intact), and the bacteria that produce them are classified as ENTEROTOXIGENIC E. Coli ( =
ETECs)
Enterotoxigenic E. coli (ETEC) cause diarrhoea without damaging intestinal

KEY POINT cell wall and without producing inflammation.
there are 2 types of cytotonic enterotoxins identified for E. coli, Heat Labile (LT) and Heat Stable
(ST) - see opposite page for mechanism of action
examples of strains of E. coli containing cytotonic enterotoxins:
Pilus Antigens Species Affected O Antigens

K88 ( = F4) piglets O138, O139, O141, O147
K99 ( = F5) calves O149
F41 piglets, calves, lambs O9, O101
REMEMBER: 1. ETECs have to attach (via their pili) before they can cause disease
2. They do not cause cell damage, just a “physiological” hypersecretory diarrhoea
3. This has important implications for therapy
it is important to remember that to determine the significance of an E. coli isolated from a case of
diarrhoea you need to isolate the organism AND demonstrate that the strain you have isolated has pili
and produces enterotoxin (ie that this particular strain is capable of causing disease)
usually demonstration of one of these is sufficient – and most frequently in practice demonstration of
the pilus antigen is performed
you need to do this is as many strains of E. coli are present in animals as part of the normal flora of
the GIT (so you will always isolate E. coli from a faecal sample), but not all animals have strains that
can cause disease
As E. coli are part of the normal flora of the GIT, it is not sufficient to
isolate E. coli from cases of diarrhoea, you must also demonstrate that the
KEY POINT appropriate virulence factors (pili, enterotoxin) are also present in that
strain to determine its clinical significance.
Mechanism of Action of the Cytotonic Enterotoxins of E. coli

for both of these toxins to act they must first bind to receptors on the surface of enterocytes. They are
then translocated into the cell where they can act
Heat Labile Toxin (LT) Heat Stable Toxin (ST)

Size (MW) 70-90kDa 25kDa
Activates ……to ↑…. Adenylate cyclase to ↑ Guanylate cyclase to ↑
intracellular cAMP intracellular cGMP
49
This Causes (this part is the net RESULT of the action of both these toxins is to inhibit
important to learn in order to the uptake of Na+ and Cl- in the villus cells and to stimulate the
understand the secretion of Cl- and HCO3- from the crypt cells. There is
pathogenesis) subsequently a net result of water loss from the enterocytes as
water “follows” these electrolytes along an osmotic gradient
Intestinal Lumen
Villus
Na+
Cl-
+ Efflux
of H20
HCO3- HCO3-
Crypt
Cl- Cl-
A ligated loop of the ileum of a rabbit

inoculated with enterotoxin showing
efflux of water
The effect of both types of enterotoxins produced by ETECs is the NET

EFFLUX of large amounts of WATER from the cells of the intestine into the
KEY POINT lumen of the intestine as well as a LOSS of ELECTROLYTES =
HYPERSECRETORY DIARRHOEA.
ii) CYTOTOXIC ENTEROTOXINS

these toxins DO damage the cells upon which they act, and the bacteria that carry them are classified
as either ENTEROPATHOGENIC E. coli ( = EPECs) or ENTEROHAEMORRHAGIC E. coli (=
EHECs)
the E. coli that produce these exotoxins AND cause disease in domestic species have been less well
characterised than ETECs (the isolates causing disease in HUMANS are better described)
the cytotoxins produced by the EPECs are called Shiga-like toxin as they act in a very similar fashion
to Shiga toxin produced by Shigella dysenteriae serotype 1
they are also called Vero toxins as they are active in Vero cells and HeLa cells
a variant of the Shiga-like toxin is suspected to play a role in the pathogenesis of some diseases caused
by E. coli including oedema disease of swine and diarrhoea in calves and rabbits
50
4. SYSTEMIC EFFECTS
Endotoxin (= Lipopolysaccharide)
endotoxin is an integral part of the cell wall of gram negative bacteria
there are three important components of endotoxin; Lipid A, core oligosaccarides, and O side chains
(see diagram above)
the O side chains are antigenically VERY diverse among and within members of the
Enterobacteriaceae; whereas the core sugars have only minor diversity
these molecules play a major role in diseases caused by the Enterobacteriaceae (especially systemic
diseases) and are frequently the direct cause of DEATH
Endotoxin plays a major role in the pathogenesis of diseases caused by

KEY POINT Enterobacteriaceae (especially systemic diseases) and may directly
contribute to the death of the animal.
Endotoxin and its specific actions will be covered later this year (in Animal Diseases)
II) Pathogenesis
ALL Enterobacteriaceae have a HOST COMPONENT to their disease production
9 Highly pathogenic (major pathogens) Enterobacteriaceae are influenced only slightly by host
factors
- these bacteria can cause disease in hosts that are only mildly compromised
- e.g some strains of E. coli
9 Lowly pathogenic Enterobacteriaceae are influenced greatly by host factors

- need a highly compromised host, and usually large numbers are required to cause disease
(lot of host factors involved in whether disease will occur)
- e.g. most strains of Citrobacter
9 Intermediate (opportunistic) pathogens are in between
Not all of the Enterobacteriaceae can cause disease, therefore

KEY POINT interpretation of the clinical significance of the organism you isolate must
also involve examination of host factors.
51
Pathogenicity of different genera within the family Enterobacteriaceae1
Common or Major Less Common Rare Pathogens Non Pathogenic

Pathogens (Opportunistic) (of humans)
Pathogens
E. coli Klebsiella Hafnia Budvicia
Salmonella Proteus Providencia Cedecea
Yersinia2 Enterobacter Erwinia Ewingella
Pantoea Kluyvera
Shigella (primates) Lecercia
Citrobacter (rarer) Leminorella
Serratia Moellerella
Edwardsiella (fish) Rahnella
Morganella Tatumella
Trabulsiella
Yokenella
Pathogenicity/Virulence Scale
HIGH LOW
Notes:
1
Enterobacteria can be arbitrarily grouped in 3 categories: major pathogens, opportunistic pathogens, and
non-pathogens. Remember, those without pathogenic significance for animals, such as Hafnia and
Erwinia, can be isolated from faeces and the environment and may contaminate clinical samples.
Therefore, it is very important to differentiate between the different genera in order to determine the
clinical significance of an isolate.
2
Yersinia are major pathogens of humans (Y. pestis is the cause of plague), but are less important in
domestic species
What are the Diseases caused by Enterobacteriaceae?
these syndromes are best discussed under individual genera

however, it is important to know that the same clinical signs can be associated with a variety of
organisms within this family
No CLINICAL SYNDROME is DEFINITIVE for a particular member of this family

– a number of different genera can cause similar diseases (ie same clinical
signs). Consequently, any investigation of these diseases requires a
KEY POINT LABORATORY COMPONENT in order to identify the specific causative agent
as this will influence your approach to treatement and control of these
diseases.
52
E. Coli Õ×Øb
E.coli is the bacterial species most commonly recovered in human clinical laboratories (and probably
veterinary) and has been incriminated in infectious disease involving virtually every human tissue and
organ system
E. coli can causes disease VERY well and are therefore VERY common causes
KEY POINT of infection in ALL species!!!!
but be careful ⇒ not all strains of E. coli can cause disease
Not all E. coli can cause disease. Therefore, interpretation of the

significance of the E. coli that is isolated from a clinical case still requires
careful examination of:
KEY POINT 1. Where it came from
2. Which virulence factors it contains
3. Its antigenic makeup
the diseases caused by E. coli are broadly grouped into:
1. Enteric Diseases
2. Non-Enteric Diseases
I) ENTERIC DISEASES
a) GROUPS of E.coli CAUSING DIARRHOEA and the MECHANISM OF DISEASE

from the stand-point of pathogenic mechanisms of disease, 4 major groups of E. coli are recognised:
i. Enterotoxigenic (ETEC)
ii. Enteropathogenic (EPEC)
iii. Enterohaemorrhagic (EHEC)
iv. Enteroinvasive (EIEC)
In addition, 2 less well defined groups of E. coli have been recognised:

i. Enteroaggregative (EAEC)
ii. Cytotoxin Necrotising Factor-positive (CNF-PEC)
i) Enterotoxigenic E. Coli ( = ETECs)

the vast majority of cases of E. coli diarrhoea in domestic species occurs in young animals and are due
to infection with ETECs
ETECs first need to attach (via pili) to produce disease
they produce cytotonic enterotoxins (see earlier in your notes)
9 Heat Labile Toxin (LT)
9 Heat Stable Toxin (ST)
these exotoxins do not result in cell damage but cause physiological changes and result in
hypersecretory diarrhoea
they are usually members of the O8; O9; O64; O101 serogroups and produce K- 88 (also called F4),
K-99 (also called F-5) or F41 pili for adherence to small intestine (ileum)
they mostly produce disease in young animals (calves, piglets, lambs, foals + puppies (?)
53
9 (note the ? is because ETECs are thought to be involved in foals and puppies but have not been
definitively proven)
to determine the significance of an isolate of E. coli from cases of diarrhoea in neonates, you must
identify the appropriate virulence factors (pili, enterotoxin).
The vast majority of cases of E. coli diarrhoea in domestic species occur in

KEY POINT YOUNG ANIMALS and are dues to ETECs!
FIGURE: The three little (dead)

pigs that died of diarrhoea due to
E. coli. Note their extremely
dehydrated appearance (they are
shrivelled up!!!) due to the large
loss of water and electrolytes in the
hypersecretory diarrhoea.
FIGURE: The post mortem of

one of these little piglets. Note the
distension of the small intestine
due to the efflux water associated
with this disease.
Distended loops of small

intestine (ileum)
ii) Enteropathogenic E. Coli ( = EPEC)

the term enteropathogenic E. coli (EPEC) is used rather loosely in veterinary medicine to refer to
some strains of E. coli that cause intestinal infections in animals (also called attaching/effacing E. coli:
AAEC)
however, by definition, EPEC refers to specific serogroups which cause diarrhoeal syndromes of
humans
these strains attach to small intestinal epithelial cells (via an adhesin called intimin) and cause
destruction of microvilli, atrophy and shedding of
enterocytes, and stunting of villi, though the nature of the toxins of EPECs is uncertain (not LT or ST)
54
this pathology results in maldigestion and malabsorption
EPECs are reported to cause diarrhoea in calves (especially in dairy operations in the U.S.) and piglets,
lambs, pups, kittens and rabbits
iii) Entero-invasive E. Coli ( = EIEC)

as the name suggests – these E. coli can actually invade the mucosa of the colon, causing an
inflammatory reaction which results in a hypersecretory diarrhoea (this is mediated by prostaglandins
and therefore is a different mechanism to diarrhoea caused by ETECs)
this mechanism is very similar to that of the Salmonella serotypes – and EIEC must be differentiated
from salmonella
these bacteria produce diarrhoea and dysentery (this occurs because there is actual damage to the
mucosa and so sloughing of the lining of the gut occurs) which is different to ETECs where there is
NO dysentery
there are certain serogroups associated with this disease (e.g. O28, 0124, 0136; O111), and many of
these E. coli’s DON’T have K antigens
a plasmid that encodes proteins that are required for “invasiveness” has recently been identified and
these proteins are antigenic – so disease can be prevented if the animals has the “right” antibodies
FIGURE: The
intestines of a calf
that has died due to
infection with
enteroinvasive E.
coli (EIEC). These
organisms can invade
the intestinal lamina
propria, with
associated cellular
damage and
necrosis. Note the
presence of catarrhal
diarrhoea as seen by
sloughing of the
mucosa resulting in
loss of fluid and
electrolytes from
the enteric surface.
iv) Entero-Haemorrhagic E. Coli ( = EHEC) or Verotoxigenic E. coli (VTEC)

these E. coli colonise the intestinal mucosa by intimate attachment to cells of the microvilli
the disease that these E. coli causes in humans includes Haemorrhagic Colitis
this disease is very important as it may progress to Haemolytic Uraemic Syndrome (HUS) or
Thrombotic thrombocytopenic purpura (TTP), which are bad diseases in people (they have a high
mortality rate)
the major role that animals play is as the primary RESERVOIR of these bacteria (mostly cattle, and to
a lesser extent pigs)
EHEC has been found in up to 4% of uncooked meat products in the USA also in water contaminated
with animal faeces
virulence of the EHEC is due to production of fimbriae and exotoxins (which are called verotoxin type
1, 2 and 2e (VT1, VT2, VT2e)
these exotoxins are enterotoxic, cytotoxic and neurotoxic
55
when absorbed into the bloodstream, these toxins are cytotoxic for endothelial cells in relatively
defined locations (e.g CNS in pigs, kidneys in humans)
vascular damage can lead to oedema, haemorrhage and thrombosis
the EHEC that cause disease usually belong to certain O and H antigens e.g. O157:H7; O87:H16;
O157:H-; and O111:NM (non motile) – of these E. coli O157:H7 is by far the best known
disease with EHECs is usually seen in children, however, elderly patients are also at risk, and EHECs
are the leading cause of acute renal failure in children
EHECs usually cause disease as isolated cases, but large outbreaks are sometimes recorded (one
outbreak in the US had 582 confirmed cases of haemorrhagic colitis (HC) with 171 hospitalisations, 41
cases of HUS and 4 deaths – all attributed to hamburgers from a single fast food restaurant chain!!!
syndromes similar to HUS have been described in cows, horses and dogs – but are thought to be
uncommon (but this may be because the syndrome is under-diagnosed – most vets don’t look for it –
and certainly don’t isolate E. coli from faeces and serotype them to see if these bacteria are involved)
in addition, strains of EHEC are involved in Oedema Disease of pigs (see later in notes),
haemorrhagic enterocolitis in calves, and post-weaning diarrhoea in pigs (see later)
v) Enteroaggregative E. Coli (EaggEC)

not invasive and only causes disease in humans
vi) CNF-EC (Cytotoxic Necrotising Factor E. coli)

these are newly described strains which have been attributed to enteritis in humans, calves and piglets,
haemorrhagic colitis in cattle, diarrhoea in rabbits, and dysentery in horses
these strains have also been isolated from septicaemia and urinary tract infections in animals and
humans
2 types of cytotoxic necrotising factors (CNF1 and CNF2) have been described, but their role in the
pathogenesis of infections is poorly understood
HOST, BACTERIAL and ENVIRONMENTAL FACTORS Influencing Production of

ENTERIC DISEASE
as with any bacterial disease, there are a number of factors that inter-relate to result in the production
of disease:
Host/Bacteria/Environmental interactions play and IMPORTANT ROLE in

KEY POINT determination of disease production by E. coli.
1. Bacterial Factors
a) Fimbriae – must be present for disease to occur
b) Enterotoxin/Cytotoxins – must be present for disease to occur
2. Host Factors
a) Age
the enterocytes in the small intestine of calves express the cell surface receptor required for attachment
of fimbriae only during their first week of age, whereas piglets retain receptors for some adhesins past
weaning (post weaning diarrhoea)
consequently, most disease caused by ETECs in calves occurs during the first couple of days to one
week of life
in older calves, ETECs usually cannot attach to the enterocytes (so can’t cause disease), but co-
infection with rotaviruses may allow ETEC to cause disease in older calves as the virus damages
enterocytes and when these cells regenerate the receptors are re-expressed
56
b) Immunity
antibodies (Ab) produced against fimbriae can attach to these adhesins and prevent adherence of E.
coli to enterocytes, therefore preventing disease
however, Ab can only attach to specific fimbriae and there is no cross protection between different
types of fimbriae
remember, neonatal calves and piglets are immunologically naïve, and require Ab from:
Colostrum
9 if specific anti-fimbriae Ab are present in colostrum, they are first absorbed (during passive
transfer of immunity) and then are resecreted from the systemic circulation back into the GIT (this
is known as “trafficking of maternal cells and antibody”)
9 these Ab can then bind to the fimbriae and prevent adherence to the enterocytes
Milk
9 Ab continue to be secreted in milk after the cessation of colostrum production, though in lower
concentrations
9 these Ab are not absorbed; thus if anti-fimbrial Ab are present, they can attach to the bacteria
present in the GIT and so prevent adherence
3. Environmental Factors
a) Numbers
as with all infectious diseases, the numbers of E. coli to which an animal is exposed is important
thus, the more pathogenic bacteria that an animal is exposed to, the more likely that these bacteria will
be able to overcome host factors and cause disease
therefore overcrowding and poor hygiene are RISK FACTORS for development of disease as they
allow a build-up in the numbers of pathogenic E. coli strains and facilitate further transmission to other
susceptible animals
b) Housing and Nutrition

stress factors such as cold ambient temperatures and frequent mixing of animals also facilitates disease
production
poor nutrition (starvation, incorrect milk diet) also encourage disease
FIGURE: A YOUNG woolly calf

(his mum is a Highland cow and
you would need this much hair if
you lived in Scotland too!). He
would be susceptible to enteric
disease caused by E. coli
(colibacillosis) due to his young
age (2 days old). This is
particularly the case if there is poor
management practices such as
overcrowding, inadequate food,
poor colostrum intake and
inadequate shelter provided by
the farmer.
57
c) SPECIFIC ENTERIC DISEASE SYNDROMES caused by E. COLI
i) Colibacillosis ( White Scours) in Calves

almost always occurs in calves that are less than 1 week of age (mostly 1 – 2 days)
infection arises from oral ingestion of sufficient numbers of pathogenic E. coli and results in profuse
watery diarrhoea (hypersecretory)
faeces may be profuse and watery, or pasty, whitish and rancid (and stick to their tails!)
diarrhoea results in dehydration and electrolyte imbalances (see earlier notes for mechanism of this
diarrhoea) which may become severe and life-threatening
in these cases the calves are profoundly depressed, inappetent, and frequently recumbent
untreated, severely affected calves die within a few days, but mildly affected calves may recover
spontaneously
ii) “Preweaning Diarrhoea” in Piglets

as with calves, this disease occurs in the very young piglet, usually in the first 12 to 24 hrs of life, and
often the entire litter is involved
piglets have profuse watery diarrhoea leading rapidly to severe dehydration, weakness and death
this diarrhoea is also found predominantly in piglets that have been born to sows without antibodies to
the specific pilus antigens that allow E. coli to attach to enterocytes (i.e. K88, sometimes K99, and
F41)
therefore, as with the disease in calves, it is good to vaccinate the sows with pilus vaccines before
farrowing
however, it can be difficult to incorporate all the pilus antigens into one vaccine (to ensure protection
against all possible strains) AND it is difficult to ensure that piglets receive both colostrum and post-
colostral Ab in the milk to be protected (if the piglet doesn’t suck very well – it is less likely to be
protected – that’s why runts die!!!)
outbreaks of diarrhoea have been reported due to “new” strains of E. coli that have not been
incorporated into vaccines
Pre-weaning diarrhoea in calves and piglets are common diseases and are
KEY POINT usually related to management factors.
iii) “Post Weaning Diarrhoea”

this syndrome is observed in older piglets (and calves), usually within a week or two post weaning and
associated with the change in diet and feeding regimens
thought mostly to involve ETECs - but others types of E. coli, such as EHEC and EPEC may be
involved as well as rotaviruses (see earlier notes for mechanism)
the reason why there is a burst of “infection” at this time is that weaning removes the influence of Ab
in the milk, just as colostral immunity (passive transfer) is waning
the ETECs have been in the GIT of the young animal the whole time, but were previously unable to
cause disease as Ab were attached to their fimbriae not permitting them to attach to the enterocytes
once these Ab are gone – the bacteria can attach and cause disease
clinical signs vary from afebrile disease with inappetence, to watery diarrhoea in severe cases
some animals may die suddenly
these cases of diarrhoea can be difficult to control, as the calves/piglets are frequently too young to
have developed their own immunity to these bacteria at the time of weaning and inducing sufficient
immunity is difficult due to interference with maternal antibodies
management is the critical issue (decreasing environmental contamination with large numbers of
bacteria)
58
iv) Oedema Disease of Swine
this is another diseases of weaner/grower pigs and usually occurs around 6 to 14 weeks of age (1-2
weeks post weaning)
outbreaks of this disease can occur or there may just be a sporadic cases, but it usually occurs in the
larger and faster growing pigs
this disease is caused by haemolytic/verotoxic (EHEC/VTEC) strains of E. coli which live in the
intestine
these bacteria produce the verotoxin called VT2e which is absorbed into the bloodstream from the
GIT, where it damages the endothelial cells of blood vessels, causing vascular damage and
perivascular oedema (as fluid leaks out of the damaged blood vessels)
consequently, you see OEDEMA as the major sign associated with this disease
9 clinical signs include oedema of the eyelids (most pronounced) and front of the face, ataxia,
convulsions, muscular tremors, posterior paresis and flaccid paralysis then death (typically within
36 hours of onset of signs)
9 on PM will see oedema of the subcutis, mesentery of the colon and greater curvature of the
stomach (classical sites for oedema)
this disease classically occurs during the post weaning period and is thought to be associated with
certain management procedures (the disease has a complex aetiology)
the procedures associated with disease are predominantly thought to be feed related (including changes
in type or amount of feed), but can also occur with vaccination, changes in temperature, changing pens
(basically stress related factors)
these factors are thought to trigger oedema disease by allowing bacterial proliferation, production of
toxin and then absorption of toxin – but the exact pathogenesis is not well defined
consequently, protection is aimed at hygiene, and managing the immediate post weaning period to
decrease stress and therefore the chance of triggering this disease
FIGURE: A (dead) pig with

OEDEMA DISEASE. The
first picture shows the head
of the pig and the second
Marked picture the spiral colon.
subcutaneous Note the oedema of the
eyelids (they are swollen).
oedema
The skin on the head has
also been sliced open to
show the extensive
subcutaneous oedema.
There is also extensive
oedema in the wall of the
colon.
59
Oedema in
the wall of
the colon
2. NON-ENTERIC DISEASES caused by E. coli

i. SEPTICAEMIA (Colisepticaemia) ×Øb Õ
very few E. coli find their way from the GIT into the bloodstream and usually septicaemia is observed
in neonates (particularly those with failure of passive transfer)
the bacteria are usually acquired directly (ie inoculated directly into the animal) such as via the
umbilicus (= “navel ill”), through the intestine (secondary to enteritis), lungs (secondary to
pneumonia), or wounds, particularly when combined with wet conditions (increase maceration) and
necrotic tissue
if the E. coli is serum resistant (ie is the correct serotype) the organism can then multiply in the blood
stream and produce disease:
9 either septicaemia/toxaemia
9 or it may localise in a variety of sites and produce localised infections there e.g. joints, lungs,
kidneys – sites where there are small capillaries to lodge in
the clinical signs will depend on whether the organism has caused an overwhelming septicaemia or
whether it has localised
clinical signs include
9 if animal is septicaemia - may see fever, depression, weakness, tachycardia and possibly diarrhoea
9 hypothermia and prostration precede death, which may occur within 24 hours
9 if animals survive septicaemic phase, frequently post-septicaemic localisation in joints results in
swelling, pain, lameness and stiff gaits in an otherwise relatively happy animal
these syndromes are particularly common in young animals with Failure of Passive Transfer, as
maternal antibodies are needed to protect the neonate from these bacteria (as long as the mothers have
been exposed to the bacteria at some stage – preferably not that long ago – so that they have developed
specific antibodies)
however, there is also a significant management component to these diseases
therefore prevention involves making sure the young animals have appropriate passive transfer, and
also preventing other stressors like exposure to adverse environmental conditions and overcrowding
susceptible animal species includes foals (E. coli is the most common cause of septicaemia in foals
world wide) calves, piglets, lambs (septicaemia is more common than enteric diseases in lambs and is
called “watery mouth”), puppies and kittens
it should be emphasised that septicaemia really is a disease of young animals (EXCEPT in POULTRY
–see later in notes)
in older animals, some compromise (e.g. immuno-compromised, surgical procedures, old age) is
usually needed for septicaemia to occur
Please Note: this doesn’t make young animals immunocompromised – just immuno-naive
60
FIGURE: A young calf
with
COLISEPTICAEMIA.
The calf was presented to
the veterinarian with
severe depression and
inability to stand or
suckle. It was also
markedly dehydrated
(because it couldn’t suck).
The calf died 2 hours
after presentation despite
the onset of therapy.
ii) CYSTITIS (= Urinary Tract Infections - UTI) ×

cystitis is an important disease of dogs (particularly young female dogs)
E. coli is by far the most common organism associated with UTI (about 40% of cases of canine
cysititis – Staphylococcus spp next with 15%, and about 20% are mixed infections)
these are usually ASCENDING infections with the causative organisms living as part of the normal
flora in the vagina, prepuce and distal urethra
disease occurs when there is bacterial colonisation of the genitalia, migration along the urethra, and
adherence to uroepithelium of the bladder (see below)
there are 3 major factors involved in the development of UTI:
1. Bacterial virulence factors
2. Numbers of bacteria
3. Host factors
1. Bacterial Virulence factors
Fimbriae (Pili)
as discussed earlier, the serotypes of E. coli that produce disease in the bladder are different to those
producing disease in the GIT
in particular, the fimbrial antigens differ as they need specific types of fimbriae to attach to the uro-
epithelium of the bladder wall (usually called “p” fimbriae)
attachment to the uroepithelium prevents the bacteria from being washed away during voiding
(urination) and allows bacterial proliferation to occur between voiding (considered to be the most
important factor in the production of UTIs)
K antigens (Capsules)
capsular antigens increase invasiveness and interfere with opsonisation and phagocytosis of bacteria by
transitional epithelial cells allowing bacteria to hang around and cause disease
61
O antigens
have been shown to decrease smooth muscle contractility, which will affect urethral peristalsis and
may allow bacteria to ascend to kidneys
Production of Ureases
some of the bacteria that cause UTI e.g Proteus, Staphylococcus but NOT E.coli, produce ureases
which can hydrolyse urea to produce ammonia
ammonia production results in an increase in urinary pH (which goes from acidic <7 which is normal
in the dog - to alkaline urine >7)
low pH of normal urine is an important defence mechanism of the urine in carnivores as it is
antibacterial
in addition the high pH occurring in the urine can damages glycosaminoglycans which protect the
mucosa from adherence of bacterial pathogens
the high pH also decreases struvite solubility allowing struvite crystals to form and deposition, which
can results in struvite calculus formation (that’s why struvite urolithiasis primarily occurs in young
females – 60% of uroliths in dogs are struvite)
the fact that E. coli does NOT produce ureases, therefore does NOT affect pH, may be used as an
indicator that E. coli (rather than Staphylococci or Proteus) is involved
2. Numbers of Bacteria
the number of bacteria isolated from urine are important for interpretation of cultures of samples
collected from dogs with suspected UTI
in healthy animals, a low number of bacteria can reach the bladder, however, these are rapidly
cleared, especially during micturition (voiding/urination)
therefore, if urine is collected by cystocentesis, very few bacteria should be isolated
however, if urine samples are collected by catheterisation or with a mid-stream voided sample, larger
numbers of bacteria may be isolated due to contamination by bacteria present in the lower urogenital
tract (distal urethra/vagina/prepuce)
in these cases, the number of bacteria isolated, together with the type of bacteria and the presence of
inflammatory cells, can assist in the interpretation of their significance
see laboratory notes for more details
FIGURE: The number of colonies

on a plate indicate the number of
bacteria present in the urine (a
colony is a clonal expansion of one
bacterium). This is only true if the
sample has been collected correctly
(ie no faecal contamination) and
processed correctly (quickly).
Enumeration of bacterial colonies is
useful for indicating the likelihood
of contamination versus infection.
In this case, there are many colonies
present, and only one type of colony
(pure culture). This is strong
evidence (along with the presence of
inflammatory cells and bacteria in the
urine) that a true infection is
occurring.
62
3. Host Factors
for UTI to occur, something is needed to alter the usual defence mechanisms of the urinary tract to
allow bacteria to ascend to the bladder and cause disease
these defence mechanisms include:
9 Voiding - is the most efficient natural defence mechanism against UTI where mechanical washout
as a result of complete voiding is responsible for removing greater than 95% of non-adherent
bacteria that gain access to the urinary bladder
9 Urethral contractions
9 Decreased epithelial receptor sites in proximal and mid urethra
9 Colonisation of urethra and genitalia with normal flora (don’t allow pathogens to colonise)
9 Mucosal secretions (contain antibody)
9 Valve-like nature of the vesiculo-ureteral junction (stops bugs going higher)
in addition the length of urethra and bacteriostatic prostatic secretions make UTI much less common in
males (of all species!!!)
in females, predisposing factors are often not required for initial UTIs to occur
whereas in male animals, or in females with recurrent cystitis, there needs to be some predisposing
factors that allow these infections to occur (e.g. things that cause interference with normal micturition,
anatomical defects, damage to mucosal barrier etc)
9 Please Note: Proteus mirabalis does exactly the same thing as E. coli when causing urinary tract
infections
iii) CANINE PYOMETRA ×

E. coli is a very important pathogen associated with pyometra in the dog
in dogs, progesterone stimulates the growth and secretory activity of endometrial glands, which in
some dogs can result in cystic endometrial hyperplasia (CEH)
oestrogen can also increase the numbers of progesterone receptors of endometrial cells, therefore dogs
on oestrogen therapy (to prevent pregnancy) have an increased risk of CEH (and pyometra)
these 2 conditions are related as CEH predisposes to bacterial invasion and therefore pyometra
there are specific E. coli (not well defined as far as their serotypes goes) that have an affinity for
canine endometrial cells which have been stimulated by progesterone
consequently, it is usually during metoestrus (= dioestrus) that you get CEH/pyometra, when you
have the luteal phase and there is increased progesterone levels
E. coli is usually the first organism to invade, but it causes damage to the uterine epithelium and
consequently allows other bacteria (including anaerobes which are part of the normal flora of the
vagina) to ascend and become involved in these infections
the pyometra may remain open or the cervix can close causing a closed pyometra and accumulation of
lots of pus – in these cases the E. coli may die and you may only be able to isolate anaerobes from the
pus filled uterus, but it was the E. coli causing the problem in the first place (not the anaerobe)
another condition observed in dogs with pyometra is renal disease (which is seen clinically as
polydipsia/polyuria) due to:
9 glomerulonephritis associated with deposition of immune complexes in the glomerular membrane
9 medullary washout – due to toxins produced by the E. coli that interfere with the renal tubular
concentrating ability in response to anti diuretic hormone (ADH)
so the thing to remember in canine pyometra, that although the bacteria did not initiate this disease per
se (needed high progesterone and CEH), they may be the cause of death of the dog!!!
Pyometra is a result of high progesterone and cystic endormetrial

KEY POINT hyperplasia (CEH). It most commonly involves E. coli, alone or in
combination with anaerobes.
63
FIGURE: A uterus obtained via surgical excision from “Fifi, the poodle. ”Fifi” was
presented due to signs of depression, inappetence, and polydipsia (drinking too much)
and polyuria (urinating too much). Radiology revealed a markedly distended viscus
(organ) in her caudal abdomen. It was suspected that this was a case of PYOMETRA and
the uterus was excised during surgery. The uterus has been cut open to show the reddish
brown fluid that was found in this distended uterus. The fluid contained many E. coli as well
as a number of different anaerobes.
iv) Septicaemia in Chickens/Turkeys/Ostriches

this is an important disease of birds where E. coli enters the bloodstream (septicaemia) usually via the
respiratory tract
this invasion may result in air sacculitis, pericarditis, coligranuloma (Hjarre’s disease =
granulomatous lesions in the epithelium of the intestine) or fibrinopurulent serositis
birds may transmit infections to their eggs with either transovarial transmission (usually more
commonly associated with Salmonella spp) or infection of the egg at the time of laying
the contamination of the outside of the egg can result in the penetration of the egg shell and yolk sac
by E. coli
furthermore if an incubator is infected, many eggs within the incubator can become infected if there is
aerosolisation of the infecting organism
the resulting infected chicks are born very weak and usually die within the first few days of birth.
v) Mastitis (Coliform Mastitis)

E. coli (and coliforms in general) are one of the most common causes of mastitis in cattle and pigs, but
no specific serotypes have been identified with this disease
coliform mastitis has an important economic impact upon the dairy and pig industries worldwide
the disease occurs opportunistically, where the source of infection is faecal contamination of the skin
of the udder and relaxation of the teat sphincter following milking (in dairy cows) increases
vulnerability to infection
cows with low somatic cell counts are particularly susceptible to infection
a particularly virulent form of the disease is PERACUTE mastitis, which usually occurs around the
time of parturition
there is a massive proliferation of organisms in the udder and release of endotoxin into the bloodstream
resulting in endotoxic shock and frequently death 24 to 48 hours
64
cows will have signs of severe depression, with drooping ears and sunken eyes
mammary secretions are watery and have white flecks
FIGURE: A cow with

PERACUTE
MASTITIS due to E.
coli. Note the
sloughing of the
affected quarter due
to this serious
infection. Yuk!!!
vii) Just about anything!

Just as with humans, E. coli can cause disease in most body systems in most domestic species
KEY POINT E. coli can cause disease in most sites in most species
OTHER ENTEROBACTERIACEAE that CAUSE DISEASE in DOMESTIC SPECIES
Salmonella
Salmonella are the other major organism belonging to the Enterobacteriaceae that cause disease in
domestic species (see table below)
a few notes regarding some specific features of Salmonella will be included to point out the differences
between these organisms and the other Enterobacteriaceae:
Salmonella Serotype Hosts Consequence of Infectin

Salmonella Typhimurium many animal species Enterocolitis + Septicaemia
humans Food Poisoning
Salmonella Dublin Cattle (host adapted) Many disease conditions
Sheep, Horses, Dogs Enterocolitis and septicaemia
Salmonella Cholerasuis Pigs (host adapted) Enterocolitis and septicaemia
Salmonella Pullorum Chicks (host adapted) Pullorum disease (bacillary white diarrhoea)
Salmonella Gallinarum Adult birds (host adapted) Fowl Typhoid
Salmonella Arizonae Turkeys (host adapted) Arizona or paracolon infection
Salmonella Enteritidis Poultry Often subclinical in poultry
Many animal species Clinical diseases in mammals
Humans Food Poisoning
Salmonella Brandenburg Sheep Abortion
Salmonella Abortus-equi Horses (host adapted) Abortion (exotic)
65
What are they?
there are 2 species in this genus

9 this is constantly being changed, but currently the only species of veterinary importance is S.
enterica
9 there are 6 subgroups of this species (don’t worry about them), again the majority of veterinary
importance belong to S. enterica subspecies enterica.
these subgroups are further divided into SEROTYPES (also called SEROVARS) where on last count
there were > 2400 serotypes (based on Kauffman-White schema used to classify these bacteria)
this schema differentiates the salmonella on the basis of their somatic (O) antigens) and flagellar (H)
antigens – and are classically named after the place that they were first identified (e.g. S enterica ss
enterica serotype Heidelberg), or the type of disease they cause (e.g. typhi) or the species that they
cause the disease (e.g. Typhimurium)
for simplicity and convenience, some just call, them by their serotype e.g. Salmonella Typhimurium
(note – there are some biovars of these serotypes – don’t worry about them)
these bacteria occur worldwide, and are parasites of the GIT of mammals, birds, reptiles, fish, insects
etc) – but they are NOT considered to be part of the NORMAL FLORA
they are excreted in the faeces, and while they are parasites of animals they may SURVIVE for long
periods in the environment e.g. in contaminated water, animal feeds, soil, raw meat and offal etc
(these sites can be major sources of infection – fomites are very important in this disease)
in these sites they may survive for up to 9 months (if damp and cool)
in addition, certain serotypes are said to be HOST ADAPTED to a certain species of animals (e.g.
Salmonella Dublin) , whilst other Salmonella are more ubiquitous and are “less reserved” about which
species they infect (cosmopolitan) e.g. S. typhimurium (most common serotype isolated in Australia)
this distinction is important as the serotypes that are “host adapted” are more likely to develop into a
CARRIER STATE in the particular host that it is adapted to – this is important as they act as a
reservoir for infection and may also have a recrudescence of disease
as with E. coli, oro-faecal transmission is the primary route of infection

in additon, Salmonella can survive a long time in the environment (months)
They use a similar range of virulence factors as reported in E. coli (e.g. endotoxin, somatic (O)
antigens, siderophores, fimbriae/pili (adhesins that make bacterial cell more hydrophobic and so allow
it to attach to negatively charged epithelial cells), and exotoxins (e.g. enterotoxins, cytotoxins, porins
– poorly described)
Resistance (R) Factors

strains that have multiple antimicrobial resistance have been desribed and are VERY important,
particularly as far as human health is concerned
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II) Pathogenesis of Infections caused by Salmonella
Salmonella require the same predisposing factors to cause disease; – virulent serotype, high numbers
of bacteria, susceptible animal (young or old, stressed, debilitated, immunocompromised, previous
antibiotic therapy etc) as for E. coli
although many aspects of the pathogenesis of salmonellosis are poorly understood, particularly the
relationship between salmonella toxins and cell damage, some of the general features associated with
virulence are known
the virulence of salmonellae relates to their ability to invade host cells, replicate in them and resist
both digestion by phagocytes and destruction by the complement components of plasma
following adherence to the surface of ileal and large intestinal mucosal cells (via fimbrial attachment),
the bacteria are taken up by these cells where they replicate in membrane bound vesicles, which often
coalesce
the bacteria are subsequently released from these mucosal cells (which may only sustain mild or
transient damage) into the lamina propria of the intestine
in this site (laminar propria), endotoxin from the cell wall of these bacteria (and perhaps other
cytotoxins), initiates a local inflammatory response
this inflammation leads to sloughing of the intestinal epithelium and erosion of the blood vessels
supplying these cells (get casts of epithelial cells in the faeces) which results in diarrhoea (which can
be high volume) and dysentery
endotoxin also mediates the endotoxic shock, which may accompany septicaemia salmonellosis
this ability to survive inside cells (ie. Salmonellae are FACULTATIVE INTRACELLULAR
PARASITES) also enables them to avoid antibodies, complement and many antibiotics whilst inside
these cells
furthermore, the invasive strains are taken up by macrophages and spread via the lymphatic system,
blood stream or both and then usually “hang out” (often for years in the CARRIER STATE) especially
in regional lymph nodes (e.g. mesenteric), spleen and liver (and gall bladder in people)
As with other organism that can survive inside host cells, Salmonella may
KEY POINT cause chronic, recurrent infections due to their ability to avoid host
defence mechanisms.
What Diseases do Salmonella Cause?
There are 3 basic clinical syndromes associated with Salmonella

infections:
KEY POINT i. Enteritis
ii. Septicaemia with localisation in distant sites (e.g. joints)
iii. Carrier State
it is recognised that healthy, adult carnivores are innately resistant to salmonellosis (the disease NOT
the organism)
Enteritis
may be acute enteritis, subacute enteritis, and chronic enteritis
enteritis due to Salmonella infection has been observed in most species of farm animals horses, cattle,
sheep, pigs, horses) irrespective of age
as discussed – it is usually associated with INVASION of the MUCOSA of the LARGE INTESTINE
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acute disease is characterised by fever, signs of depression, anorexia and profuse, foul-smelling
diarrhoea (dysentery), often containing blood, mucus and epithelial casts (due to sloughing of intestinal
mucosa)
dehydration occurs and pregnant animals may abort
on post mortem acute haemorrhage, fibrin, and increased amounts of water in the lumen of the large
bowel are observed (also losses of electrolytes - particularly sodium)
chronic salmonellosis may follow acute disease in pigs, cattle and horses where intermittent fever, soft
faeces and gradual weight loss are observed
in pigs this is often accompanied by the unusual sequelae of RECTAL STRICTURES (occurs in this
species due to unusual anatomy of rectal blood supply)
there are a number of considerations that must be made when trying to determine the clinical
significance of isolating Salmonella from animala with diarrhoea - due to the presence of CARRIERS
for example, if we have an animal with diarrhoea and we isolate Salmonella from its faeces - is this
Salmonella really causing this diarrhoea or is something else causing the diarrhoea, and this animal
was a carrier which has begun to re-excrete the bacteria due to the stress of disease?
consequently, GIT disease associated with isolation of Salmonella may be difficult to accurately
interpret
a second important consideration as far as GIT infections go – is treatment with antimicrobials
there are some that believe that if these cases of diarrhoea are treated with oral antimicrobials you may
“induce a carrier state” as the bacteria that are adequately contained in the intestine are driven into the
circulation causing a carrier (however there is NO verification of this claim)
however, oral antibiotics may disturb normal intestinal flora, increase likelihood of development of
resistance and extend duration of excretion
in general – it is better to treat with replacement fluids rather than antibiotics
Figure: Examples of Types Of Enteritis Associated with Salmonella
Enteritis – catarrhal with casts
Enteritis – catarrhal and haemorrhagic
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Enteritis – haemorrhagic
2. Septicaemia with Localisation

Salmonellae may also cause septicaemia with subsequent localisation of the organism in a variety of
organs
this syndrome predominantly occurs with host adapted species in their correct host – e.g. Salmonella
Dublin in cattle, Salmonella Choleraesuis in pigs, Salmonella Typhimurium in horses, and
predominantly in YOUNG animals
alternatively, septicaemia may result from traumatic injuries and direct injection of environmental
organisms into a tissue or the circulation, or through contamination of the umbilicus (especially in
neonates with Failure of Passive Transfer)
there are 2 basic outcomes which can result from Salmonella in the circulation
9 see septicaemia/toxaemia and perhaps death (often due to endotoxaemia) – this may occur
secondary to enteritis or due to direct inoculation into the bloodstream (primarily in neonates)
9 alternatively, the animal may “contain” multiplication in the bloodstream, but may not be able to
get rid of the bacteria completely, and the bacteria may then localise in end-organs – these animals
will present with signs referrable to the end organ in which the Salmonella has localised e.g.
swollen joints (septic arthritis), pneumonia, renal failure, meningitis, - etc
another scenario that you may see associated with either septicaemia or severe enteric disease, but in
older animals, is ABORTION – which is related to the release of endotoxin – endogenous pyrogen
finally, Salmonellosis is an important disease of birds and manifests as enteric disease, septicaemia or
a mixture of both (most common)
the bacteria associated with these infections include Salmonella Pullorum and Salmonella Gallinarum,
which can infect the ovaries of hens and be transmitted through eggs (as can Salmonella Enteritidis –
but this causes disease in humans rather than birds)
infected young chicks or turkeys huddle under the heat source and are anorexic, depressed and have
whitish faecal pasting around their vents
mortality rates are high and characteristic lesions at post mortem include whitish nodules throughout
the lung and focal necrosis of the liver and spleen
3. Carrier State
this is the final syndrome associated with Salmonella infections, though it is not actually a disease but
it is a very important facet to Salmonella infections as these carrier animals constitute the main source
of Salmonella for environmental contamination - so the importance of the CARRIER STATE cannot
be overemphasised
carriers can have :
9 Persistent Excretion (subclinical infections) – persistence of the bacteria with small numbers being
shed in the faeces
9 Intermittent Excretion (latent infections) – where the organisms are present (e.g in MLN or gall
bladder) but are not excreted. Re-excretion is often prompted by stressful events e.g. parturition,
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hospitalisation, transportation, overcrowding, concurrent disease and may or may not accompany
clinical disease
9 Passive Excretion – no invasion – just organisms passing through the GIT
the rate of carriers is very variable between species and serotypes and also is strongly influenced by
the numbers of organisms in the envrionment (e.g. geography)
the animals that carry Salmonella are asymptomatic at the time of carriage, but may have arisen after
having clinical disease, or may never have had clinical signs
whether an animal exhibits signs of disease, or become carriers, is due to the interaction between:
1) the serotypes and the animal (ie some serotypes are more likely to induce the carrier state in
certain species – host adapted),
2) the age of the animal
3) the number of bacteria ingested
A number of stress factors may reactivate infection in carriers (see list below), causing re-excretion of
Salmonella resulting in either clinical disease or inapparent shedding
FIGURE: The leg of a horse with marked subcutaneous cellulitis. The

tissue has been cut open (don’t worry - the horse is dead!) and there is a
moderate amount of reddish/orange fluid, oedema and haemorrhage in
this site. A pure culture of Salmonella was isolated from this fluid.
Stress Factors which may activate LATENT or SUBCLINICAL SALMONELLOSIS in Horses (and other
species):
Intercurrent infection Transportation
Overcrowding Pregnancy
Water deprivation Oral antimicrobial therapy
Sudden changes in rations altering intestinal flora
Surgical procedures requiring general anaesthesia
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OTHER ENTEROBACTERIACEAE that CAUSE DISEASE in DOMESTIC SPECIES
YERSINIA
Y. pestis is the cause of the plague in humans, cats and rodents
Y. enterocolitica occasionally causes enteritis (winter dysentery) in ruminants, deer, pigs, and wildlife;
causes sporadic abortion in sheep; primarily a human pathogen
Y. pseudotuberculosis causes enteritis in young animals (deer, sheep, goats, cattle, buffalo, pigs) and
subclinical infections in adults; sporadic abortion in cattle, sheep and goats, and focal hepatic necrosis
and septicaemia in guinea pigs, other laboratory animals, and caged birds
infection in small animals involves the mesenteric lymph nodes, with spread from the caseous
abscesses to the liver and spleen in particular
FIGURE: “Fluffy” the cat from the USA. Unfortunatly, “Fluffy” died of the plague!!! This disease is
caused by Yersinia pestis and is a problem in a number of regions around the world, including North
America. There are three forms of this diseases in cats (and people) bubonic, septicaemia and
pneumonic. “Fluffy” had septicaemic form, which is often rapidly fatal. On post mortem evaluation
there is evidence of focal necrotic lesions in the liver (and spleen which you can’t see). This is a
common pathological finding in this disease.
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OPPORTUNISTIC PATHOGENS
this group of enterobacteria rarely cause ENTERIC disease, but may be involved in localised
opportunistic infections in diverse anatomical locations
these bacteria are widespread in the environment (due to faecal contamination) and are available for
opportunity (provided by intercurrent infections, tissue devitalisation, inherent vulnerability of certain
organs e.g. mammary gland associated with milking)
CITROBACTER, ENTEROBACTER, SERRATIA etc

these organisms can cause a variety of opportunistic infections (but uncommonly) in a range of species
e.g. bovine mastitis, septicaemia, wound infections, respiratory tract diseases (usually secondary to
infection by other species)
KLEBSIELLA
K. pneumoniae is the main species of significance in veterinary medicine and is a significant cause of
pneumonia in animals including dogs, horses and calves
it is thought to be associated with sawdust/wood shavings and animals housed on this bedding may
have increased susceptibility
it can also be found transitorily in the upper respiratory tract and lower urinary tract, usually in small
numbers in animals
it is a heavily capsulated organism and is a common secondary invader in pulmonary disease (e.g.
secondary to Bordetella bronchiseptica infection) – but contributes significantly to disease and must be
addressed in treatment plans (particularly as may have widespread antimicrobial resistance)
this organism can cause a range of other diseases in domestic species including:
9 endometritis in mares – particularly capsule type –5 (which may be venereally transmitted)
9 suppurative infections in foals
9 mastitis in cows – particularly those that have been housed on wood shavings
9 wound infections
9 cystitis in dogs
Klebsiella has very mucoid colonies (due to heavy capsulation) and is a

KEY POINT significant cause of pneumonia in domestic species. Furthermore, this
organism may have widespread antibiotic resistance.
PROTEUS
this genus is a significant cause of cystitis in dogs (in a similar same manner to E. coli but additionally
produces UREASES therefore influences pH and crystal production – see earlier notes)
also significantly involved in otititis externa in dogs
it is postulated to cause of diarrhoea in young mink, lambs, calves, goats and puppies
it may also be isolated from a range of infections, e.g. wound, osteomyelitis etc (but care must be
taken in interpretation as it is a relatively poor pathogen and is a common contaminant)
this organism is extremely motile due to presence of flagellae – typical swarming colonies – important
for diagnosis
KEY POINT Proteus has a distinct colony morphology due to its ability to swarm.
NOTE: Morganella morganii (used to be called Proteus morganii) is a well known human pathogen. It
has been associated with ear and urinary tract infections in dogs and cats.
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FIGURE: The multiple flagellae that are FIGURE: A Proteus colony with typical
associated with Proteus spp. It is not difficult to swarming. This is a characteristic feature of
see why they are highly motile, which allows the this bacteria and is useful for assisting
to swarm on blood agar plates. diagnosis of the diseases caused by this
How would you Diagnose an Infection with Enterobacteriaceae?
Sample Collection
the type of sample obtained would depend on whether a sterile site (e.g. joint, blood, bladder) or one
containing normal flora (e.g faeces) was to be sampled
there is no point in using a sterile container to collect faeces (but you don’t want to use one that is
heavily contaminated as well!!)
Direct Examination
again, this will depend on the sample collected
samples from sterile sites may contain either only gram negative rods or they may be involved in
mixed infections
there is little value in examining faecal smears if Enterobacteriaceae are the suspected cause of disease
(you will just observe normal flora – which include E. coli etc)
Cultivation
these bacteria grow well on ordinary media, including blood agar
however, if a sample from a site with normal flora has been obtained (e.g. faeces), there is NO
POINT in culturing this sample on blood agar – the colonies of all the Enterobacteriaceae will look the
same (except Proteus) and besides the plate will be overgrown with other bacteria
therefore the specific organisms must be isolated
there are a large number of selective media (e.g. MacConkey’s agar, XLD agar) that are used for
isolation and identification of Enterobacteriaceae
these will be covered in the practical classes
remember, if you wish to prove that E. coli is the causative agent of disease, isolation of an E. coli is
not sufficient, you must also demonstrate the particular strain that you have isolated has the
appropriate virulence factors to cause the disease observed
SELECTIVE media is required to isolate specific Enterobacteriaceae from

KEY POINT sites with a nomal flora.
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Identification
all the Enterobacteriaceae are oxidase negative (this helps in differentiating this group of bacteria from
most of the other gram negative rods)
the different genera within the Enterobacteriaceae have different reactions in biochemical tests, and
these are used to differentiate this group of bacteria
9 there are available a number of minaturised systems that allow rapid identification of the
Enterobacteriaceae – see practical class
Antibody Mediated Immunity (AMI) plays a major role in infections with all the Enterobacteriaceae
however, whilst antibodies can neutralise infections – this immunity is serotype specific
therefore, unless the animal has been previously exposed to the specific serotype, then a delay in the
onset of immunity (for a primary antibody production) will occur
this is supported by the poor immunity imparted by bacterins (killed bacteria in a vaccine) to many
infections with Enterobacteriacea, especially those that cause systemic disease
alternatively, pilus vaccines have been developed and appear to have superior efficacy for enteric
disease, as they prevent adherence
these are predominantly used to immunise the mother (cow, sow) prior to parturition, and the
antibodies are then passed onto the young in the colostrum and milk
with Salmonella infections, due to the fact that they survive in cells, Cell Mediated Immunity (CMI)
plays a larger role in protection
CMI can clear infections through activation of macrophages and consequently intracellular killing
therefore, vaccines that induce CMI (i.e. live vaccines) confer better protection
there are a number of newer vaccines available in the UK and USA that are made of mutant strains of
Salmonella that are only able to multiply 6 or 7 times before they die (live avirulent vaccines)
but the bacteria are alive when inoculated (usually via ingestion), so they better promote development
of CMI
there are multiple drug resistant strains of the Enterobacteriaceae, particularly E. coli and Salmonella
therefore sensitivity testing is recommended in order to determine the antimicrobial sensitivity pattern
of isolates
Enterobacteriaceae are NOT predictably sensitive to any antibiotic,

KEY POINT therefore sensitivity testing is highly recommended.
the plasmids (R Factors) involved in antibiotic resistance may be transferred among enteric bacteria,
primarily by conjugation
within this plasmid are usually the genes for transfer of the plasmid (Resistance Transfer Factor =
RTF) and the genes for resistance to multiple drugs
because the Enterobacteriaceae are so similar, these R Factors may be transferred among the different
types of Enterobacteriaceae within the GIT where there are many opportunities for genetic
recombination
drugs that might be considered if an infection with Enterobacteriaceae is suspected include
aminoglycosides (gentamicin, neomycin), fluoroquinolones (enrofloxacin) and trimethroprim
sulfonamides (particularly for Salmonella)
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in Salmonellosis, carriers may occur even after treatment (protected against the antibiotic as they are
within cells)
in cases of systemic disease (septicaemia), antibiotics are essential , and need to be given systemically
(after sensitivity testing)
in cases of enteric disease (diarrhoea), the most important aspect of therapy is fluid and electrolyte
administration, and antimicrobial therapy is often not warranted
Fluid and electrolyte therapy is the most important aspect for treatment
KEY POINT of diarrhoea, expecially in neonates.
Fluid therapy is the most important aspect of

treatment in all cases of diarrhoea caused by the
Enterobacteriaceae (although it can be boring to
give the large volumes required to correct the fluid
deficits observed in horse!!!s). This horse is
suffering from Salmonellosis and may require up to
50 litres of replacement fluid a day! The large
bottle above the head of the horse contains these
fluids.
as the lesion in cases of diarrhoea due to ETECs does NOT involve cell death, the fluids and
electrolytes may be administered ORALLY as the intestine is still capable of water absorption
in addition, there are other mechanisms by which ions, in particular Na+, may be taken up by the
intestine
however, these mechanisms require SUBSTRATES like glucose or amino acids, for the uptake to
occur
consequently, if glucose is supplied in the oral rehydration fluid therapy, this will allow the substrate-
linked absorbtion of Na+ and will help repair the Na+ deficits that occur in this disease
finally, neonates should be maintained on milk, as this is an important source of energy – and energy
deficits are a major problem in neonates due to their high metabolic rates
however, milk and oral fluids should not be administered simultaneously, as the fluids will prevent a
proper milk clot from forming in the abomasum, which is required for correct absorption of the milk
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How would you Control Disease?
Control of these infections is based on management procedures and vaccination regimens

Vaccination administration has been discussed under immunity and resistance
Underlying predisposing factors MUST be identified and eliminated (e.g. poor housing, overcrowding,
wet/dirty conditions, poor attention to ensuring adequate passive transfer (colostrum ingestion) etc)
Dietary changes may predispose to enteric disease and therefore and changes should be introduced
gradually
REMEMBER, changes in management practices are integral to control of these diseases
animals are the major source (reservoir) of pathogenic Enterobacteriaceae that can infect humans
(mostly FOOD POISONING)
this includes Salmonella species and E. coli 0157:H7 etc
infections and epidemics are usually traceable to various food products, especially derived from meat,
eggs, milk and poultry
however humans can acquire infections DIRECTLY from infected animals, particularly those showing
signs of clinical disease (diarrhoea)
this is most important when very young children or old people are involved, as serious disease, including
death, may occur
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PSEUDOMONAS
Pseudomonas is probably one of the most misunderstood bugs around! It is a gram negative, oxidase
positive rod that likes to hang out in wet, moist conditions within the environment. It can survive very
well in these environments and doesn’t need a lot to live on! It does NOT invade tissues easily (needs a lot
of host compromise and likes best wet, moist, necrotic tissues) but when it does invade it can cause serious
diseases. This is particularly the case in compromised host tissues e.g. burn wounds. It has widespread
antibiotic resistance, and so when an infection does occur it can be very difficult to get rid of. Diagnosis
of Pseudomonas infections can be challenging and if Pseudomonas aeruginosa is isolated from a clinical
case it is important to distinguish between true infection and contamination.
Summary Table : Species of Pseudomonas that has veterinary importance, its animal hosts,
and the diseases it causes.
SPECIES HOSTS DISEASE

variety of diseases including otitis
externa, cystitis, endometritis, corneal
Wide variety of animals
ulcers, “Green Wool” and dermatitis,
Ps. aeruginosa ++++
including humans, dogs, cats,
wound infections (particularly burn or
horses, ruminants, mink etc
under bandages), nosocomial
infections
+ only species of veterinary importance
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What are they?
they are gram negative rods
they are obligate aerobes
they are motile (due to the presence of a polar
flagella)
all have pili
there are many different species of Pseudomonas,
however, only Ps. aeruginosa is of veterinary
importance
KEY POINT Only Pseudomonas aeruginosa is of veterinary importance.
if ANY other species of Pseudomonas is isolated from a sample you have submitted it is NOT
significant!!!
most members of the genus Pseudomonas live in moist or muddy soil and water
P. aeruginosa may also occur transitorily in the faeces of normal animals
KEY POINT Pseudomonas aeruginosa is UBIQUITOUS in moist environments.
environmental (exogenous) exposure of animals to this organism is constant

therefore, most infections are secondary to compromised host defence and presence of compromised
tissues
How do they cause Disease?
P. aeruginosa produces a number of virulence factors;
a) Pili
P. aeruginosa possess pili that facilitate adherence to epithelial cells and colonisation
this is especially important in the induction of corneal ulcers (tears wash away the bacteria unless they
are attached)
however, P. aeruginosa can only colonise when the fibronectin coat (which normally covers the
epithelial cell surface) is disrupted as the result of infection or mechanical trauma
colonisation of P. aeruginosa is not confined to epithelial surfaces, the organism can colonise deep
tissues as well, including tissues exposed to burns and trauma
78
b) Capsule and O antigens
as with other gram negative bacteria, the capsule and lipopolysaccharide (endotoxin) offer considerable
protection against phagocytosis
c) Protein Exotoxins
i. Exotoxin A
9 kills cells the same way as diptheria toxin does (ie. it inhibits protein synthesis by
ribosylation of host cell G proteins)
9 it is most important of the exotoxins, and toxigenic strains are more virulent than non-
toxigenic strains
ii. Exotoxin S
iii. Haemolysins
9 act synergistically with a phosphatase on lipids and lecithin to produce necrosis
9 they also break down pulmonary surfactant with resulting atelectasis
iv. Leucocidins
v. Lipase
vi. Elastase
vii. Fibrinolysin
viii. Collagenase
9 Acts to break down the collagen in tissues, including the cornea, and is responsible for the
“melting ulcers” that are seen with these infections
d) Pigments (pyocyanins)
pyocyanin reacts with oxygen to form reactive oxygen radicals that are toxic to eukaryotic and
prokaryotic organisms
P. aeruginosa protects itself from the toxic effects of pyocyanin by increasing synthesis of catalase and
superoxide dismutase
e) Bacteriocins (pyocins)
pyocins are toxic for other bacteria, and help allow Pseudomonas to have an advantage over these
bacteria when colonising an environment (including tissues)
f) Siderophores
P. aeuruginosa produces its own iron acquiring proteins pyochelin and pyoverdin
II) Pathogenesis of Pseudomonas infections

because P. aeruginosa is ubiquitous in the environment, disease determinants therefore lie largely with
the hosts and their immediate environment (this means that it is the host that dictates whether disease
occurs and not the bacteria)
Pseudomonas aeruginosa is a relatively POOR PATHOGEN that does not

cause disease easily (it does not have a good ability to invade).
It causes OPPORTUNISTIC INFECTIONS in patients with a LOT of host tissue
KEY POINT compromise, particularly if the tissue is constantly moist.
The problem with these infections is that when they do occur, they are
frequently bad due to the toxins they produce, and the fact that they are
resistant to many antibiotics.
infections are rare in healthy, normal individuals

however, in a veterinary hospital, a number of situations favour selection of this organism:
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⇒ the organism thrives in wet, poorly aerated environments within hospitals
⇒ they are especially fond of surgical areas, such as within support bags that have not been
properly dried or in hoses on anaesthetic machines that have not been cleaned and dried
properly
⇒ several human cases of severe Pseudomonas infections have been reported recently due to
contamination of endoscopes that have not been cleaned and dried properly
⇒ Pseudomonas can also survive in a number of disinfectants, particularly if they are too
dilute or too old (not been changed frequently)
these hospital situations result in an increase in number of pseudomonads in the environment of the
compromised host animal (e.g a surgical site), thereby increasing the risk of contamination which can
lead to infection
another important consideration is that P. aeruginosa usually only contaminates areas of the body that
possess reduced number of normal flora
disruption of the normal flora is almost always due to use of antimicrobial agents
since P. auruginosa is resistant to most commonly used antimicrobial agents, it can replace the normal
flora
if the site that is subsequently colonised by P aeruginosa is compromised (e.g. surgical wound or burn)
or is contiguous to a compromised site, there is the risk of a true infection developing at this site (ie
multiplication of the organism, destruction of tissues and an inflammatory response)
this is especially the case in debilitated or immunodeficient animals
tissue destruction follows liberation of exotoxin(s) and pyocyanin
HOWEVER, it is also important to remember that P. aeruginosa is a frequent contaminant in disease
processes, and isolation alone does not necessarily mean that it is significant in these situations
repeated isolation in pure culture are strong indicators of true pathogenicity
alternatively, its significance in mixed infections, particularly those involving Staphylococci or
Streptococci, may be questionable
Isolation of P aeruginosa from a sample does not mean that it is always

significant. You must identify an underlying factor that has allowed it to
cause disease, and it must be a disease that this bacteria is known to cause
in domestic species. Furthermore, this bacteria is FREQUENTLY a
contaminant at the time of collection (as they are ubiquitous and can survive
KEY POINT well in hospitals, fluids, endoscopes etc). Therefore, you HAVE to have
obtained the sample correctly (in a sterile fashion) from a normally sterile
site. If you don’t take these factors into consideration when determining the
significance of the isolation of P aeruginosa you will end up putting the
animal on expensive and totally unnecessary antibiotics.
an alternate scenario is the situation where SUPERINECTIONS occur

this is when an animal is being treated with antimicrobials due to an existing infection, but the
underlying disease process is not corrected, and as P. aeruginosa is resistant to many antimicrobials it
may secondarily infects the original site of infection, often proliferating greatly, and overtaking the
original bacteria so that it is the only bacteria involved (ie resulting in a superinfection)
KEY POINT P aeruginosa commonly causes SUPERINFECTIONS.
What Diseases do P. aeruginosa Cause?
Ps. aeruginosa can cause a wide variety of disease in a wide variety of animals
However, the pathogensis of these diseases is very similar (see above)
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The diseases that this organism has been isolated from include:
1. Otitis Externa in dogs

2. Cystitis in dogs
3. Endometritis in mares – superinfection predominantly Common and important
4. Corneal ulcers in horses
5. “Green Wool” and dermatitis in sheep
6. Wound infections in all species
7. Nosocomial infections in all species
8. Burn wound infections in people (and animals)

9. Pneumonia in people with cystic fibrosis (NOT in domestic spp) Human
10. Septicaemia in people with burns, leukemia, cystic fibrosis infections
11. Variety of other infections in many different sites in compromised hosts
12. Mastitis in cows Rare and less important

13. Septicaemia in immunocompromised animals
14. Haemorrhagic pneumonia in mink
Melting Ulcer on the cornea

of a horse’s eye
FIGURE: A corneal ulcer in a horse called “BEST BET” (the one we talked about in class).
The ulcer is associated with P. aeruginosa infection. Note the melting of the cornea which is
caused by the exotoxin produced by P. aeruginosa called collagenase (breaks down
collagen in the cornea and allows it to “melt away”). If these ulcers are not treated aggressively
(and sometimes even if they are) they can melt right through the cornea!
Sample Collection
fine needle aspirates of samples such as urine (via cystocentesis), pus, and affected tissues is the
sample collection technique of choice
81
swabs of infected tissues are inferior (except ear and eye) and care must be taken NOT to collect
swabs from draining tracts (pseudomonads love to grow up in the tissue exudates around draining
tracts but are probably NOT contributing to the disease process)
Direct Examination
Gram negative rods; may be observed either alone or as part of a mixed infection (e.g. in cases of
otitis externa)
Culture
P. aeruginosa grows well on most media – including blood agar
colonies are large, usually with a zone of beta (clear) haemolysis, and have a bluish metallic sheen
the culture has a characteristic odour (“fruity” or that of corn tortilla!) and green colour which is more
readily appreciated on nutrient agar (make sure that you can recognise these colonies during your
practical class – colony morphology gives you a good hint that you are dealing with Ps. aeruginosa)
P aeruginosa has a distinctive colony morphology, which is helpful in

KEY POINT identification of this bacteria.
FIGURE: Colonies of Pseudomonas

aeruginosa on a blood agar plate. Although
you can’t see it the colonies have a greenish
tinge!!! This feature is more apparent on
tryptose agar plates. What you can see is that
they are quite large (~ 4mm), with a rough edge
(fimbriate) and a beaten copper appearance to
the surface! If you could smell these they would
have a distinctive “fruity” odor!!! Make sure that
you can recognise these features in the
samples provided in practical classes, as they
provide a good way to identify this bacteria (ie
colony morphology helps to identify the bug).
Identification
the most important biochemical test is oxidase positive (they are rapidly and strongly positive) as this
test allows you to distinguish it from the Enterobacteriaceae
the motility test helps to distinguish Pseudomonas from other oxidase positive bacteria
specific immune responses do not seem to play much of a role in the pathogenesis of diseases caused
by P. aeruginosa or resistance to the organism
artificial protection has been shown to occur in animals vaccinated with extracts of the organism or
exotoxin A
82
treatment involves correction of host compromise and, if necessary, the use of an antimicrobial agent
Unless the underlying host compromise is corrected, antimicrobial (and
KEY POINT other) therapy will fail. Furthermore, correction of the underlying host
compromise may be sufficient in some cases to overcome infection.
infections due to P. aeruginosa may not respond well to chemotherapy - this is especially the case if
you have not corrected the host tissue compromise or if the strain is resistant
Pseudomonas aeruginosa has widespread antibiotic resistance and does

KEY POINT NOT have a predictably antibiotic sensitivity pattern.
resistance to many antibiotics is due to a permeability barrier of Pseudomonas cell wall, and to inactivation
by products encoded by plasmid-based genes (R plasmids)
P. aeruginosa may be susceptible to gentamicin, tobramycin, amikacin, carbenicillin, ciprofloxacin,
ticarcillin/clavulanic acid
these agents are used in the treatment of soft tissue infections
in cases of otitis externa – most isolates are susceptible to levels of antibiotics present in the otic
preparations which may include neomycin, polymyxin, choramphenicol or gentamicin
however, it should be noted that there are no in vitro tests that predict the susceptibility or resistance
of isolates from infectious processes that will be treated with topical preparations.
How would you Control/Prevent the Disease?
the most important consideration in preventing infection is to decrease the risk by reducing the
concentration of the organism in the environment of the patient
this may be difficult, as pseudomonads are relatively resistant to some of the commonly used
disinfectants, even in high concentration e.g. quaternary ammonium and phenolic compounds
in addition, with adequate water, P. aeruginosa can survive for long periods on water faucets, utensils,
floors, instruments (e.g. biopsy channel of endoscopes), baths, humidifiers, and respiratory care
equipment
pseudomonads are susceptible to heat (55°C for 1 hour)
control agains re-infection with P. aeruginosa also involves a reduction in cause of compromise to the
host – e.g. keeping ears dry and clean
as P. aeruginosa is an opportunistic pathogen, there is no direct transmission to humans

in addition, there is no evidence to suggest that antibiotic usage in animals has resulted in resistant strains
of P. aeruginosa that infect people
83
BURKHOLDERIA
Relative Importance
1. Glanders (caused by B. mallei) was once a widespread infection of equidae, but today it remains
important only in Asia (Mongolia and China) with pockets of activity in India, Iraq, Turkey, and the
Phillipines
9 therefore, this is an exotic disease and is included for your interest
2. Meliodosis occurs in Northern Australia (tropical regions) and cases have been reported in cats in
Sydney
3. As this is rare in southern states, you mainly need to know what are the clinical signs associated with
infection so that you can recognise potential cases and then know where to look it up!!!
Summary Table: Comparing the 2 most important veterinary species and including the disease
they cause, their geographical location, the animals affected, their normal
habitat and how they are transmitted to their host species.
Organism B. mallei B. pseudomallei

Disease Glanders Melioidosis
Present in Australia No – primarily in Asia Yes; mostly in tropical areas (above
20° latitude)
Notifiable disease in NT, Qld and
WA
Species affected
- mainly ×Øb
b ×Ø- dead end hosts Wide host range; goats, dogs,
Horses (cat, dogs, goats, camels, sheep and horses, cattle, pigs, humans
humans etc are dead end hosts)
Habitat (where Infected horses are reservoir Saprophyte; soil and water
found) Persistence depends on presence of dweller
infected horses within a population A wet environment, such as
Susceptible non equids acquire swampy terrain or rice paddies,
glanders from infected horses and horse is related to disease transmission
meat
Transmission Exposure to contaminated feed, water, Ingestion, wound infection and
fomites (ingestion). possibly arthropod bites
Sometimes through inhalation or wounds In humans, consumption of
Infectious material originates mostly from infected animal products and
respiratory tract or skin lesions airborne infections may be
important
Note: they used to be called Pseudomonas, Malleomyces, Pfeifferella, Loefferella, and Actinobacillus
What are they?
they are gram negative rods (similar to Pseudomonas to which they are related)
both species are strict aerobes
2 species of major importance (see summary table)
84
World Distribution of Glanders
B. mallei
Endotoxins and exotoxins reported, but not characterised
B. pseudomallei
exotoxin like substances reported (lethal, necrotising)
role in pathogenesis of disease not known
Facultative intracellular pathogen – survives in phagolysosomes
resistant to various defencins when inside cells
II) Pathogenesis of infections with Burkholderia

B. mallei
although toxins are suspected in pathogenesis, the exact mechanism by which they cause disease is
uncertain
primary lesions form at the point of entry e.g. the pharynx
infection spreads along lymphatics, producing nodular lesions along the way to lymph nodes and blood
stream and is the way the infection is disseminated
metastatic lesions form in lungs and other organs (e.g. spleen, liver, skin)
may also see lesions in the skin where the cutaneous form = “FARCY”
lesions occur frequently in the nasal septum and may be primary, haematogenous or secondary to
pulmonary focus
B. pseudomallei
infections are typically systemic
85
What do you see?
Glanders (B. mallei)

the basic nodular lesion that is observed in cases of Glanders is made up initially of neutrophils, fibrin
and RBCs
within the centre of these lesions, the neutrophils degenerate and this central necrotic area becomes
surrounded by epithelioid giant cells and by lymphocytes embedded in granulation tissue
near epithelial surfaces, ulceration is common
strain variation in the organism determines the suppurative versus granulomatous predominance in the
lesions
Melioidosis (B. pseudomallei)

clinical disease is usually sporadic
the host range of this organism is virtually unlimited, and avian cases have been reported
manifestations of disease depend on the extent and distribution of lesions
small abscesses may coalesce, developing into larger suppurative foci or granulomas
What Diseases do Burkholderia cause?

there are acute, chronic and latent forms of the disease
acute infections are characterised by fever, nasal discharge and lymphadenitis of the head and neck
with swelling along the upper respiratory tract
in the acute disease, upper and lower respiratory tract signs predominate and they tend to end fatally in
about 2 weeks
this form tends to predominate in donkeys and cats (felids) and to a lesser extent in mules
the pulmonary form of the disease is called “Glanders”
in horses, chronic protracted and subclinical infections are typical with signs (if present) of fever,
malaise, weight loss, persistent respiratory problems, skin abscesses and ulcers (called “Farcy buds”)
and nodular induration of cranial lymph nodes
may also see nodule formation and ulceration in nasal mucosa, lungs, and local lymph nodes
FIGURE: A “Feng Shui” a

horse from China with
“Glanders”. He has both the
pulmonary and the skin forms
of the disease. On his legs are Farcy Bud
lesions called “Farcy Buds”
which are exudative ulcerative
nodules.
86
in horses – the disease may mimic glanders
in cattle, acute and chronic infections can localise in lungs, joints and the uterus
goats suffer debilitation, respiratory and CNS disturbances, arthritis and mastitis
similar signs are seen in pigs, along with abortion and diarrhoea
dogs and cats develop febrile disease, with localising suppurative foci

needle aspirates or swabs from ulcerated nodules may reveal gram negative rods
however, glanders is an exotic diseae, therefore if the disease is suspected based on history (imported
animal) and clinical signs – the organism requires positive identification by appropriate authorities e.g.
AAHL
in addition, differentiation from B. pseudomallei is important (especially in horses where the disease
can present with similar clinical features)
an indirect immunoflourescence assay (IFA) is available for clinical material
in addition, animals with suspected glanders may be tested serologically, using a Complement Fixation
test (Mallein Test)
this is an intradermo-palpebral test which detects cell-mediated hypersensitivity, and therefore
indicates infection
this test is the basis for Glanders eradication schemes

methods for diagnosing infections with B. pseudomallei are similar to B. mallei

humoral and cell mediated responses occur
apparent recovery from glanders, including loss of dermal hypersensitivity, has been observed under
natural conditions, but without increased resistance to re-infection
no method of immunisation is known

production of complement fixing and haemagglutinating antibodies reported in infected animals
cell mediated hypersensitivities demonstrated in infected goats
How would you Treat an Infection

B. mallei is susceptible to a wide range of antibiotics e.g. sulphonamides, aminoglycosides,
chloramphenicol, tetracyclines, and erythromycin
however, although glanders is treatable by many antibiotics, treatment is inappropriate in countries
committed to glanders eradication

unpredictable sensitivity to antibiotics (therefore sensitivity testing is required)
usually susceptible to tetracyclines, chloramphenicol, trimethorprim/sulphamethoxazole and novobiocin
87

in Australia, any animal diagnosed with glanders would be euthanised (probably detected in
quarantine)
equine imports from endemic areas are mallein tested, and any reactors are destroyed.

successful vaccination of horses and zoo animals reported, but vaccines are not commercially available

human exposures are traced to acutely ill horses and may lead to acute or chronic infections
all acute infections and 50% of chronic infections were fatal prior to the advent of effective
antimicrobials

human infections range from the rapidly fatal to the subclinical
in the years 1989 – 1998 the prospective NT melioidosis study at Royal Darwin Hospital has
documented 206 culture confirmed cases of melioidosis with a average annual incidence of
16.5/100,000 people in the NT!
the overall mortality rate was 21%, with a mortality rate in septicaemic cases of 39% and in non-
septicaemic cases 4%
pneumonia was the most common form of presentation in both groups
contaminated water supply was implicated in at least 2 outbreaks of the disease
there have been a number of reports of melioidosis in survivors of the recent Tsuanami in Asia
88
CAMPYLOBACTER
Camyplobacter are small, gram negative rods that are distinguished by their curved shape. They are quite
fragile bacteria and can be difficult to grow (need micro-aerophilic conditions). They are predominantly
associated with GIT disease (mostly diarrhoea) in a variety of species and reproductive tract disease
(abortion and infertility) in production animals. In addition, they are a very common cause of diarrhoea in
humans and so there are public health concerns regarding their transmission from animals to humans.
Summary Table: Species of Camyplobacter and Arcanobacter that have veterinary

importance, their animal hosts, and the diseases they cause

++++
C. jejuni Humans, primates, (dogs, Diarrhoea
cats)
C. jejuni++++ Sheep, goats, (rarely cattle) Reproductive tract disorders
C. coli+ Humans, primates, (pigs, dogs, Diarrhoea
cats)
C. helveticus Dogs, cats Diarrhoea?
C. upsaliensis Dogs, cats Diarrhoea?
A. butzleri Humans (pigs) Diarrhoea
A. cryaerophilus+ Pigs Reproductive tract disorders
A. skirrowii+ Pigs Reproductive tract disorders
C. fetus ssp Cattle Reproductive tract disorders
venerealis+++
C. fetus ssp fetus+++ Sheep, goats (cattle) Reproductive tract disorders
NOTE: the number of + denotes the relative importance of these veterinary species within
THIS genus (NOT for all bacterial genera)
89
What are they?
the two genera Campylobacter and Arcobacter belong to the family Campylobacteriaceae and they are
very closely related
consequently they will be discussed together in this
section
they are gram negative curved rods
when 2 or more bacterial cells are placed together they
form a classical S or gull-winged shapes
they are microaerophilic, requiring an atmosphere of 3-
15% O2 and 3-5% CO2 for growth
they are oxidase positive
they are motile (with a characteristic “tumbling” motion)
there are more than 13 different species of

Campylobacter; those with veterinary importance are
listed in the table on the opposite page
the reservoir for infection with the enteric Campylobacter is the GIT of normal animals – especially
young ruminants and various birds
they can also be found in the faeces of infected animals
therefore the source of infection for susceptible animals and humans is animals and animal by-products
for example, C. jejuni has been found in milk, on poultry carcasses and in faeces of asymptomatic
dogs and cats as well as those with diarrhoea
the reservoir of the genital tract pathogen C. fetus ssp venerealis is the mucous membrane of the
preputial crypts of carrier bulls (primary source) and the mucous membrane of the vagina of carrier
cows (this is rare in cows after 1-2 breeding seasons without exposure)
the reservoir of the genital tract pathogen C. fetus ssp fetus is the intestinal tract of carrier sheep
(recovered) – perhaps through contamination from a colonised gall bladder
Arcobacter are probably acquired from infected carrier animals
Campylobacter spp causing enteric disease live in the GIT of healthy

animals
KEY POINT Campylobacter spp causing reproductive tract disease are found in either
the reproductive tract (C fetus venerealis) or the GIT (C fetus fetus and C
jejuni)
The faeco-oral route (ingestion), either directly or indirectly, is probably

the main mode of transmission for the enteric Campylobacter
KEY POINT Animals acquire the genital Campylobacter either
Venereallly - C fetus ssp venerealis
Ingestion – C. fetus ssp fetus and C jejuni .
90
although a number of virulence factors have been described for Campylobacter spp (see below), the
role of toxins (either endotoxin or exotoxins) and many of the other virulence factors is unknown in
causing disease in both the gastro-intestinal and reproductive tracts
a) Capsule
Campylobacter spp possess a glycoprotein capsule that is antiphagocytic and protects the outer
membrane from deposition of complement and the membrane attack complex
b) Pili
C. jejuni produces an adhesin (pili) that binds to fucose-containing receptors on target cells and assists
in the colonisation of the lower intestine
c) Enterotoxin
C. jejuni secretes a toxin similar in activity to cholera toxin and the heat labile (LT) toxin of E. coli
this toxin increases intracellular cAMP and causes a hypersecretory diarrhoea
d) Cytotoxins
C. jejuni also produces a number of proteins with cytotoxic activity
however, all of these toxins have a tenuous association with disease processes in animals and humans
e) Intracellular Survival
C. jejuni can survive inside mononuclear phagocytes
the exact mechanism whereby this occurs is however, unknown
II) Pathogenesis
a) Enteric Disease
C. jejuni first adheres to the cells of the small intestine, especially the distal segments
there the organism multiplies and invades the target epithelial cells (the cells with receptors for the
pili)
it is uncertain whether their exotoxins are responsible for the disease observed, however, it is likely
that the enterotoxin acts in a similar fashion to the LT toxin of E. coli in inducing increased
intracellular cAMP and therefore alterations in electroyte secretions and subsequent altered fluid
homeostasis
in addition, another cytotoxin of C. jejuni actually causes cell damage, resulting in destruction of the
mucosal epithelium and intestinal inflammation
this inflammation in turn also causes alterations in intracellular cAMP (through activation of the
arachidonic acid pathway leading to production of prostaglandins and leukotrienes, which elevate
cAMP)
faeces in animals with diarrhoea contains cell debris, mucus and blood, which sometimes may be seen
grossly
little is known about the interaction of Arcobacter and the intestinal tract of the host
b) Genital Tract Disease

the pathogenesis of these diseases differs depending on the species of Campylobacter and host species
91
i) Cattle – C. fetus ssp venerealis
these organisms are introduced into a susceptible female by an infected bull at coitus
the organisms remain at the cervicovaginal junction until the end of oestrus, then they multiple at
this site and when the conditions are suitable they move into the uterus
further multiplication and perhaps active invasion results in uterine inflammation with resultant
endometritis and cessation of pregnancy
these animals will then return to oestrus
this cycle continues until the animal has mounted a sufficient immune response to eliminate the
agent from the uterus
subsequently, the animal conceives and pregnancy goes to term
ii) Sheep and Goats – C. fetus ssp fetus and C. jejuni

sheep and goats are infected following ingestion of either of these organisms
they then gain entry to the blood stream (how is unknown) and localise in the pregnant uterus,
especially in the latter stages of pregnancy
incubation may be as long as 2 months, but eventually the placentitis which results causes infection
of the foetus and amniotic fluid and abortion
the placenta, fluids, and foetus contain large numbers of organisms and act as a source of infection
for susceptible animals
therefore abortions, when they do occur, usually occur as “abortion storms” after a few scattered
abortions
the liver of aborted foetuses may have characteristic “donut” shaped necrotic foci – which help in
the diagnosis
cattle are rarely infected with C. fetus fetus or C. jejuni and when they are, sporadic abortions are
observed
¾ mastitis has also been reported in cattle associated with C. jejuni infection
abortion in dogs has been associated (very rarely) with C. jejuni infection
iii) Pigs – Arcobacter cryaerophilus

9 pigs are associated with a variety of reproductive abnormalities associated with Arcobacter spp
(mainly cryaerophilus) but the pathogenesis is poorly understood
What Diseases do Campylobacter and Arcobacter Cause?
1. Enteric Disease – Diarrhoea

Campylobacter are one of the major causes of diarrhoea in people (it is the most common cause of
Traveller’s diarrhoea in people worldwide)
Campylobacter are a MAJOR cause of diarrhoea in people and are a public

health risk.
KEY POINT
Animals and birds are the source of infection (either directly or indirectly)
for people. However, companion animals are rarely involved in transmission.
however, the role of C. jejuni (and other species of Campylobacter) in inducing diarrhoea in domestic
species is less well defined
the faeces of ~10% of asymptomatic dogs, and 5% of asymptomatic cats are found to contain C.
jejuni
9 this % may be higher in animals obtained from the pound
although this organism has been isolated from dogs and cats with diarrhoea, its role in disease
production is unclear
92
however, it is thought to cause diarrhoea in young (<6 months) puppies and is manifest as mucus-
laden, watery or bile-streaked diarrhoea (with or without blood and leucocytes in the faeces)
in cats, clinical signs of campylobacteriasis is poorly documented and probably does not occur in the
absence of other pathogens
C. jejuni is thought to be associated with diarrhoeic conditions in cattle, goats, pigs, lambs, mink and
ferret
FIGURE: A toilet near a stream in

Ecuador. You may need to use
this if you get Campylobacter
diarrhoea when travelling!!!! By
the way – it is not recommended
that you drink the water
downstream!
2. Reproductive Tract Disorders
The most important disease in animals caused by Campylobacter spp is

KEY POINT ABORTION in ruminants .
a) Cattle
the disease is mainly seen in beef cattle, since the agent is killed by the techniques used to prepare and
store semen for AI (therefore there is little disease in dairy cattle)
the early stage abortions caused by Campylobacter in cattle is mostly seen as return to service (repeat
breeding) and prolongation of the oestrus cycle (up to 60 days; 21 days is normal)
this is manifest as prolonged calving intervals and extended calving periods
occasionally, sporadic abortions later in gestation do occur
b) Sheep and Goats

usually see abortion storms in these species
the liver of the aborted foetus may have
characteristic “donut shaped” lesions
the gall bladder of sheep (and goats?) may
become colonised with C. fetus fetus and this may
become a source of infection for susceptible stock
FIGURE: Two dead lambs that were aborted during

an abortion storm in a flock of sheep. Note that they
are quite late foetuses as C. fetus fetus causes late
term abortion in sheep and goats (unlike C. jejuni
abortion in cattle which occurs in early pregnancy and
is seen as return to service).
93
FIGURE: The liver of an
aborted lamb foetus showing
characteristic donut shaped
lesions.
c) Pigs
late term abortion and infertility have been recorded
1. Sample Collection
Enteric infections
faecal samples are obtained for diagnosis of C. jejuni diarrhoea in people, but are frequently of
little value in domestic animals due to the high carriage rate in healthy animals
however, faecal cultures are frequently performed by commercial laboratories!
Reproductive tract infections

Cattle:
in bulls samples are best taken from the prepuce
in cows, the anterior vagina and cervix should be sampled
in both cases – 10% of the herd should be sampled (or 20 animals if herd is large)
Sheep:
samples from the liver and abomasum of aborted foetuses are most rewarding
the placenta and foetal fluids usually have Campylobacter but are also usually contaminated with
other normal flora and are therefore difficult to culture
Pigs:
Arcoabacter have been isolated from stomach, kidneys and placenta of aborted foetuses
2. Direct Examination
smears may be stained with gram stain and reveal slender, curved rods;
9 in cattle, numbers of organisms are often low (and difficult to detect among the normal flora)
9 in sheep, numbers of organisms are higher, especially in the foetal stomach (abomasum)
9 if the liver lesions are present a presumptive diagnosis may be made
9 wet mount preparations may be useful in both species where a typical “tumbling” motility may
be observed
94
Characteristic morphology of the bacteria (curved rods) and motility
(tumbling motion) may be used to assist in the diagnosis of these infections.
KEY POINT Furthermore, classic gross pathological lesions, (“donut” shaped lesions on
the liver) on aborted foetuses may also assist in a presumptive diagnosis.
3. Culture
specific media for Campylobacter may be used and assist isolation
microaerophilic conditions are required for growth of Campylobacter, whereas Arcobacter spp are
aerotolerant
4. Identification
curved rods that are oxidase positive may allow a presumptive diagnosis
other biochemical tests would not be applicable in a veterinary practice
5. Serological Testing
antibody in cervicovaginal mucus has also been used in serological assays to assist diagnosis of
reproductive tract infections in cattle and sheep
development of an active immune response results in the uterus being cleared of the organism, which
usually occurs within a year of infection
opsonising antibodies, as well as antibodies to the pili, capsule and flagella all assist in the clearance of
these organisms
in gastro-enteritis, circulating antibodies develop as a result of infection, but their role in clearance of
the bacteria is not clear (the disease is usually self-limiting before the appearance of antibody)
enteritis produced by C. jejuni is usually self-limiting and therefore antibiotics are usually not
indicated
if the disease is more severe (e.g. in young puppies), macrolide antibiotics (erythromycin) are the
drugs of choice
in reproductive tract disorders, bulls can be treated with streptomycin
antibiotic treatment of cows is however, is usually unrewarding
abortion storms in sheep may be stopped by giving antibiotics (penicillin is the drug of choice), and
aborting ewes should be isolated from the rest of the flock
both forms of the disease are best controlled by prevention
Prevention is the best way to control both forms of the disease caused by
KEY POINT Campylobacter spp and is preferred to treatment in herd situations.
95
in cases of enteric disease – meticulous care should be given to hygiene procedures (such as hand
washing, cleaning and disinfection)
this is especially the case if immunodeficient people are likely to be at risk, as infection in this group
of people can be very severe
in the case of bovine abortion due to C. fetus ssp venerealis, the use of young bulls (who are less
susceptible to infection) will decrease the prevalence of reproductive tract disease in cattle, and these
should be only bred to cows with a known breeding and disease history
if the infection becomes established in a herd, AI may be used to control and eliminate the disease (C.
fetus ssp venerealis can be eliminated from a herd whereas C. fetus ssp fetus cannot)
vaccines are available and have been used in both cattle and sheep to control outbreaks of disease in
endemic herds or flocks; but long lasting immunity is not achieved
vaccination should be performed yearly
ANTIBIOTIC Don’t use antibiotics indiscriminantly,

especially in cases of diarrhoea in small
GENERIC animals!!!! They are RARELY indicated.
it is now recognised that C. jejuni is a leading cause of diarrhoea in humans and that a variety of
animal species may be the direct source of infection for people (as well as animal products being an
indirect source)
puppies and kittens pose a greater risk for transmission of the organism, especially those animals with
diarrhoea
9 these animals should be removed from households with at risk individuals until the diarrhoea has
resolved
9 however, they still remain an uncommon source of infection for humans
the caeca of ~ 50% of chickens contain C. jejuni, and these organisms contaminate the environment
during the slaughtering process
9 therefore just about all chicken meat found in stores will be contaminated!!!
9 this is considered to be the major source of infection for people
96
from between 2-100% of healthy cattle can shed C. jejuni and may explain outbreaks of diarrhoea
following ingestion of unpasteurised milk
other species of Campylobacter have been isolated from faeces of dogs and cats, and are thought to
have (rarely) the potential for causing diarrhoea in people
C. fetus ssp fetus (but not C. fetus ssp venerealis) has been shown to be an infrequent cause of
systemic infection in humans including abortion, meningitis, pericarditis, endocarditis, peritonitis,
salpingitis, septic arthritis, and abscesses
veterinarians, farmers, abattoir workers and others associated with sheep and cattle are at a greater risk of
acquiring infections
97
LAWSONIA
Lawsonia are a newly recognised pathogen that we still can’t grow on conventional media (agar places
etc). They predominantly cause disease in pigs, but are increasingly being recognised as a cause of GIT
disease in other species as well (e.g. foals, puppies, and a bunch of pocket pets). Their importance to the
pig industry is so great that they are the most common reason for use of in-feed antibiotics (which has
significant implications for human health!). The diseases they cause in pigs are Porcine Proliferative
Enteropathy and Proliferative Haemorrhagic Enteropathy. These diseases remain difficult to diagnose,
and serological and molecular techniques are predominantly used over more conventional methods of
diagnosis of bacterial diseases.
Summary Table : Species of Lawsonia that has veterinary importance, its animal hosts,
and the diseases it causes.
SPECIES HOSTS DISEASES
Pigs (also affects foals, 2 syndromes recognized in pigs:

hamsters, rats, guinea Porcine Proliferative Enteropathy (PPE)
pigs, sheep, deer, emus Proliferative Haemorrhagic Enteropathy
L. intracellularis+
and ostriches, rabbits, (PHE)
ferrets, dogs and Diarrhoea, poor growth, and intestinal
macaques) haemorrhage recognized in other species
+ the only species of veterinary importance
98
What are they?
Lawsonia are gram negative curved or sigmoid shaped bacilli.

they require microaerophillic atmospheric conditions (<8% O2)
they are obligate intracellular bacteria (ie they must live inside cells – therefore you can’t grow them
on agar plates or in liquid medium)
they are motile by unipolar flagellum at some stage in life cycle
they are acid fast with modified Ziehl Nielsen stain
this is a new genus, with a new species:

9 Lawsonia intracellularis
Lawsonia intracellularis is a newly recognised pathogen that causes disease

KEY POINT predominantly in pigs.
its closest relative is Bilophila wadsworthia (free-living anaerobic human pathogen), and Desulfovibrio
spp. (sulfate reducing anaerobe)
in the GIT of infected carrier pigs
There are 4 main routes of infections for pigs:
1. Faecal-oral route between pigs

9 infection from sow to piglet
9 infection between in-contact pigs
2. Spread of contaminated faeces

9 L. intracellularis is able to survive in faeces, and infect susceptible pigs after 2 weeks in storage
3. External vectors
9 rats and mice have been infected with pathogenic porcine strains of L. intracellularis.
4. Introduction of new breeding animals

9 infected animals may be introduced into susceptible herds or vice versa
virtually nothing is known about the cellular products of Lawsonia, except that they contain endotoxin
as part of their cell wall, which probably aids virulence of this organism
99
II) Pathogenesis
other microbial flora are necessary to produce an optimal environment for entry and replication of L.
intracellularis
once they have established an infection in the small intestines, they cause an increase in mitotic activity
of crypt enterocytes associated with their presence
the proliferation of immature enterocytes, which don’t differentiate into absorptive cells as they move
up the villous, leads to extension and crowding of the villous-crypt structure and ultimately weight loss
in pigs as they lose proper digestive function
L. intracellularis is shed in the faeces, probably within sloughed cells (which can be detected by PCR
in faeces)
What Diseases does Lawsonia Cause?

L. intracellularis is a significant cause of variation in weight gain in the weaner pigs world wide.
in addition, outbreaks of L. intracellularis infection in minimal disease herds leads to death of breeding
and finishing animals
it is the main reason for in-feed antibiotic medication of grower and finisher pigs
Treatment of L intracellularis infection is the PRIMARY REASON for in-feed

KEY POINT antibiotic medication of grower and finisher pigs.
the estimated prevalence of previously infected herds is >90% in the US and UK

in addition, the estimated prevalence of actively infected herds is 5-45% world wide
L. intracellularis can cause diarrhoea and poor growth in pigs, hamsters, rats, guinea pigs, foals,
sheep, deer, emus and ostriches
it also causes intestinal haemorrhage in pigs, foals, hamsters, blue and red fox, rabbits, ferrets, dogs,
and macaques
disease in pigs is common and expensive to control
disease is also costly in fur producing animals
outbreaks of disease in laboratory animals can affect research outcomes
there are 2 syndromes recognized in pigs:
1. Porcine Proliferative Enteropathy (PPE)

9 which causes diarrhoea and reduced weight gains in younger pigs
9 grossly there is thickened mucosa of the terminal ileum and colon with necrotic material overlying
the mucosa and enlarged lymph nodes
9 this syndrome is also called intestinal adenomatosis and necrotic enteritis
2. Proliferative Haemorrhagic Enteropathy (PHE)

9 causing intestinal haemorrhage, tarry faeces and occasionally sudden death in finisher and breeding
pigs
9 grossly there is thickened mucosa of the terminal ileum and frequently a blood clot in the lumen of
the gut
in both conditions there is proliferation of crypt epithelial cells of the ileum, which leads to crowded
and branched crypts consisting of immature intestinal epithelial cells
within these sections of the ileum, special stains (Warthin starry silver stain) demonstrate curved
bacteria within apical cytoplasm of proliferating cells
there are numerous mitotic figures in crypt and villous cell
there is little inflammatory response
100
FIGURE: Three little pigs with Lawsonia infection. Note the
fact that they are very skinny (for pigs) due to weight loss and
that they have a typical pot bellied appearance.
Pot Belly!
Thin!!!
FIGURE: The ileum of one of the little pigs with

Lawsonia intracellularis infection. Note the grossly
thickened mucosa of the terminal ileum and colon
(porcine proliferative enteropathy).
101
this organism is an obligate intracellular bacteria, therefore they cannot be grown in routine media
used to diagnose most bacterial infections
diagnosis of infection therefore relies on evidence of disease; demonstration of the organism, and/or
demonstration of antibody:
1. Clinical signs
9 diarrhoea, poor growth and inappetance in weaner/grower pigs (6-20 weeks old) are suggestive of
infection
9 however, these signs are not specific for L. intracellularis as infection with Serpulina pilosicoli and
Salmonella sp. cause similar clinical signs
2. Demonstration of the Organism
a) Histopathology
9 the presence of curved intracellular bacteria within proliferating epithelial cells of the ileum, as
demonstrated by Warthin Starry stain, modified acid fast stain, or by immunostaining is diagnostic
of infection
9 however, lesions may have resolved before necropsy
b) DNA probes
9 detection of L. intracellularis DNA in faeces and/or intestinal mucosa is a moderately sensitive
method for detection of infection
c) PCR amplification
9 detection of L. intracellularis DNA (after PCR amplification) in faeces and/or intestinal mucosa
indicates current infection and is a very sensitive method of detection
9 this method can detect as few as 103 bacteria per gram of faeces
d) Fluorescent Antibody stain

9 alternatively, fluorescent antibodies that are specific for L. intracellularis can be used to detect
organisms in faecal smears, and has been used to detect current infections
e) Demonstration of Antibody - Serology

9 indirect fluorescent antibody (IFA) test which detects serum IgG antibodies to L. intracellularis has
been used to detect infected pigs
9 it indicates previous infection (had time for antibodies to be produced)
pigs infected with L. intracellularis when young develop mild clinical disease, clear the organism and
develop immunity to re-infection
however, the mechanism of this resistance is not known
How would you Treat and Control an Infection

there are a number of methods used to treat or control infections:
102
Antibiotics
9 continuous or strategic in-feed medication with antibiotics (predominantly tetracyclines or
macrolides) are used commonly
Possible vaccines
9 attenuated whole bacterial cultures have been used
9 a genetically engineered DNA vaccine in a viral vector is currently being developed
Development of immunity
9 pigs infected with L.intracellularis when young develop mild clinical disease and develop
immunity to re-infection – therefore in some piggeries this strategy is used to prevent disease but
not infection
Remember - treatment of L. intracellularis infection is the primary reason

KEY POINT for in feed antibiotic medication of grower and finisher pigs.
None
103
HELICOBACTER
Helicobacter are spiral shaped, gram negative rods which only grow under microaerophilic conditions.
They are one of the few bacteria that can survive in the hostile environment of the stomach and it Is here
they are thought to cause disease (their role in disease in domestic species is currently controversial, but
they have definitely been associated with serious disease (gastric ulceration) in humans). A number of
specific diagnostic tests have been developed for these infections (following on from those developed in
humans). There are some public health concerns regarding these organisms as companion animals (in
particular cats) may be a reservoir of infections for humans.
Summary Table: Species of Helicobacter that have veterinary importance, their animal hosts, the
primary site where they are normally found, and the diseases they cause
SPECIES HOSTS SITE DISEASE

H. pylori human, cats, stomach chronic superficial gastritis, gastric
macaques ulcers, gastric neoplasia (humans)
gastritis in cats (catteries)
H. felis cats, dogs, humans stomach gastritis (? – subclinical)
H. bizzozeronii* dogs, cats, stomach gastric coloniser only
humans, monkeys
H. suis pigs stomach gastritis (?)
H. mustelae mink stomach gastritis (?)
H. canis dogs, humans intestines, gastritis (?)
liver (dog)
H. pullorum poultry, humans intestine,
liver (chicken)
H. rappini sheep,, humans, intestine, stomach Abortion, hepatic necrosis (sheep),
dogs liver (sheep) intestinal disease (humans),
asymptomatic in dogs
Note*: An organism called H. heilmannii and Gastrospirllium hominis are now thought to be the same organism as H.
bizzozeronii.
104
What are they?
Helicobacter spp are curved to spiral-shaped gram negative rods

9 the degree of spiral formation varies between species
they are microaerophilic (they do not grow under aerobic or anaerobic conditions and achieve optimum
growth in high humidity and 5%CO2, 90%N2 and 5%H2)
Helicobacter are curve to spiral shaped gram negative rods that are
KEY POINT microaerophilis.
FIGURE: Scanning electron

micrograph of Helicobacter felis
isolated from lesions in the stomach
of a cat. The role of these bacteria
in causing disease in domestic
species remains controversial.
Helicobacter are related to Campylobacter and Arcobacter

there are ~31 species of Helicobacter that have been named to date and which infect a variety of
animal species
however there is much confusion in the literature (and among scientists!) as to the nomenclature and
which species of Helicobacter are found in which host species
9 many were previously classified as Campylobacter
9 reports of Helicobacter spp in dogs and cats sometimes refer to them as gastric Helicobacter-like
organisms (GHLO) rather than listing them as individual species
the species of veterinary importance, their animal hosts and the diseases that they cause are listed in
the summary table:
gastric helicobacters reside in the gastric mucus layer of various mammals (including dogs, cats,
ferrets, monkeys, cheetahs, pigs, cows, and foxes)
the ecological niche of the intestinal helicobacters is the crypts of the colon and caecum, and in some
cases the organisms also colonise the bile canaliculi of the liver
the gastric Helicobacters are highly prevalent in dogs and cats with between 90-100% of clinically
healthy cats, and 67-100% of clinically healthy dogs infected
105
animals raised in closed colonies (e.g. pounds or kennels) usually have prevalence rates approaching
100% and is usually higher than pet cats or dogs
some Helicobacters colonise specific hosts, whereas others are capable of infecting a numbers of
different animals species
animals and birds may be the reservoirs for zoonotic transmission
Helicobacter are the only bacteria that can colonise the harsh environment
KEY POINT (low pH) of the stomach. Some species of Helicobacter may instead colonise
the colon, caecum and liver.
both oral-oral and faeco-oral routes probably occurs in transmission of gastric Helicobacter
in intestinal Helicobacter, transmission is via faeco-oral route only
there is considerable debate as to whether viable Helicobacter exist in the environment (for example
water sources), even temporarily, and whether these are a potential source for transmission
however, the recent isolation of H. pylori from surface water in the USA and Sweden would suggest
that this is a possible source, and has public health implications (these organisms are quite resistant to
chlorine)
KEY POINT Oral-oral and faeco-oral routes of transmission occur.
relatively little information is known regarding the virulence factors of Helicobacter spp, and the
information that is known is primarily for H. pylori
most reports have focused on H. pylori’s ability to induce gastric inflammation by either disruption of
the gastric mucosal barrier (through cytotoxins or disrupting phospholipases) or by altering the gastric
secretory axis (decreasing somatostatin release, inducing hypergastrinaemia, diminishing
responsiveness of parietal cells and so on)
a) Cytotoxins
9 a vacuolating cytotoxin is produced and is thought to play a role in the H. pylori induced peptic
ulcers, and H. pylori associated chronic atrophic gastritis, a pre-cancerous gastric lesion
9 this toxin, when purified and inoculated into the stomach of mice, will induce acute gastric
erosions
b) Urease
9 the production of urease by Helicobacter results in ammonia production as a by product of
metabolism
9 this is irritant to the gastric and intestinal mucosa
9 thus, the presence of these urease producing bacteria in stomachs and liver may damage cells
adjacent to the colonising bacteria
9 in addition, urease is required (along with flagellae) to sustain colonisation of Helicobacter spp in
the gastric mucosa
106
c) Superoxide Dismutase and Catalase
9 these enzymes have evolved as adaptive mechanisms of the bacteria to minimize oxidative damage
by host cell enzymes
9 in addition, the products of the recA gene, play a role in the repair of DNA and therefore allow
the bacteria to repair themselves in the harsh environment of the stomach
II) Pathogenesis of Helicobacter infections

disease in animals may be defined as tissue injury or clinical manifestation:
9 this fundamental difference is important when discussing infections in dogs and cat
9 this is because the relationship between Helicobacter spp and gastric inflammation in dogs and cats
is unresolved, with inflammation or glandular degeneration accompanying infection in SOME but
NOT ALL subjects
9 there appears to be some association between the number of Helicobacter and the presence of
lymphoid follicles in cats but not in dogs
The exact role of Helicobacter in the induction of gastric inflammation in

KEY POINT dogs and cats remains controversial.
What Diseases do Helicobacter Cause?
in humans, extensive evidence exists implicating H. pylori in the pathogenesis of chronic superficial
gastritis and in formation of peptic ulcers
infection with Helicobacter spp also predisposes humans to development of gastric cancer (carcinoma
and lymphoma) – but the precise mechanism of carcinogenesis is unclear
IN HUMANS there is good evidence that H. pylori is the cause of chronic

KEY POINT superficial gastritis and peptic ulcers and that these diseases may be the
precursors to gastric cancer.
considerable controversy exists as to whether Helicobacter spp cause disease in dogs and cats
to date, relatively little attention has been paid to the pathogenicity of species other than H. pylori and
the consequences of a Helicobacter infection in healthy and sick animals is far from clear
in addition, information gathered in H. pylori infected humans is frequently directly transcribed to
animals (particularly dogs and cats) without consideration of the different Helicobacter spp involved,
or the response of the non-human host
The evidence that Helicobacter spp cause disease in domestic species (in
particular dogs and cats) is far more tenuous than in humans. Despite this
KEY POINT fact, H. pylori is widely reported in the literature as a cause of gastritis, and
is commonly diagnosed and treated in small animal practice in Australia.
107
Gastritis
in dogs and cats it would appear that Helicobacter spp can induce a mild to moderate chronic gastritis
with glandular degeneration and infiltration of lymphocytes and plasma cells with lymphoid
hyperplasia
however, normal gastric secretory function is present in these animals
the evidence for disease in cats is more compelling (slightly!), with more severe gastritis, characterised
by glandular degeneration and marked lymphoid follicular hyperplasia with infiltration by neutrophils
and eosinophils, has been observed with H. pylori infections
however, it should be emphasised that more research is needed to better define the relationship
between these bacteria and clinical disease in domestic species
clinical signs that have been attributed to infection (in both cats and dogs) include chronic vomiting,
weight loss, and in some cases severe emaciation and diarrhoea
these signs have been attributed to the Helicobacter as these organisms were observed in gastric biopsy
samples of cats and dogs with GI illness
however, the direct cause-and-effect relationship to clinical illness is usually not ascertained!!!
the approach to diagnosis of Helicobacter in humans and domestic species differs to most other
bacterial infections in as much as the organism is rarely cultured (as it is difficult) and diagnosis is
usually made through direct visualisation of the organism or through identification of their products
(urease)
much research has been performed on isolation and/or identification of this organism in human
samples because of its importance in human health, however, the significance of isolation and
identification of Helicobacter spp in domestic species remains largely unresolved
Diagnosis of Helicobacter infection in dogs and cats most commonly relies on

direct visualisation (usually via histology) of the bacteria in conjunction with
KEY POINT histological evidence of chronic gastritis and lymphoid aggregates in
animals with clinical evidence of GIT disease.
Diagnostic tests for Helicobacter spp may be divided into:

9 Non-invasive tests
9 Invasive tests
108
TEST MEASURES
Non - Urea breath test Both measure breakdown of urea by urease produced by
Invasive Helicobacter. Mainly used to determine response to therapy in
Blood test humans.
Serology Antibodies to Helicobacter

Invasive Rapid Urease Test All of these tests are aimed at identifying Helicobacter in the
Biopsy and Histopatholgy stomach of suspected cases. Consequently, samples from the
Touch cytology stomach have to be obtained, usually via gastroscopy (invasive).
Culture However, as most dogs and cats are colonised by Helicobacter
PCR spp, the significance of their identification remains controversial.
Electron Microscopy More recently, a visual scale for histologically assessing
canine gastric biopsy specimens has been introduced (similar
to that used for humans) which assesses the degree of gastritis
and lymphoid follicular hyperplasia and offers promise for a more
systematic approach to determining the relevance of the
organisms present. However, as the lesions are not evenly
distributed in the stomach, multiple biopsies are necessary
(fundus, cardia, and antrum-pylorus area), which is quite
invasive.
although humans and animals mount a significant systemic IgG response and local IgA response to
these organisms, these antibodies are not protective, and the bacteria persis in the mucous layers or
closely adhered to the gastric epithelium and protected from the gastric acid milieu
the guidelines for the National Institute of Health in the USA state that all patients with peptic ulcers
and H. pylori infection should be treated
however, no therapy is recommended for H. pylori infected asymptomatic people
a triple therapy regimen using a combination of antibiotics and anti-ulcer medication for 1 – 2 weeks is
widely used in humans and shows eradication rates of >90%
the drugs used in this regimen include amoxicillin (or azithromycin or tetracycline), metronidazole,
and bismuth subsalicylate (or cimetidine, ranititidine, omeprazole, lansoprazole = acid-secretory
inhibitor drugs)
whether antimicrobial therapy should be instituted in domestic pets with gastritis or ulcer disease is
presently unknown, and few controlled, randomised blind therapeutic trials have been published for
dogs and cats (although similar regimens to that used in humans is a common treatment for this disease
in small animal practice!!!)
in addition, in most dogs and cats, if the animal is re-tested for the presence of the bacteria after
cessation of therapy, they are frequently positive
whether this is due to therapeutic failure or re-colonisation is not known
there are currently no vaccines available for this disease
109
cats have been shown to harbour H. pylori in their stomachs and are therefore regarded as potential
natural reservoir for the organism and a zoonotic risk
however, studies of cat owners have demonstrated no greater likelihood of H. pylori antibodies than
non-cat owners, suggesting that transmission between species does not occur
alternatively, it has been suggested that the H. pylori found in cats is actually a anthropozoonosis
(reverse zoonosis where cats get infected by human-derived bacteria), as stray cats apparently rarely
have this species of Helicobacter
other Helicobacter spp (particularly H. bizzozeronii/H. heilmannii), which are predominantly found in
dogs and cats and not people, may potentially be transmitted to people
there has been a couple of documented cases of this occurring in a cat or dog owning people
however it is thought that this is a very unusual occurrence
110
PASTEURELLACEAE
KEY POINT The Pasteurellaceae is a family not a genus.
The family Pasteurellaceae comprises 4 genera:

1. Pasteurella spp.
2. Mannheimia
3. Actinobacillus spp.
4. Haemophilus spp.
Note - there have been frequent name changes among
these 4 genera!!!!
they have a number of common characteristics –
which is helpful to remember for this group of
bacteria
they are all gram negative rods (mostly short rods or
coccobacilli
they are all facultatively anaerobic
they are all oxidase positive; this biochemical test helps to differentiate them from the
Enterobacteriaceae (which are oxidase negative)
they are all non-motile
most of the Pasteurellaceae are commensal parasites of animals (ie part of the normal flora or present
on carrier animals)
each genera will be discussed separately in this section of the notes
111
PASTEURELLA + MANNHEIMIA
Summary Table: The species of Pasteurella, the animals they infect, the site they are normally found
(source) and the diseases they cause.

+
P. aerogenes pigs GIT Gastroenteritis and abortion
P. anatis+ ducks GIT
P. avium+ fowl URT
P. bettyae+ humans URT Bartholin gland abscesses; septicaemia
(humans and infants)
P. cabali++++ horses URT Pneumonia
P. canis++++ dogs URT Pneumonia, mouth disease
P. dagmatis+ dogs URT
P. gallinarum+ fowl URT
M. granulomatis+ cows URT Granuloma
M. haemolytica++++ ruminants URT Pneumonia
P. langaaensis+ fowl URT
P. lymphangitides+ cows Bovine lymphangitis
P. mairii+ pigs Abortion in sows, septicaemia in piglets
P. multocida++++ Ruminants, URT Pneumonia and other respiratory
pigs, cats, dogs, conditions, mouth disorders; bite wound
rabbits, humans infections in cats, dogs, and humans
P. pneumotropica+ rodents URT Pneumonia
P. stomatis+ dogs URT Pneumonia in dogs, other mouth disorders of
dogs
P. tetudinis+ turtles URT
Bibersteinia . ruminants URT Pneumonia
trehalose+
A. ureae+ humans URT
P. volantium+ fowl Wattle
s
bacterial genera)
112
PASTEURELLA
What are they?
they are gram negative coccobacilli or short
rods
they have bipolar staining in smears of
exudates (only the tips of the cells stain if
use Giemsa or Diff Quik stain)
KEY POINT Bipolar staining is useful for identification .
there are currently 16 recognised species (see summary table) – however, they are constantly being re-
classified!!!
Note:
9 P. haemolytica and P. granulomatis have been reclassified as Mannheimia
9 P. lymphangitides should belong to the Enterobacteriacea but has not been re-classified
9 P. ureae has been reclassified as Actinobacillus
9 P. multocida is the type species and the most important pathogen of the Pasteurella genus
¾ It has been divided into a couple of subspecies – don’t worry about these!
9 the other important species are indicated by ++++ in the summary table
9 HOWEVER, in a clinical situation there is usually no advantage in determining the species of
Pasteurella (AS LONG AS YOU HAVE COLLECTED AN APPROPRIATE SAMPLE AND
NOT JUST COLLECTED NORMAL FLORA!!!)
Differentiation to the species level is rarely required for Pasteurella spp.

However, for correct interpretation of an isolate, it is important that you
KEY POINT have collected the appropriate sample in the appropriate fashion and not
collected normal flora (including Pasteurella spp).

Pasteurella have a world-wide distribution
they are normal flora (commensals) of upper respiratory tract and oral cavity of a wide variety of
species (e.g. cattle, sheep horses, pigs, dogs, cats, rabbits, chickens, turkeys, monkeys lions,
panthers, people, turtles etc, etc)
there are some species difference as to exact location (e.g. P. multocida is found in the oral cavity of
cats and dogs, but in the nasal cavity of pigs)
carriage of the organism may be widespread among a population (e.g. P. multocida in cats) or may be
rare (exceptional) as with P. multocida in fowl cholera or haemorrhagic septicaemia
113
in fowl cholera, one host species may serve as the reservoir for another (e.g. ducks or wild water birds
may be the reservoir for infection in domestic chickens)
Pasteurella are part of the normal flora of the upper respiratory tract and
KEY POINT oral cavity.

they don’t have to – they are frequently already there!!! (endogenous infections)
Most infections are ENDOGENOUS, therefore disease results from

KEY POINT opportunistic infections.
alternatively, infections may also be acquired by inhalation or ingestion

9 this occurs in groups of animals in which some of the animals do not have the disease producing
strains as part of their normal flora and other animals do
organisms may also be instilled (e.g bite wounds or scratches)
the Pasteurella are OPPORTUNISTIC PATHOGENS, frequently requiring a change in HOST
FACTORS +/- ENVIRONMENTAL FACTORS to cause disease
however, in some diseases (fowl cholera and bovine haemorrhagic septicaemia), host and
environmental factors play less of a role in the induction of disease, the strains of organism involved
are not usually found as part of the normal flora (therefore direct transmission plays a larger role) and
environmental contamination contributes to indirect transmission within a population
FIGURE: Louis Pasteur, who is considered one of the forefathers of modern day
microbiology as well as the person after whom this family and genus is named.
Notice that he is holding a couple of rabbits (a lot of which he killed in the name
of science!). But this is quite apt as rabbits have a lot of diseases associated
with Pasteurella spp.
114
a) Endotoxin
as with other gram-negative infections, endotoxin undoubtedly plays a role in the pathogenesis of
infection due to Pasteurella spp, as it initiates and promotes the inflammatory cascade
in addition, it is directly toxic to respiratory tract epithelium and it also decreases the quantity of
quality of pulmonary surfactant which alters pulmonary mechanics and gas exchange
in acute diseases, the predominant lesions (widespread haemorrhage and necrotic foci) is presumed to
be due to endotoxin
b) Fimbriae
probably all Pasteurellaceae produce adhesins (fimbriae)
the expression of these proteins is probably reliant on environmental cues – that is the adhesins are
expressed when the bacteria inhabits epithelial surfaces, but repressed when the micro-organism is
inside the host, where adherence to a phagocytic cell would be disadvantageous
they aid adherence to epithelial surfaces, and therefore assist colonisation
c) Capsule
as with other bacteria that possess a capsule, the capsules produced by Pasteurella spp (particularly P.
multocida), plays many roles, the most important of which include:
9 interference with phagocytosis (antiphagocytic)
9 protection of the outer membrane from the deposition of the membrane attack complexes generated
by the activation of the complement system
9 it serves as an adhesin for respiratory tract epithelium in the case of some avian strains of P.
multocida
the amount of capsule produced is inversely proportional to the amount of iron available
9 so more capsules is produced in vivo (where there is less iron), and when it is needed by the
bacteria
9 in P. multocida, five different capsular polysaccharides (serotypes) have been described (A, B, D,
E, and F) where there is a difference in pathogenicity between these different capsular serotypes
(but it is not important to remember these!!!):
i) Capsule Type A
9 some of these serotypes are part of the normal flora of the upper respiratory tract of a number of
species and are primarily associated with respiratory disease in stressed animals
9 other strains of this serotype are more pathogenic and are only found in the respiratory tract of
carrier animals which have survived infection and act as the reservoir for infections in susceptible
animals that are in contact
9 these serotypes have been isolated from a wide variety of infections (see later)
ii) Capsule Type B

9 these serotypes are found in the nasopharynx of carrier animals only and cause disease in newly
susceptible in-contact animals
9 they cause Haemorrhagic Septicaemia in ruminants in Asia
iii) Capsule Type D

9 these serotypes are found as normal flora in respiratory tract and oral mucosa of dogs and cats
9 produce disease only when instilled in abnormal areas e.g. dog/cat bite wounds
9 Type D isolated from pneumonia and atrophic rhinitis in pigs
9 may be also be involved in disease in stressed animals within the respiratory tract e.g. sinusitis,
pharyngitis, otitis media (rarely)
115
iv) Capsule Type E
9 found in the nasophaynx of carrier animals only (see type B above)
9 causes Haemorrhagic Septicaemia in ruminants in Africa
v) Capsule Type F
9 these capsular serotypes have been recovered from turkeys
9 their role in disease is not clear
d) Siderophores
because iron is an absolute requirement growth requirement, Pasteurellaceae must acquire this
substance if they are to exist within the host
some avian strains of P. multocida produce a siderophore (= multicidin) that they use to acquire iron
other non-avian Pasteurella acquire their iron by binding transferrin-iron complexes to their surface by
way of an outer membrane protein whose production is regulated by the concentration of iron
e) Exotoxins
a heat labile exotoxin (pmt or Pasteurella multocida toxin) is produced by some strains of P.
mutlocida -particularly type D isolated from pigs
this exotoxin is dermatonecrotic, cytotoxic and osteolytic – playing a role in the pathogenesis of the
disease atrophic rhinitis of pigs
it is closely related to the cytotoxic necrotising factors produced by some strains of E. coli
it is also mitogenic for osteoblasts
other strains of P. multocida produce hyaluronidase (which may assist spreading of infections through
tissues) and neuraminidase (which may have a role in colonisation of epithelial surfaces)
An exotoxin, produced by P. multocida type D, plays a role production of

KEY POINT lesions observed in atrophic rhinitis in pigs.
II) Pathogenesis
as with other bacterial infections involving normal flora – some change in the host/parasite relationship
must occur for disease to occur
As Pasteurella spp are part of the normal flora, there must be a change in
KEY POINT the host/parasite/environment relationship to allow Pasteurella spp to
cause disease.
frequently this altered relationship is a result of environmental stressors (such as transportation or

cold weather) or concurrent viral infections
these stressors allow proliferation of Pasteurella in the upper airways and also enable migration to
the lower airways
their pili allow them to attach to the epithelial cell surfaces
the capsules and leukotoxins help to subvert phagocytic activity and inflammatory responses, and
endotoxin has a role in the lesions produced
the lesions vary with site of infection, virulence of strains, and host resistance
in septicaemia in ruminants and birds (fowl cholera), vascular damage results in haemorrhage and
fluid loss but little cellular inflammatory response
in these species, focal necrosis in parenchymatous organs or ulceration of mucous membranes may
occur and in ruminants generalised haemorrhagic lymphadenopathy may be observed
in most cases of pneumonia, inflammatory cells are prominent, with RBCs, neutrophils and
mononuclear cells appearing and predominating successively
the tissue appearance changes accordingly from reddish black to red, pink and grey
116
necrosis, abscess formation and fibrin deposition occur and may vary in severity
in chronic fowl cholera, caseopurulent inflammation occurs in joints, middle ear, ovaries or wattle
infections may remain localised or may spread and/or develop into septicaemia in some cases
(particularly in compromised hosts)
Infections with Pasteurella are usually characterised by fibrino-purulent

KEY POINT inflammation.
What diseases do Pasteurella Cause?

disease to P. multocida occur worldwide and occur in virtually all species of animals
this bacteria can cause a wide range of disease and may be the primary agent, but more frequently it is
a secondary invader when resistance of the animal is reduced by various stressors (e.g. transport) or
other pathogenic agents (e.g. viruses, bacteria, mycoplasma)
the diseases that are associated with Pasteurella spp infection fit into 4 broad categories:
1. Septicaemia Cattle, Birds, Rabbits, Pigs, Humans

2. Bronchopneumonia Cattle, Sheep, Goats, Pigs, Horses, Rabbits, Dogs, Cats,
Humans
3. Rhinitis Piglets, Rabbits
4. Wound Infections Cat, Dogs, Humans
the pathogenesis of these primary disease syndromes overlaps
1. SEPTICAEMIA
the strains of P. multocida responsible for septicaemia are present in carrier animals
under stress these carriers excrete large numbers of organisms and if the animals are crowded there
will naturally be a large load of organisms shed into the environment
if at this time these carrier animals are introduced into group of animals where there are susceptible
animals present (i.e. those which do not have antibodies to the specific serotype of this organism),
these naïve animals will become infected, and a proportion will develop disease
this disease is usually an acute septicaemia
some animals survive septicaemia, develop solid immunity and become carriers in turn
septicaemia results from invasion by the Pasteurella through penetration of the pharyngeal mucous
membranes
alternatively, septicaemia may develop subsequent to a localised infection (e.g. pneumonia, wound
infection), usually in compromised hosts
2 examples of specific septicaemic syndromes includes;
i) Haemorrhagic Septicaemia in Cattle (P. multocida capsule types B + E)

9 this disease occur in migrating wild herbivores in Africa (due to serotype E) and in water buffalo
in the monsoon regions in Asia (due to serotype B)
9 if the animals are stressed, carriers will excrete lots of organisms which infect susceptible animals
9 transmission is by aerosol, but the environment aids the spread e.g. high humidity and temperature
allows the organism to enter the respiratory tract where the organisms are able to invade the
mucous membranes of the pharynx
9 these organisms are highly pathogenic and they quickly multiply and invade the blood stream,
where they multiply further with development of septicaemia
9 they result is haemorrhage, oedema on serous and mucosal surfaces and in lymph nodes, spleen,
lungs and other organs (i.e. typical endotoxic shock)
117
9 the organisms also excretes a necrotising exotoxin which is also thought to contribute to the
clinical signs
9 clinical signs will include high fever, depression, subcutaneous oedema, hypersalivation, diarrhoea
or sudden death
9 all excretions and secretions are highly infectious
P. multocida capsular types B and E are exotic to Australia, therefore the

KEY POINT disease Haemorrhagic Septicaemia is exotic to Australia.
FIGURE: Water buffalo in Pakistan. This species can succumb to haemorrhagic

septicaemia caused by P. multocida type B and large losses of precious cattle
can occur during outbreaks. As with other diseases produced by Pasteurella spp,
stress plays a major role in inducing these infections.
ii) Fowl Cholera in wild and domestic Fowl

9 fowl cholera is most commonly caused by P. multocida capsule Type A, where the more pathogenic
strains of this serotypes can result in acute to peracute septicaemia in turkeys, water fowl and chickens
(and other birds occasionally)
9 the organism is transmitted by oral and nasal secretions from carrier animals which contaminate
drinking water and food and infection is acquired by ingestion or inhalation
9 clinical disease is precipitated by stress such as overcrowding, laying or moulting or severe climatic
changes
9 a common scenario occurs when wild birds carrying the pathogenic serotypes migrate to Australia,
become stressed and shed bacteria into environment of local birds who succumb to the infection
9 the peracute form can result in mortality rates of up to 60% without preceding clinical signs
9 the acute type of disease is seen as listlessness, anorexia, diarrhoea and nasal and ocular discharges,
which may last for several days, and may account for up to 30% mortality rates
9 subacute and chronic forms of the disease also occur (P. gallinarum may be isolated from chronic
cases)
118
2. RESPIRATORY TRACT INFECTIONS (Bronchopneumonia):
a variety of respiratory tract infections are associated with P. multocida infections in a range of species
capsule serotypes A, and occasionally type D, are implicated in bacterial pneumonia in a number of
species including cattle, pigs, rabbits, horses, dogs, and cats
disease may also results from extension of organisms to other sites e.g. middle ear
examples of respiratory tract infections include:
i) Shipping Fever in Cattle

9 may be caused by P. multocida type A (but more commonly involves Mannheimia haemolytica –
see later under Mannheimia)
9 this disease is a major cause of economic losses, particularly in the feedlot industry – where it is
the most common cause of mortality in North America (and probably Australia)
Shipping Fever is on of the MAJOR causes of ECONOMIC LOSS for cattle

KEY POINT producers worldwide.
9 the organisms responsible for these infections are present as normal flora in the upper respiratory
tract (URT = naso and oropharynx), though the numbers of these bacteria (particularly M.
haemolytica) may be very low
9 however, the numbers of these organisms increases, especially in response to stressful events
9 the disease is predominantly observed in young animals (6-8 months) and in the presence of
stressful situations (transportation, assembly, handling, starvation, dehydration, overcrowding,
chilling, overheating, excess or irregular high energy feed, poor ventilation, castration, dehorning
etc) or due to the presence of co-operating organisms e.g. viruses (IBR, PI3), mycoplasma, or
other bacteria
9 it is seen as a severe lower respiratory tract disease (fibrinous pneumonia, bronchopneumonia or
pleuropneumonia)
FIGURE: Lungs from a weanling

angus steer that had been recently
transported. Just about the whole
lung is abnormal, with marked
reddening and consolidation evident.
These lungs are stuffed and it is not
surprising that the steer died. P.
multocida type A or Mannhemia
haemolytica (see later in these notes)
are most commonly involve, although
viral infections are a common
predisposing cause (along with the
stress of transportation)
9 although P. multocida (or M. haemolytica) are frequently the secondary invaders in this disease, it
is these bacteria that contribute significantly to the pathogenicity of these infections and result in
severe illness and death
P. multocida and M. haemolytica are the major contributors to the clinical

KEY POINT disease observed Shipping Fever – although they are frequently secondary
invaders.
119
9 clinical signs usually occur 1 – 2 weeks after the stressful event, and are marked by high fever,
inappetence and listlessness
9 the respiratory signs (nasal discharge and cough) are few and variable; but usually become more
pronounced as the disease progresses (and the fever abates)
9 abnormal lung sound are predominantly heard in the cranio-ventral lung lobes (apical lobes),
which are the first and most severely affected
ii) Snuffles in Rabbits (Rhinitis)

9 snuffles is a mucopurulent rhinosinusitis of rabbits due to P. multocida
9 this disease develops under stressful conditions (e.g. pregnancy, lactation or mismanagement)
9 complications include bronchopneumonia, middle and inner ear infections, conjunctivitis and
septicaemia
9 P. multocida is a major pathogen of rabbits and may be involved in other syndromes in this species
including orchitis, balanoposthitis and pyometra
FIGURE: “Pete” the white rabbit who has

“Snuffles”. You can see why this disease got its
name – “Peter” has a mucopurulent discharge
from its nose and which causes him to make a
snuffling noise!!! Infections with P. multocida
are very common in rabbits and should be high
on your list of differential diagnosis when you
meet them in practice!
iii) Atrophic Rhinitis in Pigs

9 atrophic rhinitis is a disease of young pigs (3-7 weeks)
9 it is due to P. multocida type D (and occasionally type A) which are carried in the nasal cavity of
clinically normal pigs
9 the disease is a result of the combined infection of the nasal turbinates with Bordatella
bronchiseptica and toxigenic strains of P. multocida
9 certain strains of type D have been shown to produce a heat-labile exotoxin, which is
dermonecrotic and cytotoxic and results in turbinate bone destruction
9 however, this toxin can only induce lesions within the nasal cavity if the nasal mucosa is firstly
irritated
9 this irritation is caused by an unrelated dermonecrotic toxin produced B. bronchiseptica
9 in addition, high concentrations of ammonia (that are found in pig production units), may act
synergistically with the P. multocida toxin
9 the underlying lesion in atrophic rhinitis is a chronic rhinitis accompanying disturbed osteogenesis
adjacent to the inflamed areas
120
9 increased osteoclastic and diminished osteoblastic activity destroys the turbinates and bones of the
snout, resulting in distortion of facial structures
9 histologically, fibrous tissues replaces osseous tissue
9 bone atrophy is accompanied by inflammation of varying acuteness
9 clinical signs in these young piglets include sneezing, epistaxis, and tear duct obstruction (seen as
staining of the face)
9 skeletal abnormalities produce lateral deviation of the snout or wrinkling due to rostrocaudal
compression
9 the piglets may go onto develop pneumonia – caused in part due the fact that they no longer have
turbinates, which are part of the natural defence mechanisms of the respiratory tract
9 atrophic rhinitis has also been reported in rabbits
FIGURE: The snout of a pig in cross

section. This pig was suffering from
Atrophic Rhinitis which is caused by a
combination of Pasteurella multocida
type D and Bordatella bronchiseptica
(see another picture of this under
Bordatella). Note the atrophy of the
turbinate bones in the nose of this pig.
An exotoxin produced by P. multocida
type D plays a major role in the production
of this disease
3. OTHER INFECTIONS
as P. multocida is an inhabitant of the oral flora of a large number of different species, this organism
may be commonly involved in contamination and infection of BITE WOUNDS and ABSCESSES (they
are the most common organism isolated from bite wound infections along with strict anaerobes)
infections with P. multocida are particularly common as a result of cat and dog bite wounds and cat
scratches, and therefore are most commonly seen in cats, dogs (involved in fights) and also humans
bite wound infections are more common in cats than dogs as their skin is tougher and more elastic and
therefore seals over the contaminated puncture wounds more readily
infections are often polymicrobial (involving more than one type of bacteria) and usually include strict
anaerobic species (also found in the oral cavity of the biting animal)
Bite wound infections are common in cats and dogs and are usually
KEY POINT polymicrobial – involving P. multocida, P. canis and strict anaerobes (e.g.
Bacteroides spp).
the size and degree of abscess formation depends on a number of factors including the overlying skin
tension, amount of dead space, and gravitation of exudate below the point of penetration
abscesses are most commonly found around the face, legs, back and base of the tail
the clinical signs of abscess formation reflect the site and severity of the infection and may include
noticeable swelling with few other signs of illness, or more extensive infection is associated with
fever, anorexia, depression and regional lymphadenopathy
121
the abscesses begin as firm tissue swellings (which are hot and painful to touch) which mature to a soft
fluctant central area, which discharges (if possible) to an external site
the discharge is often foul smelling (due to the presence of anaerobes) and red-brown in colour
these infections usually remain localised (the sick animals are toxaemic not bacteraemic), however in
compromised hosts they can progress to more serious diseases e.g. septicaemia, peritonitis,
endocarditis, etc
P. multocida may also be isolated from wound infections (due to licking), foreign body lesions and
infections of serous cavities (e.g. pyothorax) in dogs and cats
other diseases associated with P. multocida in domestic species include sporadic encephalitis, abortion
and urinary tract infections (UTI)
FIGURE: “Deuteronome”, a 9 year old intact male cat who has a large fluctuant swelling
underneath his chin. “Deuteronome” is a bit of a fighter, and his reward is a nice big abscess
associated with a bite wound. This is probably polymicrobial (involving a number of different bacteria
that originate in the mouth of the biter), but commonly involves P. multocida.
Large fluctuant swelling under the jaw of

“Deuteronome”. This lesion developed after being bitten
in a fight!@!!
How would you diagnose an Infection?

fine needle aspirates of abscesses (wound infections) are the optimal sample to obtain
in cases of pneumonia – transtracheal washes (samples obtained from the trachea in a sterile fashion by
going directly into the cervical trachea and therefore avoiding the normal flora of the upper respiratory
tract) may be obtained in live animals, or alternatively samples via a guarded catheter through an
endoscope may be obtained
in animals that have recently died (< 2-4 hours) or have been euthanised, swabs taken in the
appropriate fashion (see PM laboratory notes) may be used for cultivation
in birds, blood may be collected for direct examination and cultivation if septicaemia (fowl cholera) is
suspected
122
it is important to transport the samples as quickly as possible to the laboratory, and without cooling
(unlike most bacteria) as these organisms do not survive very well during transportation
smears of exudates, tracheal wash samples and blood may be stained with Giemsa or Diff Quik and
examined for bipolar organisms
their presence is suggestive (but not unique) to Pasteurella
on gram stain, Pasteurella do not look distinctive
Small rods that show bipolar staining Red Blood Cells
FIGURE: Blood smear stained with Giemsa (similar to Diff Quik)

showing the bipolar staining of the organims (Pasteurella spp).
2. Cultivation
Pasteurella grow best in presence of blood or serum and are small, clear colonies after 24 hours
incubation
no haemolysis is observed (as opposed to Mannheimia haemolytica)
3. Identification
there are a number of biochemical tests that are used to differentiate this genus and include the oxidase
test and motility
they are oxidase positive (as compared to Enterobacteriaceae which are negative)
they are non motile (as compared to Pseudomonas which is motile)
for further tests and description of colony morphology etc see practical class notes

immunity to infection with Pasteurella spp is predominantly humoral (Antibody Mediated Immunity)
and plays a significant role in protection against haemorrhagic septicaemia in ruminants and fowl
cholera
the type specific capsular antigens are the major immunogens in haemorrhagic septicaemia
123
with other forms of pasteurellosis, the picture is less clear, and both antitoxic and antibacterial
antibody appears to be important

Pasteurella spp, especially P. multocida from food producing animals, have become increasingly
resistant to penicillin (less so strains from dogs, cats and horses), tetracyclines, and sulphonamides
this is because of widespread usage of these drugs in order to treat pneumonic pasteurellosis in this
species (and to treat infections in ruminants in general)
Increasing RESISTANCE is being observed in Pasteurella spp and is the result

KEY POINT of heavy antibiotic usage in ruminants.
resistance is usually associated with R plasmids

the gene encoding resistance to tetracyclines is unique to Pasteurella (but there are other forms of
tetracycline resistance)
strains from carnivores, horses and humans have less resistance (due to less pressure) and are
generally susceptible to most antibiotics, except aminoglycosides
9 penicillin is usually the drug of choice in these species
strains from food producing animals have more variable antibiotic resistance patterns (see above)
in these species, cost of the drug, and withdrawal times are additional considerations
9 ceftiofur, tilmicosin, florfenicol, tetracyclines (and potentiated sulphonamides and Penicillin G if
susceptible) have been used to treat infections
9 tilmicosin and florfenicol however, are not available in Australia
in birds, sulphonamides, penicillin G, fluoroquinolones (!!!) and tetracyclines may be used to treat
fowl cholera
oral therapy using sulphonamides, tetracyclines (and in pigs and poulty penicillin) are used for mass
medication via feed or water, both therapeutically and prophylactically
How would you Control against Disease?

disinfectants, heat and U.V. light are rapidly lethal for Pasteurella spp
however, P. multocida may survive for months in dead bird carcasses
vaccines including bacterins (whole, killed vaccines), live attenuated vaccines and subunit vaccines are
available and have been used for the prevention of shipping fever and fowl cholera respectively with
varying degrees of success
immunisation is more dependable for control against bovine haemorrhagic septicaemia (because of the
specific capsular types involved), and atrophic rhinitis
management practices directed at reducing stress are important in the prevention of pasteurellosis in
livestock

Pasteurella multocida has also been isolated from a wide variety of infections in humans
infection of wounds with P. multocida after bites or scratches usually involve animal isolates (ie
isolates found in the mouths of dogs, cats, horses etc)
in general, those involved with cat bites or scratches are more likely to cause infection and cause more
serious infections (this will be discussed in greater detail in lectures and notes on zoonotic infections in
semester II)
124
all animal bites should be treated rapidly and appropriately
other human infections with P. multocida usually do not have an animal source
Dog and cat bite wounds are also

commonly infected when humans are
bitten (especially cat bite wounds). So
when you become vets – make sure that
you are aware of these infections and get
appropriate treatment (and this does
NOT mean treating yourselves!!!).
FIGURE: Mannheimia haemolytica is an important pathogen of sheep and

goats and is the predominant species involved in pneumonia in these animals
(Pasteurella are rarely involved). This is “Molly” the ewe with her baby
“Mollette”. Unfortunately, both “Molly” is not that well – see the next page!
125
MANNHEIMIA
What are they?

until recently, this organism was called Pasteurella haemolytica
there were 2 different biotypes (A and T) recognised which differed in pathogenicity, antigenicity,
biochemical activity and species in which disease is produced (A = cattle, T = sheep)
9 Biotype A – has now been reclassified as Mannheimia haemolytica
9 Biotype T – has now been renamed as Pasteurella trehalosi
Mannheimia haemolytica is found as a commensal of the nasopharynx of some normal cattle and sheep
there are currently 11 serotypes based on capsular antigens

part of the normal flora of the upper respiratory tract of ruminants

the resident flora are usually involved in disease
many of the virulence factors for P. multocida are also involved with M. haemolytica infection
however, this bacteria has an additional virulence factor that is very important in the pathogenesis of
the disease produced by this organism:
Leucotoxin
some strains of P. haemolytica produce a powerful exotoxin called leucotoxin (lkt) which belongs to an
important group of exotoxins called RTX toxins
the P. haemolytica leucotoxin is capable of killing alveolar macrophages and other ruminant leucocytes
if it is in high concentration
it also causes down regulation of MHC type II surface proteins on macrophages and therefore affects
their ability to present antigen to immune cells
the leucotoxin thus aids the pathogenicity by destroying a primary defence mechanism of the lungs
(phagocytes) and by impeding specific development of immunity
destruction of leucocytes also increases the inflammation by release of acid and enzymes from
leucocytes
in low concentrations, M. haemolytica leucotoxin activates macrophages (rather than kills them) which
results in release of tumor necrosis factor (TNF) and IL-1
these cytokines cause stimulation of PMNs leading to the release of H2O2, which in turn is converted to
hydoxyl radicals by alveolar epithelial cells; these radicals kill cells and result in the accumulation of
oedema fluid and fibrin
126
What Diseases does Mannheimia haemolytica cause?
1. Shipping Fever
Bronchopneumonia (“shipping fever” in cattle or “enzootic pneumonia” in sheep and goats) is the
most important infection caused by M. haemolytica
the same disease may be produced in cattle by P. multocida type A in cattle, but in sheep and goats
only M. haemolytica is usually involved
the disease has been described in the notes on Pasteurella (see above)
the bacteria may have either a primary or secondary role in the development of the severe, fibrino-
necrotic pneumonia
2. Septicaemia
M. haemolytica also causes septicaemia in nursing lambs, which is frequently co-incident with a
change in diet
in these cases the organisms, that are already present in the tonsils, multiply and invade the adjacent
tissues and thus gain access to the bloodstream
this disease resembles bovine haemorrhagic septicaemia, except intestinal involvement is often absent
and the morbidity and mortality rates are much lower
3. Mastitis
P. haemolytica also causes severe necrotising, gangrenous mastitis in ewes (often called “blue bag”)
nursing lambs introduce the organism from their nasopharynx and provide the mechanical trauma of
the teat required for invasion (it often occurs late in lactation as the lambs are bigger and more capable
of inducing bruising and trauma)
severe mastitis results (parts of the udder may undergo necrosis and sloughing – hence “blue bag”) and
acute systemic reactions may accompany the disease of the udder due to endotoxaemia, with death
possibly ensuing
a similar disease occurs in cattle, though less commonly than in sheep
FIGURE: The udder of “Molly” and it

is pretty obvious that she is suffering
from mastitis. In sheep this can be
due to Mannheima haemolytica. This
disease is also called “blue bag”
(because the udder becomes a blue
colour due to the severe necrosis
and sloughing of the skin). Trauma
induced by “Mollette” whilst suckling is
a common instigating cause. The
bacteria are part of the normal flora of
the URT and GIT of the lambs and can
be inoculated into the traumatised
udder at this time.
127
How would you diagnose an Infection?
as for P. multocida except M. haemolytica colonies are haemolytic on blood agar

Calves naturally exposed to M. haemolytica or vaccinated with attenuated live organisms, develop
antibodies to all surface antigens and the leukotoxin, and are resistant to experimental challenge
further studies have demonstrated that antibodies to the leucotoxin (cytotoxin) and capsule induce the
best protection, and calves with low antibody titres to the leucotoxin are more likely to die from
pneumonic pasteurellosis
however, if antibodies to the capsule only are produced, the organisms may get phagocytosed but the
exotoxin is concentrated intracellularly and is more potent
therefore, most current vaccines include only the leucotoxin

as for P. multocida infections in ruminants
remember, antimicrobial resistance to commonly used antibiotics in ruminants (e.g. penicillin,
tetracyclines and sulphonamides) may occur
therefore susceptibility testing is recommended if a serious outbreak occurs
empirical therapy with ceftiofur (a 3rd generation cephalosporin), tilmicosin or florfenicol has been
used with success in North America (but only ceftiofur is available in Australia)
How would you Control the Disease?

there have been many attempts to produce an effective vaccine for Shipping Fever
currently, the recommendation for feedlot cattle is vaccination twice at a14 day interval with M.
haemolytica leucotoxin extract vaccine, with the second dose at least 14 days before arrival in the
feedlot
however, variable results have been produced with this regimen, and the search for the optimal
vaccine is ongoing
one recent development is the incorporation of the M. haemolytica leucotoxin in transgenic white
clover in order to produce an edible vaccine! (ref: Infect Immun 2001; 69:5786—93)
chemoprophylaxis (antimicrobial administration) does appear to decrease disease in animals introduced
to a feedlot
for example, in a recent paper it was reported that florfenicol administration at the time of arrival at a
feedlot significantly reduces the incidence of respiratory tract disease and delays the time of onset of
disease, whereas vaccination with a bacterin-toxoid and a modified live leucotoxin deficient strain had
no effect on the development of respiratory tract disease (AJVR 2002; 63:251-256)
preconditioning programs (preparing weaned calves for a feedlot environment) have also been used
(with variable success) in order to decrease the economic impact of this disease
combined vaccination programs including vaccines for IBR, PI3 and Haemophilus somnus together
with M. haemolytica vaccine have also been used to decrease the overall incidence of bovine
respiratory disease
128
ACTINOBACILLUS
Although Actinobacillus are genetically distinct from Pasteurella (that is why they are a separate genus!),
they are phenotypically very similar, live in similar environments and are associated with similar diseases.
Summary Table: Species of Actinobacillus that cause disease in domestic species, the animal that they
infect and the diseases that they cause.
BACTERIA HOST DISEASE

A. lignieresii+++ ruminants pyogranulomatous processes (e.g. “wooden
tongue”)
A. equuli++++ horses septicaemia (“Sleepy foal disease”), peritonitis
(pigs)
A. equuli ss haemolytica+ horses Pneumonia
A. suis++ pigs septicaemia, pneumonia, localised infections
A. pleuropneumoniae++++ pigs porcine pleuropneumonia
A. capsulatus+ rabbits Arthritis
A. salpingitidis+ chickens Salpingitis, peritonitis
A. seminis+ sheep Epididymitis
bacterial genera)
A. salpingitidis and A. seminis do not belong to the genus Actinobacillus and will probably be
reclassified soon!
129
What are they?
they are gram negative, small rods (coccobacilli)
they are facultatively anaerobic
almost all species are urease (+ve)
the species of veterinary interest are listed in the table (with weighting for their relative importance):

worldwide distribution
occur as commensal parasites (normal flora) on mucous membranes of the gastro-intestinal tract,
respiratory tract and genital tracts of various spp
A. lignieresii mouth + GIT cattle and sheep
A. equuli + A. suis-like mouth, tonsils + GIT horses
A. suis mouth + URT pigs
A. pleuropneumoniae URT pigs
Actinobacillus are normal flora – found on the mucous membranes of the

KEY POINT oropharynx, nasopharynx and GIT.
How do the enter the Host?
except in neonates, most infections with Actinobacillus are probably endogenous
KEY POINT Most infections are ENDOGENOUS.
in addition, they are not invasive organisms – therefore they have to be instilled before they produce
disease
they are opportunistic pathogens, causing disease when their host’s integrity is compromised, as with
trauma, immaturity or other stressors
trauma to mucous membranes of ruminants by rough feed may cause the herd outbreaks (and the
“appearance” of being a transmissible disease)
How do they cause Disease?
a) Endotoxin and Capsules
as with other members of the Pasteurellaceae, virulence is associated with endotoxin production and
sometimes capsule (polysaccharide) formation (capsules are only found in A. pleuropneumoniae)
endotoxin is also thought to be play a role in the vasculitis observed in the glomeruli of the kidney
associated with A. pleuropneumoniae infections
protective immunity may be conferred by antibody to the casule – though immunity is serotype specific
(8 serotypes identified)
b) Exotoxin (Haemolysin)
equuli and A. lignieresii produce an RTX haemolysin, which destroys RBCs and WBCs
130
however, they produce very small amounts of this toxin and so are not very haemolytic on blood agar;
nor do they destroy many WBCs within the lesions that are produced (PMNs do not appear very
“toxic” in these infections)
in contrast, A. suis also produces an RTX haemolysin and A. pleuropneuomoniae produces 3 different
RTX toxins - these 2 species produce a lot of haemolysin
the RTX toxin kills macrophages and neutrophils at high concentrations and stimulates an oxidative
burst at lower concentrations (see earlier under Mannheimia) – it is an important virulence determinant
– especially of A. pleuropneumoniae
the presence of exotoxins in other Actinobacillus species has not been determined
The RTX leukotoxin of A. pleuropneumoniae is an important virulence

KEY POINT determinant.
II) Pathogenesis
the bacteria are present as part of the normal flora in the oral cavity, gastro-intestinal and respiratory
tracts
transition to a pathological state requires a change in host parasite relationships
9 stress e.g. overcrowding
9 at birth (naïve host)
9 entry to site where it is normally not found (e.g. penetrating wounds – typical scenario with A.
lignieresii)
What Diseases do they Cause?

in general, diseases caused by Actinobacillus spp are suppurative to granulomatous, and may resemble
“actinomycosis” (but don’t get the 2 genera confused – Actinomyces are gram positive rods!)
Infections caused by Actinobacillus spp appear as suppurative to

KEY POINT granulomatous lesions.
FIGURE: “Millicent”
the jersey cow.
“Millicent” has a
discharging sinus on
the side of her face and
is also having trouble
eating lately.
Subsequently, she has
lost weight as well as
decreasing her milk
production (a bad thing if
you are a dairy cow).
131
i) A. lignieresii
this species causes disease in cattle and less commonly sheep and rarely pigs, dogs, horses and humans
the organism is probably inoculated by trauma (e.g plant thorns), and then initiates disease
the lesions consist of multiple granulomatous abscesses, most frequently around the head and neck
region, but may also be seen in the upper alimentary tract e.g. rumen, reticulum, and may extend to
the liver
the chronic granulomatous disease, which is most commonly seen in the tongue of ruminants, is called
“Wooden Tongue”
The disease WOODEN TONGUE is caused by A. lignieresii and occurs in

KEY POINT ruminants (predominantly cattle).
these lesions begin as a firm nodule that eventually ulcerates and discharges a thick, viscous white-
green pus that contains small (<1mm) grey-white granules = “sulphur granules” or “club colonies”
other genera that produce similar “club colonies” (and therefore which you need to differentiate from
Actinobacillus if you see these colonies) include Actinomyces and Staphylococcus (see class practical
notes for details)
regional draining lymph nodes may also be affected in cases of actinobacillosis, as the organism is
spread via the lymphatics
lesions may also involve the lungs and less frequently other internal organs
lesions in sheep may be seen in the lungs and mammary glands
in all species the course of the disease is slow and protracted and healing is also slow
A granulomatous abscesses
within “Millicent’s” tongue
FIGURE: Unfortunately “Millicent” was culled. A post mortem examination

revealed not only a large abscess within her check and which was draining to
the outside via a sinus tract, but also multiple granulomatous abscess within
her tongue.
132
ii) Actinobacillus pleuropneumoniae
this bacteria was formerly called Haemophilus pleuropneumoniae
it is a major cause of pneumonia in pigs, particularly young grower and finisher pigs (2-6 months of
age)
it is not considered part of the normal flora, but can be isolated from nasal mucosa and tonsils of
healthy carrier pigs in infected herds
infection is spread by contact (direct and indirect) and is via inhalation
in newly infected herds, morbidity and mortality may be very high, but in endemic herds morbidity
and mortality rates are much lower, although infection rates remain high (pigs become infected but
they do not get disease)
the spread of infection is favoured by crowding and poor ventilation
the organisms enters the lower respiratory tract and susceptible pigs develop a severe fibrinous,
necrotizing, haemorrhagic pleuropneumonia
the clinical signs vary with the immune status and environmental stress of the pig, and may be
peracute, acute, subacute or chronic
in the majority of cases the clinical course is 1-2 days and includes fever, anorexia, reluctance to
move, and respiratory distress (laboured respiration with exaggerated abdominal components –
“thumps”)
a blood stained frothy discharge from the nose and mouth is characteristic
however, even in the most extensive pneumonia, the bacteria are rarely isolated from tissues outside
the respiratory tract except in the young piglet without maternal antibodies where septicaemia is
occasionally seen
abortion may occur, but this is probably due to the effects of the fever and/or toxaemia
the disease may be diagnosed using serology (ELISA) or by cultivation of samples obtained from the
lungs of diseased pigs at post mortem
however, nasal swabs may be used to detect carriers in a herd situation (for eradication of the disease)
and serology is also useful for monitoring infections in herds (for control measures)
iii) A. equuli
this species is pathogenic for horses (predominantly), pigs and occasionally calves
the mode of infection is probably via ingestion, though infection of the umbilicus or placental
transmission have also been recorded
in addition, there is some speculation that Strongylus vulgaris larvae may carry the organism and this
may be the route of infection for GIT disease (peritonitis) and prenatal infections in foals (mares
become bacteremic and the organism is then transferred to the foal in utero)
There are 3 primary disease syndromes associated with A. equuli
1. Septicaemia in foals (“Sleepy Foal Disease” or “Navel Ill”)

9 foals are usually infected at birth (or less commonly prenatally) and clinical signs are observed
within a couple of days (fever, inappetence, prostration)
9 stressed foals become septicaemic and may die within 48 hours of birth
9 the foals that survive usually develop multi-focal abscesses in organs such as kidneys and joints
(see multiple swollen joints)
9 this organism may also occasionally cause septicaemia in piglets and calves
133
FIGURE: The kidney from a week old quarter
horse foal with “Sleepy Foal Disease”.
Haematogenous dissemination of
Actinobacillus equuli results in microabscesses
throughout the body, including the kidney.
These appear as very small, creamy dots in the
cortex of the kidney. You would expect this
region to be affected as it the organisms lodge in
the glomeruli and form an infection (and
inflammation) at this site.
2. Peritonitis
9 this disease primarily occurs in adult horses
9 there may be a history of stress (e.g. transportation) but this is not necessary
9 there is some speculation that there is an association with migrating strongyle larvae
9 the horses develop a severe peritonitis with moderate to marked peritoneal effusions with very high
WBC counts present, though low numbers of bacteria are observed in these fluids
9 clinical signs include inappetence, fever and mild colic
FIGURE: A large volume of peritoneal fluid

obtained from “El Karim” a 14 year old Arab
gelding with signs of mild colic. The fluid is a
yellowish/reddish/brown colour, which is typical
of infections of the peritoneal cavity (peritonitis)
caused by Actinobacillus equuli. There was
a very high WBC count present in this fluid
(>250 x 109 WBC/L), although the neutrophils
were not demonstrating any toxic changes. “El
Karim” responded very well to antimicrobial
therapy (Penicillin G) and lots of TLC.
3. Pneumonia
9 Actinobacillus spp is commonly found as part of the mixed bacterial infections (together with
Streptococcus spp and Pasteurella spp) in lower respiratory tract infections in horses
9 Both A. equuli and A. suis-like bacteria have been isolated from this condition
iv) A. suis
is not a common cause of disease, but has been associated with septicaemia and other infectious
processes in young piglets and occasionally abortion in sows
134
How would you Treat these Diseases?
as with some of the Pasteurella and also Mannheimia, many strains of A. pleuropneumonia are
developing increasing resistance to antibiotics (and there are different patterns of resistance depending
on the geographical location)
hence, antimicrobial sensitivity testing is recommended
empirical therapy with ceftiofur, penicillin, (or fluoroquinolones) may be used whilst waiting for
results of sensitivity testing
however, results of treatment are frequently disappointing because of the severity of acute disease and
the persistence of infection in recovered pigs (which may then act as carriers and be the source of
infection for other pigs in the herd)
in finishing units, where the disease was confirmed and sensitivity testing performed, twice daily
injections early in the course of the disease was found to be superior for treatment of the disease to
mass medication in feed or water
for other species of Actinobacillus – penicillin is the drug of choice
Penicillin is the drug of choice for infections for all Actinobacillus spp
KEY POINT except A. pleuropneumoniae.
How would you Control these Diseases?

the only disease caused by Actinobacillus for which there are recognised control programs is porcine
pleuropneumonia caused by A. pleuropneumoniae (this is indicative of the prevalence and therefore the
economic importance of the various diseases)
there are basically 2 options for the control and prevention of porcine pleuropneumonia:
9 control at an economical level using good management combined with possible use of vaccines
9 eradication of the infection from the herd
vaccines for A. pleuropneumoniae exist and are effective, but are only serotype specific (there are 12
serotypes) – however, some cross protection between serotypes does exist
as this organism is not part of the normal flora (unlike the other species of Actinobacillus) eradication
is possible – mostly through depopulating the entire herd and then repopulation with animals from
herds that are clinically and serologically negative for A. pleuropneumonia
evidently, strict biosecurity must then be maintained to avoid re-introduction of the agent
135
HAEMOPHILUS AND HISTOPHILUS
Haemophilus and Histophilus are the last of the genera in the family Pasteurellaceae that we will discuss.
They are different to the other 3 genera in that they require specific factors (X+/-V) within the media for
them to grow. In addition, they really like microaerophilic conditions. Hence they can be more difficult
to isolate than the other 3 genera. Similar to the other Pasteurallaceae they like to hang out in the
respiratory tract and cause disease in this site. However, unlike the other, some species of Haemolphilus
also like to cause in the genital tract or even the brain!
Summary Table: Species of Haemophilus and Histophilusthat cause disease in domestic species, the
animals that they infect, the site that they are normally found (source), and the
diseases that they cause.

Haemophilus parasuis pigs URT Bronchopneumonia, Glasser’s disease
(polyserositis)
Avibacterium poultry URT Infectious Coryza
paragallinarum++++
Histophilus somni+ cattle URT, Pneumonia, thromboembolic meningo-
genital encephalitis (TEME), arthritis, reproductive
failure (abortion)
Haemophilus dog genital Neonatal infections, vaginitis, balon
haemoglobinophilus
136
What are they?
they are small, gram negative rods and filaments

they facultatively anaerobic, but most grow better with increased CO2
usually require complex or preformed growth factors provided in blood for growth
9 X factor = protoheme, and/or

9 V factor = NAD
9 see practical notes for details of species requirements
9 commercial discs of X and V factors are available
Haemophilus spp require X +/- V Factors in the media for growth – therefore
KEY POINT cannot be cultured on routine blood agar.
even after specific growth factors have been provided, growth is best on complex media e.g. chocolate
agar (basically blood agar where the RBCs have been lysed)
if put Haemophilus onto media deficient in one or both of these factors, Haemophilus colonies will
cluster around containment colonies of other bacteria which produce the critical factors in excess
(“satellitism”)

these organisms are commensals on mucous membranes of the upper digestive, respiratory and genital
tracts of certain species (generally host specific):
Respiratory tract H. parasuis pigs

H. paragallinarum poultry
Histophilus somni cattle
H. parainfluenzae cats, cattle, sheep
Genital tract Histophilus somni cattle

H. haemoglobinophilus dogs
as with P. multocida, amongst Haemophilus there are strains which are part of the normal flora (e.g.
H. parasuis, H. somnus etc) and others which are only found in carrier animals (e.g. H.
paragallinarum in poultry)
Most Haemophilus spp are part of the normal flora of the respiratory or
KEY POINT genital tract of animals.
How do they Enter the Host?

the most frequently route of entry is by inhalation (exogenous route) due to close contact
but infections may also be endogenous (already there!)
indirect transmission is likely during epidemics
137
a) Endotoxin and Capsule
as with other members of the Pasteurellaceae, virulence is associated with endotoxin production and
anti-phagocytic capsule (polysaccharide) formation
endotoxin binds to macrophages (through LPS binding proteins) and causes release of the pro-
inflammatory cytokines IL-1 and TNF
b) Immunoglobulin Binding Proteins

virulent strains of H. somni are resistant to killing by complement proteins, whereas commensal
strains are not
evidence suggests that this serum resistance is related to the presence of immunoglobulin-binding
proteins (bind Ig in a way that they then can’t fix complement and therefore avoid complement
mediated killing)
surface fibrils are also thought to play a role
c) Exotoxin
like Pasteurella spp, Haemophilus gallinarum produces a heat stable exotoxin which kills macrophages
in the lower respiratory tract and exacerbates bronchopneumonia
H. somni and H. parasuis also produces an exotoxin that is cytotoxic to endothelial cells
II) Pathogenesis
diseases associated with Haemophilus are not very different clinically to Pasteurella
young or naïve animals are most susceptible
stress and crowding play a role
Haemophilus spp usually gain infection through the mucous membranes of the nasopharynx
if multiplication is not checked, it may lead to pneumonia, bacteraemia, joint infections and meningitis
all infections have a suppurative component
there are many Haemophilus spp that may be found on domestic animals and poultry, but serious disease is
associated with only a fewe)
i) Haemophilus parasuis
this organism is part of the normal flora of the nasopharynx of pigs
7 serotypes and 2 biotypes have been identified:
9 biotype I – normal animals
9 biotype II - disease in young pigs and carrier state
H. parasuis can cause bronchopneumonia secondary to viral infection (just as with Pasteurella
infections)
other bacteria (e.g. Pasteurella, Mycoplasma) may participate
however, in young, weaned pigs organisms can invade the mucosal barrier of the oropharynx and enter
the bloodstream resulting in septicaemia and localisation in serosal and synovial cavities – Glässer’s
Disease
Glasser’s Disease (polyserositis) is an important disease of young, weaned

KEY POINT pigs caused by Haemophilus parasuis.
138
some of the affected pigs may die of septicaemia, but most develop a serofibrinous serositis (pleuritis,
peritonitis, pericarditis, arthritis, meningitis)
this is associated with biotype II
the mechanism of invasion is not known, but stress factors are often identified (weaning, transport,
management stress)
the disease strikes sporadically within days of the stressful event
morbidity and mortality are generally low in endemic herds (due to widespread acquired resistance –
disease occurring in weaned pigs as maternal antibody is waning), but may be high in previously
unexposed herds (pathogen free piggeries)
clinical signs included fever, inappetence, respiratory and abdominal distress, lameness, convulsions
or coma, and widespread discolouration of the skin
death is probably due to endotoxic shock as evidenced by DIC present (e.g. glomerular thrombus
formation and microthrombi in other organs)
ii) Avibacterium paragallinarum

Used to be called Haemophilus paragallinarum
this organism is the cause of Infectious Coryza of chickens
it is an acute, contagious upper respiratory tract infection of chickens of all ages and is characterised
by nasal discharge, sneezing, swelling of the sinuses and oedema of the head
there is a catarrhal inflammation of these sites with marked heterophil infiltrates
the lower respiratory tract may also be affected (air sacculitis), and rales may be heard in these
chickens
chronic forms of the disease also exist
in general, there is high morbidity, but low mortality – but can cause significant economic losses due
to decreased growth and egg production
in addition, superimposed infections with mycoplasmas and helminth parasites exacerbate and prolong
outbreaks
recovered chickens become carriers (remember they are not considered to be normal flora) and may
shed for a long time
Japanese quail are also highly susceptible to infection
FIGURE: A piglet (“Yet another Babe!”) that has septicaemia caused by

Haemophilus parsuis. This infection is called “Glasser’s Disease”. Note
there are multiple dark reddish discoloured areas of the skin associated
with disease (due to widespread vascular damage therefore leakage of RBCs
in skin and subcutaneous tissues).
139
iii) Haemophilus somni
Used to be called 3 separate bacteria: Haemophilus somnus, Haemophilus agni, Histophilus ovis
this organism is a common cause of disease of cows in the US, particularly in feedlot cattle, but occurs
less commonly in Australia
it is part of the normal flora of male and female bovine genital tract, and less commonly in respiratory
tracts
spread is by contact and it appears that calves are affected early in life from their dams
there are 4 main syndromes associated with infection, but a range of other conditions have been
reported:
1.Thromboembolic Meningoencephalitis (TEME)

9 seen in feedlot, weaner calves
9 crowding, intercurrent disease and exhaustion can precipitate the disease
9 invasion of the genito-urinary tract or respiratory tract results in septicaemia and localisation in
cerebral tissues
9 H. somnus adheres to the vascular endothelial cells, resulting in damage and exposure to
subendothelial collagen
9 this causes platelet aggregation, coagulation and thrombus formation (which are all the effects of
endotoxin release) with subsequent infarcts in the brain and cerebellum
9 the pre-encephalitic phase is marked by high fever, clinical signs in the encephalitic phase depend
on the area of the brain that is affected (motor and behavioural abnormalities may develop)
FIGURE: The brain of a cow suffering from thromboembolic meningoencephalitis

(TEME). This is due to infection with Histophilus somni. There are multiple small red
areas of haemorrhage throughout the brain which are associated with vasculitis, thrombus
formation, and infarcts (with subsequent leakage of RBCs into the parenchyma of the brain.
Multiple small red areas within the brain which are

small areas of haemorrhage associated with
vasculitis and thrombotic infarcts
140
2. Respiratory Disease
9 similar pathogenesis to “shipping fever” caused by Pasteurella spp
9 results in pneumonia +/- septicaemia
3. Arthritis
9 joint infections and arthritis may result from septicaemia
4.Reproductive Failure
9 significant cause of early abortion, cervicitis and endometritis in cows
9 vaginal discharge is commonly observed
iv) other species of Haemophilus

poorly characterised “species” of Haemophilus have been isolated from rams (used to be called H.
ovis or H. agn – now identified as Histophilus somni) with epididymitis and sheep with respiratory and
mammary infections
H. haemoglobinophilus is a commensal of the lower genital tract of dogs and is thought to cause
cystitis (very rarely!!!), and neonatal infections (ditto) – its role in balanoposthitis and vaginitis, where
it is frequently found, is uncertain (to say the least)
An unnamed species of Haemophilus is found in cats associated with conjunctivitis
Immunity and Control

immunity to Haemophilus is thought to be predominantly humoral (AMI) – at least in mammals – but
the role of CMI is unknown
specific capsular antigens are considered important in protection, but most species are antigenically
heterogeneous
bacterins against H. paragallinarum, H. parasuis and H. somnus have been used with variable success
(they can prevent serious disease but not infection
in the case of H. somnus infection, they have only been useful for prevention of TEME and not the
other syndromes
in addition, production of vaccine is complicated by the heterogeneity of strains
control of infectious coryza may be assisted by elimination of carrier birds or depopulation of infected
flocks (unlike the other species of Haemophilus where the bacteria are part of the normal flora)
How would you treat an Infection?
most species of Haemophilus are susceptible to penicillin, ceftiofur and tetracyclines

in cases of TEME, timely therapy and maintenance of treatment are critical for recovery
Tilmicosin has also been used to treat shipping fever (where Haemophilus was thought to be involved)
in the USA
for birds with fowl coryza – sulphonamides or erythromycin in the feed has been used
141
BRUCELLA
Brucella are gram negative coccobacilli (short rods!) which are strictly aerobic. There are 6 species of
Brucella, only 2 of which occur in Australia (thanks to a very expensive eradication scheme in which B.
abortus was eradicated from Australian cattle and buffalo). They are associated with reproductive tract
and systemic infections of a variety of species (Brucellosis). Some of these infections have a significant
economic impact in the countries where they occur. In addition, a number of these organisms have
zoonotic potential and cause serious infections in humans.
Summary Table: Species of Brucella, the animals they infect, whether they are zoonotic, whether they are
present in Australia, and the diseases they cause
SPECIES ANIMAL ZOONOTIC In Australia DISEASE

B. abortus+++ Cattle (bison, camel, yes no abortion, orchitis,
water buffalo, yaks, epididymitis, fistulous
horses) wither)
B. melitensis+ Goats, sheep yes no abortion, mastitis,
(camels, alpacas, orchitis, epidymitis,
llamas)
B. suis+ Pigs (hares, reindeer, yes yes abortion, orchitis,
caribou, cattle) epididymitis,
arthritis
B. ovis+ Sheep no yes epididymitis (abortion is
rare)
B. canis+ Dogs yes no abortion, epididymitis,
osteomyelitis,
discospondylitis,
meningoencephalitis
B. neotomae Wood rats no no None
Notes: the number of + denotes the relative importance of these veterinary species within THIS genus (NOT for
all bacterial genera)
9 B. ovis and B. suis are the only species present in Australia and only B. ovis has economic significance for
the agricultural industries (B. suis is not present in commercially reared pigs)
9 B. suis however, has important zoonotic significance (but not B. ovis)
142
What are they?
Brucella are gram negative coccobacilli (easily mistaken for cocci)

they are strict aerobes
they are oxidase positive (except B. ovis)
they are non-motile
there are six species of Brucella – see table above for details
bovine brucellosis is a major cause of economic losses to the cattle industries (both dairy and beef)
worldwide
in addition, it is a significant cause of zoonotic infections, particularly in veterinarians, farmers and
abattoir workers
HOWEVER, it has been eradicated from Australia and the OIE recognises Australia as brucellosis-free
(very important for our export markets)
Brucellosis, caused by B. abortus, B. canis and B. melitensis are all EXOTIC

KEY POINT DISEASES in Australia.
B. suis and B. ovis are found in Australia; however, B. suis is only found in feral pigs and not in the
domestic pig populations
it is primarily of concern as a zoonotic disease
Brucella spp are obligate parasites and require an animal reservoir for maintenance in a population
Brucella are obligate parasites of animals and are found as facultative

KEY POINT intracellular parasites, predominantly in the cells of the reticulo-
endothelial system and the reproductive tract.
host preference is exhibited by the different species of Brucella (see table above); however a broad
host range has been demonstrated for some species (e.g. B. suis has been isolated from cattle and
sheep)
all species are pathogenic, facultative intracellular organisms with a predilection for reticuloendothelial
cells and the reproductive tract
Brucella are disseminated by direct or indirect contact with infected animals

ingestion is the most common route of entry; although infections may also be acquired by exposure of
the mucous membranes of the conjunctiva, genital tract, respiratory tract and skin directly to the
organism
KEY POINT The most common route of entry to a host is via INGESTION.
the most common source for exposure in cases of B. abortus, B. melitensis, B. suis and B. canis is
through aborted foetuses, the placenta, and post abortion uterine fluids
143
genital tract infections in cattle are routinely cleared within 30 days after calving, and cows are not
considered infectious for other cattle after this time
genital infections in pigs, may persist longer than this
milk (of cows and goats) is another source of infection for calves and kids via ingestion
direct in utero infection has also been documented
infection of the accessory sex glands in males allows dissemination of infection in semen – and
venereal transmission of B. suis, B. ovis and B. canis is common
urine is another source of infection of B. canis in dogs
a) Intracellular Survival++++
all Brucella can survive inside phagocytes and so avoid killing by antibody and/or antibiotics and this
is a major pathogenic mechanism as it allows persistence of this bacteria in the host
various mechanisms are employed by Brucella to allow for survival and multiplication inside these
cells
9 they inhibit phago-lysosomal fusion
9 they suppress the myeloperoxidase H2O2-halide system
9 the production of superoxide dismutase and catalase may play a role in defence against
oxidative killing
in addition, heat stress proteins are thought to help avoid intracellular killing by the host’s hydrolytic
enzymes, oxygen radicals and myeloperoxidase systems in the phagolysosome
b) Endotoxin
is thought to enhance intracellular survival (see above)
c) Porins
these are proteins in the outer membrane of Brucella and are thought to stimulate delayed type
hypersensitivity reactions
d) L forms
these cell wall variants may exist and may play a role in persistence of infections in the host
II) Pathogenesis
following exposure, Brucella penetrate intact musosal surfaces
9 in the intestine, the epithelium over the Peyer’s patches are the preferred site for entry
after penetration of the mucosal barriers, organisms may be engulfed by phagocytes, where specific
receptors on the surface of macrophages appear to mediate attachment and uptake
various mechanisms exist to allow intracellular survival (see above)
the organisms then (as either free bacteria or within phagocytes) localise to the regional lymph nodes
where they proliferate and infect other cells
alternatively, they may be killed at this stage, and the infection is terminated – some cattle appear to be
innately resistant to infection
9 this resistance appears to be related to the ability of their macrophages to contain the organism
from the regional lymph nodes, the Brucella disseminate haematogenously and localise in the reticulo-
endolthelial system (RES) and reproductive tract
9 there is a preferential localisation in the reproductive tract of pregnant animals as “factors”
(e.g. erythritol) in the allonatoic fluid within the gravid uterus can stimulate growth of Brucella
infection subsequently spreads to the foetus, though the exact mechanism of abortion is not known but
probably results from:
144
9 interference with foetal circulation due to existing placentitis
9 the direct effect of endotoxin
9 foetal stress from the inflammatory response of foetal tissues
Brucella also localise in the genital tract of males – though the exact mechanisms is not known
it is in the cells of the RES and the reproductive tract that this organism persists, sometimes for the
lifetime of the animal
9 for example, humans with brucellosis are infected for life and have life-long recrudescence of
their illness!!!
What Diseases does Brucella Cause?
KEY POINT The generic term for diseases caused by Brucella spp is BRUCELLOSIS.
susceptibility to infection depends on age, sex, breed and pregnancy status

9 young animals are more resistant to infection and frequently clear infections (but latent infections
can occur)
9 sexually mature animals are much more susceptible to infections, regardless of gender, and most
animals infected as adults remain infected for life
1. GENITAL TRACT DISEASE ×

the primary clinical manifestation of brucellosis are related to the reproductive tract
The most common clinical signs associated with Brucella infections are
KEY POINT related to the GENITAL TRACT.
in general, animals do not exhibit overt systemic illness (ie fever, depression)
in females – abortion is the most common manifestation of infection – though this varies between
species of Brucella
in males – epididymitis and orchitis are usually observed (again may vary) and may result in decreased
fertility or sterility
a) Brucella abortus
In females:
9 abortion is the most common manifestation of infection
9 usually no premonitory signs are shown
9 it usually occurs in the 5th month or later of gestation
9 retained placenta may be a complication
9 females usually only abort once, presumably due to acquired immunity
In males:
9 epididymitis and orchitis are the most common presenting signs
9 lesions are usually unilateral, but may be bilateral
B abortus remains a VERY important cause of reproductive tract disease in

KEY POINT cattle worldwide
b) Brucella melitensis
145
causes a similar disease in goats (and sheep) to that observed in cattle with B. abortus, except that
acute mastitis often develops
there are frequently palpable nodules in the udder and milk is clotted and watery
9 that is why goat’s cheese is a good source of B. melitensis for people!!!
c) Brucella canis ×
abortion in bitches occurs around 50 days
dogs with B. canis develop scrotal swelling as a result of fluid accumulation in the tunica
“Buster” the Beagle who has an embarrassing problem!!!. He has orchitis and epidymitis associated with
Brucella canis infection. Note the swelling of the testicles (they are also very red – but you can’t see
this!!!). This is an exotic disease in Australia.
These testicles now removed! Note the particularly swollen epididymis.
Swollen Epididymes
146
d) Brucella ovis
rarely causes abortion in ewes
infections in rams predominantly affect the epididymis with testicular lesions being uncommon
9 mature rams with lesions in the epididymis are usually due to B. ovis, whereas in younger rams
are usually due to other organisms that cause epididymitis
2. NON-GENITAL TRACT DISEASE

there is a variety of syndromes that may develop in many animal species
a) B. suis b
pigs may develop arthritis and lumbar spondylitis
b) B. canis ×b
dogs may develop meningoencephalitis, osteomyelitis, discospondylitis, and anterior uveitis (often the
initial presenting sign in dogs)
c) B. abortus b
may cause hygromas in cattle
may also cause “poll evil” and “fistulous withers” in horses (usually after contact with infected cattle)
FIGURE: “Harry” the eventer. He has

a marked discharge coming from the
area of his withers. This is due to an
infection of the underlying
ligamentum nuchae and vertebrae
(therefore he has desmitis,
osteomyelitis and spondylitis) with B.
abortus and is called “Fistulous
Withers”.
FIGURE: Poor “Harry’s” head. He has also has a condition called

“Poll Evil”. This is the same condition as above, just affecting his
the ligamentum nuchae as it attaches to the occipitus and also
involving the atlas (first cervical vertebra)!
147
Sample Collection
great care should be taken if working with suspected infected tissues due to the zoonotic potential of
this organism
this is a notifiable disease if infections in cattle (B. abortus), goats (B. melitensis) or dogs (B. canis)
are suspected
Suspected cases of bovine, caprine or canine brucellosis should be

KEY POINT immediately NOTIFIED to appropriate authorities and care should be taken
in handling specimens due to their zoonotic potential.
appropriate samples for diagnosis depends on the species involved and the disease manifestation
live animals
9 vaginal secretions, semen, discharge from abscesses, milk samples, blood (from dogs due to the
prolonged bacteraemia observed in this species)
9 in males – material from the epididymis, testis and accessory sex glands should be examined
dead animals
9 spleen, liver, udder and multiple lymph nodes (e.g. supramammary in dairy cows)
aborted foetus
9 abomasal fluid and placenta are the preferred samples
FIGURE: The placenta from cow with placentitis due to B. abortus. Samples from infected
cotyledons would be suitable for cultivation. However, care must be taken in obtaining these samples due
to the serious zoonotic potential of this bacteria (fortunately it is now exotic to Australia, but if you live in
the UK, the USA or Mongolia you will still come across this bacteria!).
148
examination of gram stains of the foetal stomach contents from an aborted foetus is frequently
rewarding
large numbers of gram negative coccobacilli will be observed
a modified acid fast stain (Ziehl Nielsen) may also be used to demonstrate Brucella
organisms can be detected in semen but are usually present in low numbers
otherwise, Brucella are difficult to detect in smears of samples
2. Culture
samples may be cultured onto blood agar or selective media
this would be performed in a specialist laboratory due to the exotic nature of these diseases
3. Identification
this would be performed at an appropriate reference laboratory
4. Immunodiagnosis
antibody detection is commonly used for diagnosis of brucellosis and control programs
samples that are tested include blood, milk and occasionally semen
in Australia, sheep are the only animals that are routinely tested
9 rams are tested for antibodies to B. ovis using either CF or ELISA
a number of immunodiagnostic tests have been developed for cattle sera:
9 tube agglutination
9 plate agglutination
9 rose Bengal plate or card tests
9 rivanol agglutination and 2-mercaptoethanol agglutination
9 complement fixation
9 ELISA
usually a highly sensitive but less specific test is used for screening purposes; and are followed by
more specific tests for confirmation purposes
for milk samples, the Brucella milk ring test is used
a similar approach to that used in cattle is also used for goats and sheep for B. melitensis
in pigs, these tests can be used to monitor piggeries (herds) but are not sensitive for individual animals
(due to variable antibody production)
9 this is not an issue in Australia, as B. suis is not found in domestic piggeries
in dogs, screening is by the rapid slide agglutination test (RSAT), which is sensitive but not very
specific
FIGURE: A Rose Bengal Test – used to detect

antibodies in the milk of cattle. This is not a
particularly specific test – but was good as a
screening test for dairy herds. Remember, if cattle
have antibodies to this bacteria – then they are likely to
be still infected as adult animals do not clear the
infection.
Positive agglutination in these 2 milk

samples indicating the presence of
antibodies to B. abortus
Negative agglutination in these 2 milk

samples indicating the lack of antibodies
to B. abortus
149
5. Nonculture Detection Methods
a number of non-culture methods, including PCR, immunoperoxidase staining, DNA probes, and co-
agglutination have been used to detect Brucella in tissues and fluids
evidence indicates that antibody against Brucella play both a protective and detrimental role
low levels of IgM and IgG cause complement-mediated lysis of the bacteria
however, higher levels of IgG antibodies appear to act as blocking antibodies that modulate the ability
of complement to mediate lysis
this probably accounts for the lack of correlation between protection and antibody titres
in addition, the blocking antibodies mediate opsonisation, which actually facilitate cellular uptake by
phagocytes, to which they have adapted by intracellular survival
these phagocytes that cannot eliminate infection play a role in the dissemination of organisms to other
parts of the body, and to the persistence of infection
effective immunity is primarily cellular in nature (cell-mediated immunity)
specifically sensitised T cells release cytokines that activate macrophages, which in turn can kill
Brucella by oxygen intermediates
a more effective immunity develops if animals are infected prior to sexual maturity
as a general rule, treatment of livestock is not attempted because of eradication programs in place
(cattle, goats, etc)
tetracylines, and streptomycin have been used to treat B. ovis infections in rams with variable results –
in general response to therapy is poor as the organisms are intracellular, and abscesses and fibrosis in
the lesions make antibiotic penetration poor
in dogs, long term therapy with tetracylines (doxycycline, minocycline) and streptomycin in
combination has been used
treatment failures are common
treatment should also consist of neutering affected animals
approaches to control and prevention of brucellosis depends on the animal species, Brucella spp,
management practices and availability and efficacy of vaccines
Brucella abortus
approaches to control of bovine brucellosis have adopted one or a combination of the following:
9 immunisation alone (with attenuated strains of the bacteria e.g. strain 19)
9 testing and removal of infection animals in conjunction with an immunisation program alone
9 testing and removal of infected animals without immunisation
the choice of program depends on the level of infection in a region or country, and the economic
feasibility of implementing the program
Australia implemented a successful eradication program based on first immunisation and testing
followed by testing and removal without immunisation
In 1989 Australia was declared brucellosis free and has maintained that status since
150
Brucella ovis
removing infected rams and preventing new infections in rams are the main means of controlling B.
ovis infections in a flock
all rams should be palpated for epididymal lesions at least twice a year before the breeding season and
rams with palpable lesions culled
serological tests (ELISA and CF tests) are used to identify infected rams without lesions
vaccination can be employed but its efficacy is limited and it interferes with serological identification
no effort is made to control infections in ewes as although they are transitorily infected (and therefore
may play a role in transmission), they will naturally eliminate infection by the next breeding season
Brucella canis
efforts to control canine brucellosis involves serological testing of dogs prior to breeding
males may also be evaluated by palpation of their genitalia (epididymis and testes) prior to mating
humans may acquire infections with B. abortus, B. canis, B. melitensis and B. suis
the severity of disease differs for the different species of Brucella (B. melitensis is considered to be the
worst, followed by B. suis, B. abortus and B. canis)
B. ovis and B. neotomae do not infect humans
Brucellosis is primarily a disease of the reticuloendothelial system in humans, with a mild
lymphadenopathy, splenomegaly and hepatomegaly commonly observed
clinical signs are generally non-specific and include alternating fever and chills, night sweats, fatigue,
muscle and joint pains and backaches
depression and insomnia are common and frequently make this a very debilitating disease
accidental inoculation with live Brucella vaccines can result in disease
151
BORDETELLA
Bordetella are small gram negative rods that cause important infections in dogs and pigs. They love the
respiratory epithelium and are obligate parasites of this site (ie they cannot live anywhere else). However,
they are NOT part of the normal flora, rather are maintained either on carrier or diseased animals. The
most important species in this genus is B. bronchiseptica and it is associated with upper and lower
respiratory tract disease in a variety of species including dogs (where it causes Canine Cough) and pigs
(where it causes Atrophic rhinitis). Both of these infections are contagious, and so appropriate quarantine
measures should be implemented in outbreaks to prevent further spread of infection.
Summary Table: Species of Bordatella, the animals they infect, the site they are normally found
(source) and the diseases they cause
SPECIES HOSTS SOURCE DISEASE

Bordetella Dogs, pigs, other URT Infectious tracheitis (Canine
bronchiseptica+++ species cough/Kennel cough), atrophic
rhinitis, pneumonia
Bordetella avium Turkeys URT Rhinotracheitis
Bordetella parapertussis Sheep URT Pneumonia
Bordetella pertussis Humans URT Whooping cough
152
What are they?
Bordetella are pleomorphic coccobacilli (short rods to coccoid forms)

the species of veterinary importance are motile
the species of veterinary importance have been included in the table opposite:
strains of B. bronchiseptica from different animal species (e.g. pigs, dogs, horses) appear to be
different
they are primarily found as parasites of the ciliated respiratory epithelium (ie in the parts of the
respiratory tract that is covered by ciliated epithelium)
they are NOT regarded as part of the normal flora, but can be found in the nasopharynx of healthy
animals (these animals are CARRIERS)
B. bronchiseptica occurs in wild and domestic carnivores (dogs, cats), pigs, rabbits, wild and
laboratory rodents (guinea pigs, rats) and occasionally horses, other herbivores, primates and turkeys
Bordetella bronchiseptica may be isolated from the respiratory epithelium

KEY POINT of a wide variety of animals and is maintained in CARRIER ANIMALS.
the organism is maintained in the respiratory tract for up to 3 months after recovery in dogs (despite
the production of local antibody) and for up to 19 weeks in recovered cats
these animals are usually the initial source of infections (and this is important information to give to
your clients as it determines the time that an animal has to be quarantined from other susceptible
animals e.g. at dog show!!!)
in pigs, the ultimate source of infection are carrier sows, in which the carrier rate declines with age
B. bronchiseptica may also be found occasionally in the environment (soil and/or water), but it is
thought to be short-lived in this environment
Recovered dogs and cats can harbour B. bronchiseptica in their respiratory

KEY POINT tract for 3 and 5 months respectively. This is important with regard to
CONTROL of disease and prevention of spread to naïve animals.
infections with B. bronchiseptica may be endogenous or exogenous

9 the bacteria may have been harboured on the respiratory epithelium of the diseased animal
(endogenous infection) or it may be directly or indirectly transmitted from a carrier to another
animal (exogenous infection)
inhalation is the principle mode of transmission in exogenous infections where the spread may be via
direct contact with sick dogs, or indirectly through aerosolised microdroplets from infected dogs or
contact with infected fomites
appropriate quarantine measures should be implemented to prevent transmission in outbreaks of
disease
153
Infection with B. bronchiseptica in dogs is CONTAGIOUS and measures to
KEY POINT prevent the spread of infections are important for control of disease.
in B. avium infections, spread is via contamination of water and litter
a) Fimbriae
these bind to sialic acid residues on the ciliated epithelium and allow the Bordatella to colonise the
respiratory tract
in addition, B. bronchiseptica has an affinity for respiratory mucus, which helps it to adhere to and
colonise the nasal mucosa
b) Endotoxin
the exact role of endotoxin in disease produced by B. bronchiseptica is not well defined
c) Exotoxins
a dermonecrotic toxin is produced by B. bronchiseptica and this toxin shares homology with the CNF-
1 toxin of E. coli but it is different to the dermonecrotic of Pasteurella multocida type D
9 it is at least partially responsible for the lesions produced in atrophic rhinitis
9 the toxin impairs the ability of osteoblasts to differentiate, which leads to turbinate atrophy
an adenylate cyclase is also produced which is capable of altering some cellular functions of host cells
including phagocytosis and intracellular killing
9 it also immobilises the respiratory tract cilia causing ciliostasis
a range of other exotoxins are also produced by B. bronchiseptica and include a haemolysin, proteases,
and a tracheal cytotoxin (toxic to tracheal epithelium)
II) Pathogenesis
once inhaled, B. bronchiseptica preferentially attaches to, and replicates on, the cilia of respiratory
epithelium
the production of exotoxins by the bacteria during this time leads to cilial paralysis, and inflammation
if phagocytosis occurs (and it sometimes doesn’t as the adenylate cyclase produced by B.
bronchiseptica may interfere with this function), the organism can survive within the phagolysosome
or it can escape into an endocytic compartment and therefore avoid fusion with lysosomes
9 thus B. bronchiseptica has several mechanisms to avoid killing by phagocytes
because of its ability to colonise the respiratory tract, and to produce a variety of potent toxins that
impair phagocytic function and induce ciliostasis, B. bronchiseptica is uniquely capable of facilitating
colonisation of the respiratory tract by other opportunistic pathogens
B. bronchiseptica causes mild to moderate disease of the AIRWAYS.

However, its major role is to provide conditions that allow other, more
KEY POINT significant pathogens, to colonise the lower respiratory tract and therefore
cause more serious disease.
154
FIGURE: “Big Boy”, an 8 year old
Great Dane. He had been boarded at
a local kennel 3 weeks previously and
subsequently developed a bad
cough. Now he is lethargic and
inappetent and is having trouble
breathing.
What Diseases does Bordetella Cause?
1. Canine Infectious Tracheobronchitis ×

(= ITB, “Canine cough”or “kennel cough”)
this is an acute, contagious respiratory infection of dogs characterised by sudden onset, paroxysmal
coughing (“goose-honking” cough) with variable amounts of mucus, and naso-ocular discharge
kennel cough is considered to be among the most prevalent infectious disease of dogs worldwide
Canine infectious tracheobronchitis is one of the most COMMON infectious

KEY POINT diseases of dogs worldwide!.
the clinical signs are attributed to infection by one or a combination of bacterial and/or viral agents
that colonise the epithelium of the upper respiratory tract, trachea, bronchi, bronchioles, and
pulmonary interstitium
respiratory signs in dogs known to have single agent infections are generally mild, and frequently self-
limiting, but multiple-agent infections usually result in more severe disease
in the majority of cases, B. bronchiseptica initiates the damage to the respiratory mucosa with
secondary colonisation by other pathogens:
9 viruses may be involved, particularly canine parainfluenza virus, but also canine adenoviruses 1
and 2, and canine herpesvirus
9 Mycoplasma are also frequently involved
9 other bacteria may also be involved and include Streptococcus, Pasteurella and occasionally the
Enterobacteriaceae (E. coli, Klebsiella)
outbreaks of canine ITB are relatively common and can reach epidemic proportions when dogs are
housed in high-density population environments such as pet shops, boarding kennels, and commercial
kennels
although the host range of B. bronchiseptica includes cats, horses, and rodents, most outbreaks are a
result of direct dog-to-dog or airborne contact with respiratory secretions
although infections are transmitted rapidly and efficiently in high density populations with high
morbidity, death associated with respiratory infection (either due to B. bronchiseptica alone or with
other agents) is uncommon, particularly in adult dogs
however, more severe disease is frequently observed in dogs that have received no prior or vaccine
exposure to the various agents that cause canine ITB
155
Bordetella bronchiseptica may remain in the airways of recovered dogs for
KEY POINT up to 3 months after cessation of clinical signs. This is important for ongoing
transmission of the organism.
FIGURE: The lungs from “Big Boy”. His infectious tracheobronchitis had progressed to severe
pneumonia. Note the cranioventral distribution of the lesions (the darker bits of lung!) that is typical of
pneumonia caused by inhaled bacteria.
Pneumonic areas
of lung
(consolidated)
2. Atrophic Rhinitis of Pigs

the lesions associated with atrophic rhinitis have already been described in the notes on Pasteurella spp
B. bronchiseptica alone causes temporary turbinate atrophy by disturbing osteoblast physiology, but
these lesions are usually self-limiting
as with kennel cough, B. bronchiseptica prepares the respiratory epithelium for establishment and
activity of toxigenic P. multocida type D
this disease predominantly affects young pigs (<6 weeks), when osteogenesis and bone remodelling is
most active
156
Bordetella bronchiseptica induces the initial changes in the respiratory
KEY POINT tract of pigs, but Pasteurella multocida is required to produce the severe
lesions that are observed in ATROPHIC RHINITIS.
FIGURE: The nose of a pig (“Not Another Babe!!!”) which has been cut through cross sectionally. “Not
Another Babe” was suffering from atrophic rhinitis (also see earlier in the discussion of Pasteurella). The
severe distortion of the nasal septum and the erosion of the turbinates are evident in this picture.
Distortion of the central Atrophy of the nasal

nasal septum. turbinates
3. Upper respiratory tract disease and pneumonia Ø

B. bronchiseptica can also cause pneumonia in other species including rabbits, guinea pigs (an
important cause in these 2 species), cats and horses
in cats (predominantly kittens), B. bronchiseptica can cause upper and lower respiratoy tract disease as
evidences by pyrexia, sneezing, nasal discharge, submandibular lymphadenopathy and rales on
auscultation
coughing may occur, but is not as evident as in dogs
bronchopneumonia may also occur, especially in kittens, and mortality is higher in these cases
cats, like dogs, carry the organisms asymptomatically following recovery
pneumonia has also been recorded in a range of other species (e.g. horses), and it is now gaining
acceptance in these species that it is likely to be a primary pathogen
in some cases, direct transmission from dogs is suspected
157
4. Coryza of Turkeys
this is an acute disease of turkeys characterised by suppurative rhinitis, sinusitis, tracheitis,
bronchopneumonia, and airsacculitis and is caused by B. avium
frequently a presumptive diagnosis is made in cases of canine ITB and atrophic rhinitis due to the
classical clinical presentation
in cases of canine ITB, if an animal is very ill (febrile, inappetent, signs of depression), cultivation of
lower respiratory tract secretions may be indicated in order to determine the bacterial species involved
and their antimicrobial sensitivity pattern
samples of the upper respiratory tract (e.g. nasal or nasopharyngeal swabs) are NOT appropriate
in cases of atrophic rhinitis, swabs of the nasal cavity are indicated in order to demonstrate the
presence of B. bronchiseptica and P. multocida
in cases of canine ITB, the presence of an inflammatory reaction (neutrophils) together with gram
negative rods would be observed in lower respiratory tract (tracheal) secretions
3. Culture
B. bronchiseptica will grow on blood agar and is a strict aerobe
selective media is also available to assist isolation of B. bronchiseptica from nasal swabs in suspected
cases of atrophic rhinitis (so that B. bronchiseptica may be differentiated from normal flora of the
nasal cavity)
4. Identification
the presence of oxidase positive, motile, gram negative rods assists in their identification
colony morphology will help to differentiate from Pseudomonas spp.
there are a range of other biochemical tests that can be used to confirm your diagnosis but these are
not readily used in veterinary practice
B. bronchiseptica can parasitise dendritic cells, which results in decreased immune responses due to
inefficient antigen presentation
however, when immunity does develop to B. bronchiseptica it appears to be predominantly antibody
mediated, and local antibody is believed to prevent B. bronchiseptica colonisation in dogs
no other immune responses have been shown to be protective
dogs that have recovered from B. bronchiseptica infection are highly resistant to re-infection for at
least 6 months
in uncomplicated cases of canine ITB, the value of antibiotic therapy appears to be limited (but there is
some evidence that they may reduce the length of time of coughing and may help prevent the
development of secondary pneumonia)
158
in these cases, empirical therapy with trimethoprim sulphonamides, amocixillin-clavulanate or
doxycline may be indicated
in complicated cases of B. bronchiseptica infection, where pneumonia has developed, these organisms
are not predictably sensitive, and drug resistance has been demonstrated
therefore, antimicrobial sensitivity testing should be performed on isolates obtained from these cases
in addition, the use of antitussive agents (cough suppressants) and bronchodilators have been used in
conjunction with antibiotics in uncomplicated cases of canine ITB
atrophic rhinitis is not treatable and control should be aimed at prevention
EMPIRICAL THERAPY is indicated in uncomplicated cases of Canine Cough

in dogs. However, in more severe cases with progression to pneumonia, B.
KEY POINT bronchiseptica does NOT have a predictable sensitivity patter and antibiotic
sensitivity testing should be performed.
vaccines have been used to prevent infection in dogs and pigs

for dogs, both parenteral and intranasal (live attenuated) vaccines are available for administration
either alone or in conjunction with other vaccines against the upper respiratory tract viruses e.g. CAV-
2, canine parainfluenza virus and canine distemper virus
vaccination has been shown to be beneficial in control of kennel cough and it is recommended that
puppies receive an initial vaccine and booster (2-4 weeks apart) and are then re-vaccinated annually
depending on risk
9 the intranasal vaccine only requires one initial dose and then annual re-vaccination
in addition, before known or potential exposure to other dogs (e.g. boarding) a single booster vaccine,
administered intranasally, is recommended at least 5 days prior to exposure if the dog has not been
vaccinated in the previous 6 months
KEY POINT VACCINATION is recommended for dogs to prevent Canine Cough.
Intramuscular or intranasal vaccines are available for kennel cough
159
in pigs, vaccines (bacterins) are usually administered to pregnant sows to provide colostral immunity
to piglets
these vaccines may also include toxigenic strains of P. multocida
alternatively, piglets may be directly administered vaccines (either bacterins or live avirulent vaccines
administered intranasally)
prophylactic treatment of piglets with sulphonamides in feed or water and elimination of carrier sows
from the herd based on results of culture of nasal swabs have also been used to control atrophic rhinitis
in piggeries
QUARANTINE/ISOLATION MEASURES
environments in which transient dogs are housed in adjoining kennels are conducive to efficient and
rapid transmission of agents capable of causing canine cough
although important in preventing infections, vaccination may not guarantee protection against
development of signs, particularly in high density populations
in these situations adequate ventilation from 12 to 20 exchanges of air per hour are recommended to
try to decrease the spread of airborne infectious agents
in addition, because airborne transmission is common, dogs suspected of having contagious respiratory
infections should be ISOLATED when signs first develop in an effort to limit exposure to susceptible
dogs
thorough cleaning of housing facilities, where the infected dogs were kept, is necessary to prevent
further spread of canine cough
disinfection of kennels is best performed with fresh bleach (sodium hypochlorite), chlorhexidine or
benzalkonium solution
in addition to extensive cleaning, individual dogs are treated as necessary to manage clinical signs
although it can be tried, there is no evidence that IN vaccination, administered when clinical signs are
first detected, will alter the course of an outbreak
once an outbreak has begun, isolation of the entire kennel for up to 2 weeks may be the only
reasonable, most efficacious way of containing further infections
and remember, recovered dogs may carry the organism in their respiratory tract for up to 3 months,
which needs to be conveyed to owners of breeding or show dogs
finally, if possible, all new dogs entering a stable kennel population should be isolated for 4 weeks
prior to introduction to the kennel in order to observe the dog and implement worming, vaccination,
and if appropriate, selected laboratory screening procedures
Some general principles for Environmental Management of Kennels include:

1. Regular cleaning and disinfecting schedule should be established
2. Surfaces should be cleaned of wastes and other organic material before disinfectants are applied.
Organic materials compromise the effectiveness of many disinfectants
3. Disinfectants and other cleaning products such as soaps should be combined only by, or on
direction of, the manufacturer. Inappropriate combinations can cause loss of effectiveness. Dogs
should be removed from pens that are being disinfected and reintroduced when pens are dry.
4. Many products are more effective in hot water and less effective in hard water or when combined
inappropriately with other agents.
5. Bedding should be clean and dry. Access by insects, rodents and birds should be prevented.
Bedding should be changed as often as needed to maintain sanitation. Some litters of puppies may
require multiple changes daily.
6. Feed should be stored in containers that phohibit access by insects, birds and rodents.
7. Faecal waste should be disposed of dily, and waster containers should be disinfected frequently.
Faecal waste should not be disposed of within animal areas.
8. Equipment such as heating and cooling systems and vacuums should be cleaned and serviced
regularly by qualified persons to avoid creating habitats for micro-organisms
160
9. Stored equipment, debris, weeds and other items not critical to kennel function should not be
allowed to accumulate.
B. bronchiseptica can cause pneumonia, bronchitis, sinusitis, septicaemia, endocarditis, peritonitis,

meningitis, and wound infections in humans
infections occur predominantly in immunocompromised individuals or pre-existing disease (e.g.
chronic bronchitis and pneumonia)
evidence for direct transmission of B. bronchiseptica from pets to people is however, largely
circumstantial
161
MORAXELLA
Moraxella are small gram negative rods that cause the important bovine disease called Infectious Bovine
Keratoconjunctivitis (or Pink Eye). The bacteria are normally found on the conjunctiva and upper
respiratory tract of healthy, carrier cattle and are therefore already present ready to cause disease. A
number of risk factors can predisposes cattle to disease (e.g. dust, white eyes) and the bacteria need to
possess both pili and haemolysin to be able to cause disease. There is a vaccine available to help farmers
control this important disease, but it is only partially effective as it is based on pili, which can vary
between strains and protection is strain specific.
Summary Table : Species of Moraxella that has veterinary importance, its animal hosts, and the
disease it causes.
Pink Eye (bovine infectious

Moraxella bovis++++ Cattle
keratoconjunctivitis = IBK
162
What are they?
Moraxella are gram negative coccobacilli to short rods

they are non-motile
there is only one species is of veterinary importance; Moraxella bovis
KEY POINT The ONLY species of veterinary importance is Moraxella bovis.
this species is found world wide

it is a commensal (parasite) of the mucous membranes of the bovine conjunctiva and upper respiratory
tract (nasopharynx) of healthy, carrier cattle
Moraxella bovis may be found on the conjunctiva and upper respiratory

KEY POINT tract of healthy, carrier cattle.
infections may be endogenous, or exogenous (transferred from healthy carriers)

dissemination is by direct and indirect contact, including flying insects (predominantly flies) and
possible airborne transmission
a) Fimbriae
these structures mediate attachment to the conjunctival and corneal epithelium
non fimbriated strains of M. bovis cannot cause disease
there are a number of immunologically distinct fimbriae (7) and they form the basis of serotyping of
M. bovis
b) Haemolysin
this exotoxin causes lysis of both RBC, WBCs (phagocytes) and also bovine corneal epithelial cells
non-haemolytic strains of M. bovis are not pathogenic
163
FIGURE: An electron microscopic picture of Moraxella bovis showing their fimbriae. There are 9
different types of fimbriae (serologically distinct). Fimbriae are required for attachment to the corneal
epithelium and subsequent colonisation. Disease is produced by the haemolysin, which is also capable
of lysing bovine corneal epithelial cells as well as phagocytes.
E.M. showing the pili of M. bovis
II) Pathogenesis
Environmental factors play a role in the onset of disease and include ultraviolet irritation, flies, dust
and woody pasture plants
in addition, factors such as lack of eyelid pigmentation (white eyes) and prominent placement of the
eyes are predisposing factors
all of these factors may cause epithelial damage and therefore allow adherence of the organism to the
corneal epithelium
the bacteria then produce a cytotoxin which destroys phagocytes as well as bovine corneal epithelial
cells which induces inflammation (predominantly neutrophilic)
this results in the production of the lesions observed which ranges from mild corneal oedema and
epiphora to severe oedema, corneal opacity, vascularisation, ulceration and in some cases rupture
concurrent infections with viruses (IBR and adenoviruses), and Mycoplasma species may complicate
the disease
What Disease does Moraxella Cause?
1. Infectious Bovine Keratoconjunctivitis (= IBK or “Pink Eye”) in Cattle

this is a highly infectious disease of cattle, mostly affecting beef cattle
it causes significant economic losses in cattle industries world wide due to decreased weight gains
when infections occur
“Pink Eye” is a major disease of beef cattle and is responsible for

KEY POINT SIGNIFICANT economic losses.
164
following colonisation of the cornea and conjunctiva by M. bovis, the initial clinical signs of IBK are
ocular discomfort with profuse lacrimation, photophobia, epiphora and blepharospasm
commonly the infection then causes conjunctivitis which manifests as a hyperaemic and oedematous
conjunctiva, accompanied by a profuse serous ocular discharge, which later may become purulent in
nature
keratitis (corneal ulceration) usually becomes evident within 48h of initial ocular discomfort, and an
area of corneal opacity becomes progressively oedematous, enlarges rapidly and may develop into an
ulcer with denuded areas of cornea covered by fibrin
the more advanced ulcers take 4-6 weeks to heal, whereas less advanced cases of conjunctivitis and
keratitis may recover within 2 weeks due to rapid corneal vascularisation
these cases show either complete recovery or minimal corneal scarring
young animals are more commonly affected (due to immunological naivety)
the prevalence of disease is greatest in summer and early fall (high UV radiation, dusty etc)
the lesions produced have been described under pathogenicity
healing of the corneal ulcers takes several weeks, and corneal scarring may also result
deaths (due to ascending infection) are rare
FIGURE: “Flo”, who has a corneal that is white instead of clear. Corneal ulcers allow the uptake of
water into the cornea (as well as having an inflammatory infiltrate) and that is why the cornea appears white.
“Flo” would also have blepharospasm (trying to shut her eye all the time, and due to “Pink Eye”.
Corneal Ulcer
a presumptive diagnosis is usually made on the basis of clinical signs
if an outbreak has occurred, isolation of the organism may be attempted in order to perform antibiotic
sensitivity testing
swabs of the corneal ulcer (peripheral swabs are best)
gram negative coccobacilli, together with neutrophils, may be observed on smears of exudate
confirmation of the presence of M. bovis must be made by cultivation
3. Culture
M. bovis grows only aerobically on blood agar
165
4. Identification
not applicable for veterinary practitioners
antibody is produced during infection, with secretory IgA predominating locally

temporary resistance to re-infection follows recovery, but is short acting
the relative roles of local versus systemic and humoral versus cell mediated immunity have not been
well defined in this syndrome
this is a self-limiting disease, and recovery commonly occurs without treatment intervention, though
treatment does decrease the risk of complications and scarring
affected animals should be placed in a dark stall, free from dust and flies
topical antibiotics should be applied – where cloxacillin or long acting tetracyclines are considered to
be the drugs of choice
this may be administered as a subconjunctival deposit (cloxacillin), locally, or systemically
(oxytetracycline)
the use of eye patches, is also of benefit in these infections to protect the eye from sunlight
FIGURE: This is “Millicent” and she is modelling

the eye patch that is available for treatment of
IBK in cattle. The eye patch prevents exposure
to sunlight (and therefore exacerbation of the
disease) and flies (and therefore helps prevent
spread). In some case these eye patches are also
impregnated with antibiotics.
there are a number of vaccines available for control of IBK including bacterins (killed whole cell
vaccines) and pili based vaccines
the pili based vaccines are more effective than bacterins, but are serotype specific (therefore all 7
serotypes would require inclusion in a vaccine for complete coverage)
166
development of vaccines based on the haemolysin are a likely future prospect, and offer cross-
protective immunity amongst all strains of the organism
fly control should also be implemented to help prevent spread
none
167
TAYLORELLA
Taylorella equigenitalis is a small gram negative rod that causes an important disease in horses –
Contagious Equine Metritis. This disease is exotic to Australia (but is present in most countries with
which we trade horses). It is a venereal disease, with transmission usually occurring from carrier stallion
to susceptible mares. This results in a purulent endometritis which can be detected as copious vaginal
discharge. Due to the exotic nature of this disease, all horses entering Australia on a permanent basis must
be cultured for T. equigenitalis and be negative on at least 3 consecutive occasions.
Summary Table : Species of Taylorella that has veterinary importance, its animal hosts, and the
diseases it causes.
T. equigenitalis++++ horses contagious equine metritis
168
What are they?
Taylorella are gram negative coccobacilli

they are facultative anaerobes, but grow best with increased CO2
there is only one species of veterinary importance – Taylorella equigenitalis
KEY POINT Taylorella equigenitalis is the only species of veterinary importance.
this bacteria is only found as a obligate parasite of the equine genital tract, and is maintained in a
population in carrier stallions or mares
donkeys may harbour a CEM-like bacteria, and donkey strains may infect horses and vica-versa
the organism is EXOTIC to Australia and North America, but is found in Europe
outbreaks of this disease have occurred in Australia and North America due to the introduction of
carrier stallions or mares
Taylorella equigenitalis is EXOTIC to Australia, but outbreaks have occurred

KEY POINT with the introduction of carrier stallions and mares.
CEM is one of the equine diseases that is most likely to be introduced to Australia, and therefore all
veterinarians in equine practice should be aware of this disease
T. equigenitalis is transmitted to mares or stallions during coitus (venereal transmission) –

predominantly from stallions to mares
the organism is found on the surface of the penis, in the preputial smegma, and in the urethral fossa in
carriers stallions
the infection can also be transmitted between stallions and mares by attendants, fomites and especially
instruments during mating
alternatively, the infection may be transmitted during artificial insemination (AI)
KEY POINT Taylorella equigenitalis is transmitted venereally.
a) Fimbriae
have been observed in some strains
their relationship to pathogenicity is not well defined
169
b) Immunoglobulin Binding Proteins
there is some evidence that strains of T. equigenitalis produce immunoglobulin-binding proteins that
are similar to those of Haemophilus somnus
these proteins bind Ig in a way that they then can’t fix complement and therefore avoid complement
mediated killing
II) Pathogenesis
the organism is maintained in a population on carrier animals
9 foci of carriage in mares is the clitoral fossa and sinuses and in stallions the foci are the
prepuce, urethra and urethral fossa and sinuses
within a few days of exposure of a naïve mare to an infected carrier stallion, a purulent endometritis
develops with variable amounts of exudate
the main damage is to uterine epithelium which becomes covered by neutrophilic exudate
the epithelium is eroded or undergoes severe degenerative changes
at this time there may be a mucoid to mucopurulent vaginal discharge of variable abundance (usually
observed several days after service)
the uterine infection usually subsides spontaneously within several weeks, when endometrial repair is
complete and there is no lasting impairment of breeding performance
however, mares may go on to become long term carriers, and thus transmit the infection back to naïve
stallions
although infection has been demonstrated in placenta and new born foals, abortion is thought to be rare
stallions do not develop any signs of illness, but remain as carriers of the agent indefinitely
What Diseases does Taylorella Cause?
Contagious Equine Metritis (CEM)
this is an acute, suppurative, self-limiting infection of

the uterus, cervix and vagina of mares
it causes temporary infertility in mares (failure to
conceive), and can be a significant cause of economic
losses in the horse breeding industry in countries in
which this organism is found
it is highly contagious disease
infection is followed by long term carriage of the
organism, predominantly in stallions, but also in some
mares
the clinical signs and lesions produced have been
described under pathogenesis
FIGURE: “Molly” the 5 year old warmblood mare.

She has a marked purulent vaginal discharge, which
is not good if you are a brood mare. She was bred
approximately 2 weeks ago to a recently imported
warmblood stallion. What would you do if you were
called out to this case?
170
the presence of copious mucopurulent vaginal discharge in a mare(s) that has been recently served to
an imported stallion should warn the attending veterinarian of the possibility of this disease
The typical clinical signs associated with T. equigenitalis infections should

KEY POINT WARRANT NOTIFICATION of appropriate authorities in order to confirm or
rule out a diagnosis of CEM.
diagnosis of CEM, or identification of carrier stallions or mares requires demonstration of T.

equigenitalis in the genital tract
swabs of uterine discharge (in clinical cases) or of the clitoral fossa in suspected carrier mares, or the
urethral sinus, fossa glandis and prepuce in the suspected carrier stallion should be obtained
all stallions and mares to be used for breeding purposes must be swabbed at least three times one week
apart, with no growth of T. equigenitalis obtained, before they can be imported to Australia on either a
temporary or permanent basis
All horses imported into Australia for breeding purposes must be shown to
KEY POINT be negative for carriage of T. equigenitalis.
small gram negative coccobacilli may be observed in gram stains of exudates obtained from infected
mares together with many neutrophils
samples should be transported to an appropriately qualified laboratory (in Australia they will be sent to
the Australian Animal Health Laboratory – AAHL) for confirmation of the diagnosis
3. Culture
T. equigenitalis are relatively fastidious bacteria and require chocolate agar for growth (they require X
factor but not V factor)
in addition, growth requires the
presence of 5 – 10% CO2
4. Identification
not applicable for veterinary
practitioners
FIGURE: After “Molly” gets over her infection, you

need to show that she is not a carrier. To do this
you have to sample the clitoral fossa and have
three consecutive negative swabs. Note the
very fine swab being used (paediatric swab) as it
is difficult to get into this site!!!
171
recovered horses show increased resistance for several months (if get re-infected during this time they
show milder clinical signs and fewer bacteria)
after this time, the mare may become re-infected with bacteria
the cause of this increased resistance is not known, but is possibly antibody mediated
uterine infusions of antibiotics (ampicillin or penicillin) for 5-10 days may be used in cases of CEM
carrier stallions may be treated with chlorhexidine (disinfectant) and nitrofurazone for at least 5 times
in order to treat the carrier state
this is a notifiable disease in Australia

the disease has been previously introduced on at least one occasion to Australia (in 1977) and was
eradicated by a successful but very costly campaign
no cases of CEM have been reported since 1980, and Australia is officially CEM-free since 1985
bacterins have been used in countries in which CEM is endemic, but they appear to have limited
efficacy
test and treatment are most commonly used in these countries to control outbreaks of disease
the organism is fragile, and cannot persist in the environment for long periods (usually only a couple
of days) which assist the control of outbreaks

none
172
SECTION 3
Gram Positive Rods
173
174
ACTINOMYCETES
KEY POINT Actinomycetes is a FAMILY not a genus.
the family Actinomycetes comprises 5 genera of veterinary importance:

1. Actinomyces
2. Nocardia These 3 genera are filamentous branching rods
3. Dermatophilus spp
4. Arcanobacterium
5. Actinobaculum
Some members of this family have as a common characteristic the ability to form filamentous,
branching rods which is USEFUL for DIAGNOSIS
Actinomyces, Nocardia and Dermatophilus are gram positive,

KEY POINT filamentous, branching rods.
these bacteria are sometimes called “higher bacteria” because they have some of the cultural and
morphological features of fungi
9 extensive filamentation
9 branching
9 usually the production of aerial hyphae on their colonies and these aerial hyphae have
asexual spores (conidia)
within this family, the pathogenic patterns of Actinomyces and Nocardia are similar, particularly in
small animals
however it is clinically important to differentiate between them as the 2 genera can cause similar
syndromes, BUT they have a different treatment (Actinomyces is susceptible to penicillin; Nocardia
has widespread resistance) and different prognosis (infections due to Actinomyces have a better
prognosis than Nocardia)
It is important to DIFFERENTIATE between Actinomyces and Nocardia as

KEY POINT their treatment and prognosis are different.
175
TABLE: This table shows the contrasting characteristics between Actinomyces and pathogenic
Nocardia . These are important for diagnostic laboratories as they allow us to differentiate
between these 2 genera, which has important clinical implications.
Characteristic Actinomyces Nocardia

Bacteria Atmospheric requirement facultative or obligate strict aerobe
anaerobe
Growth on Sabaroud’s - +
Modified Acid Fast - +
Cell Wall Characteristics Lysine + Mycolic Acid +
Source Endogenous Exogenous
Clinical Disease often mixed infections usually single isolate
Lymph Node Involvement Rare Common
Granules in exudate (“sulphur”) Common Rare
Penicillin Susceptible Resistant
176
ACTINOMYCES
Actinomyces are gram positive, branching rods that like to hang out in the oral cavity of a wide range of
species. Hence they are perfectly placed to cause disease in this site and are the cause of a disease that is
very aptly named “Lumpy Jaw”. This disease is found in many species, including ruminants, humans and
kangaroos. It can also cause infections of serous surfaces (or serositis). Infections with Actinomyces
species can be suspected when typical hard “granules” are seen in exudates from infected animals. These
granules are also called “club colonies” due to the shape of the bacterial colonies when viewed under a
microscope. Infections with Actinomyces spp can be difficult to treat due to their ability to form cell wall
deficient variants.
Summary Table : Species of Actinomyces that have veterinary importance, their animal hosts, and

++++
A. bovis cattle (humans, marsupials – “Lumpy Jaw” (bovine actinomycosis)
particularly kangaroos and wallabies)
A. viscosus+++ dogs (cats, horses, cattle, humans) canine actinomycosis: localised pyogranulomatous
lesions of the skin, pyothorax +/- peritonitis,
lymphangitis, disseminated lesions (rare)
cutaneous pustules in horses; abortion in cattle
A. hordeovulneris+++ dogs serositis (pleuritis, peritonitis, arthritis,
discospondylitis), cutaneous and visceral
abscessation, lymphangitis
A. israelii+ humans (cattle, pigs) granulomatous abscesses and osteomyelitis in
humans, mastitis in sows
A. lignieresii+ dogs, cats rare infections
A. meyeri cats rare infections
bacterial genera)
177
What are they?
gram positive rods belonging to the family Actinomycetes

the rods may show branching
9 branching is especially evident in exudates from
lesions
9 in these samples the rods are often mildly beaded
due to uneven staining (these are NOT spores)
but which is in contrast to Nocardia spp which are
often markedly beaded
9 they can also appear as short, club-shaped
(diptheroid) rods (so appear similar to
Arcanobacterium spp – see later)
they are non-acid fast (unlike Nocardia that are
modified acid fast)
they are either facultatively anaerobic (A. viscosus,
A. naeslundi and A. hordeovulneris – most also preferring increased CO2) or strict anaerobes (A.
bovis, A. israeli, A. meyeri)
there are a number of different species, many of which are found as commensals as part of the normal
flora of the oropharynx
the species that are commonly associated with disease in animals are listed in the summary table
most Actinomyces are found on oral mucous membranes and tooth surfaces of mammals (except A.
hordeovulneris which is thought to live on awns in the seed head of grasses of the genus Hordeum –
called Foxtails in the USA)
they are also found secondarily in the GIT
Actinomyces spp are found in the oral cavity of cattle, dogs, cats, goats, sheep, monkeys, rabbits,
squirrels, hamsters, and marsupials
Most Actinomyces are NORMAL FLORA of the oral cavity of mammals,

KEY POINT including humans.
most infections caused by Actinomyces are endogenous in origin

9 they are caused by introduction of a commensal strain into susceptible tissues of the host
9 except for the occasional isolation of A. israelii (human strain) from dogs and cattle, there is little
to suggest interspecies transmission
Actinomycosis is regarded as a non-communicable (non-contagious) infection
most infections with Actinomyces are initiated with wounds of the mucous membranes of the upper
digestive system (e.g. puncture wounds due to sticks, rough feed etc)
bites are another (rare) means of transmission and may involve non-host strains
Infections with Actinomyces are ENDOGENOUS in origin and are non-

KEY POINT communicable (not contagious).
178
II) Virulence Factors

Actinoymyces are gram positive rods – therefore endotoxin is NOT present in their cell wall
exotoxins have not been demonstrated
surface fibrils on A. viscosus may play a role as an adhesin for host cells or other bacteria (“co-
aggregation”)
in addition, surface antigens are thought to play a role in stimulating chemotaxis of neutrophils and
mitogenic (lymphocytic proliferation) activity
III) Pathogenesis
Actinomyces evoke a pyogranulomatous reaction by unknown mechanisms
because of the organism’s normal habitat, infections are, by necessity, somehow linked to
oropharyngeal area
they are opportunistic pathogens dependent on mechanical disruption of the normal mucosal barriers in
order to invade tissues
in these tissues, bacterial colonies are formed and trigger a suppurative response in this region
peripheral to this reaction, a granulomatous, mononuclear infiltrate occurs which is surrounded by a
fibrous reaction
the abscesses usually have one (or several) sinus tract which carries exudate to the outside
the exudate from these tracts often containing small granules (those associated with A. bovis are
sometimes called “sulphur granules” because of their yellow colour, whereas A. viscosus are whitish
soft, grey granules)
9 these are masses of bacteria colonies that are surrounded by a microscopic fringe of “clubs”
consisting of mineral and possibly antigen-antibody complexes
9 their clinical significance is that they help to indicate a possible infection with Actinomyces spp
(but be careful – other bacteria can also induce “granule” production e.g. Actinobacillus and
chronic Staphylococcal infections called botryomycosis)
infections are spread by direct extension, or more rarely by haematogenous means
in these more distant sites they can produce abscesses, empyema or suppurative serositis (infection of
serosal surfaces)
most infections with Actinomyces species are polymicrobial and their pathogenicity is dramatically
increased in mixed infections
Infections with Actinomyces are commonly POLYMICROBIAL and are

KEY POINT characterised by a PYOGRANULOMATOUS response.
179
FIGURE: An expensive Hereford bull called
“Ralph” who has a very large lump on the
side of his jaw. This is a case of “Lumpy
Jaw” (most appropriately named!) and is due
to Actinomyces bovis infection.
Big Lump on the

jaw of this bull
What Diseases does Actinomyces Cause?
1. “Lumpy Jaw”
this is an infection of the mandible, or less commonly the maxilla, of cattle, (similar infections occur
in humans, kangaroos and wallabies, dogs, cats, and sheep)
it is usually caused by A. bovis but also occasionally A. isrelii
the bacteria are introduced from their location in the oral cavity into the alveolar or para-alveolar
region of the jaw either following trauma to the oral mucosa or during tooth eruption
in this site they initiate a chronic osteomyelitis which eventually leads to replacement of normal bone
by porous bone, which is laid down in an irregular fashion that is honeycombed with sinus tracts
containing pus
clinically, the swelling is initially painless, but may become painful with time; there may be
dislodgement of teeth, inability to chew, mandibular fractures and usually there are a number of
fistulas that are discharging to the surface
the lesions may expand locally, but there is little tendency for vascular dissemination
FIGURE: The mandibles from another infected bull and demonstrates the invasion of the bone that
occurs in these cases associated with chronic osteomyelitis.
Lytic bony lesions in

both mandibles due
to chronic
osteomyelitis
180
2. Abscesses ×Ø b
Actinomyces spp may be isolated from soft tissue infections of many species (including dogs, cats,
horses and ruminants)
in general, these are in the form of localised granulomatous abscesses, primarily in the skin and
subcutaneous tissues
often they are a result of bite wounds, and contain mixed microbial flora
in horses they have been isolated (usually together with Brucella abortus) from supra-atlantal and
supraspinous bursitis (= “poll evil” and “fistulous withers” respectively)
in addition, Actinomyces species have been isolated from cervical abscesses in horses – which requires
differentiation from the lesions produced by Streptococcus equi subspp equi
3. Serositis and Discospondylitis ×Ø
Actinomyces spp are common isolates from suppurative serositis of dogs and cats
usually this involves the thorax (pyothorax), with or without extension into the abdomen (peritonitis)
and occasionally retroperitoneally
in the thoracic cavity, pyogranulomatous lesions are observed on the surface e of the thoracic organs
and there is an accumulation of pleural and pericardial fluid containing soft, grey-white granules –
these lesions closely resemble those found with canine nocardiosis
the main clinical finding in these cases is respiratory distress
in dogs, infection is frequently associated with foreign bodies, particularly migrating grass awns
9 the most common type of grass awn involved belong to the genus Hordeum (“foxtails”) and
therefore one of the Actinomyces spp that is frequently isolated from these lesions is called A.
hordeovulneris; the other most common species isolated is A. viscosus
9 these grass awns sometimes lodge near vertebrae, causing actinomycotic discospondylitis
in cats, pyothorax and subcutaneous “fight wound” abscesses are the most common disorders in which
Actinomyces spp are involved, and frequently as part of a mixed infection
in dogs and cats, Actinomyces may also cause a nodulo-ulcerative lymphangitis which is
predominantly cutaneous in distribution
4. Mastitis and Abortion

pyogranulomatous mastitis has been reported in sows due to infection with Actinomyces spp
Signalment and Clinical History

as with other gram positive rods, clinical presentation, species affected, and type and location of
lesions may suggest a presumptive diagnosis
Signalment, history and clinical signs often provide a PRESUMPTIVE

KEY POINT DIAGNOSIS for infections caused by gram positive rods.
Sample Collection
for a definitive diagnosis fine needle aspirates from unopened lesions or tissues should be obtained
swabs from draining tracts are NOT acceptable
samples containing “granules” are usually preferred
Direct Examination
suspected exudates should be examined for “granules”
181
“Granules” may be observed in the resulting exudate and can help with
KEY POINT diagnosis.
9 in cases of A. bovis infections these are frequently yellowish in colour (hence they are called
“sulphur”), but may also be white, tan or grey (especially if A. viscosus is involved)
9 they may be up to several mm in size and can vary in firmness
9 if granules are observed they can be carefully washed in saline, and placed on a slide, squashed
(carefully) by a coverslip, and examined for the presence of the “clubs” at the periphery (these are
caused by a gelatinous sheath and the deposition of calcium phosphate around the terminal
filaments of the Actinomyces colony
9 in addition, a gram stain of this preparation can be made, and the presence of gram positive
branching rods determined
Culture
most strains from animals do not require strict anaerobic conditions, but grow better with increased
CO2
Identification
in cattle, a strong presumptive diagnosis can usually be made based on the clinical signs, presence of
sulphur granules in discharges, and demonstration of gram positive branching rods
in dogs and cats; differentiation between Actinomyces and Nocardia is clinically important as the both
may cause similar diseases – suppurative serositis – but have different antibiotic sensitivity patterns
(see earlier table for differentiating features)
FIGURE: Gram stain of the exudate from the jaw of “Ralph” the bull. Note the fine branching rods
together with cellular debris (inflammatory cells).
Gram positive branching rods
It is important to DIFFERENTIATE between Actinomyces and Nocardia in

KEY POINT samples obtained from dogs and cats due to different antimicrobial
sensitivity patterns.
determination of the species of Actinomyces is not important in most clinical cases as it does not alter
treatment, control or prognosis of these diseases
in addition, many animal isolates cannot be assigned to existing species
182
infected humans show cell-mediated and humoral immune responses

9 circulating antibody produced during infection confers no protection
9 specific resistance, if any occurs, is probably cell-mediated
9 in addition, Actinomyces are killed by phagocytes (do not survive intracellularly)
treatment of bovine actinomycosis involves surgical drainage, iodine compounds and antibiotics
9 accessible soft tissue lesions should be drained
9 iodine may be given orally daily or intravenously weekly
9 treatment must be interrupted if signs of toxicity (hypersalivation, anorexia, vomiting) occur by
can be resumed some weeks later
9 antibiotic therapy usually consists of penicillin (+/- aminoglycoside – the contribution of which to
resolution is dubious)
the resolution of the bacterial infection will not restore normal bone structure in these animals, but
further degeneration of the jaw can occur
in dogs and cats, treatment consists of drainage, lavage, and removal of foreign bodies together with
long-term antibiotic therapy (weeks to months)
9 large doses of penicillin or a penicillin derivative (e.g. ampicillin) is the drug of choice and no
resistance to this antibiotic has been demonstrated
9 poor penetration of antibiotics into the dense granulomatous tissue reaction necessitates prolonged,
high doses
9 alternatively, erythromycin, rifampin, cephaloridine, minocycline imipenem, and tetracyclines
have been used successfully
9 aminoglycosides and fluoroquinolones are ineffective
the propensity of Actinomyces spp to form cell-wall-deficient variants (L-forms) can make treatment
difficult in all species and may result in failure to respond to therapy
however, success rates of >90% have been recorded in dogs with actinomycosis with appropriate
therapy
FIGURE: This is “Bob” the

Bull Mastiff. He was
presented to you because
of a large swelling under
his jaw. As part of your
therapeutic plan you
drained the abscess,
obtaining some of the
purulent material in the
process. You also
extracted a grass awn
from the abscess during
the surgery. In addition,
you placed a Penrose
drain to assist drainage of
the abscess. The results
of your culture and
sensitivity testing
revealed Actinomyces
hordeovulneris which
was sensitive to
Penicillin.
183
there is no vaccine available

avoidance of rough pastures in cattle, and where foxtails are prevalent, conscientious grooming should
occur
there are no reports of actinomycosis being transmitted from clinically infected animals to humans or
to other animals
nevertheless, human infections with Actinomyces spp does occur, and people handling infected tissues
or discharges should take appropriate precautions
in addition, infection with Actinomyces spp has been recorded in people bitten by dogs and cats
184
ARCANOBACTERIUM / ACTINOBACULUM
Summary Table : Species of Arcanobacterium and Actinobaculum that have veterinary importance,
their animal hosts, and the diseases they cause.

Arcanobacterium cattle (sheep, goats, wild abscesses in lung, pericardium,
pyogenes1+++ ruminants, camels, pigs, endocardium, pleura, peritoneum, liver,
humans) joints, uterus, renal cortex, brain, bones,
and subcutaneous tissues
abortion
mastitis
Actinobaculum suis2+ pigs cystitis
1
NOTE: Arcanobacterium pyogenes used to be classified as Corynebacterium pyogenes and then Actinomyces
pyogenes
2
Actinobaculum suis is a strict anaerobe that causes cysititis in sows. Their normal habitat is the preputial
mucosa of boars. They are transmitted to sows during coitus. Boars are rarely affected, but sows may develop cystitis
3-4 weeks after mating. Anorexia, arching of the back, dysuria and haematuria are prominent signs. If both kidneys are
involved, death may result. Diagnosis, treatment and control are similar for C. renale infection of cattle (bovine
pyelonephritis). They will not be discussed further in these notes.
185
ARCANOBACTERIUM
This is a new named group of bacteria, the most important of which used to be called Corynebacterium
pyogenes and then Actinomyces pyogenes (you will see these names in old text books). Now it has a new
genus all by itself! This bacteria is an important pathogen of ruminants, but can cause disease in a wide
variety of species. The infections caused by A. pyogenes are characterised by purulent inflammation (and
it is classified as one of the “pyogenic” bacteria). They are short, gram positive rods, that are similar in
shape to the diptheroid bacteria. Actinobaculum suis causes cystitis in sows.
What are they?
Arcanobacterium are gram positive, pleiomorphic rods – see description for diptheroids
9 their gram reaction may be unstable (similar to Streptococci) and so they frequently stain gram
negatively
there is only one species of veterinary importance: Arcanobacterium pyogenes
Arcanobacterium pyogenese is the only species of veterinary importance in

KEY POINT the genus Arcanobacterium.
A. pyogenes is found on nasopharyngeal mucosa of susceptible species, particularly cattle, sheep and
pigs
KEY POINT Arcanobacterium pyogenes is commensal (normal flora) on it host species.
A. pyogenes
most infections are probably endogenous arising from commensal organisms
in summer mastitis, cow-to-cow spread, aided by flies, is thought to occur
a) Exotoxins
a 58 kDa haemolytic exotoxin (pyolysin) lyses RBCs, has dermonecrotising activity and is thought to
have a role in the pathogenesis of disease produced by this organism
pyolysin is similar to other exotoxins produced by other gram positive bacteria (e.g. Clostridium and
Listeria)
this bacterium also produces a protease and neuraminidase, neither of which have a defined role in
virulence
186
II) Pathogenesis of Arcanobacterium infections
A. pyogenes is a common cause of suppurative lesions in many domestic species worldwide, especially
cattle, pigs and sheep
infections are frequently complicated by other potentially pathogenic commensals, especially non-spore
forming anaerobes (e.g. Bacteroides, Fusobacterium, Porphyromonas, Prevotella and
Peptostreptococcus)
the lesions observed are abscesses, empyemas or pyogranulomatous
abscesses are often heavily encapsulated
any offensive odours are contributed to the anaerobic component of infections
FIGURE: “Gertrude”, a 3 year old

Friesian cow. “Gertrude” was
presented because of weight loss and
a decrease in milk production.
Multiple biochemical analysis revealed
elevated hepatic enzymes (GLDH,
ALP) and bilirubin.
1. Cattle
because A. pyogenes is part of the normal flora of susceptible species, disease prevalence is sporadic
and is governed by precipitating stress or trauma
in cattle, A. pyogenes is involved in most purulent infections of traumatic or opportunistic origins
Arcanobacterium pyogenes causes opportunistic infections and is a

KEY POINT COMMON cause of purulent infections in cattle.
the lesions produced may be localised, regional or metastatic

common sites for localisation are the lymph nodes, lung, pericardium, endocardium, pleura,
peritoneum, liver, joints, uterus, renal cortex, brain, bones, and subcutaneous tissues (ie many
different tissues may be involved!!!)
this bacteria is also associated with pyometra, metritis, abortion and mastitis
9 “summer mastitis” is a communicable disease among pastured dairy cattle during their dry cow
period and is most prevalent in northern Europe (Britain and Ireland)
9 it often takes a destructive course, causing abscess formation and sloughing
9 bacteria implicated in its aetiology included A. pyogenes, S. dysgalactiae and non-sporing
anaerobes (Peptostreptococcus indolicus)
A. pyogenes also occurs in association with anaerobes in other mixed infections such as foot abscesses
187
FIGURE: “Gertrude” was subsequently euthanised and post mortem revealed a large whitish abscess
within the parenchyma of the liver and which is extending to the surface. Arcanobacterium pyogenes
was isolated in pure culture from this abscess.
Liver abscess due to infection

with Arcanobacterium pyogenes
Sample Collection
aspirates collected in a sterile fashion should be obtained from abscesses, lymph nodes or other
infected tissues
Direct Examination
gram stained smears from exudates/aspirates (lymph nodes or abscesses), tissues or tracheal washes
reveal gram positive short rods (usually a mixture of morphological forms including cocci, short and
long rods)
there may be other bacteria (mixed infections), especially in cases of A. pyogenes
The presence of diptheroid shaped rods in the smears of aspirates from

KEY POINT lesions may allow a presumptive diagnosis in many of these infections.
188
Arcanobacterium pyogenes which WBCs and other
are very short gram positive cellular debris
coccobacilli
FIGURE: Gram stain of the purulent exudate that was obtained from the abscess in
“Gertrude’s liver (on the previous page). Note that the bacteria are very short rods to
coccobacilli which is the typical morphology of Arcanobacterium pyogenes.
Culture
this group of bacteria grow on blood agar in air
frequently the first sign of a positive culture is a hazy haemolysis along streak lines after 24 hours of
aerobic incubation
cultures may take up to 48 hours to produce discernible colonies, and colonies are usually small
Identification
classical clinical signs, the presence of gram positive rods (diptheroids) and typical colony morphology
is frequently sufficient for a presumptive diagnosis
biochemical characterisation is usually only performed in referral laboratories
Interpretation
care must be taken in the interpretation of isolates of diptheroids from clinical samples as there are
many diptheroid bacteria that do not cause disease in animals or humans but occur in animals as
commensals
therefore this type of bacteria are common contaminants
immune responses to A. pyogenes are not well understood

infection confers no useful resistance
189
incision and drainage of abscesses are essential

although many antibiotics, including penicillin, are effective in vitro, medical treatment alone is
usually non rewarding due to inadequate delivery of the drug to the centre of the lesions
in addition, infections are commonly mixed, therefore a broader antimicrobial coverage may be
required
infections due to A. pyogenes are difficult to control as they are opportunistic

no vaccines are available, although a pyolysin vaccine is being developed – its usefulness remains to
be determined
A. pyogenes has been recovered occasionally from suppurative conditions in humans and
circumstances point to opportunistic infections
190
NOCARDIA
Nocardia are really interesting bugs that can cause disease in a wide variety of animals. However, they
don’t cause disease easily! And so significant underlying host compromise should be looked for if a
Nocardial infection is diagnosed. Like Actinomyces, they are gram positive branching rods, which
frequently have a very beaded appearance. The reason for this “beading” is that stains find it difficult to
penetrate the cell wall of these bacteria due to the relatively high lipid content (remember most stains are
aqueous based). These lipids also help the bacteria to survive inside the host, including inside host cells!
The lesions caused by Nocardia tend to be chronic and pyogranulomatous in nature and can be found in a
variety of organs (especially the thorax in cats and dogs). Unfortunately, Nocardia infections can be very
difficult to treat (due to their intracellular nature and antimicrobial resistance) so the prognosis of infected
animals is guarded.
Summary Table : Species of Nocardia that have veterinary importance, their animal hosts, and the
diseases they cause.
++++
N. asteroides Probably all animal species pneumonia and suppurative pleuritis with
(“N. nova” is included But most commonly dogs, empyema is the most common finding; but
in this “group” of cats and horses dissemination to other sites such as liver,
bacteria called a kidneys, bones, joints, and rarely the CNS can
species!) also occur
nocardial mycetoma have also been reported
N. braziliensis+ dogs (cats, horses, humans) localised granulomatous abscesses, pyothorax
+/- peritonitis, lymphangitis
N. otitidiscaviarum+ dogs, (humans) serositis, pyothorax, discospondylitis,
(previously called abscesses, lymphangitis
N. caviae)
NOTE:
9 N. asteroides and N. brazilensis are actually probably a heterogenous collection of Nocardia

9 “Nocadia nova” is part of this collection, and is the most frequent Nocardia isolated from cats and dogs
191
What are they?
they are gram positive branching rods

9 branching is especially evident in exudates from lesions
9 in smears of exudates they appear as long, slender branching filaments with a tendency to fragment
into rods and cocci
9 the rods may also be “beaded” due to uneven staining
9 they can also appear as short, club-shaped (diptheroid) rods

when cultured, these organisms produce aerial filaments which
may form spores
there are MANY species of Nocardia but only three species of
veterinary importance have been identified (see summary table)
pathogenic nocardiae are saprophytes found in many climates in soils, water and on decaying
vegetation
they may be found there either as part of the indigenous flora of the soil/water or as contaminants
pathogenic nocardia are found worldwide
there are many species of non pathogenic Nocardia that may also be found in soil and water
Pathogenic Nocardia spp are saprophytes and live in the soil, water and on
KEY POINT plants. Therefore infections are ENDOGENOUS.
infections are exogenous

there are 3 main routes of infection:
a. inhalation
b. trauma (inoculation through puncture wounds)
c. ingestion
dust, soil and plant material serve as fomites
bovine mastitis may be introduced via the teat canal and can be disseminated by equipment and
personnel
192
i) Virulence Factors
a) Mycolic Acid and Cell Wall lipids
mycolic acid is a cell wall constituent of coryneform bacteria (such as Corynebacteria, Rhodococcus
and Mycobacteria) as well as Nocardia
this cell wall constituent, together with other nocardial lipids, is thought to play a role in virulence by
helping pathogenic nocardiae survive within phagocytic vacuoles by preventing phagosolysosome
formation
other cell wall lipids may trigger granulomatous reactions
variations between strains and growth phases in cell envelope constituents are paralleled by changes in
virulence and infectivity
b) Superoxide Dismutase and Catalase

superoxide dismutase and catalase are thought to assist in the virulence of pathogenic Nocardia by
helping to minimize oxidative damage to the bacteria by host cell enzymes (myeloperoxidase system)
and therefore protecting against intracellular killing
Pathogenic Nocardia spp are FACULTATIVE INTRACELLULAR BACTERIA that

avoid killing by host phagocytes. They do this as once they are ingested
KEY POINT they inhibit phagosome-lysosome fusion and inactivate the
myeloperoxidase system.
ii) Pathogenesis of Nocardia infections
Infections with Nocardia spp are OPPORTUNISTIC and usually require MAJOR
KEY POINT HOST COMPROMISE (e.g immunocompromise or underlying systemic disease)
or inoculation of large numbers of bacteria.
similar to Actinomyces spp, the Nocardia spp are involved in pyogranulomatous and generalised
suppurative processes
these infections usually only occur in immunocompromised animals/people or animals that are exposed
to large numbers of bacteria
nocardiosis is primarily a suppurative process with variable granulomatous features
local wound infections commonly extend to the local lymph nodes, or may spread by contiguousness
infections can be regionalised or disseminated with haematogenous dissemination possibly resulting in
osteomyelitis and widespread abscess formation
CNS involvement is rare in animals
exudates are sanguinopurulent (look like thick “tomato soup”) and sometimes contain small (<1mm
diam) soft granules (“sulphur-like granules”) which consist of bacteria, neutrophils and debris, but
lack the microstructure of true sulphur granules (club colonies)
What Diseases do Nocardia Cause?
disease due to pathogenic nocardiae is found worldwide suggesting constant exposure

probably all animal species are susceptible to infections, but dogs are most commonly observed with
disease
193
disease has also been reported in humans, cats, horses, goats, sheep, pigs, primates, rabbits, marine
mammals, birds and fish etc
in humans, disease is associated largely with immunodeficiencies and this association has also been
established in horses, dogs and cats
1. Dogs and Cats ×Ø

these species can develop debilitating, febrile illnesses, usually secondary to an immunosuppressive
event (e.g. post immunosuppressive viral infection – FIV, FeLV, canine distemper)
the disease is uncommon in dogs and rare in cats
the disease is also most common in young animals (pups)
pneumonia and suppurative pleuritis with empyema is the most common finding; but dissemination to
other sites such as liver, kidneys, bones, joints, and rarely the CNS can also occur
case fatality rates exceed 50%
in addition, solitary extrapulmonary nocardiosis occurs infrequently as an cutaneous or subcutaneous
abscess (nocardial mycetoma or“actinomycotic mycetoma”) in dogs and cats
2. Horses
local or generalised infections have been reported – but are very rare
usually, infection is associated with severe systemic disturbances (e.g. combined immunodeficiency in
arab foals, or equine Cushing-like syndrome)
FIGURE: “Mo” unfortunately doesn’t survive (despite heroic efforts on your behalf).
On post mortem you find that his chest is full of a fluid that looks like “tomato soup”
(ie it has a reddish/brown colour and a thick consistency). Furthermore his lungs don’t
look good (there are areas of consolidation and reddening, and there are also multiple
grey-white nodules throughout the lung, especially subserosally. The local hilar lymph
nodes are all grossly enlarged). In other words “Mo” had empyema and pneumonia.
Emypyema of the thorax
194
3. Cattle
b) a) Bovine Mastitis
9 nocardial infection is an economical important form of bovine mastitis
9 it is most often traceable to unsatisfactory hygienic practices, and the organism is often first
introduced into the udder with intramammary mastitis infusions applied in the “dry cow period”
9 acute disease is triggered with the onset of lactation, when milk flushes the organism from limited
foci through the lactiferous duct system and results in fever, anorexia and abnormal milk secretion
(often reddish/brown in colour)
9 the affected gland is swollen, hot, and painful
9 discharging fistulous tracts may develop and lymphadenopathy is common
9 occasionally, disseminated nocardiosis may occur in these cases
9 the affected gland usually becomes chronically affected and non-functional and the prognosis for
the cow is guarded to poor
9 fatalities (5-10%) may occur during the acute stage or upon rupture of the udder
9 “outbreaks” of nocardial mastitis may also occur when nocardiosis occurs in one animal and
spreads in the course of the milking operation
c) Bovine Farcy
9 this disease is restricted to the tropics (most often reported in the Sudan) and is primarily attributed
to Mycobacterium farcinogenes, but N. farcinica, has also been implicated
9 this is a chronic suppurative infection usually starting as small cutaneous nodules, often of the
medial aspect of the lower limb and on the neck
9 lesions may progresses to involve the lymphatic vessels of the extremities or the head region and
their associated lymph nodes
9 gradually the nodules enlarge and coalesce, and may then ulcerate and discharge sinus tracts along
the path of infection, although the affected animal usually remains in good health (unless
dissemination to internal organs occurs)
4. Other Species
lymphadenitis, pneumonia, abortion, and mastitis have been reported in pigs, sheep, cattle and goats
in addition cases observed in birds, whales and dolphins have mostly involved the respiratory tract
with signs of systemic dissemination
Sample Collection
samples of exudates collected aseptically should be submitted for gram stain and cultivation
Direct Examination
smears made from aseptically collected samples contain branching, gram positive filaments along with
shorter coccobacillary forms
however, these bacteria cannot be distinguished from Actinomyces spp based on a gram stain (unless
an acid fast stain is performed – and even then the three species of veterinary importance have variable
degrees of acid fastness)
Culture
specimens for culture should not be chilled or frozen
nocardial colonies have a distinct appearance on blood agar and assists with identification of this
organism
9 slow growing (can only see colonies after a couple of days, but will grow even at 10C)
195
9 colonies are dull and opaque with waxy to velvety surface which become wrinkled with age
9 hard to dislodge with a loop
FIGURE: A smear from a colony

of Nocardia spp showing the
typical fine, filamentous rods
with branching. In addition
“beading” of the rods can be
seen because of uneven uptake of
the stain into the filaments (this is
due to the relatively high lipid
content of the cell wall of these
bacteria).
Identification and Interpretation

the fact that they fail to grow anaerobically helps distinguish them from Actinomyces spp
identification of the species of the species of Nocardia is difficult, but is of clinical importance in
samples obtained from dogs and cats as N. asteroides and “N. nova” species have different
antimicrobial sensitivity patterns and different prognosis (see later under therapy)
isolation of a pure culture of Nocardia spp, even in low numbers, from a closed lesions is significant
however, as these bacteria are ubiquitous in soil, care must be taken in interpretation of isolates
obtained from ulcerated skin lesions (if suspect a mycetoma) or the respiratory tract (present
transitorily)
antibody and cell mediated immune responses, including hypersensitivity reactions, commonly develop
during nocardial infections
antibody apparently confers little protection; specific resistance is largely cell-mediated
antibiotic therapy of nocardial mastitis may produce temporary relief of clinical signs and cessation of
shedding, but no permanent cure of this disease has been recognised
in other species, surgical debridement of granulomatous lesions and drainage of effusions is an
essential component of therapy
antimicrobial sensitivity tests should be performed as these bacteria do not have a predictable
sensitivity pattern and different species of Nocardia have different sensitivity patterns
9 unfortunately, sensitivity testing is difficult to do (send it to a specialist laboratory) and so often
empirical therapy is performed
9 sulphonamides, either alone or in combination with trimethoprim, have been found to be the most
successful drugs to date for N. asteroides infections
9 amikacin, imipenem-cilastatin, cefotaxime, ampicillin, tetracyclines (minocycline and doxycycline)
have also been used to treat infections
9 amoxicillin-clavunate, penicillin and fluoroquinolones are NOT effective against many of the
Nocardia spp
196
9 for N. nova infections there is good reported responses to ampicillin therapy
9 furthermore, these infections are reported to respond better than other Nocardia spp (at least the
isolates obtained at Sydney Uni!)
Nocardia do NOT have a predictable antimicrobial sensitivity pattern.

Furthermore, different Nocardia have different sensitivity patterns and
KEY POINT response to therapy Therefore, it is worthwhile determining the species of
Nocardia involved in an infection.
antibiotics must be continued for a prolonged period of time (up to 12 weeks for cutaneous lesions, 6
months for uncomplicated pulmonary lesions and 12 months for systemic infections!!!)
the prognosis for cases of nocardia is poor – one review in dogs found ~50% of dogs were died and
~40% were euthanised!!!
however, early diagnosis and multidrug therapy (combination of antibiotics) may improve these figures
in addition, recent reports of N. nova infections suggest that this species may be more responsive to
antimicrobial therapy
In cases of Nocardial infections, antibiotic therapy must be continued for

prolonged periods of time. Even then, the prognosis for infections with
KEY POINT Nocardia is guarded to poor (frequently due to the underlying diseases which
allowed Nocardia to cause an infection in the first place!).
no practical immunisation method is presently available and it is unlikely to be developed due to the
sporadic nature of this disease
control of mastitis involves removal of infected animals, thorough disinfection of premises (chlorine
disinfectants and benzalkonium chloride) and scrupulous milk hygiene
there have been no reported cases of human nocardiosis acquired by direct contact with an infected dog
or cat
however, there are several documented cases of cutaneous nocardiosis transmitted to humans by the
scratch or bite from clinically healthy cats and dogs
in these cases, the Nocardia spp are ubiquitous in the soil, and have contaminated the claws and teeth
of cats and dogs – however, the risk of contracting nocardiosis from dogs and cats is no greater than a
puncture wound whilst gardening
immunosuppressed people should take special precautions when handling dogs or cats with nocardiosis
197
DERMATOPHILUS
Dermatophilus is another interesting bacteria, as it is the only one with a type of life cycle. Consequently,
there are a number of different morphological forms of D. congolensis and all of these can be seen in
exudates from lesions. Not surprisingly (considering its name) Dermatophilus predominantly causes skin
infections! However, in order for Dermatophilus to be able to cause disease there needs to be some form
of host compromise, frequently provided by environmental conditions. The most common environmental
risk factor is rain, hence the common name for this disease being “Rain Scald”. The best way to treat
these infections is by getting the animal out of the rain (!), mild scrubbing with disinfectants can also help
to get rid of the scabs (which harbour the bacteria) and dry out the lesions.
Summary Table : Species of Dermatophilus that has veterinary importance, its animal hosts, and the
diseases it causes.
cattle, sheep, goats, horses, (dogs, cats,

Dermatophilosis (exudative
D. congolensis++++ pigs, humans - much less common in
dermatitis)
these species)
198
The LIFE CYCLE of Dermatophilius
o this starts with a reproductive unit called a zoospore, which are small and motile
o these zoospores germinate to produce a germ tube which is about 1um thick
o the germ tube elongates and thickens, dividing both transversely and longitudinally and forming a
strand several layers thick
o this strand is enclosed within a gelatinous sheath, and the constituent cells become coccoid as they
differentiate into multi-flagellated zoospores
o the zoospores are then liberated as the strand disintegrates, completing the life-cycle
Motile Zoospore
Formation of germ tube

Release of motile zoospores
Elongation and division

of germ tube
Differentiation into
motile zoospores
FIGURE: A Giemsa stain of a sample obtained from the scab on a calf (see clinical case). Note the
“railroad track” morphology of the filamentous rods, which is due to division of the rods in several planes
(to ultimately form the zoospores). You should also be able to see that the rods are branching.
199
What are they?
Dermatophilus are gram positive, branching, filamentous rods

they are strictly aerobic
they are not acid-fast (unlike Nocardia that are modified acid fast)
there is only one species; Dermatophilus congolensis
unlike other bacteria, Dermatophilus has a type of “life cycle” – see opposite page
D. congolensis is an obligate parasite of the skin of animals – it does not multiply saprophytically (in
the soil), but may exist there transitorily
its reservoir is infected animals (carriers)
cattle, sheep, goats and horses are common hosts, but the disease has also been diagnosed in pigs,
dogs, cats, turkeys, primates, humans and wild mammals (including marine mammals)
D. congolensis is found worldwide, but the disease has the greatest economic impact in tropical Africa
Dermatophilus is an obligate parasite of the SKIN of mammals and the

KEY POINT RESERVOIR for infections are infected animals (usually cattle, sheep, goats
or horses).
spread is by direct and indirect contact with reservoir hosts, fomites or insects (flying and nonflying,
biting and non-biting)
injury by thorny plants and shearing cuts may create portals of infection or inoculate the agent
moist conditions promote its dissemination (see later under pathogenesis)
Spread is by direct or indirect contact with reservoir hosts, fomites or

KEY POINT mechanical transmission by insects.
i) Virulence Factors
none identified
ii) Pathogenesis of Dermatophilus infections

infection with D. congolensis is characterised by an exudative dermatitis
infection is usually confined to the living epidermis, and does not penetrate into the dermis (except in
cats where subcutaneous infections have been described)
as access to the epidermis is restricted by the presence of hair, wool, sebaceous secretions and the
stratum corneum, the ability of the organism to initiate an infection relies on disruption of these by
either soaking or trauma
200
Dermatophilosis is a classical example of the relationship between
KEY POINT host/parasite/environment interaction. It is an opportunistic pathogen
that can only cause disease if environmental and host conditions allow it to!.
once deposited in the epidermis, the motile zoospores responds to a CO2 gradient and “home” to
deeper layers
within these layers the zoospores germinate to form long germ tubes and filaments within the
epidermis and hair follicles
the body responds with the infiltration of PMNs which form a layer of cells under the infected
epidermis
beneath this forms new epidermis, which is in turn invaded by the zoospores
the eventual result is a thick scab with layers of neutrophilic exudate and infected keratinising
epidermis
What Diseases does Dermatophilus Cause?
D. congolensis causes the Dermatophilosis which is an exudative dermatitis in a wide variety of

species
9 also called “Streptothricosis” or “Cutaneous Streptothricosis” in all species, but particularly
cattle
9 “Rain Scald”, “Rain Rot” and “Greasy Heel” (in horses)
9 “Lumpy Wool”, “Mycotic Dermatitis”and “Strawberry Footrot” (in sheep)
the prevalence of dermatophilosis in all species depends on the presence of infected animals, the
dissemination of the bacteria (e.g. by arthropods or thorny plants) and the epidermis of susceptible
hosts being accessible to invasion due to trauma or wetting
although disease affects animals of all ages, it is more prevalent and more severe in young animals
in all species the primary lesions are painless and non-pruritic
the scabs are easily lifted off by the hair, which protrudes from both surfaces, leaving a moist,
erythematous (reddened) depressed area in the skin (characteristic of this disease)
soaking provides the necessary wetting to prepare for invasion, and therefore lesions are
predominantly found where the skin is wet and remains so (e.g. top-line of horses (equine “rain
scald”), feet and legs (ovine “strawberry footrot” and equine “greasy heel”), face and scrotum
(“mycotic dermatitis” in sheep)
in addition, prolonged wetting favours their expansion, and biting arthropods may also extend infection
to sites that are not wet (e.g axilla, flank, ventral abdomen)
the extent of the lesions may vary from a few scabby areas of roughened hair or “lumpy wool” to
widespread loss of epidermis, which is then susceptible to secondary infection – this progressive form
of the disease is mainly found in cattle in Africa, though death has also been reported in sheep and
goats
in sheep, neglected cases of “lumpy wool” may lead to complete solidification of the fleece
lesions in other sites (not skin) have been observed in cats and include the tongue, urinary bladder,
lymph nodes and subcutaneous tissues
9 they are probably associated with puncture wounds of cats in an immunodeficient state
201
FIGURE: The back of a calf with
dermatophilosis. Note the big
scab that the guy has plucked off its
back! Below these scabs the skin is
pink and moist (which is Big scab!
characteristic of these lesions and
helps to differentiate it from other
skin lesions such as Dermatophyte
infection). The calf had been tied up
to a tree and left out in the rain for
several days (obviously this picture
wasn’t taken recently).
Moist, erythematous (red)

region underlying the scab
Sample Collection
scabs should be collected from animals with lesions
they are usually easy to pick off, and hair protrudes from their surface
as the surface of the skin is not sterile – sterile techniques are not necessary
9 in this case diagnosis is reliant on demonstrating the presence of a specific agent and not merely
the detection of bacteria (as is the case if sampling a sterile site)
Direct Examination
smears of the ground up scabs should prepared and stained with gram stain and Diff Quik
typical phases of the life cycle should be observed (in conjunction with many neutrophils) and includes
9 large gram positive cocci in packets (=zoospores)
9 multicellular branching filaments – these filaments have characteristic traverse and longitudinal
divisions that result in 3-8 paired rows of coccoid elements
9 detection of these forms is diagnostic
202
Branching of the Filamentous rod with typical
filamentous rod “railroad track” appearance
FIGURE: A Giemsa stain of a sample obtained from the scab on the calf. Note the “railroad track”
morphology of the filamentous rods, which is due to division of the rods in several planes (to
ultimately form the zoospores). You should also be able to see that the rods are branching.
Culture
this is often not necessary if diagnostic forms (gram positive branching, multicellular filaments) are
observed in smear preparations
however, frequently in subacute and chronic cases, bacterial elements may be rare, especially the
branching rods
in these cases, cultivation may assist in the detection of D. congolensis
the organisms grow best in the presence of CO2 (5-10%) on blood agar
haemolytic colonies appear within 48 hours and gram stain will demonstrate zoospores and branching
elements
Identification
this is rarely required in practice (smears +/- typical colony morphology are generally sufficiently
diagnostic) and would be performed by a specialist laboratory
antibody is widespread among cattle in endemic areas

however, it is unknown whether antibody mediated immunity or cell mediated immunity plays the
major role in protection
animals can remain infected for long periods; however, when they are cleared of infection, re-infection
usually doesn’t occur
203
acute cases are often self-limited, and resolve with the drying of the skin
the mild cases also respond to grooming, moving of the animal to a sheltered position and removal of
the scabs
9 clipping as much hair as you can and bathing in organic iodides or dilute chlorhexidine solutions
can help to remove the scabs – this should be done daily until resolved)
Mild cases of dermatophilosis do not require systemic antibiotic therapy.

KEY POINT Topical therapy with disinfectants, together with appropriate management
(avoiding initiating cause) will usually resolve the lesions.
severe cases can be treated with systemic antibiotics – penicillin G is the drug of choice, although a
combination of penicillin and streptomycin is said to be more effective
tetracyclines have also been used in cattle, and ampicillin in cats (for treatment of deeper infections)
persistent cases on the lower limbs of horses (“greasy heel”) are helped by keeping dry and antiseptic
washes (e.g. dilute chlorhexidine solutions)
vaccination trials in Africa have been inconclusive

there are no vaccines currently available in Australia
control is reliant on management procedures such as minimising skin trauma, exposure to rain (provide
shelter) and to arthropods
FIGURE: A young Arab colt with

Dermatophilosus. Treatment and
control of this infection would
include the use of daily antiseptic
washes then drying these lesions
and making sure that the colt did not
graze in paddocks with long grass.
people are accidental hosts of D. congolensis (therefore it is considered to a zoonotic disease)

people handling infected carcasses or tissues can develop pustular dermatitis, although the disease in
humans is extremely rare
the disease in humans resembles that in domestic species and will spontaneously resolve in 1-2 weeks
however, you should remember that the scabs that are removed from affected animals contain
infectious organism – therefore they should be disposed of appropriately
9 these scabs are potentially infectious for other animals as well as human
204
DIPTHEROIDS
Summary Table: Species of Corynebacterium/Rhodococcus with veterinary importance, their animal

hosts, and the diseases they cause.
GENUS and SPECIES HOSTS DISEASES

Corynebacterium sheep, goats, horses, caseous lymphadenitis (CLA),
pseudotuberculosis+++ humans lymphangitis, pigeon breast
Corynebacterium renale+ cattle (other pyelonephritis

ruminants_
Rhodococcus equi+++ horses, cats pneumonia, wound infections,
lymphadenitis
Note: the number of + denotes the relative importance of these veterinary species within THIS genus (NOT
Note: Rhodococcus equi used to be classified as Corynebacterium pyogenes
205
“Coryneform” (or Diptheroid) bacteria
“Coryneform” bacteria (also called “Diptheroids) are a group of bacteria with the common feature
being pleomorphic, gram positive rods
Coryneform (Diptheroid) bacteria are a group of bacteria that share a

common morphology – short, club-shaped, gram positive rods. Although
KEY POINT Arcanobacterium pyogenes and Actinobaculum suis belongs in the
Actinomycete family – they also have a diptheroid morphology.
they are non-sporeforming and non-motile

the typical shape of this group of bacteria is that of short, club-shaped rods
however, they also often occur in packets of parallel (“palisades”) or criss-crossing cells (“Chinese
letters”) that included coccoid, rod, club and filamentous shapes
this pattern is called “diptheroid” – which is named after the type species C. diptheriae an important
human pathogen
“Diptheroid” bacteria occur in packets of parallel (“palisades”) or criss-

KEY POINT crossing cells (“Chinese letters”) that include coccoid, rod, club and
filamentous shapes.
there are 4 genera and 5 species of diptheroids that cause disease in animals, 4 of which have
significant importance (see table on opposite page)
1. Corynebacterium
2. Rhodococcus
3. Arcanobacterium1
4. Actinobaculum1
1
these genera have already been discussed in the family Actinomycetes
the term “coryneform” is often used synonymously with “diptheroid” to refer to the typical shape of
these bacteria
however, in some instances coryneform is used to collectively discuss bacteria with cell wall
constituents including mycolic acid, meso-diamino-pimelic acid, and arabinogalactan. In this case,
Corynebacterium is a coryneform as well as Mycobacterium, and Nocardia, but not Rhodococcus and
Arcanobacterium as they lack these cell wall constituents
206
CORYNEBACTERIUM
Corynebacteria are very small, club-shaped gram positive rods. This genus used to contain a number of
important bacteria that have since been classified elsewhere (e.g. A. pyogenes, R. equi). However, it still
contains the bacteria C. pseudotuberculosis which is the cause of an important disease of ruminants
“Cheesy Gland”. This is a very apt name for this infection, and the infected lymph nodes (the bacteria
most commonly causes lymphadenitis) look just like a lump of soft, crumbly cheese! It is an economically
important disease of ruminants, for which a vaccine is commonly used to help control losses.
Corynebacterium renale is another species in this genus and its claim to fame is as the cause of
pyelonephritis in cattle.
What are they?
Corynebacterium are gram positive, pleiomorphic rods – see description for diptheroids
their gram reaction may be unstable (similar to Streptococci) and so they frequently stain gram
negatively
there are 2 species of veterinary importance:
1. C. pseudotuberculosis
2. C. renale
9 the bacteria known as C. renale is probably 3 different species of bacteria
1. C. renale or type I
2. C. cystidis or type II
3. C. pilosum or type III
9 however, differentiation of these three types is not clinically relevant
C. pseudotuberculosis
is found in the GIT of normal sheep and goats, the soil of sheep/goat pens and in lesions in infected
sheep or goats
the source of infection is discharges from ruptured abscesses or nasal and oral secretions (if pulmonary
abscesses exist)
the habitat of the equine strain is not known
the current view is that C. pseudotuberculosis is an animal parasite and only an accidental soil
inhabitant – however, the organism can survive in soil for up to 8 months and so contaminated soil
may the source of infection
C. renale
members of this group of bacteria inhabit the lower genital tract of cattle and sometimes other
ruminants
they are maintained in carrier cows or bulls
when implicated in urinary tract infections in other species (e.g. horses, dogs) it is not known if the
reservoir of infection is the lower urinary tract of these species
Many diptheroids are found as commensals on animals including

KEY POINT Corynebacterium spp.
207
C. pseudotuberculosis usually enters through breaks in the skin, and is therefore strongly associated
with trauma to the skin as occurs at shearing, docking, mulesing etc
in addition, entry through intact skin has been postulated, though the skin still requires some
alterations, such as when it becomes wet and macerated as occurs at dipping
C. renale
many clinical infections are probably endogenous arising from ascending infection of commensal
organisms
organisms may pass between animals by direct and indirect contact
an exotoxin with phospholipase D activity can:
9 lyses sheep and bovine RBCs and endothelial cells
9 produces dermal necrosis in rabbits
9 is lethal in various experimental animal models
a 40kDa serine protease is also produced (corynebacterial protease 40 = CP40)
virulence is attributed to the exotoxin and possibly to the serine protease and cell wall lipids
in addition, this bacteria is a facultative intracellular parasite whose resistance to phagolysosomal
disposal is related to its surface lipids
KEY POINT Corynebacterium pseudotuberculosis is a facultative intracellular parasite.
C. renale
both pili mediated attachment to urothelium and urea hydrolysis are considered critical in the
development of disease
the urease breaks down urea with production of ammonia
it is the ammonia which is thought to initiate the inflammatory process and in addition causes
suppression of antibacterial defences, possibly through complement inactivation by ammonia
clinically we observe highly alkaline urine (pH >9.0) (though herbivore urine is normally mildly
alkaline)
many bacteria that cause disease in the urinary tract produce urease (e.g. Proteus, Staphylococcus
intermedius); however, C. renale produces urease in very high concentrations!!!
II) Pathogenesis of Diptheroid Infections
the lesions observed in C. pseudotuberculosis infections are thick walled abscesses
the development of disease (“caseous lymphadenitis”) is usually traced to skin infections, though
subsequently the organism rapidly localises in the local lymph nodes
after introduction to the lymph node, the agent elicits a diffuse inflammation, followed by formation of
an abscess that coalesces and undergoes encapsulation
208
the nature of the pus varies largely with age of the lesion, which grossly appears creamy to dry and
crumbly (“cheesy”)
old abscesses consist of dead macrophages with peripheral neutrophils, giant cells, and a heavy fibrous
tissue capsule
inflammatory cells traverse the capsule peripherally, adding a layer of suppuration and a new capsule
several such cycles give the lesions, especially in sheep, an “onion ring” appearance
the disease may subsequently disseminate first through the lymphatics and subsequently
haematogenously, to produce abscesses in many lymph nodes and organs
lesions almost always contain just C. pseudotuberculolsis
C. renale
in cattle, the process is an ascending urinary tract infection, with the bacteria attaching via pili to the
urothelium of the bladder and initiating a cysitits
this proceeds to a chronic pyelonecrotic process which attacks successively the bladder, ureter(s),
renal pelvis, and renal parenchyma
Infections with all diptheroids (Corynebacterium, Arcanobacterium,

Rhodococcus) usually produces abscesses. Characteristic lesions involve
KEY POINT suppurative processes, usually with a granulomatous component
(PYOGRANULOMATOUS).
1. C. pseudotuberculosis
a) Sheep and Goats - “Caseous Lymphadentitis”

caseous lymphadenitis is one of the most important bacterial infections of small ruminants
in abattoir surveys in Australia 26-54% of adult sheep were infected!, and a similar prevalence has
been observed in sheep in North and South America
the prevalence of this disease increases with age and most forms of infection are chronic
in sheep, shearing, docking, dipping and high dust are significant risk factors for the development of
infectionclinically, there is palpable enlargement of one or more of the superficial lymph nodes, which
may rupture to reveal creamy to caseated pus with no odour
the lesions the general health is unaffected unless dissemination occurs to other lymph nodes, viscera
or the CNS, causing progressive debilitation (“thin ewe syndrome”)
Caseous lymphadenitis is a COMMON and IMPORTANT bacterial infection in

KEY POINT small ruminants and the development of infection is associated with
shearing, docking and dipping.
209
FIGURE: A lymph node from a goat
like “Billy”. The goat also had a big pre-
scapular lymph node that developed
after he was shorn. A pure culture of C.
pseudotuberculosis was isolated from
the node (therefore this is a case of
caseous lymphadenitis). It is easy to
see from this picture why this disease is
also called “cheesy gland” as the lymph
node is taken over by this thick, yellow,
cheesy pus!!! Smears of this pus reveal
numerous diptheroid gram positive
rods, some of which are intracellular.
b) Horses (and Cattle)

there are 2 forms of the disease seen in horses:
i)
“Ulcerative lymphangitis”
9 this is a rare infection of horses (and cattle) in Australia
9 it is seen as an ascending lymphangitis, usually involving the hind legs starting at the fetlock
9 the disease usually progresses towards the inguinal region and is marked by swelling and abscesses
which rupture to leave ulcers along the course of infection
9 haematogenous dissemination is rare
FIGURE: “Neddy”, an Arabian gelding. He has ulcerative lymphangitis. Note that the infection has
followed the lymphatic chain and is involving both the lymphatic vessels (lymphangitis) as well as the lymph
nodes (lymphadenitis). C. pseudotuberculosis was isolated from this infection.
Infected lymph node

( = lymphadenitis)
which is swollen
Infection of the lymph vessels

( = Lymphangitis)
210
ii) “Pigeon Breast or Pigeon Fever”
9 this form of infection with C. pseudotuberculosis is only reported in the USA and predominantly
in Western states (mainly California)
9 the geographic distribution is probably due to strain variation
9 the disease is seen as abscesses, usually in the muscles of the chest and caudal abdominal region of
the horse
9 the infective mechanism is poorly understood, but the peak season (autumn) and geographical
restriction (mainly California) suggest an arthropod vector
9 horses with “pigeon breast” will have swollen pectoral regions that are painful; in addition they
may be lame depending on the size and location of the abscess
9 septicaemia occurs rarely, but may result in abortion, debilitation, and death
9 superficial lesions resolve slowly after drainage
2. C. renale
a) Cattle - “Pyelonephritis”
in cattle, the process is an ascending urinary tract infection, beginning with cystitis, which proceeds to
ureteritis, and pyelonephritis
it is usually observed in cows near parturition, appearing as an opportunistic infection by commensal
organism present in the vagina
bulls are rarely affected, but are commensal hosts of all three types of C. renale (and can transfer the
organism to cows during coitus)
rectal palpation reveals a thickened bladder and ureteral wall, distended ureters, and enlarged kidneys
with obscured lobulations
early cases show pollakiuria, haematuria, and increasing degrees of abdominal pain
chronic infections progress to debilitation and death due to uraemia
b) Sheep (“Pizzle Rot”)

in male sheep, infection with C. renale is usually associated with posthitis or “pizzle rot”
however, this disease is multifactorial, and requires the presence of a number of factors, as well as
urea-splitting organisms (often, but not always C. renale) to produce disease
the disease occurs typically in animals on rich legume pastures that is high in protein (which increases
the urea excretion) and oestrogens (which cause preputial swelling and urine retention in the sheath)
“pizzle rot” is a necrotising inflammation of the prepuce and adjacent tissues in rams and wethers,
where ammonia is thought to initiate the inflammatory process in a site that is constantly irritated by
urine
clinically, pustules and scabs are observed at the preputial orifice, with extension into the internal
prepuce in severe cases
in these cases, urethral obstruction may develop
the disease is occasionally observed in bulls and goats
Sample Collection
fine needle aspirates collected in a sterile fashion should be obtained from abscesses, lymph nodes or
other infected tissues
Direct Examination
gram stained smears from exudates/aspirates (lymph nodes or abscesses) reveal gram positive
diptheroids (usually a mixture of morphological forms including cocci, short and long rods)
211
frequently intracellular bacteria are observed, particularly with C.pseudotuberculosis as these bacteria
are facultative intracellular organisms
The presence of diptheroid shaped rods in the smears of aspirates from

lesions may allow a presumptive diagnosis in many of these infections.
KEY POINT Intracellular bacteria are frequently observed in cases of C.
pseudotuberculosis infections.
Culture
cultures may take up to 48 hours to produce discernible colonies, and colonies are usually small
Identification
Interpretation
care must be taken in the interpretation of isolates of diptheroids from clinical samples as there are
many diptheroid bacteria that do not cause disease in animals or humans but occur in animals as
commensals
therefore this type of bacteria are common contaminants
Care must be taken when interpreting the significance of isolation of

KEY POINT diptheroids as they are also common contaminants.
Other Methods of Diagnosis

serological tests have been developed for C. pseudotuberculosis, but they are of limited value in the
diagnosis of infections
the role of antibody and cell-mediated responses that occur during infection are undefined
antibody to exotoxin (phospholipase D) limits dissemination of abscesses, however, in some cases of
caseous lymphadentitis the disease can be progressive (suggesting limited usefulness of antibody), and
equine abscesses can recur
C. renale
no useful immunity develops in the course of infection
serum antibody is present and antibody coating (mostly IgG) of bacteria occurs in the urine in bovine
pyelonephitis (not cystitis), but has no beneficial role
in sheep and goats, antibiotic treatment is ineffective
abscesses are usually excised or drained
212
equine abscesses are handled surgically and prolonged penicillin therapy is used to prevent or treat
disseminated disease
C. renale
members of the C. renale group are susceptible to penicillin, but antibiotic therapy is successful only
in the early stages of infection
in these cases, a prolonged course of penicillin is required
ovine posthitis is treated surgically and with local antiseptic preparations
Drainage or excision of lesions is the most helpful thing to do for infections

by diptheroids (where possible!) Where this is not possible, long term
KEY POINT antimicrobial therapy is required. In general, the more chronic the
lesions, the more guarded the prognosis, and the less effective the
therapy.
in sheep and goats, control is aimed at limiting exposure be segregation or culling of affected animals
and at scrupulous sanitary care during activities like shearing, dipping and surgical procedures
there is a combination bacterin-toxoid vaccine available in Australia which may be helpful in limiting
infections, but is not entirely protective
a modified live vaccine shows promise
C. renale
dietary restrictions and testosterone administration have been used to control pizzle rot in sheep
disease is opportunistic in cattle, and no control measure are employed
C. pseudotuberculosis may rarely cause infection in people and usually follows animal contact (e.g.
shearers are at increased risk)
infection probably arises through contamination of cuts or wounds in people, and mostly is seen as a
benign lymphadenitis
the organism has been isolated from the milk of infected goats when mammary lymph nodes are
infected
C. renale has never been isolated from human infections
213
RHODOCOCCUS
Rhodococcus are also classified as diptheroid bacteria (due to their short, club like morphology) but like
members of the family Actinomycetes they have a higher lipid content in their cell wall. In addition, they
can survive inside cells and therefore the typical lesion associated with these bacteria is pyogranulomatous
inflammation. These lesions are most commonly observed in foals, and in particular in their lungs. This
disease is called “Rattles” due to the harsh respiratory sounds made by infected foals! This organism also
can infect a range of other animals (and adult horses) but usually requires a degree of host compromise
(or tissue compromise). As with other bacteria that can survive intracellularly, these bacteria can be
difficult to treat, particularly as foals are often quite severely affected by the time disease is detected.
Long term, often expensive, antimicrobial therapy is often required to get resolution of disease.
What are they?
Rhodococcus are gram positive, pleiomorphic rods – see description for diptheroids
9 their gram reaction may be unstable (similar to Streptococci) and so they frequently stain gram
negatively - particularly in samples from clinical exudates
there are many species of free living Rhodococcus but only one species has veterinary importance,
Rhodococcus equi
KEY POINT Rhodococcus equi is the only species of veterinary importance.
R. equi is found in the soil and also animal manure and probably secondarily in the intestines of
mammals and birds
UNLIKE OTHER DIPTHEROIDS, R. equi is a SAPROPHYTE and is normally

KEY POINT found in the environment.

infection with these soil organisms is acquired by:
9 inhalation
9 ingestion
congenital infections have also been recorded via umbilical or mucous membrane exposure
214
FIGURE: “Little Mo”, a 2
month old palomino foal.
Obviously, “Little Mo” is a
little stunted for his age!
His owner tells you that
“Little Mo hasn’t been
growing well lately, and
appears a bit dull and
lethargic.
a) phospholipase C
a diffusible R. equi “factor” (a phospholipase C) lyses RBCs in synergy with phospholipase D of C.
pseudotuberculosis (this is used to help identify these bacteria)
however, its role in the production of disease is not understood b) Capsule
these bacteria have a capsule which helps prevent phagocytosis
despite the presence of a capsule, these bacteria may be opsonised by complement C3b (generated by
the alternate pathway) in which case they attach to macrophages by way of the Mac-1 complement
receptor
once phagocytosed, the organism inhibits phagolysosomal fusion and therefore survives intracellular
(facultative intracellular parasite)
c) Vap
virulent strains of R. equi contain a high molecular weight plasmid that encodes many proteins
including a 15-17kDa protain called Vap (virulence associated protein)
this presence of this protein correlates with the ability of strains of R. equi to cause disease, but the
exact function of this protein is not known
Rhodococcus equi are FACULTATIVE INTRACELLULAR PARASITE – surviving

KEY POINT in macrophages by inhibiting phagosomal-lysosomal fusion.
II) Pathogenesis of Rhodococcus Infections

the lesions of R. equi are abscesses and granulomas
the elements of pyogranulomatous inflammation included macrophages and giant cells, together with
neutrophils, that may predominate in caseopurulent portions
215
1. Bronchopneumonia in Horses (“Rattles”)

foal pneumonia (rattles) is the most common manifestation of infection in horses
it is a significant cause of morbidity and mortality in foals world-wide, including Australia
“Rattles” is a SIGNIFICANT cause of severe pulmonary disease in foals

KEY POINT world-wide.
FIGURE: The lungs from “little Mo”.

The lungs are full of pyogranulomatous
abscesses. Unfortunately, “little Mo” did
not have a lot of respiratory reserve! and
collapsed when stressed whilst being
herded in order to send him to the vets!
Muliple abscesses in
the lungs of a foal
R. equi is part of the equine environment and its concentration varies with the history of equine use on
a property
on problem farms there are thought to be more virulent strains present, although the correlation
between the presence of virulent strains and the presence of disease is not strong, consequently other
factors are probably involved in development of disease
most, if not all foals are exposed to R. equi, but relatively few foals succumb to the disease process
it is thought that susceptibility coincides with the fading of maternal antibody and precedes natural
immunisation by subclinical exposure; however recent studies suggest that foals become infected at a
very young age but do not develop disease until later (possibly when maternal antibody is waning)
the peak seasonal distribution is summer, and is attributed to the abundance of susceptible foals and
heat and dust which imposed added burdens on respiratory tract defences
the disease only occurs in foals aged between 1 to 6 months of age (it is not observed in
immunocompetent adults)
it is important to remember that the disease has a long incubation period, and we usually only observe
clinical signs (e.g. inappetence, depression, fever, tachypnoea; rarely nasal discharge and lung sounds
may not be present) when there are severe and extensive lung lesions
a suppurative, pyogranulomatous bronchopneumonia is observed in these cases with large abscesses in
the lungs and hilar lymph nodes
occasionally there is haematogenous spread with localisation in joints, bone, skin and spleen
(extrapulmonary disease)
in addition, many foals have aseptic polyarthritis and uveitis due to the deposition of antigen-antibody
complexes (type III hypersensitivity reaction)
216
it is important to differentiate between this reaction and localisation of the organism in the joint (septic
arthritis) as the treatment and prognosis is different
Swelling of joints may be observed in foals with “Rattles” and may be due to
either dissemination of infection to the joint (septic arthritis) or a type III
KEY POINT hypersensitivity reaction (aseptic arthritis). It is IMPORTANT to
differentiate between these 2 disease processes.
some foals may develop a gastro-intestinal form with ulcerations in the intestine and abscesses in the
mesenteric lymph nodes – the mode of infection in these cases is thought to be ingestion (versus
inhalation for the pulmonary form of disease)
the prognosis varies indirectly with the age of the foal at the time of onset of clinical signs and is
poorest for those showing signs at less than 2 months of age
in these cases, the case fatality rate exceeds 50%
R. equi may also cause placentitis and abortion in mares, but this is very unusual
2. Other species (cats) Ø

R. equi has been isolated from both granulomatous lesions in the cervical lymph nodes in pigs as well
as normal pigs – therefore its pathogenic roles is disputed
sporadic suppurative infections have been described in diverse mammals including cats, ruminants,
koalas, crocodiles and alligators and humans
in Australia, these infections are most commonly observed in cats
the infection begins as a localised lesion (pyogranulomatous skin disease and cellulitis) which
commonly spreads to local lymph nodes, and sometimes the lungs and uterus
FIGURE: The paw of “Mouser”, a 17 year old DSH cat.

The paw was generally swollen and there multiple
draining tracts with a seropurulent discharge coming
from the sinuses. The owner noted that the lesions had
developed over a long period of time (or at least that is
what she tells you). The cat was lame on this leg, and
the local lymph node (pre-scapular) was also swollen.
A pure, heavy culture of R. equi was obtained from a
fine needle aspirate of one of the paw abscesses as well
as the local lymph node.
Draining tracts
Pyogranulomatous skin disease, cellulitis, and lymphadenitis have been

KEY POINT observed in CATS associated with R. equi infection.
217
fulminating bacteraemias have been observed in crocodiles
Sample Collection
in the case of R. equi infection in foals, a transtracheal wash is the preferred method of sample
collection if confirmation of a diagnosis of R. equi is warranted (care must be taken as frequently
infected foals have severe respiratory compromise!!!)
fine needle aspirates collected in a sterile fashion should be obtained from abscesses, lymph nodes or
other infected tissues (e.g. bones) if these are present in the foal or other species (e.g. cats)
blood cultures may also be indicated if septicaemia is suspected
Direct Examination
gram stained smears from tracheal washes or exudates/aspirates (lymph nodes or abscesses) reveal
gram positive (and/or negative) diptheroids (usually a mixture of morphological forms including cocci,
short and long rods)
frequently intracellular bacteria are observed as these bacteria are facultative intracellular organisms
The presence of diptheroid shaped rods in the smears of tracheal washes

KEY POINT allows a presumptive diagnosis in many of these infections.
Culture
R. equi colonies are larger than the other diptheroids
and are characteristic pink (usually becomes obvious in older cultures) and very mucoid (looks like
“pink spit”)
FIGURE: A blood agar plate showing the

characteristic colony morphology of R. equi. The
colonies are pink (trust me!) and are very mucoid
(which allows them to coalesce or join together) and
they look like “pink spit”.
Rhodococcus equi has a characteristic colony morphology – they look like

KEY POINT pink spit !!!!
Identification
218
Other Methods of Diagnosis
serological tests have been developed for R. equi, but they are of limited value in the diagnosis of
infections
a PCR has been developed for R. equi, but its clinical application has not been determined
radiology is of use in some cases (as foals are sufficiently small that this is possible, unlike adult
horses)
in addition, ultrasonography is being used for early detection of R. equi lesions in foals, but is time
consuming and expensive
in addition it relies on the abscesses involving tissues that are adjacent to the pleura
functional CD4+ T cells (TH1) are necessary for protective immunity in R. equi infections as they
“activate” macrophages by way of gamma interferon
in addition, antibody (probably against the virulence associated proteins - Vaps) are needed since
maternally derived antibodies as well as passively administered antibody appear to be protective
thus, both cell-mediated and humoral immunity are important in conferring protection against disease
horses past infancy are immune to disease, therefore they must receive immunising exposures to R.
equi during their infancy (first 6 months of age( which results in protective humoral and cell-mediated
responses
both AMI and CMI appear to be involved in this protective response which enables macrophages to
kill infecting organisms
the prognosis in cases of R. equi pneumonia is always guarded

chest radiographs give valuable prognostic clues - animals with nodular or cavitary lung patterns and
hilar lymph node enlargement respond less frequently to therapy than those with diffuse alveolar or
interstitial reactions
antibiotics are necessary in these cases and the preferred treatment is erythromycin in combination
with rifampin due to the ability of these antibiotics to penetrate cells (remember these bacteria are
facultative intracellular organisms)
these antibiotics must be given together, due to propensity of organisms to develop resistance to
rifampin if this antibiotic is given alone
9 fatal cases of hyperthermia have been recorded in foals and associated with erythromycin therapy
9 an alternate macrolide called azithromycin may therefore be used, but this is VERY expensive
(and may also cause hyperthermia)
a prolonged course of antibiotics is usually required (4-6 weeks) and antibiotics should be continued
until clinical signs have resolved and the foal has a normal WBC count and fibrinogen level
however, these antibiotics are expensive, and therapy with neomycin, ampicillin (alone or in
combination) or trimethroprim-sulphonamides may be a cheaper (though less effective) alternative
prevenative measures include ensuring colostrum intake, dust control, and removal of foals from
contaminated grounds
prophylactic antibiotic therapy has been used in endemic regions, but are not recommended due to the
possibility of the development of resistance
219
early diagnosis (ultrasonographic examination) and prompt treatment have been effective measures
against mortality on some large studs
there are recent suggestions that immunoprophylaxis may be of benefit
9 vaccinating mares with the organism has not shown to be an effective way of increasing the levels
of specific antibodies in colostrum
9 however, giving hyper-immune plasma (plasma from horses immunised with either whole R. equi
or recombinant virulent associated proteins – Vap) to foals does confer some protection against R.
equi induced pneumonia in foals
9 consequently, some stud farms administer this plasma in the first month of life
R. equi can cause serious disease in humans; where it causes severe pneumonia and disseminated
infections, particularly in immunocompromised individuals
affected foals may be a source of infection for people, though often non-virulent strains (Vap negative)
of the organism are isolated from human infections
soil is the more common source of infection for humans, though higher numbers of bacteria may be found
on farms where there is a high prevalence of infection in horses
220
LISTERIA
Listeria are short, gram positive rods, that are quite uniform in shape (regular). They are widely
distributed in nature, found both as commensal flora of animals (enteric carriers) as well as in soil, silage,
effluent, and water sources. In addition, they like cold temperatures, and this can be used to enhance
isolation of these organisms! Add these bacteria to your list of facultative intracellular parasites. There
are 2 main forms of disease associated with L. monocytogenes (the only species of significant veterinary
importance); a visceral form and a neural form. The neural form of the disease has the name “Circling
Disease” and predominantly occurs in ruminants. In addition, it may cause abortion in these species.
Listeria can also cause significant disease in humans, though it is mainly as a food borne pathogen
(especially soft cheese and other dairy goods) rather than direct transmission from infected animals.
Summary Table : Species of Listeria that have veterinary importance, their animal hosts, and the
diseases they cause.
L. monocytogenes++++ sheep, cattle, goats (dogs, cats, neural form (meningoencephalitis)

horses rare)
cattle, sheep, horses, dogs, visceral form (septicaemia and abortion)

cats, humans, pigs, birds
ocular form (endophthalmitis)
sheep, cattle goats
L. ivanovii+ cattle, sheep abortion
L. innocua Sheep Meningoencephalitis (rare)
221
What are they?
Listeria are short, gram positive rods,

which are regular but can be club shaped
(and so mistaken for diptheroids)
they are facultative anaerobes, but grow
best with increased CO2
they are non-spore-forming and motile
there are 6 recognised species of Listeria,

but only Listeria monocytogenes has major
human and veterinary importance:
L. ivanovii, and L. innocua may also cause

disease in ruminants, however,
differentiation to a species level is seldom
of clinical significance in practice situations
Listeria have a world wide distribution - especially in temperate climates

they have been isolated from soil, silage, sewage effluent, decaying vegetation, stream water and over
50 different animal species including ruminants, pigs, horses, dogs, cats, and various species of birds
animals and humans are asymptomatic enteric carriers of Listeria and in some areas up to 70% of
humans are reported to be faecal carriers of this bacteria (however a survey in Japan found on 0.9% of
dogs and no cats were carriers)
the organism may also be isolated from genital secretions and nasal mucus of apparently healthy
animals
Listeria is also isolated from raw or processed food (which has public health importance), including
meat, seafood, vegetables and dairy products
the widespread distribution of environmental and animal associated Listeria, make localising the source
of infection in a particular outbreak difficult
Listeria are ubiquitous in the environment and are carried in a wide

variety of animals as asymptomatic enteric carriers. Poor silage and food
KEY POINT items are a common source of the organisms in outbreaks of disease for
ruminants and people respectively.
222
FIGURE: A diagram of the possible ways that Listeria can be
maintained in the environment and then transmitted to humans and
animals.
most infections are thought to be exogenous

the route of infection may differ between the 2 forms of the disease (visceral and neural)
visceral infections are always thought to arise from ingestion of soil organisms or contaminated feed;
damage to the intestinal mucosal integrity is not necessary
poor quality silage , with pH >5.5 is commonly implicated and accounts for listeriosis often being
referred to as “silage disease”
an aymptomatic carrier can be a source for further contamination of the environment and therefore the
indirect source of infections
in the neural forms of the disease, infection is thought to result either from septicaemic spread (after
ingestion) or arise via spread of the organism along branches of the trigeminal nerve after entry
through the mucous membranes of the eye, nose or oropharynx
Listeria may have different routes of entry into the host for the 2 forms of
KEY POINT disease it causes (visceral and neural).
a) Facultative Intracellular Parasite (Haemolysin)
pathogenic Listeria species can survive inside cells (both phagocytes and non-phagocytes)
a haemolysin of L. monocytogenes (lysteriolysin O), which resembles streptolysin O, it thought to aid
intracellular survival of Listeria in cells by lysing phagosome and ferritin vesicles
ivanolysin, another cytolysin, is the counterpart in L. ivanovii
production of a phospholipase C and lecithinase also appears to be important in mediating membrane
lysis
223
KEY POINT Listeria is a FACULTATIVE INTRACELLULAR PATHOGEN.
II) Pathogenesis of Listeria Infections

Visceral Disease
in these cases, exposure to Listeria occurs via the oral route (ingestion)
entry into intestinal epithelial cells or M cells is mediated by internalin, a surface protein, and its
interaction with host receptors
after passage through the intestinal barrier, Listeria can be observed in phagocytic cells within the
lamina propria
further dissemination occurs via the bloodstream (septicaemia) with septic embolisation of many
organs including the CNS
in the different tissues, Listeria can be internalised by phagocytic cells or by non-phagocytic cells
through induced phagocytosis
after internalisation the organism escapes the phagosome, becomes associated with actin filaments in
the cytoplasm, and propels itself to the cell’s plasma membrane via actin polymerisation
in this way, it is able to pass to neighbouring cells in plasma membrane protrusions and thus avoid
host defence mechanisms
clinical signs are due to the degree of intestinal inflammation and the sites of embolic microabscess
formation
Neural Disease
an alternative route of entry into an animal (predominantly ruminants) is via damaged oral, nasal or
ocular mucosal surfaces and within neural sheath of peripheral nerve endings, particularly the
trigeminal nerve
in this way the organism may gain entry to the CNS, where it produces disease
organism have been demonstrated in the myelinated axons of the trigeminal nerve and cytoplasm of
medullary neurons (so most of the pathology is found in the brainstem)
in both forms of the disease, small abscesses develop at the site of deposition of the bacteria, which
may or may not be visible grossly
the general diseases caused by Listeria spp are called “listeriosis”

ruminants are the most frequently affected domestic species, but infection of many other species has
been reported including turkeys, sheep, dogs, cats and humans
there are 2 main forms of listeriosis which are the visceral form (septicaemia and abortion) and the
neural form (meningoencephalitis)
stress factors predisposing to clinical disease included nutritional deficiencies, environmental
conditions, underlying disease, and pregnancy
cases are usually sporadic, but may involve up to 5% of cattle herds or 10% of sheep flocks over a
period of time (e.g months)
the disease is more commonly observed in winter and spring in temperate climates
Two forms of Listeriosis may be observed; visceral and neural. The latter
KEY POINT form is predominantly observed in ruminants.
224
1. Visceral Form (Septicaemia and Abortion)
this form of the diseases is predominantly seen in neonates and is the most common presentation in the
horse (although it is a very rare disease in this species!) and the dog and cat
in dogs and cats clinical signs include fever, diarrhoea and vomiting
neurological signs have been apparent in some cases (due to septicaemic spread to the CNS)
unusual locations for infection have been reported (very rarely) in dogs and cats (e.g. peritonitis due to
a plant awn migration through the bowel, front paw abscess following an insect bite)
chinchillas are particularly susceptible to septicaemia
abortion is most commonly observed in ruminants, but also occurs in other species (e.g. dog)
in cattle and sheep, abortion is the usual manifestation of L. ivanovii infection, but L. monocytogenes
may also be involved
the abortion is usually late term – after 7 months in cattle or 12 weeks in sheep – and the foetus may
be macerated or delivered weak and moribund
retained placenta and metritis may result
systemic signs are rare in the cow unless the foetus is retained and triggers a fatal septicaemia
although abortion is usually sporadic, abortion rates of up to 10% have been recorded
it is uncommon to find the encephalitic (neural) form and abortions occurring in the same outbreak
2. Neural Form (“Circling Disease”)

the encephalitic form is sometimes called “circling disease” and is the most common form observed in
ruminants
in cattle the disease is subacute to chronic
clinical signs include signs of depression, anorexia, tendency to circle in one direction, head pressing
or turning the head to one side, unilateral facial paralysis, and bilateral keratoconjunctivitis
similar signs are seen in sheep and goats, but the course is usually more acute and frequently fatal
in these species “star gazing” (due to their head being stretched back) is a common presenting sign
“Circling Disease” is the neural form of listeriosis and is most commonly

KEY POINT observed in ruminants.
FIGURE: “Noddy” the merino

lamb. “Noddy” is demonstrating
the typical “star gazing”
posture that is adopted with
the neural form of listeriosis.
This is due to the fact that he has
a meningoencephalitis.
Note the arched back
225
Sample Collection
as with other gram positive rods – the clinical signs and signalment often lead to a presumptive
diagnosis of listeriosis
confirmation of the diagnosis requires samples to be obtained from affected individuals, the samples
collected depending on the clinical signs and availability
samples can include CSF, blood, brain (PM tissues – often the case in septicaemic animals!), spleen,
liver, abomasal fluids and/or meconium of aborted foetus or weak neonates
A presumptive diagnosis can be frequently made based on history,

KEY POINT signalment, clinical signs or post mortem findings in diseases caused by
gram positive rods.
FIGURE: Although it is unusual, some of

the ewes in the flock that “Noddy”
belonged to also aborted. This is the
liver from one of the aborted foetuses.
Note the characteristic small white
abscesses that are found throughout
the parenchyma.
Direct Examination
a direct smear may reveal many, small, gram positive rods in cases of septicaemia or abortion
only a low number of organisms (or none) are observed in the encephalitic form
negative findings are inconclusive
Culture
the organisms grows readily on blood agar, but prefers increase CO2
isolation of the organism, especially from cases of encephalitis, may be enhanced by “cold
enrichment”, where the tissue is stored at 4°C and then subcultured weekly for up to 12 weeks
selective media is available for cases where contamination with other bacteria is likely
Identification
biochemical tests may be used to definitively identify Listeria spp, and are performed in specialist
laboratory practices
Serology
is not useful for diagnosis of Listeria infections due to cross-reactivity with other gram positive
bacteria
226
as a facultative intracellular parasite, Listeria is primarily contained by cell-mediated responses

humoral factors may play some limited role in host defence
underlying host immunosuppression, especially of the cell-mediated immune responses, appears to be
an important factor in the intracellular persistence of the organism and in the development of clinical
listeriosis
Listeria is sensitive to penicillin, macrolides, aminoglycosides, trimethroprim sulphonamides,

tetracyclines and rifampin
however, treatment may be ineffective, especially in neural form cases in ruminants (especially in
sheep)
timely treatment in these cases is important and the outcome is usually better
a combination of gentamicin and ampicillin is reported to have the best outcome in canine cases of
listeriosis, although trimethroprim sulphonamide and rifampin is said to have better penetration to the
CNS if there is neural involvement
Penicillin is the drug of choice for the treatment of listeriosis in ruminants,

KEY POINT however advanced cases rarely respond to therapy.
no immunising preparation have met with success

killed preparations have been ineffective, while live, attenuated vaccines afforded some protection in
sheep
in addition, vaccination may not be warranted due to the sporadic nature of the disease
control measures include reduction or elimination of feeding of silage, particularly poor quality silage
all forms of stress should be minimised
affected animals should be isolated and infected material disposed of properly
Listeria monocytogenes is a significant cause of disease in humans

the major human cases of listeriosis are associated with immunosuppressed individuals, the elderly, the
very young and pregnant women
in addition, the organism has been passed to infants in breast milk
meningitis is the most common form of listeriosis in humans, but other manifestations such as
endocarditis, oculoglandular disease, and dermatitis have been reported
most human cases occur in urban environments in the summer
in general, there is little evidence for animals being a direct source of the infection in human cases,
though there are occasional reports of listerial dermatitis in veterinarians and farmers after handling
tissues from listerial abortions (seen as papular lesions on hands and arms)
otherwise, animal handling is an unlikely source of infection for humans; but animal products may be
a source
227
for example, human epidemics have been traced to food sources of animal origin, including milk, soft
cheeses, and liver pate. Coleslaw made from cabbage originating from a farm with an outbreak of
ovine listeriosis has also been reported
in many instances, post processing contamination is found to be the source of Listeria in cases of food
poisoning – where frequently there is the opportunity for selective growth of L. monocytogenes to occur
during long periods of refrigeration
228
ERYSIPELOTHRIX
Erysipelothrix are another group of short gram positive rods (in fact they look just like Listeria!). These
bugs are important pathogens of pigs, where they cause the impressive disease called Diamond Skin
Disease. Actually, this is a cutaneous manifestation of a systemic disease and the organism basically
causes septicaemia (and other syndromes more commonly associated with this such as endocarditis and
arthritis). It can cause similar diseases in other species (e.g. dogs) and also disease in humans and marine
mammals (where it likes to hang out in fish slime!). Infections may be difficult to treat and high
doses/long term therapy may be required.
Summary Table : Species of Erysipelothrix that have veterinary importance, their animal hosts, and

++++
E. rhusiopathiae pigs (sheep, dogs, cats, turkeys, there are 4 syndromes: septicaemia, a
marine mammals, humans) generalised skin form, arthritis, vegetative
endocarditis
E. tonsillarum++ dogs septicaemia, endocarditis (arthritis)
NOTE: the number of + denotes the relative importance of these veterinary species within THIS genus
(NOT for all bacterial genera)
229
What are they?
Erysipelothrix are small, gram positive rods
9 they are regular in shape - unlike the

diptheroids and actinomycetes, which vary in
their shape and therefore are called
“irregular”
they are facultative anaerobic, but prefer

environments with 5-10% CO2
they are non-motile, non-spore-forming
there are 2 species of veterinary importance:
9 Erysipelothrix rhusiopathiae
9 Erysipelothrix tonsillarum
the second species has only been recently described, and is frequently misclassified as E. rhusiopathiae
– however, differentiation to a species level is seldom of clinical significance in practice situations
Erysipelothrix are small, gram positive rods that are facultatively

KEY POINT anaerobic.
Erysipelothrix can be isolated from a wide variety of environmental settings (e.g. sewerage effluent,
abattoirs, surface slime of fresh and saltwater fish, soil
however, its saprophytic nature is in question
it has also been recovered from the alimentary tract (GIT) and mucous membranes of many different
mammals and birds, and can be isolated from the tonsils of apparently healthy pigs, which are thought
to be the primary reservoir
The upper GIT of pigs is thought to be the RESERVOIR for Erysipelothrix, but
KEY POINT this organism is commonly isolated from a wide range of environments
including soil and the surface of fish and crustaceans.
transmission is mostly by ingestion of contaminated material (e.g. soil, surface water, fish meal etc) or
after direct contact with infected pigs
wound infections and arthropod bites are other possible routes of entry
KEY POINT Erysipelothrix gains entry to the host primarily via INGESTION.
230
a) Neuraminidase
strains of E. rhusiopathiae vary in their virulence, where virulent strains produce high levels of
neuraminidase, which is thought to be an important virulence factor in acute septic infections
this enzyme cleaves sialic acid present on cell surfaces, leading to vascular damage and hyaline
thrombus formation
in addition, antibodies to neuraminidase are protective against experimental infections in mice
Capsule
some strains are thought to have a capsule which plays a role in prevention of phagocytosis
II) Pathogenesis of Erysipelothrix Infections

there are 4 main syndromes observed with E. rhusiopathiae infections in domestic species:
9 septicaemia
9 a generalised skin form
9 arthritis
9 vegetative endocarditis
Erysipelothrix regularly invades the bloodstream, and whether disease develops and the type of disease
that is observed depends on the virulence of the strain and the immune status of the host
in addition, these various forms may occur separately, in sequence or together within a herd of
affected individuals
1. Septicaemia
9 acute septicaemia can result in death of the animal
9 alternatively, the with less virulent strains, the organism may localise in either the skin, joints or
the heart to cause the chronic forms of this disease
9 the subsequent localisation of the bacteria dictates what form of the chronic disease is observed
clinically:
2. Skin Form
9 partial immunity of the host and low virulence of the strains appears to account for the localised
skin form mostly seen in pigs
9 lesions are probably the result of thrombus formation following immune complex deposition
(Arthus reaction)
3. Arthritis
9 localisation of E. rhusiopathiae in joints of pigs firstly leads acute and then chronic arthritis with
fibrinous exudation and pannus formation
9 subsequent damage to the articular cartilage is an immunological response to the persistent
bacterial antigens in synovial tissues and it is chondrocytes that are responsible for the chronic
articular changes rather than the bacteria per se
4. Valvular Endocartiditis
9 is presumably initiated by bacterial emboli which lodge on the heart valves
9 subsequent vascular inflammation results in chronic changes and damage to heart valves
Four syndromes are observed with Erysipelothrix infection; septicaemia,

KEY POINT dermatopathy, arthritis, and endocarditis. The syndrome that occurs
depends on the stain of the bacteria and the immune status of the host.
231
What Diseases does Erysipelothrix Cause?
the disease is generally termed “erysipelas”

pigs are the most commonly affected species and are also most severely affected by this organism
however, a range of other species have also been reported with infection including sheep, turkeys,
dogs, cats, marine mammals and humans
1. Pigs
pigs less than 3 months and over 3 years of age are least susceptible
passive immunity (for piglets less than 3 months) and active immunity (for pigs older than 3 years)
probably accounts for this age-related susceptibility
predisposing factors include environmental stress, dietary changes, fatigue and subclinical aflatoxicosis
the 4 syndromes are observed in this species:
a) Acute Septicaemia
this syndrome is mostly observed in pigs and is rarer in other species
pigs dying of acute erysipelas infections exhibit haemorrhages of the gastric serosa, skeletal and
cardiac muscles, and renal cortex
congestion of the lungs, liver, spleen, skin and urinary bladder is also observed
the haemorrhages are due to vascular damage with microthrombi present, and a mononuclear cell
infiltrate is observed in most cases
clinically, pigs present with fever, anorexia, signs of depression, vomiting, stiff gait and reluctance to
walk
infrequently, sows may abort as a result of infection
if untreated, this form of the disease has a high mortality rate
b) Cutaneous Disease – “Diamond Skin Disease”

skin lesions may be observed in the acute septicaemic form and are seen as urticarial lesions, which
may be palpated (because they are raised) before becoming visible
when visible they are pink, or in severe cases purple, especially on the abdomen, thighs, ears and tail
in severe cases the skin becomes necrotic and is sloughed
in less severe forms of erysipelas in pigs, lesions are limited to the skin but may be accompanied by a
mild fever
these skin lesions have a red to purple rhomboidal shape – therefore the disease is called “diamond
skin disease”
lesions may progress to necrosis or they may resolve, leaving a mild scruffiness to the skin
mortality is seldom seen in this form of the disease
“Diamond Shaped” lesions are the classical form of the skin disease caused
KEY POINT by Erysipelothrix.
232
FIGURE: A pig with the Diamond
skin disease. This is the skin
manifestation of systemic disease,
where the organism first produces
a septicaemia and subsequently
localises in the skin. In addition,
part of the lesions are caused by
an immune mediated mechanism
(Arthus reaction).
Skin lesions on a pig with Diamond

Skin Disease. It is obvious how this
disease got its name!!!
a) Arthritis
acute synovitis usually proceeds to more chronic articular changes, though clinically the chronic form
may be observed without any evidence of acute disease
this form of the disease is more commonly seen in older animals
the synovial membranes become hyperplastic with villous formation and these are infiltrated with
mononuclear cells and is seen as marked periarticular fibrosis
spreading of granulation tissue over articular surfaces and erosion of articular cartilage may also occur
(this is called “pannus formation”)
ankylosis of the joint may be ultimate outcome
clinical signs include limping, stiff gait and enlargement of the affected joints
b) Endocarditis
in valvular endocarditis, the mitral valve is most commonly involved with development of large,
valvular, vegetative lesions due to fibrin deposition and connective tissue proliferation
emboli may produce infarcts in the spleen and kidney
clinically, signs of cardiac insufficiency or sudden death are observed
233
Pannus formation in the joint of a pig
chronically infected with E. rhusiopathiae
FIGURE: The joint of a pig with arthritis due to Erysipelothrix rhusiopathiae. Note the pannus
formation (spread of granulation tissue) starting where the joint capsule inserts and spreading into the joint.
In chronic cases of Erysipelothrix infection, arthritis or endocarditis may be

KEY POINT found.
2. Sheep
polyarthritis is the most common presentation of Erysipelothrix in sheep
entry is thought to be through the umbilicus or wounds associated with castration, docking, shearing or
dipping
affected animals have a stiff gait and, often, swollen joints
they may have trouble getting up and down
a cutaneous infection is also observed post dipping in sheep
pneumonia has been reported in ewes
3. Dogs ×
all isolates of Erysipelothrix that have been isolated from canine cases of this disease have been E.
tonsillarum (although they were originally identified as E. rhusiopathiae)
in dogs, septicaemia and endocarditis are most commonly observed, although arthritis is also reported
clinical signs in this species in include intermittent fever, shifting leg lameness, and recent onset of
heart murmur
4. Turkeys
Erysipelothrix infection in birds, especially turkeys, is usually observed as a septicaemia
the male bird is usually affected, possible through fight wounds
alternatively, females may be infected if inseminated with contaminated semen (this is though to be an
important source in females)
turkeys develop cyanotic skin, become droopy and may subsequently die!!!
a swollen cyanotic snood, if present, is considered pathognomonic
mortality rates range from 2-25%
chronic manifestations in turkeys included vegetative endocarditis and arthritis
turkeys with endocarditis appear weak and emaciated or die suddenly without prior signs
234
other avian species that have been reported to be infected include chickens, chukars, ducks, emus,
parrots, pheasants and peacocks
5. Marine mammals
due to the presence of Erysipelothrix spp in marine environments, it is not surprising that
these mammals may also be infected
septicaemia and urticaria have been reported in dolphins, porpoises, sea lions and walruses due this
species and they are often fatal
Sample Collection
specimens are collected from appropriate sites according to the clinical signs observed (e.g. joints)
blood cultures collected from several affected animals are useful in diagnosing septicaemia or
endocarditis
alternatively, necropsy samples may be used for isolation and diagnosis of infections – samples should
include liver, spleen, kidney, heart and synovial tissues
recovery of the organism from skin lesions is also possible
in the more chronic forms of the disease, cultures from joints or heart valves is often less successful
Direct Examination
gram stains of collected samples reveal uniform, relatively short gram positive rods
however, a negative result does not preclude infection with E. rhusiopathiae
FIGURE: A gram stain of E. rhusiopathiae. Note the fact that they are very short!
Very short, and lightly staining

gram positive rods
235
Culture
this organism will grow on routine media (blood agar) if incubated at 37C and 10% CO2
alternatively, media containing aminoglycosides and vancomycin may be used to selectively grow this
bacteria in contaminated samples
colonies are non-haemolytic and very small at 24 hours
Identification
this bacteria most closely resembles Listeria spp and the fact that it is non-motile and catalase negative
will help differentiate this, but is usually performed in a specialist diagnostic laboratory
immunological responses are partially responsible for lesions observed in the skin and the joint of
infected animals
persistence of antigen in the joint tissues is thought to act as a chronic stimulus for immune reaction
and development of arthritis
in addition, autoimmune process, secondary to the erysipelas infection, may be responsible for some of
the chronic joint changes
Erysipelothrix is susceptible to a wide range of antibiotics, but penicillin is usually the drug of choice
in contrast, it is resistant to aminoglycosides and sulphonamides and occasional resistance to the
macrolides has been observed
treatment with penicillin for at least 5 days is effective against the acute form of the disease in pigs
tetracyclines and tylosin are alternative, although some resistance to tetracyclines and some macrolides
has been reported
Penicillin (or Ampicillin) is the drug of choice for Erysipelothrix infection,

KEY POINT but may need to be given long term in high doses and for prolonged periods
in cases where endocarditis and/or arthritis are suspected.
the organism is very resistant to drying and is very long lived (one broth culture remained viable for
17 years)
the organisms also withstands salting, pickling and smoking and can survive for up to 6 months in pig
faeces, cadavers and fish slime in cool temperatures
however, it is killed by moist heat
good sanitation and nutrition are beneficial in preventing outbreaks in both pigs and turkeys
infected carcases should be disposed of in a proper manner and replacement animals isolated for at
least 30 days before introduction to the herd
in turkeys, penicillin is also the drug of choice which may also be given in the drinking water for
prophylaxis
vaccination is recommended for pigs and turkeys in areas with a previous history of erysipelas:
9 a live, attenuated vaccines and bacterins have been used for vaccination in pigs and turkeys
236
9 while effective against the acute forms, neither type appears to be highly protective against chronic
erysipelas
9 in addition, certain strains of Erysipelas have been refractory to vaccine-induced immunity
humans can become infected with E. rhusiopathiae, where the most commonly recognised form of
infection is “erysipeloid”, a self-limiting infection of the skin, usually involving the hand
don’t confuse this with the human disease “erysipelas” which is a streptococcal infection (because of
the pink/red skin lesions observed)
most human infections are acquired through occupational exposure, and therefore pig veterinarians,
fishermen, abattoir workers, butchers or pig hunters are most at risk
the organism usually enters via abrasions in the skin, and after 1-5 days of incubation a painful
erythematous lesion develops at this site
usually the lesions are self-limiting (by 1-3 weeks) however, more severe disease such as endocarditis,
septicaemia and arthritis have also been recorded
infected dogs do not seem to pose a public health hazard
FIGURE: The finger of a person with erysipelas. This infection could have been caught whilst handling
infected animals or alternatively whilst patting a dolphin, whale or fish!!!
237
BACILLUS
Bacillus anthracis is one of the few obligate pathogens in the bacterial world. This is particularly the case
for highly susceptible species, such as ruminants. Fortunately, humans (and companion animals) are less
susceptible to this bacteria, but high numbers of the organism or its spores will overcome this lack of
susceptibility (hence its use as a WOMD!). They are gram positive, blunt-ended, spore forming rods.
Their spores are highly resistant to environmental degradation and can live for years, even in the
extremely unfriendly Scottish coastline! The disease Anthrax has major significance, not because a lot of
animals die annually from this disease (in fact relatively few animals die every year from anthrax in
Australia), but because it CAN cause extremely severe disease in many animals. Hence, the disease is
notifiable, and strict quarantine precautions are put into place in any suspected or confirmed cases of
anthrax. Fortunately, a vaccine is available, which can help limit the losses in the case of an outbreak of
anthrax.
Summary Table: Species of Bacillus that have veterinary importance, their animal hosts, and the diseases
they cause

++++
B. anthracis cattle, sheep, horses, goats, pigs, dogs, anthrax
cats, mink, humans (other carnivores and
herbivores)
B. cereus++ cattle, humans, mastitis, abortion, food poisoning
B. subtilis+ humans, animals uveitis, conjunctivitis
B. licheniformis+ cattle, sheep sporadic abortion
B. larvae bees American foulbrood
Bacillus spp very common laboratory contaminants none!!!
238
What are they?
these bacteria are large, gram positive rods with square ends
they produce highly resistant endospores
9 there are only 2 spore-forming gram positive
rods: Bacillus and Clostridia
9 therefore this morphological feature can be used
to help identify these 2 genera
9 to differentiate between these 2 genera, growth
aerobically and anaerobically is useful, where
Clostridium spp will only grow anaerobically
whilst Bacillus spp will all grow in air
(aerobically)
9 in addition, for Bacillus spp produce catalase
(positive) whereas Clostridia do not
most species of Bacillus are aerobic (some are
facultatively anaerobic e.g. B. anthracis)
B. anthracis (the most important species in this genus) has a capsule which only forms in vivo, and
assists identification of this organism
there are about 60 different species, but only a couple cause disease in domestic species and humans
Bacillus anthracis is by far the most important species of Bacillus and will
KEY POINT be discussed in detail in these notes.
short notes on other pathogenic Bacillus spp are provided at the end of these notes
in addition, Bacillus spp are common laboratory contaminants and so care must be taken in the
interpretation of any Bacillus isolate
soil is the source of B. anthracis for herbivores

other species, including humans, are exposed via infected animals and animal products (e.g. fleeces
are a potential source especially for sheep shearers (“wool sorter’s disease”), also hides, bone etc)
B. anthracis is found worldwide as spores (the non-vegetative form of the bacteria)
spores are produced for survival in adverse environmental conditions (high temperatures and
dessication) and have been shown to survive greater than 100 years on some Scottish Islands!
once the soil is contaminated with spores, for example at autopsy, it is potentially contaminated for a
very long time (hence don’t do autopsy on suspect anthrax cases out in the paddock)
spores are brought to the surface by flooding, excavation, subsidence, or earthworms!
areas in non-endemic regions may also be contaminated with spores by floods, industrial effluent from
rendering plants, tanneries, carpet mills (from spores in the wool), or wherever carcasses are salvaged
bone meal may also be the source of infection if fed to animals
in Australia there are limited areas that are normally suitable for survival of anthrax spores within the
soil
9 this region is termed the “anthrax belt” and comprises parts of the western plains region of NSW
and the Goulburn Valley region in Victoria
9 soil in these regions are alkaline, rich in calcium and nitrate, have a high moisture content with a
pH range of 5-8
9 in these areas, anthrax has a low and decreasing prevalence and the disease usually only occurs
sporadically
239
9 however, larger outbreaks do occasionally occur, as in the Goulburn Valley in Victoria in 1997
(see later under Control and Prevention for more details)
9 occasional, and very rare, outbreaks have also been reported in SA, Tas, Qld and WA (never
reported in the NT)
The SOURCE of infection for herbivores is usually soil. Cases of anthrax

KEY POINT occur sporadically in Australia and are mostly restricted to cattle or sheep
that live in the “anthrax belt” of NSW and Victoria.
other Bacillus spp are ubiquitous and are found widely in soil, air, dust and water
this is why they are common laboratory contaminants
There are many non-pathogenic Bacillus spp and they are UBIQUITOUS in the
KEY POINT environment and are common laboratory contaminants.
FIGURE: Map of Australia

showing areas involved in
the anthrax belt.
anthrax spores are spread by birds, animals and within dust

these spores germinate and bacteria multiply when exposed to high temperatures, moist conditions and
when there is a lack of other soil-living bacteria
9 this is particularly the case in alkaline soil (pH 5-8) and after heavy rain following drought
9 these conditions also favour spore survival and so increase contamination of the environment with
anthrax spores and therefore increase the likelihood of an anthrax outbreak
B. anthracis spores are transmitted to vertebrate hosts by:
1. ingestion (oral mucous membranes)
9 ingestion of contaminated feed, particularly if animals are grazing abrasive material which cause a
break in oral mucosa, may cause infection as the spores invade this animal at these sites
9 this is the most common mode of infection for herbivores and carnivores (e.g. dogs, pigs)
9 this mode of infection is unusual in humans
2. through wounds, scratches or occasionally unbroken skin
240
9 this is the most common mode for human infections (“malignant carbuncle”)
3. mechanical transmission through biting insects is rare for all animals
4. inhalation, which predominantly occurs in human infections
The 4 modes of transmission of anthrax include ingestion, via wounds or

KEY POINT scratches, mechanical transmission with biting insects or inhalation.
outbreaks of anthrax in cattle and sheep usually begin with a few cases contracted from the soil
from these cases, excretions (or PM discharges) may seed the area and secondary cases occur
There are 2 major virulence factors of B. anthracis – the capsule and an exotoxin
only encapsulated and toxigenic strains are virulent and these are classified as “obligate pathogens”
(one of the few bacteria that are “true obligate pathogens” – that is why it is a good agent for
bioterrorism!)
Strains of B. anthracis that are encapsulated and toxigenic are OBLIGATE

KEY POINT PATHOGENS.
1. CAPSULE
B. anthracis produces a large polypeptide (poly-D-glutamic acid) capsule and is the only Bacillus spp
to do this
therefore, the presence of a capsule is frequently used to help a diagnosis of anthrax
however, B. anthracis loses its capsule when cultured on routine agar plates, so the organism cannot
be distinguished from others members of the genus Bacillus unless specific tests are performed (see
later under “identification”)
9 cultivation of the organism on specialised media and with high CO2 will cause capsule production
the gene encoding the polypeptide capsule is found on plasmid pXO2
the capsule confers resistance to phagocytosis, but antibodies to the capsule do not elicit protection
241
Activation of Oedema Factor and/or Lethal Factor
PA binds to receptor on
Cell Cell
Cell
PA protein
Receptor on Surface of Cell

Unknown protease cleaves
PA to make a smaller protein
Either EF or LF can now

bind to Pa on cell surface
Cell
Cell
Pa EF or Pa
protein LF protein
EF or LF toxin can now be

internalised into the cell
Cell
Pa
EF or protein
LF
Toxin is NOW active!
242
2. EXOTOXIN
the anthrax exotoxin is complex and consists of 3 protein components I, II and III which are encoded
on a plasmid pXO1; each of the 3 components is necessary for toxicity
loss of this plasmid (which occurs if the bacteria is cultured at 42°C) results in loss of toxigenicity
the 3 components include:
Component I is the Oedema Factor (EF) produce OEDEMA

Component II is the Protective Antigen (PA)
Component III is the Lethal Factor (LF)
Activation of Oedema Factor and/or Lethal Factor (see previous page)

the oedema factor (EF) and lethal factor (LF) both require activation for toxic activity
they are activated in a similar way (see diagram on opposite page):
9 protective antigen (PA) binds to specific receptors on the surface of cells
9 part of the PA (which is an 83kDa protein) is cleaved off by an unknown protease to produce a
63kDa protein
9 this smaller protein can now bind to either oedema factor (EF) or lethal factor (LF) and thus allow
internalisation of these 2 components of the toxin into the cell, where they are subsequently
activated
Method of Action of Toxins (Oedema Factor and Lethal Factor)
i) Oedema Factor (see next page)

oedema toxin is an adenylate cyclase which is activated by host cell calmodulin
9 the oedema toxin causes increased cAMP production, where the increased concentration of cAMP
in the cell results in active secretion of Cl- and HCO-3 and prevents absorption of Na+ and Cl-
9 therefore WATER is driven out of the cell (along an osmotic gradient) and causes oedema of the
surrounding tissues
9 neutrophils are the principal target of oedema factor, which severely inhibits their function
ii) Lethal Toxin

the exact mechanism of cellular injury is not known but results in cell death (cytotoxicity) and also
apoptosis (programmed cell death) of leucocytes
in addition, lethal toxin causes release of large amounts of IL-1 and TNF from macrophages
the generalised release of IL-1 causes increase in vascular permeability, capillary thrombosis, and
widespread damage to the reticulendothelial system resulting in death of the animal from secondary
changes including diffuse oedema, tissue damage, acute renal failure, anoxia, shock and death
Oedema toxin is an adenylate cyclase and cause efflux of water out of

cells. Lethal toxin induces a generalised release IL-1 from macarophages
KEY POINT and this results in a secondary inflammatory cascade that ultimately is the
cause of the animal’s death.
243
Method of Action of Oedema Factor in Cells
ATP cAMP
Oedema Toxin converts ATP to cAMP (ie is an adenylate cyclase)
Ç cAMP causes:
Cl-
Inside Outside
Cell HCO3 Na+ Cell
Cl-
H2O
Net Efflux of Water from
the Cell
Note: This is the same principle of action of a number of different toxins that act in the GIT to
produce diarrhoea.
For example, the heat labile enterotoxin (LT) produced by E. coli. However LT of E. coli differs to the
oedema toxin of B. anthracis in as much as it acts on cellular adenylate cyclase to cause conversion or
ATP to cAMP, rather than being an adenylate cyclase itself (like the oedema toxin)
Similarly, inflammation of the mucosa and submucosa of the intestine can cause increased prostaglandin
synthesis, which results in ↑ cAMP concentrations and electrolyte excretion.
In all cases water is drawn out of the cell along with electrolytes resulting in diarrhoea
Alterations in intracellular cAMP concentrations is COMMON to the

pathogenesis of many locally acting enterotoxins and/or inflammation of
KEY POINT the intestine. The ultimate result being excretions of electrolytes and
water with resultant DIARRHOEA.
II) Pathogenesis of Bacillus anthracis Infections

the first thing that occurs in anthrax is that the spores usually enter through breaks in the mucous
membranes of the oral cavity or the skin and germinate at the site of entry
the resulting vegetative bacteria then undergo proliferation and if the temperature and bicarbonate
concentrations are suitable (usually those found in vivo ie within the tissues, are suitable), the genes
encoding the capsule and toxins are turned on
this results in toxin production, where oedema toxin is the first toxin elaborated and gelatinous oedema
at the site of inoculation is observed
244
the inflammatory reaction at this stage are minimal
infection subsequently may disseminate to the reticuloendothelial sites (liver, spleen, blood vessels)
and when these sites become saturated, a terminal bacteraemia occurs with enormous numbers of
bacteria within the circulation
the end result of infection is dependent upon the species infected as there is a difference in the innate
susceptibility of individual species to the different toxins produced
There are species differences in susceptilibilty to ANTHRAX where cattle

KEY POINT and sheep are highly susceptible, humans, horses and goats moderately
susceptible, and pigs and dogs are relatively resistant.
i) Animals resistant to establishment of infection

animals in this group include pig, dog and cat
these animals need to be exposed to a high spore dose (large numbers) to become infected
they are most likely to acquire disease by eating carcasses/sniffing infected blood and excretions of
infected animals (which have high numbers of organisms/spores)
once infected, however, these animals are very sensitive to the toxins produced, but particularly to the
oedema toxin and the effects of this toxin are often seen first (so need to act when these signs are seen)
ii) Animals susceptible to establishment of infection

animals in this group include herbivores and humans
these animals require only a tiny spore dose to succumb to infection (e.g. may acquire 1-2 spores in a
wound from a grass seed or wounds in mouth obtained from grazing on stubble)
these animals are resistant to the effects of toxin until levels of toxin are high (particularly the lethal
toxin) and which results in sudden death
so frequently infected animals in this group are presented as sudden death or rapidly dying animals (ie.
bacteria enter multiply toxin builds up kills host)
tend to see less effect of oedema toxin in this group of animals
FIGURE: Ruminants (Cows and Sheep, but not Goats) with anthrax commonly
present as sudden death. This was one of 5 hereford steers that were found dead
on a Farmer Brown’s property located on the western plains of NSW. What would
you do if you were called to this farm?
245
FIGURE: Carnivores (Pigs and Dogs) with
anthrax commonly present with localised
oedema, especially around pharynx.
Note: Pharyngeal swelling due to oedema
What Diseases does Bacillus Cause?
1. BACILLUS ANTHRACIS - ANTHRAX

what we see clinically in cases of anthrax will vary between different species:
i) CATTLE and SHEEP

9 these are the most susceptible species
9 once spores have been ingested the incubation period is ~1 to 5 days, though once clinical signs
are first observed there is a rapid (few hours to 2 days) progression of the disease
9 these species are often found dead or rapidly dying with the peracute or acute form of the disease
(septicaemic form)
9 if clinical signs are observed they can include fever, abortion, haematuria, haemorrhagic diarrhoea
and often regional oedema
9 bleeding at the body orifices is commonly observed
9 post mortem findings include widespread haemorrhages; a black, engorged friable spleen; tarry,
non-clotting blood; and the absence of rigor mortis
9 occasionally ruminants may exhibit localised oedema or ulcerative skin lesions and recover
Sudden death of cattle or sheep is the MOST COMMON FORM of anthrax

KEY POINT observed in Australia.
2. HORSES and GOATS

9 although these are also herbivores, these species are much less sensitive to infection
9 horses usually develop colic and diarrhoea and oedema may be observed in dependent part of the
body (ventral abdomen, lower limbs) or at the site of infection (throat or intestine)
9 this localised throat swelling may sometimes cause death by asphyxiation
9 occasionally septicaemia may be observed in these species (as with ruminants)
3. CARNIVORES (Pigs, Dogs, Cats, Mink)

9 primarily show the effects of oedema toxin and the length of time to death takes longer (chronic or
subacute forms)
9 in these species a localised form is most common
246
9 this usually involved the pharynx (pharyngitis) with extensive swelling and haemorrhage of mouth
and throat observed
9 obstructive oedema may also cause asphyxiation in these species
9 ulcerative haemorrhagic enteritis sometimes occurs
9 dogs, cats and mink are less susceptible than pigs, but develop similar lesions
9 occasionally, massive exposure through ingestion of tainted (contaminated) meat may lead to
septicaemia and rapid death
Anthrax is much less common in horses, goats, pigs and carnivores and may
KEY POINT present as localised disease of the pharynx with various degrees of oedema.
FIGURE: The udder of a “Bessie” the Jersey cow with gangrenous mastitis due
to infection with B. cereus. The udder is grossly distended and dard red in
color. The teat is beginning to slough and the rest of the udder will probably
slough off (if “Bessie” doesn’t die first!!!).
FIGURE: These are 4 milk samples

from “Bessie’s” 4 quarters. The one
on the far right is from the affected
quarter. It is a typical “port wine”
colour! But I don’t recommend that
you drink it!!!
247
2. OTHER BACILLUS SPECIES
a) Bacillus cereus
B. cereus causes gangrenous mastitis in cows
this organism enters via the teat canal, frequently as a consequence of treatment for other cause of
mastitis
9 the organism can cause a “superinfection” due to it relatively widespread resistance to commonly
used antibiotics
9 alternatively, udder surgery may predispose to infection
gangrenous mastitis arises from bacterial multiplication and results in milk that is dark red in colour
(called “port wine” milk)
only one quarter may be affected with the other 3 quarters normal
however, frequently this condition is fatal due to massive tissue destruction in the affected quarter(s)
and resultant toxaemia
in addition, B. cereus can cause opportunistic infections, including abortion, in animals
B. cereus is also responsible for food poisoning in humans manifested by vomiting and diarrhoea
the toxins involved include an emetic toxin (cereulide) and 3 secretory enterotoxins
b) Bacillus subtilis
this bacteria has been implicated in conjunctivitis and uveitis in animals and humans
c) Bacillus lichenformis
this organism is widespread in the environment and is associated with food spoilage
it has also been recognised as a cause of abortion in cattle and sheep
in parts of Britain, multiple abortions have been linked to feeding of spoiled silage or mouldy hay
because this organism is ubiquitous – it is only of diagnostic significance when isolated in heavy, pure
culture from foetal abomasal contents
d) Bacillus species
there are many species of Bacillus and these are ubiquitous in the environment
in addition they are a common source of laboratory contaminants
it is important not to confuse these contaminants with pathogens
the disease anthrax primarily occurs in the anthrax belt, but any rapidly fatal disease in herbivores
should be checked for anthrax by a blood smear
anthrax is a notifiable disease, and an investigation of suspected cases is frequently performed by the
Department of Agriculture, with the assistance of the notifying veterinarian
Sample Collection
every effort should be made to diagnose this disease before the carcass is opened
B. anthracis generally doesn’t sporulate in tissues and the vegetative bacteria (which are present in the
unopened carcass) will be rapidly destroyed (within 1-2 hours) at ambient temperatures
however, if the carcass is exposed to the atmosphere (for example during an autopsy) heavy
contamination of the environment with spores can occur
alternatively, spores may get into the environment if blood oozes from the carcass or if there is
predation of the carcass
these spores are extremely resistant and will survive many years in the environment, providing a
continual source of contamination of the environment with this organism
if anthrax is suspected, a sample of blood from the ear vein (or other superficial vessels) should by
carefully collected
248
alternatively, aqueous humor may be collected and has the added advantage of remoteness from early
sources of post mortem contamination with Clostridia spp
if the carcass is opened (using the right precautions) splenic impression smears are a good source of
the organism
precautions against contamination of the environment are important and are discussed in more detail
under “Control”
Direct Examination
a presumptive diagnosis can be made by examining peripheral blood smears or smears from the spleen
stained with gram stain or a special capsular stain
9 the capsular stain (McFadyean’s new methylene blue) can be used on clinical samples (e.g. blood
smears, splenic impression smears) as they stain the capsule as a pale pink halo around the light
blue, large, square ended rods (bacilli) and which assists identification
9 in addition, B. anthracis is often present as chains of gram positive rods, which are not spore-
forming (spore production does not occur in vivo, only after the carcass is exposed to air) and
which can assist in identification
9 a diagnosis of anthrax is frequently made on the results of blood smears
but care must be taken to differentiate the large gram positive rods observed from other species of
Bacillus and from Clostridia spp
9 these are common PM invaders and can quickly invade tissues in a dead carcass
9 however, these bacteria do not have a capsule and this is used to differentiate B. anthracis
9 it is best to do a smear on fresh carcasses to avoid the presence of PM invaders and to ensure that
the capsule is still present
if you are able to diagnose anthrax on the spot, it is then possible to institute appropriate carcass
disposal (bury at least 6 feet in lime), quarantine procedures and notification of authorities and is better
than diagnosis after an autopsy, which requires a bigger clean up
FIGURE: A smear of blood taken from the ear vein of one of Farmer Brown’s cow
with suspected anthrax. You can see B. anthracis in a blood smear stained with
McFadyean’s stain. Note the light pink staining capsule around the organism.
Red Blood Cells
B. anthracis
Note: Large rods with fuzzy capsule around them

249
A presumptive diagnosis of anthrax can be made in the field (or practice
laboratory) on the basis of signalment, history, clinical signs and the
KEY POINT presence of large, blunt-ended, capsulated rods in blood smears obtained
from a peripheral vein. A post mortem examination should not be routinely
performed if anthrax is suspected.
Culture
B. anthracis grows on common media, but definitive identification requires specific tests
this would routinely be performed in a government laboratory
Identification
identification of a specific bacteriophage
alternate tests for identification (PCR, lectin binding, fluorescent antibody, or mouse inoculation) may
also assist identification but are performed in specialist laboratories
in most species, immunity is directed against the Protective Antigen (PA)

in this case antibodies attach to this protein and prevent binding to host cells, therefore the Lethal
Factor or the Oedema Factor cannot bind, be internalised and be activated
the capsular polypeptide does not stimulate protective antibody
B. anthracis is very susceptible to penicillin (can distinguish B. anthracis from other Bacillus spp by
its susceptibility to penicillin)
consequently, this is the drug of choice in animals suspected of having anthrax (including humans) if
they haven’t already died! (early administration of high doses may be effective)
other antibiotics that may be effective in therapy included fluoroquinolones (hence the huge increase in
share prices for the company that produces ciprofloxacin after September 11), tetracyclines,
streptomycin, and erythromycin
treatment should continue for at least 5 days
in some areas, antiserum is given simultaneously, but it is not available in Australia or the USA
in peracute or acute cases of anthrax, treatment is often unsuccessful
apart from Bacillus anthracis (which is sensitive to penicillin), other species of Bacillus are
unpredictably sensitive to antibiotics (see later for B. cereus)
Penicillin is the drug of choice for B. anthracis. Other Bacillus spp are NOT
KEY POINT predictably sensitive to antibiotics.
Australia has a detailed national procedure for dealing with anthrax (and other emergency diseases)
and these protocols must be followed strictly in cases of an outbreak
anthrax is a notifiable disease subject to compulsory government controls including quarantine,
disposal of carcasses, and vaccination
250
the vaccine that is utilised in Australia is based on the spores of B. anthracis (Sterne’s non-capsulated
avirulent spore vaccine) which gives moderate protection
in Australia, it is generally only given to animals at risk during outbreaks
9 for example in the 1997 outbreak of anthrax in the Goulburn Valley, Victoria, anthrax was
diagnosed on 83 farms and killed 202 head of cattle
9 in this outbreak, 600 farms were quarantined and 80,000 head of cattle were vaccinated (had to get
more vaccine in from overseas as ran out in Australia)
9 a more recent case of anthrax occurred in the same region in April, 2002 but only 1 steer was
diagnosed with disease although all 31 head of cattle on the property were subsequently vaccinated
and the property quarantined
9 in addition the carcass was burnt on the site and the ashes buried, the site where the animal died
was disinfected with 5% formaldehyde, any milk from infected animals was discarded, and cattle
on the adjoining properties were vaccinated and underwent constant surveillance
movement of animals and their waste products, feed and bedding from affected and adjacent premises
is prohibited
personnel implementing control measures should wear protective clothing and footwear which must be
disinfected before leaving the affected farm
foot baths containing sporicidal disinfectants (5% formalin or 3% peracetic acid) should be placed at
entrances to affected farms
contaminated buildings should be sealed and fumigated with formaldehyde before bedding is removed,
then once bedding is removed, the building should be sprayed with 5% formalin and left for at least 10
hours before final washing
immediate disposal of carcasses, bedding, manure, fodder and other contaminated material is
mandatory – carcasses should be incinerated or buried deeply away from water courses
contaminated material and equipment must be disinfected with 10% formalin or, if appropriate,
incinerated
scavenger animals should not be allowed access to suspected carcasses and insect activity should be
minimized by application of insecticides on and around the carcass
in contact animals should be isolated and kept under close observation for at least 2 weeks
prevention of anthrax is difficult due to the sporadic nature of the disease (therefore long term
vaccination is usually not warranted due to the relatively high cost of the vaccine) and the longevity of
the spores in the environment
anthrax is a zoonotic infection, but exposure to a common site is the most common source of infection
for humans rather than direct contact with infected animals
nevertheless, you should take care if handling animals suspected of having anthrax, particularly if you
have cuts or wounds on your hands
in general human exposures are contracted in occupations dealing with animals and animal-derived
material such as imported hides, wool and bone
3 forms of the disease have been described in humans, but only the first form has been described in
Australia:
1) cutaneous (“malignant carbuncle”)

9 this form results from percutaneous introduction of spores
9 this is a localised ulcerative inflammatory lesion that is covered by a black scab (“eschar”)
9 possible complications include subcutaneous oedema and septicaemia
9 this form is observed in the majority of “natural” cases of human anthrax (>90% of cases)
9 it can result in ~20% mortality rates if untreated
251
2) pulmonary (“woolsorter’s disease)
9 if a lot of spores are acquired by inhalation this form can result (e.g. when opening mail
containing anthrax spores!!!)
9 pulmonary oedema, haemorrhagic pneumonia and sometimes meningitis are observed
9 this form is rapidly lethal (approaching 100% mortality rate) unless treated early
3) intestinal anthrax
9 haemorrhagic enteritis can result from ingestion of the spores
9 this form is rarely observed in humans
252
CLOSTRIDIUM
There are several important diseases caused by this genus of bacteria and it is important that you can
recognise the disease syndromes.
TABLE: Clostridial Species Of Veterinary Importance
SPECIES DISEASE
C.botulinum**** Botulism
C.tetani**** Tetanus
C.perfringens ****
Type A
Malignant oedema, enterotoxaemia, enteritis + post-injection myositis in
Type B horses
Type C Lamb dysentery, haemorrhagic enteritis
Type D Struck, haemorrhagic enteritis in foals/piglets
Type E Enterotoxaemia
Haemorrhagic enteritis in calves
C.novyi****
Type A Malignant oedema, big head
Type B Black disease
C.haemolyticum* Bacillary haemoglobinuria
C.septicum**** Abomasitis =Braxy (sheep),
Malignant oedema (sheep, cattle)
C.chauvoei**** Black leg, malignant oedema
C.sordelli**** Malignant oedema
C.spirofrome** enterotoxaemia
C.difficile** Enterocolitis in foals, rabbits, guinea pigs, pseudomembranous colitis in
humans
C.colinum* Necrotic enteritis and hepatitis in game birds
C.piliforme* Acute hepatic necrosis and enterocolitis
253
• Clostridia are gram positive, endospore forming, anaerobic rods
• They are commonly found in the soil or gastrointestinal tract of animals
• They produce three major types of disease
- Histotoxic
- Enterotoxic
KEY POINTS: - Neurotoxic
• Malignant oedema, enterotoxaemia (by Clostridium perfringens), Black Leg,
Black disease, botulism and tetanus, are the major disease syndromes caused
by this genus of bacteria and knowledge of the pathogenesis of disease,
means of diagnosis, treatment and management/prevention is essential.
What are they?

These bacteria are usually large, strictly anaerobic, gram positive RODS
some are pleomorphic (boat shaped, citron form (like a lemon), filaments)
in certain stages of growth they may appear gram negative or gram variable (mottled, beaded) but all
have a gram positive cell wall
Most strains are strict anaerobes but some are aerotolerant
Catalase negative, oxidase negative, non-capsulated
Of the pathogenic species, all members are motile by peritrichous flagellae, with the exception of C.
perfringens (non motile).
Produce ENDOSPORES that may be ovoid or spherical.
Sporulation occurs in all members but the readiness with which it occurs varies from strain to strain. eg
C.perfringens only produces spores on certain media and then inconsistently. In the early stages of spore
formation, the position of the spore is often marked by an area of intense staining - but as it matures, the
spore presents a colourless centre surrounded by a peripherally staining ring.
Clostridia are gram positive, spore forming, anaerobic bacterial rods which
KEY POINT
produce disease due to production of potent exotoxins
Where do they live?

Their distribution varies with the species involved but may be found in soil, freshwater and marine
sediments.
Some appear to be common inhabitants of the intestinal tract of man and other animals. For some
species, the intestinal canal is the main habitat and their presence in soil is due to faecal contamination.
They persist in ecological niches with a suitably low oxidation-reduction potential, and can survive
adverse environments as SPORES.
What do they need to grow?

Free oxygen inhibits the growth of Clostridia, although they vary in their sensitivity to killing by
atmospheric oxygen. Prefer 2% to 10% CO2.
Most Clostridia will grow in an incubator if the oxidation-reduction potential is sufficiently low in the
medium. This can be done by adding reducing substances, some of which act mainly by absorbing
oxygen, others act by establishing a low Eh after molecular oxygen has been nearly used up or
removed by mechanical means. Cooked meat medium affords excellent conditions for anaerobic
growth.
Will grow on nutrient agar without the addition of blood or serum but growth is enhanced by
fermentable carbohydrate eg. glucose.
To stop swarming of these motile bacteria during primary isolation, agar is increased to 4%.
254
Single spores and spores within the sporangium of Clostrium tetani
How well do they survive?

As vegetative cells they are no more resistant to heat than any other vegetative organism.
In the sporing stage all members have a pronounced but variable resistance to heat. Spores of
C.botulinum, for example, withstand boiling for 3 to 4 hours. C.novyi spores are less resistant than
C.botulinum. Boiling for less than 5 minutes destroys C.perfringens spores. It is the presence of
these resistant spores that makes autoclaving an essential part of sterilization of instruments for
surgery.
All Clostridia are sensitive to PENICILLIN although their sensitivity to other antimicrobial agents is
NOT predictable. Resistance plasmids have been recognized in pathogenic Clostridia as well as soil
saprophytes.

This depends on which major disease syndrome we are discussing (see individual diseases)

Clostridia produce a large variety of different EXOTOXINS, which have a wide range of properties.
Exotoxin production may be encoded on genomic DNA, but more frequently is associated with
plasmids or phages.
There are many ways of discussing Clostridia and the diseases they cause. One way is to study the
organisms individually and to some extent one must do this as the diseases they cause may vary
widely. The other way is to look at them in groups according to the general type of disease condition
occurring. We will focus on the later.
Clostridial diseases can be divided into three broad categories:
HISTOTOXIC –
toxin in tissues (muscle, udder, liver)
ENTEROTOXIC –
toxin in blood but absorbed from intestines
NEUROTOXIC –
toxin in nerves
The major disease syndromes caused by Clostridium species are

KEY POINT botulism, tetanus, malignant oedema, black leg, black disease and
enterotoxaemia
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What diseases do Clostridia produce and what is their pathogenesis?
I) HISTOTOXIC DISEASE
Any tissue in the body may be involved in histotoxic disease but the main tissues are
MUSCLE
LIVER
UDDER
INTESTINE
Organisms involved:
C.perfringens,
C.chauvoei,
C.novyi,
C.sordelli,
C.septicum,
C.haemolyticum
These Clostridia are found exogenously from soil or endogenously from the intestinal tract.
For disease to occur these bacteria require entry to the tissues following trauma, local multiplication
and the production of toxin. This results in local and systemic tissue damage and rapid death
The mere isolation of a potentially or known pathogenic organism from a wound does NOT necessarily
mean this is the culprit. You must look to see if it is contamination or if some multiplication is
occurring. If the latter is occurring, a number of things may be happening:
1. Simple colonisation where the presence of the organism is of little consequence. There may be a little
mild inflammation only.
2. Where organisms are contributing to the disease at the site:

(a) anaerobic cellulitis - in this situation, the organisms are taking advantage of the devitalised tissues,
multiplying, producing gases and toxins which cause local inflammation.
(b) gas gangrene - more extensive and serious condition where multiplication of organisms and toxins
causes local extension and exacerbation of the lesion and where general systemic effects of toxin and
tissue necrosis result.
a) GAS GANGRENE
(aka: myonecrosis or malignant oedema)
The major type of histotoxic disease involves muscle and is termed MYONECROSIS, GAS GANGRENE
or MALIGNANT OEDEMA. Toxins and enzymes are produced during multiplication of the organisms
and damage is done to muscle and associated tissues locally. Systemic absorption of toxin leads to death.
The name “gas gangrene” is due to the production of gas by the Clostridia (mainly hydrogen and
nitrogen), leading to the classical “crepitus” (feels like “bubble wrap” when touch you the skin)
The basic requirement for tissue damage is the presence of the organisms in the tissue and the right
conditions for the germination of spores and continued multiplication of the organisms. This is generally
provided by some external contamination of the tissue (through wounds, compound fractures, etc), or
ingestion of the offending organism and some damage and stasis to the gut to allow the organisms to
proliferate extensively. The organisms then multiply, produce their toxins and enzymes. The lesion spreads
by continued growth of the bacteria in conditions created by toxin activity ie. necrosis resulting from reduced
blood supply and reduced tissue oxidation - reduction potential which follows oedema. This exacerbate the
original lesion and the the toxic products of the bacteria and the affected tissues are absorbed systemically
and death may occur - sometimes before any clinical signs are evident in the affected individual.
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C.perfringens is the major cause of myonecrosis/gas gangrene - accounting for over 70% of cases. It may be
involved alone or in combination with other species. The pathology of gas gangrene due to C.perfringens is
probably the result of alpha toxin but other proteases, eg. DNases, collagenases, may be involved.
Types of Diseases
Gas gangrene mainly C.perfringens
(aka myonecrosis or malignant oedema) (also C.chauvoei, C.novyi ype A, C.septicum, C.sordelli)
Anaerobic cellulitis various Clostridial spp.
Black leg (in cattle) mainly C.chauvoei
Black disease C.novyi type B
Big head mainly C.novyi type A
Bacillary haemoglobinuria C.haemolyticum (rare in Aust. so vaccine not available)
b) ANAEROBIC CELLULITIS
This is a less aggressive form of myonecrosis/gas gangrene. However, it may progress to gas gangrene with
time. At the time of presentation, animals with acute cellulitis feel well, while there are usually more
systemic signs with gas gangrene.
A common predisposing event is the injection of irritant substances eg. vitamins, corticosteroids, iron
supplements – esp. in pigs and horses. Therefore, it is important to disinfect skin surfaces prior to giving
intramuscular injections. It is also essential to use aseptic technique when accessing medication from multiuse
vials as contamination of these medications can provide a source of clostridia. If the area where the injection
has been given becomes sore/swollen later that day – DON’T DISMISS IT. Anaerobic cellulitis may occur
from infection with a wide range of Clostridial species.
257
Myonecrosis = Malignant oedema = Gas gangrene
Spores enter wound often secondary to trauma
If conditions are right germination of spore takes place
EXOTOXINS especially alpha, cause muscle necrosis allowing more replication of bacteria
Factors such as collagenase and gas cause further muscle damage
Exotoxins and tissue products are absorbed into circulation causing widespread damage to
endothelial and blood cells, liver and muscle
DEATH
c) BLACKLEG
The name 'blackleg' is specifically reserved for emphysematous myonecrosis due to C.chauvoei, mainly in
cattle (but sometimes sheep) and frequently in the absence of apparent penetrating wounds. Blackleg most
frequently occurs in young well-fed cattle less than 3 years of age. The hindlimb muscle mass is most
frequently affected but has also been reported in myocardium, diaphragm or tongue. Clinical signs may
include fever, anorexia, depression and lameness but sudden death without observed clinical signs is
common.
C.chauvoei may inhabit the intestines. The route of infection to the muscles is not entirely known but is
suspected to be seeded by spores from the gastointestinal tract. One theory is that spores lie dormant in the
muscles until conditions are suitable for spore germination. Damage to the muscle from blunt trauma,
overexercise or phlebotomous insects have been considered initiating triggers of local anoxia that allow the
germination of spores. Acute ingestion of large numbers of the bacteria is also considered possible inciting
cause of dissemination.
This is really just a form of myonecrosis due to proliferation of bacteria in muscle, but is considered
separately due to consistent bacteria involved; the age and species of animal affected (catlle less than 3 years)
258
and the frequent absence of obvious wounds. The monospecific nature of the infection is also used as
evidence for the dormant spore theory. However, most cases of blackleg do have other Clostridia present as
well. The advocates of the spore theory suggest they are simply post mortem invaders.
Death can occur within 24 hours of inoculating the organisms, but may take several days. There is crepitant
swelling over the affected muscle mass (usually hindquarters, shoulder, or neck). Grossly, affected muscle is
black, disrupted by gas bubbles, haemorrhagic at the periphery of the lesions and has a rancid or sweet,
sickly odour. Microscopically there is little evidence of white blood cells as they are destroyed by the potent
exotoxins. Destruction of the muscle fibres due to oedema, gas production ad haemorrahge is visible
microscopically. The a-toxin produced is necrotsing, haemorrhagic and letham, while the role of the β-toxin
(DNase), γ-toxin (hyaluronidase) and Ω-toxin (haemolyisn) are of uncertain importance.
C.septicum, C.perfringens, C.novyi, C.sordelli, C.histolyticum can be involved in similar disease.
FIGURE: Gross appearance of an animal with

blackleg at post mortem. Note the haemorrahic
and darkened appearance of the hindlimb
muscular. Photo courtesy of Peter Windsor,
from OLIVER collection
d) BIG HEAD
Infection of rams generally associated with C.novyi type A (but other species may be involved as well).
Characterized by swelling of the head, neck and/or cranial thorax, due to oedema. Results from head butting,
which allows entry of soil organisms into the subcutis of the head.
e) INFECTIOUS NECROTIC HEPATITIS (“BLACK DISEASE”)
(×Ø )
C.novyi type B is the cause of "Black Disease" in sheep. Lesions are seen in the liver of sheep (and on rare
occasions in pigs, cattle, horses, dogs, cats). It has been suggested that the spores of C.novyi are lying
dormant in the liver waiting for the appropriate damage to be inflicted, which allows them to germinate. The
most usual trigger in sheep is the wandering of liver fluke larvae. However, neoplasms and other tissue
destructive lesions may result in the multiplication of C.novyi and death (eg cats and dogs). It is much more
likely that the organisms gain entry to the liver along with the wandering parasites that bring the bacteria
with them from the intestine. In those cases where there has been an association with neoplasms, there has
been an intimate connection from the liver to the intestine - often via the bile duct.
The name “Black disease” arises from the darkened undersurface of the skin of affected animals (especially
seen after drying of the hide), due to engorgement of subcutaneous blood vessels.
In C.novyi disease, the alpha toxin (which is a phospholipase c) is the major cause of the lesions seen
(haemolysis, necrosis, and death). Prevention of the disease in sheep involves control of liver fluke (ie. the
snail intermediate hosts) as well as using C.novyi type B vaccine (usually as part of a multicomponent
vaccine). Treatment of affected sheep is not feasible. Valuable cattle, if diagnosed early, might be treated
with high doses of penicillin +/- antitoxin.
259
f) BACILLARY HAEMOGLOBINURIA (“Red Water”)
Bacillary haemoglobinuria is caused by infection with C.haemolyticum (previously known as C.novyi type
D). This bacterium probably has world-wide distribution, but is not as abundant as other Clostridia within the
intestine or soil. This disease is reported mainly in USA but is rare in Australia (so vaccine is not available
here). Most frequently seen in cattle, less commonly in sheep.
Bacillary haemoglobinuria is most common in well nourished cattle and other ruminants, greater than one
year of age. The mode of transmission is ingestion. Organisms are thought to reach the liver
haematogenously. If there is damage to the liver (eg. liver fluke infestation), the organisms multiply in the
anaerobic environment.
Toxin produced is a beta toxin (phospholipase C) – it is lethal, necrotising and also a potent haemolysin.
Affected animals have fever, abdominal pain and dark red urine (haemoglobinuria). Subcutaneous oedema
sometimes present. Usually die of anoxia (caused by the acute haemolytic anaemia) in 1 to 4 days. Again,
control of liver fluke important. Bacterins are available overseas, but immunity is usually of short duration.
Pathogenesis of Black Disease
Organism are ingested and spores lodge in the liver
Migrating liver fluke larvae cause local necrosis
Lower redox potential allows germination and multiplication
Toxins, especially alpha, enter circulation
Increased capillary permeability, damage to muscles including heart.

Congestion of subcutaneous vessels
Sudden Death
Diagnosis Of Histotoxic Clostridial Disease

In cases of sudden death, specimens should be collected as soon as possible, as Clostidial organisms from the
gut are common post-mortem invaders. Diagnosis depends mainly on:
1. Clinical features and pathology - gas, necrosis, haemorrhage, toxaemia and death.
2. Visualisation of organisms in tissues - Impression smears should be made of necrotic muscle, liver,
or other affected tissue. Diff Quik (Giemsa) stains will show large, thick rods that may or may not
have spores. Gram staining will show these as being gram positive, gram variable or gram negative.
260
On careful inspection, a range of other organisms may also be seen.
3. Fluorescent antibody (FAb) tests performed on impression smears for C.chauvoei and C.septicum
(which share spore antigens) used to be available through EMAI but are no longer available.
Culture is rarely performed for diagnostic purposes in cattle and sheep. In horses and small animals where
the condition may be seen early, culture may be attempted as confirmation of the gram stain and often as part
of a negligence claim.
Treatment And Control

If an animal is diagnosed with histotoxic clostridial disease in time (ie. before it dies!), urgent and aggressive
debridement of the wound should be implemented. It is necessary to remove all the necrotic tissue in which
the organisms are multiplying, and to re-establish blood supply to the affected area. In human patients,
placing them in a HYPERBARIC OXYGEN chamber (higher than atmospheric pressure) has been found
useful and may help to avoid amputation of a limb and decreases mortality. This is only available through
human hospitals usually. In small animals, the use of intranasal oxygen may assist recovery by increasing
the inhaled oxygen from 16% to 40%.
Treatment has to be given early to animals and should include PENICILLIN and ANTITOXIN (at least in
humans). If the condition is left even a few hours without appropriate therapy, the animal will die before
the diagnosis is confirmed. In our animals, the antitoxin is usually not administered as huge doses are
required and there may not be sufficient time to prevent the effects of the already elaborated toxins.
Therefore the hallmarks of treatment are aggressive and urgent debridement, oxygen therapy, antibiotics
(penicillin) +/- antitoxin (if available).
As many of these affected animals may simply be found dead, the only effective management of this type
of disease is preventive vaccination programs. Bacterins have been produced for some of these conditions
and organisms. eg C.chauvoei, C.novyi, C.septicum. These are routinely given to production animals as
a combined clostridial vaccine (5 in 1 or 6 in 1) administered early in life. These vaccines are both
antibacterial (prevent multiplication of the organism) and anti-toxic (stop the effects of the toxin).
Naughty vet is
not wearing
gloves or
protective
This ram had been on supplemental

feed during the previous week. He
dropped dead while entering the yards
and a post-mortem was done
immediately. Note the distended,
261darkened loops of intestines.
II) CLOSTRIDIA causing ENTEROTOXAEMIA or ENTEROPATHY
The Clostridia most frequently responsible for enteric disease or enterotoxaemia are C.perfringens (Types
A to E), C. difficile, C.spiroforme, and C.colinum. Of these, C.perfringens types B, C and D are the
most important. C.perfringens is widely distributed in the environment (soil and faeces) and the intestinal
tracts of humans and animals. Therefore infection can occur exogenously or endogenously.
Types of Disease
Enterotoxaemia of sheep, goats (Pulpy Kidney) C.perfringens type D
“Enterotoxaemia” of calves and lambs C.perfringens type E
Dysentery/enteritis (any animal species) mainly C.perfringens types A, B, C and E;

C.difficile; C.colinum
“Struck” C.perfringens type C
Enteropahy or Enterotoxaemia of rabbits C.spiroforme and sometimes C. perfringens

type E
Abomasitis (Braxy or Bradsot) C.septicum
DISEAESES ASSOCIATED WITH Clostridium perfringens

ENTEROTOXAEMIA of SHEEP and GOATS
(“PULPY KIDNEY”)
This is a special category of disease that manifests as a SYSTEMIC (TOXAEMIC) DISEASE - even though
the multiplication of the organisms occurs locally in the intestine it seems to produce very little damage at
that site.
It is a disease of sheep and goats found worldwide. It is particularly a problem with feedlot lambs fed rich
rations of grains (“overeating disease”, feedlot animals) or lambs (3-10 weeks old) suckling heavily lactating
ewes grazed on lush pastures. The major toxin elaborated by C.perfringens Type D, which is the most
common cause of pulpy kidney in Australia, is the epsilon toxin.
Pathogenesis
The disease is initiated by conditions such as overeating or continued feeding on rich diets which cause gut
stasis and therefore multiplication of C.perfringens in the small intestine rather than in the normal location of
the large intestine. It is a “true toxaemia” with little evidence of enteritis, despite multiplication of the
organism at this site.
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Pathogenesis of enterotoxaemia of sheep and goats
Change in food conditions eg rich diet, increases amount of undigested starch in intestines and
reduces peristalsis
Rapid proliferation of C.perfringens Type D, normally present in the intestines in small numbers
Epsilon prototoxin released and activated by trypsin and chymotrypsin
Large amount of active epsilon toxin accumulates and increases intestinal permeability. Epsilon toxin
is absorbed into circulation
Epsilon toxin increases capillary permeability in brain causing oedema, degeneration and necrosis of
neural tissue. Also causes damage to capillaries in Loop of Henle and to the renal tubules resulting
in glycosuria
Lesions in brain stimulate catecholamine release from adrenal medulla, causing glycogenolysis and
hyperglycaemia
Death with convulsions and hypovolaemia, oedema of lungs, pericardium and other cavities
C.perfringens type D produces a protoxin of the epsilon toxin, which, in the presence of the correct
amounts of trypsin or pepsin is converted to an active toxin. This protoxin of course, is also normally
produced in the large intestine but there is not sufficient trypsin there to make it active. In the small
intestine there are specific receptors on the enterocytes that facilitate absorption of the toxin into the
circulation.
Once in the circulation, the toxin causes necrosis of cells and damage to vascular endothelial cells,
resulting in the local liquefactive necrosis of brain tissue, necrosis of renal cortex (“pulpy kidney – this is
the result of rapid post-mortem autolysis in th toxin damaged tissue), and perivascular oedema in meninges
and brain. Oedema may occur in cardiac muscle and the lungs. Effusions are often found in the
pericardial sac and peritoneal cavity. The effects of the epsilon toxin on the central nervous system and
other tissues cause sudden death, preceded occasionally by convulsions and other neurological signs.
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C.perfringens is a rare cause of disease in adult cattle, deer, camels and horses. It can be found in suckling
calves and results in similar signs to that seen in sheep. In goats however, catarrhal, fibrinous or
haemorrhagic enterocolitis are seen with C.perfringens Type D.
Diagnosis Of Pulpy Kidney

1. Visualisation of large numbers of the organisms in the upper small intestine may be a useful
indicator. Collect samples quickly after death, otherwise organisms from the large intestines will enter the
small intestines and multiply.
2. Identification of the epsilon toxin in the small intestinal contents (last 1 to 1.5 m of ileum) or serum
of affected individuals is required (counterimmunoelectrophoresis or ELISA tests used).
Treatment and Prevention

There is no specific treatment, as animals are usually found dead.
Routine vaccination with a toxoid (active against the effects of the epsilon toxin) should be carried out to
ensure that the flock is protected. Immunity is usually of short duration. Vaccinate ewes before lambing to
confer some maternal Ab to lambs. Then vaccinate lambs at 1 to 2 months of age and again 4 weeks later.
As part of the management strategy, it should be kept in mind that susceptible animals should be managed to
ensure that stasis of the gut is kept to a minimum (ie move to poorer pasture; provide roughage etc).
Occasionally a chronic neurological manifestation of enterotoxaemia is seen: focal symmetrical
encephalomalacia (FSE), which is characterised by haphazard roaming, blindness, head pressing and inability
to eat. Low levels of immunity due to inadequate vaccination are thought to contribute to the pathogenesis of
this disease. Toxin is not detectable in the circulation by the time the disease is apparent.
TOXINS PRODUCED BY C. perfringens

Type A α where α alpha
Type B α, β, ε β beta
Type C α, β ε epsilon
Type D α, ε ι iota
Type E α, ι
Types A-E Enterotoxin
Over 15 exotoxins produced by C.perfringens have been described but a definitive role in pathogenesis has
been demonstrated for only a few.
The α toxin is produced in varying amounts by almost all types of C.perfringens. It is a phospholipase
(lecithinase) and is capable of hydrolysing membrane phospholipids in RBCs, WBCs, platelets, endothelial
cells and muscle cells. It is haemolytic, necrotising and lethal.
The β toxin is a protein responsible for necrosis of intestinal mucosa and possibly CNS signs. Produced by
some strains of type B and C. Mode of action unknown.
The precise biological activity of the ε (epsilon) toxin has not been determined, but it is necrotising +
highly lethal. Increases intestinal permeability + is toxic to the CNS. Produced by strains of type B + D
as a protoxin.
The ι (iota) toxin is necrotising and lethal. Produced only by type E. Mode of action unknown. Causes
necrosis of intestinal mucosa.
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DYSENTERY / ENTERITIS
Severe diarrhoea and dysentery may be associated with C.perfringens types A, B, C and E. It is more
common in young animals (calves, lambs, foals, piglets) although it is also reported in adults.
Clostridium perfringens Type A

Type A is associated with food poisoning in humans – (3rd most common cause of food poisoning in the USA
after Salmonella and Staphylococcus aureus), but usually not serious. Generally associated with improperly
cooked meat products. Abdominal pain and diarrhoea generally occur 7 to 15 hrs after eating suspect food.
Enteric disease associated with Clostridium perfringens Type A is reported in ruminants, poultry, dogs,
neonatal foals, and neonatal pigs.
In ruminants affected animals are depressed, anaemic, icetric and haemoglobinuric. Death may occur after
only 6 to 12 hours with large numbers of bacteria found in the small intestines. It is more common in beef
and dairy cattle specially those raised for veal. Contamination of colostrum,or ingestion of large volumes of
icy cold colostrum are known predisposing causes. Gross lesions including haemorrhagic gastroenteritis with
dilated, thickened, emphysematous walls may be present. Microscopically, the intestines are necrotic and
haemorrhagic with large number of rod shaped bacteria dn gas bubbles in the mucosa/submucosa.
Necrotic enteritis by type A or C is seen in domestic poultry as well as captive an dfreeliving wild birds.
Contaminated soil, dust, litter or feed are the likely source of infection. Clinical signs can vary from poor
feed efficiency to diarrhoea to sudden death.
In neonatal foals, haemorrhagic diarrhea results from C.perfringens type A, leading to diffuse necrsis of the
villous mucosa, hyperaemia and haemorrhage of the lamina propria, submucosa and subserosa. A similar
disease is seen in neonatal pigs.
In dogs, watery to mucoid diarhhoea with blood is frequently seen and can lead to peracure death with
haemorrhagic mucosal necrosis.
Clostridium perfringens Type B

Haemorrhagic enteritis in lambs, goats, calves and foals.
Clostridium perfringens Type C

Disease is typical seen in newborn animals due to the absence of a substantial competing normal flora.
Alterations in flora seen in sudden dietary changes in another cause.
A disease in adult sheep called “Struck” occurs in cold environments (mainly UK) – results when they pick
at feed through the snow. The cold material causes gut stasis, leading to C.perfringens type C overgrowth in
the abomasums and small intestines, leading to mucosal necrosis and toxaemia often without dysentery or
diarrhoea. Sheep die so suddenly it is as though they have been struck by lightening. Vaccines have been
developed but as incidence is sporadic, control does not usually involve use of a vaccine.
Clostridium perfringens Type E

The so called “enterotoxaemia” caused by C.perfringens Type E is more probably a HISTOTOXIC disease
as the alpha and iota toxins produced cause necrosis of intestinal mucosa which results in dysentery. This
disease is reported in the USA, UK and Australia.
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DISEAESES ASSOCIATED WITH OTHER CLOSTRIDIA
C.spiroforme
This organism causes enterotoxaemia of rabbits and laboratory rodents. This bacteria is not part of the rabbits
normal flora but is acquired from the environment. Poor hygiene, stress and diet influence the likelihood fo
disease. Antibiotic therapy or the weaning process upsets caecal microflora and allows the organism to
proliferate. Clinical signs include a hugely dilated fluid filled caecum. Large numbers of the spores are seen
in the caecal contents. Unlikely most other Clostridia, C.spiroforme have a loosely coiled, spiral morphology
of end-toend aggreagations of semicircular cells.
C.difficile b
C.difficile has been isolated from marine sediment, soil, sand, hospital environments, faeces of non-
diarrhoeic humans, camels, donkeys, dogs and cats (up to 39% prevalence), domestic birds, cattle.
Pseudomembranous colitis in humans, pigs and foals. Produces two toxins – toxin A (an enterotoxin –
causing fluid accumulation in the bowel) and toxin B (a lethal toxin). Associated with use of antibiotics (most
frequently ampicillin, clindamycin and cephalosporins) or some anti-cancer drugs. Hospitalised patients
frequently become colonised with this organism (up to 46% of adults treated with antimicrobials). Symptoms
range from mild diarrhoea to toxic megacolon and bowel perforation.
Hamsters and guinea pigs treated with antibiotics can develop a fatal enteric disease. It is known to cause
enterocolitis of various severity in horses, dogs (frequently pups), rabbits and neonatal piglets (1-7days)
C. colinum
Ulcerative enteritis in birds (quail disease)
C.septicum
Abomatitis (aka Braxy or Bradsot) is a disease of sheep, mainly in UK and Europe. Usually associated with.
Arises after eating frozen succulent feed which damages the abomasal and duodenal walls and allows
invasion of the ingested clostridial organisms. This results in necrosis and haemorrhagic oedema of the
abomasal and duodenal walls.
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NEUROTOXIC DISEASE
Neurotoxic clostridia cause disease by the production of potent exotoxins that act on the nervous system.
Two very important diseases are included in this group: botulism and tetanus.
BOTULISM
×b Õ
Botulism is a neurological disease caused by Clostridium botulinum. Other clostridia (eg. C.barati and
C.butyricum) can also produce the disease botulism if they acquire the genes via transfer of either phages or
plasmids.
C.botulinum is most commonly found as spores, which are widely but unevenly distributed in the soil and
aquatic sediments worldwide. Under appropriate environmental and physical conditions (12oC to 35oC, low
oxygen tension, high quantities of organic matter), the spores germinate and the vegetative bacteria produce a
toxin that is released upon cell lysis. When animals die, the spores of C.botulinum which are commonly
found in the gastrointestinal tract, germinate and produce toxin. Carrion eaters may ingest the toxin directly
from the source or the toxin may persist in the environment.
Botulinum toxins
Eight immunologically distinct types of botulinum exotoxin have been identified (A, B, C1, C2, D, E, F and
G). Seven of these are pharmacologically similar neurotoxins. One of them (C2) is not a neurotoxin but
causes increased vascular permeability. The practical implication is that protection against intoxication by
one C.botulinum type does NOT confer protection against the others.
Not all types produce intoxication in all animal species, some species (eg cattle) need only protection against
types B, C, & D while A is most frequently implicated in disease of man (60% of cases in the USA). The
reason for host specificity is not known. Note the absence of cats in the icons above. As the superior
species on the universe, cats are very resistant to the effects of botulinum toxin unless injected
subcutaneously (in horrible 1950s experiments). Botulism has however been reported in domestic cats but is
extremely rare (only 2 reports in the worldwide literature – one with type E (Haugaard et al 1974) the other
with type C (Elad et al 2004). It has been reported in lions but jaguars eating the same food were unaffected.
Pigs are also rarely affected.
One strain of C.botulinum usually produces one type of toxin, although strains that produce multiple toxins
have also been identified. All the botulinum toxins are released from C.botulinum as INACTIVE
PROTOXINS. Some strains are activated to full toxicity by their own proteolytic enzymes eg A, B, F. Some
are only partially activated by the bacteria’s own proteolytic enzymes – and then require exogenous proteases
(trypsin or chymotrypsin) for full toxicity. Some strains that do not produce proteolytic enzymes themselves
require totally exogenous proteases for full activity of the toxin.
Toxins vary in their resistance to heat that is important for some types of intoxication.
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Pathogenesis of Botulism
Toxin is absorbed from its site of entry to the body

(usually the gastrointestinal tract, but possibly also wounds)
Transported to neurones via the blood stream.
Circulating toxin reaches susceptible nerve endings but can only bind at the neuromuscular junctions
or nerve-nerve junctions (cholinergic junctions of peripheral nerves)
The toxin acts presynaptically and blocks acetylcholine release.
Flaccid paralysis, respiratory depression, possible vascular thrombus formation
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Botulinum toxin types and the species of animal they affect
DO NOT MEMORISE THIS LIST
TOXIN TYPE SPECIES OF ANIMAL

A humans, chickens, goats, mink
B humans, horses, cattle, goats, mink
C1 wild water fowl, chickens, cattle, horses, mink
C2 cattle, horses, sheep
D cattle, horses, dogs, sheep
E humans, gulls
F humans
G humans
Pathogenesis of botulism
Botulinum toxin is one of the most powerful biological toxins known. 1mg contains 120,000,000 mouse
lethal doses and less than 1μg is a lethal dose for humans. Toxin is absorbed from its site of entry to the
body (usually the gastrointestinal tract) but it may enter via wounds (see later). Once absorbed, it is
transported to neurones via the blood stream. Circulating toxin reaches susceptible nerve endings but can
only bind at the neuromuscular junctions or nerve-nerve junctions (cholinergic junctions of peripheral nerves)
by use of specific gangliosides which differ among nerve fibres and toxin types ie. the receptors may not be
the same for all toxin types and the ganglioside receptors vary amongst different nerve endings.
The toxin binds to receptors, enters the nerve cell and acts presynaptically to block acetylcholine release.
This manifests as flaccid paralysis.
The botulinum paralysis develops in three steps:
1. Fixing or binding of toxin (which occurs rapidly and irreversibly) to ganglioside receptors on the
pre-synaptic nerve terminal. Binding occurs externally since the presence of antitoxin can prevent it.
2. Once toxin has bound, there is a rapid internalisation process on the pre-synaptic nerve terminal,
involving receptor-mediated endocytosis of the toxin.
3. The final blocking step prevents the release of acetylcholine (Ach) from vesicles within the motor
end plates. This neuromuscular blockade results in FLACCID PARALYSIS. The mechanism by
which Ach release is inhibited probably varies between the toxin types, but the end result is the
same.
As well as the neurotoxin, C.botulinum possesses a haemagglutinating factor that has been associated with
vascular thrombus formation. Respiratory failure is the usual cause of death.
The toxin that causes botulism may be acquired in three main ways:
1. FORAGE POISONING (FOOD POISONING) – This is the most common form of intoxication in
most domestic species eg. horses, cattle, sheep, dogs. The toxin is preformed in food and is subsequently
ingested. This usually involves high protein foods (which are rich in proteases) or foods contaminated by
rotting cadavers (dead rats or cats), vegetation or other foodstuffs. The rotting provides the nutrients and the
enzymes, if required, to enable full activation of the toxin before it is ingested. Animals can acquire this
form of botulism from improperly stored silage, silage or hay contaminated by the bodies of small rodents
which are a good source of protein and enzymes, or from carcasses etc. The latter is a problem in
269
phosphorus deficient areas, as animals (esp. cattle) will frequently eat bones of dead animals (pica) in an
attempt to alleviate the deficiency. On occasions, reports of botulism in animals have come from animals
standing in floodwaters and drinking water containing dead animals (and of course toxin).
Human intoxications usually come from specific foodstuffs that have not been processed correctly (eg canned
fish or meat). This is especially true for home preserved products which were not high in acid or sugar eg
carrots, meats. C.botulinum spores are highly heat resistant and may survive the “sterilising” process (eg.
can survive boiling for more than an hour). The anaerobic environment produced by canning may further
encourage proliferation of organisms. Most of the outbreaks seen in humans are due to type A, with smaller
contributions from B and E.
Vast losses of wild fowl (ducks, pelicans, etc) sometimes occur as a result of toxin produced in masses of
decomposing algae and waterweeds. Wild carrion eaters, pigs and some carnivores (dogs, cats) appear to be
relatively resistant.
2. WOUND BOTULISM – Rare form of intoxication. C.botulinum multiplies in the contaminated

wound under anaerobic conditions, toxin is elaborated + is absorbed into the bloodstream + circulated to
other parts of the body including nervous tissues. Types A (humans) + B (horses) have been associated with
wound botulism. In horses, it has been reported as a consequence of injections, castration + umbilical
infections. In humans, has been associated with intravenous drug use.
3. TOXICOINFECTIOUS BOTULISM - (infant botulism; shaker foal syndrome)

This condition has been reported in children between 3 and 26 weeks of age and in foals less than 8 months
old - usually 3 - 4 weeks. The disease is contracted by ingestion of spores from food such as honey. The
organism colonises and produces protoxin in the intestinal tract. The toxin is activated by intestinal proteases
and is subsequently absorbed into the systemic circulation. Infants and very young animals are generally
susceptible to colonisation of the intestine by C.botulinum because the intestine does not contain the intestinal
microflora to prevent the growth of C.botulinum. Spectrum of clinical disease ranges from mild illness to
sudden death. The possible role of infant botulism in SIDS has been suggested and is under investigation.
A similar condition has recently been recognised in adult humans in the US, especially associated with
abdominal surgery and antibiotic therapy, but is very rare.
In horses, usually attributed to type B toxin. In human infants, associated with types A, B, C, E and G.
Clinical signs
In general it is seen as an ascending flaccid paralysis. Tendon reflexes in companion animals are
diminished or absent, but response to painful stimuli may persist. Ultimately death is caused by respiratory
paralysis and failure. Signs of decreased cholinergic function may also be observed (eg. constipation,
urinary retention, decreased salivation and lacrimation, dilated pupils).
In chickens and wild birds, the disease is known as “limber neck”. Birds will be seen with their heads or
necks dragging on the ground.
In horses, clinical signs may vary with the type of toxin ingested. Signs are predominantly related to
progressive muscular paralysis (general weakness, dysphagia, decreased tongue tone and tongue may hang
out the side of the mouth, altered gait, slowness in eating, colic or inability to rise).
Type D botulism occurs in cattle with pica in South Africa, Texas, South America. Cattle are recumbent
and drooling, with laboured breathing.
In dogs, botulism is caused by type C toxin. Dogs develop progressive, symmetric, ascending weakness
from hindlges to forelegs that can result in quadrplegia and recumbency with loss of vocalisation.
270
Perception of pain is maintained but the ability to move in response to a painful stimuli is lost. The
severity of clinical signs varies with the amount of toxin ingested and individual susceptibility.
Diagnosis
A tentative diagnosis can usually be made based on history and clinical signs. The possibility of botulism in
animals or birds should be entertained whenever a number of animals or birds show paretic signs especially
if they are being fed wet mash or swills containing high concentrations of proteins and carbohydrates. Make
sure you keep a sample of the food source for identification of the toxin.
There are no significant findings on blood chemistry profiles, radiographs or cerebrospinal fluid analysis
unless there are secondary complications. Electromyography (EMG) findings in dogs are variable. At
autopsy, no abnormalities are detected either grossly or histologically.
Confirmation of the diagnosis of botulism is based on finding toxin in the serum, faeces, vomitus or
samples of suspected food. Samples need to be collected as early as possible. Large volumes of samples
are needed to detect the toxin eg 10ml serum, 50g faeces, vomitus, or food. Detection of C.botulinum
spores from the GUT of affected animals is also considered for diagnosis but is inferior to detection of
toxin.
The previously popular mouse bioassay whereby samples were injected into mice to see if they died of
botulism have understandably become out of favour due to the obviously animal ethics issues. This is
being replaced in most places with ELISA or PCR.
Treatment and Prophylaxis

Treatment is not usually attempted in large animals unless signs are mild. In humans or small animals,
treatment includes:
a) Antitoxin (if available) – must be given early in disease and must be of the correct Type eg most
cases of C.botulinum in dogs are type C therefore the equine product with antibodies to types A,B
and E is of no use.
b) Supportive therapy - fluids, rest, nutritional support, artificial ventilation.
c) Antibiotics – while metronidazle or penicillin based antibiotics are given in an attempt to reduce any
intestinal population of C.botulinim, this is done with the understanding that most cases of botulism
are the result of ingestion of preformed toxin.
The neuromuscular blockade associated with botulinum toxin is irreversible, and new neuromuscular
junctions need to be created for recovery of muscle function (which may take weeks to months). During this
time the animal needs intensive supportive care to ensure they don’t get pressure sores, aspiration
pneumonia, anoxia due to decreased ability to inhale (weak muscle) etc
Active immunity to C.botulinum is directed against the toxin (toxoid) and is specific for the antigenic types
of toxin. There is a vaccine against C.botulinum types C and D available for use in cattle in Australia.
Botulism may be avoided by ensuring good nutrition (so that stock are not attracted to bones or carrion);
not feeding spoilt silage or hay; preventing access to drinking water fouled with dead animals; preventing
dogs from getting access to carcasses (by burning).
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TETANUS
Tetanus is an afebrile (ie no fever) neurological disease caused by the toxins of C.tetani.
Spores of C.tetani are found worldwide in the soil (especially heavily manured soils) and in the intestine of
man and other animals, particularly horses (so the story goes). The vegetative organism produces terminal
spores within a swollen sporangium, giving the distinctive “drumstick” appearance.
Antigenic structure
There are at least 10 antigenic types of organisms. However, the toxins formed are all pharmacologically
identical and are neutralised by antitoxin prepared against toxin from any one antigenic type ie although
strains of organism may produce toxin of variable toxicity, the toxin from all is the same.
Pathogenesis of tetanus
Spores of tetanus are found in the soil where they may reside for a long time. The spores are introduced into
the animal via some wound that is usually described as a deep penetrating wound with minimal trauma. The
wound is considered to need to be deep to enable sufficient anaerobic conditions to allow sporulation to
occur. However, any wound that compromises the blood supply to an area would allow sporulation of
tetanus spores. The feet of horses are considered less well supplied with blood and are thus favoured places
for instilled tetanus spores to germinate. Has been associated with teething pups raised in filthy conditions. It
is considered that minimal multiplication of tetanus organisms is required to produce large amounts of tetanus
toxin. The delay between entry of spores and the appearance of clinical signs can be explained by the
gradual development of conditions suitable for spore germination and movement of toxin from the wound to
CNS.
C.tetani produces two toxic substances:
a) a haemolysin (tetanolysin or cytotoxin)
b) a potent lethal toxin (tetanospasmin or neurotoxin)
272
The elaborated tetanus protoxin is converted to its active toxin by tissue proteases. Tetanus neurotoxin is
synthesised as a single chain polypeptide and nicked by proteases at a peptide bond into two dissimilar
polypeptide chains with M Wt. 100 kDa and 50 kDa, held together by a disulphide bridge - the 'so-called'
extracellular toxin. Tetanus toxin has a high affinity for di and tri- sialogangliosides in cells from all parts
of the nervous system but preferentially the spinal cord. Tetanus toxin acts centrally rather than locally. It
accesses the CNS by transportation within the axons of motor and sensory nerve fibres (intra-axonal
retrograde transport).
Motor neurones normally receive excitatory stimulation eg from sensory neurones or excitatory interneurones
at the same time as inhibitory impulses from inhibitory interneurones. The nature of the excitatory
transmitter is not known but the inhibitory transmitters are glycine and GABA. Motor neurone stimulation
depends on the outcome of these opposing excitatory and inhibitory influences.
The tetanus neurotoxin acts specifically on the inhibitory synapses, preventing the release of glycine and
GABA from the inhibitory interneurones in the brain and spinal cord. The lack of inhibition leads to over-
excitation of motor neurones that manifests as increased muscle tone, rigidity and spasm (SPASTIC
PARALYSIS).
The binding of tetanus toxin to presynaptic sites of inhibitory neurons is irreversible. Recovery depends on
production of new axon terminals.
Tetanus toxin can also affect the autonomic nervous system.
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Pathogenesis of Tetanus
Spores are introduced into the animal via deep penetrating wound with minimal trauma.
Germination of the spore occurs, leading to production of small numbers of Clostridium tetani vegetative
bacteria which produce LARGE volumes of tetanus toxin at the deep wound
Tetanus toxin is transported within the axons of the peripheral motor and sensory fibres (intra-axonal
retrograde transport) to the CENTRAL NERVOUS SYSTEM
Tetanus toxin has a high affinity for di and tri- sialogangliosides in cells from all parts of the nervous system
but preferentially the spinal cord. Tetanus toxin acts centrally rather than locally.
The tetanus neurotoxin acts specifically on the inhibitory synapses, preventing the release of glycine and
GABA from the inhibitory interneurones in the brain and spinal cord. The lack of inhibition leads to over-
excitation of motor neurones that manifests as increased muscle tone, rigidity and spasm (SPASTIC
PARALYSIS).
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Immunity to Tetanus
Natural immunity
Mammals vary in their susceptibility to tetanus. The resistance is related to the inability of the toxin to
penetrate and bind nervous tissue, because direct CNS injections of toxin produce equivalent signs in
different species.
1. Mice, guinea pigs, rabbits, monkeys, man and horses are highly susceptible and will succumb to low
doses of toxin. Horses are the most susceptible.
2. Dogs, cats, cattle, pigs, sheep, goats are much less susceptible.
3. Most birds and cold-blooded animals are extremely resistant.
Acquired immunity
1. Passive - antitoxin administered to neutralize the toxin. This can be used to give temporary
protection from challenge. Used in man and also horses if the vaccination history is questionable and the
horse has received a wound. Only exceptionally high doses of antitoxin may be of any consequence to
neutralise unbound toxin in an animal that is already clinically affected.
2. Active - Immunisation with formolised toxin (toxoid) in an adjuvant. This is essential for horses
among our domestic animals. Sheep and cattle are also immunised routinely.
Because the most likely wound to lead to tetanus is the one that is never noticed or predicted, it is important
that susceptible animals have current vaccination status. (NB: only about one third of the horse population in
Australia is regularly vaccinated)
Diagnosis
Usually made on clinical grounds alone. Isolation of organisms is usually extremely difficult or impossible
even if the location of the wound is known (little multiplication). May follow castration, shearing, docking,
mulesing, injections etc. Docking by use of elastic band ligatures is especially hazardous.
Clinical signs
Incubation period usually 3–10 days but could be as long as several months. Usually the classic spastic
paralysis is seen. May be described as ascending or descending – descending paralysis is more commonly
seen in humans and horses. Consequently, the first clinical signs observed may be protrusion of the
nictitating membrane across the eye, followed by spastic paralysis of the fore and hind limb muscles. There
is an unsteady, straddling gait and the head and tail are held stiffly extended. Later, animals will fall over,
still in a state of tetany, and be unable to get up again. Affected animals are hyperaesthetic and a sudden
noise or touch induces spasm. Tetany of the masseter muscles is known as “lockjaw”. Dogs frequently get a
typical “worried” expression due to pricking of the ears, and wrinkling of the skin on the forehead.
In all species, advanced disease is characterised by convulsive contractions of voluntary muscles, with
resultant extensor rigidity and a “saw horse” stance. When death occurs, it is due to spasms of muscles
involved in respiration.
May occasionally present as localized disease. This form is sometimes seen in dogs, or more rarely cats. In
these cases, the effects of the toxin are localized to the synapses that are closely supplied by the lesion. This
can develop due to the partial resistance to the effect of the toxin observed in these species.
There is a high incidence of tetanus in pigs following castration, lambs following castration, shearing,
docking, vaccinating, and injections, cattle: genital tract post-partuition and post-castration and neonatal
tetanus via umbilical cord causing neonatal death in all species including humans
275
Treatment
If a wound that may be contaminated with tetanus spores is noticed soon after it has occurred, tetanus can be
prevented by ensuring that an anaerobic environment does not develop. Debridement of all necrotic tissue,
exposure to oxygen and use of chemoprophylaxis is important. In practice, the use of penicillin (both locally
within the wound, and systemically) and injection of antitoxin should be practiced in any animal where there
is no history of current vaccination - especially amongst the most susceptible species eg horses. Antitoxin
cannot neutralise the toxin already fixed to the nervous tissue but is able to neutralize toxin that is still in the
muscle tissue. Be mindful that medications may not get to intended sites when blood supply and tissue
penetration is poor as in areas of necrosis, which is why debridement is needed if the site of origin can be
identified.
Muscle relaxation may be achieved by use of diazepam, acetyl promazine or one of the longer acting
barbiturates until symptoms abate. Affected animals should be kept as quiet as possible and supportive
treatment such as i/v fluids administered if the animal is unable to eat or drink. The mortality rates reported
in humans vary between 40-80%. The prognosis for animals with localized tetanus is good.
Prevention
At risk animals can be vaccinated with a TOXOID. This should be done routinely in horses due to their
marked susceptibility to tetanus. This is usually done every second year in Australia. Management issues
include minimising the presence of objects eg barbed wire or nails that may cause the initial injury.
Vaccination of cattle and sheep is included as part of a 5 in 1 vaccine.
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MYCOBACTERIUM
It would appear that to be a world authority on Mycobacteria,

you need to be handsome, entertaining and answer to the
name …… Richard
Lecture 1
Dr Richard Malik, a feline specialist and world authority on mycobacterial diseases in cats and dogs will
give the second lecture, concentrating on disease syndromes seen in small animals. Comprehensive notes
are included in this handbook. Richard has just completed a chapter on mycobacterial diseases in dogs and
cats that will appear in the Third edition of “Green’s Infectious Diseases of the Dog and Cat” which will
be released later this year and will be an updated version of the notes provided here.
Lecture 2
This lecture will be given by Professor Richard Whittington, a world authority on Johne’s Disease and
Chair of Farm Animal Health. Lecture notes will be provided in advance. Unfortunately due to printing
deadlines, the notes were unable to be incorporated in this handbook. Richard Whittington in his lectures
will give you a global and historical perspective on mycobacterial diseases across all species including
humans and then will focus on bovine tuberculosis and Johne’s disease.
277
MYCOBACTERIAL DISEASES OF CATS AND DOGS IN AUSTRALIA
Richard Malik, Patricia Martin, Denise Wigney, Geraldine Hunt, Carolyn O’Brien and
Daria Love
University Veterinary Centre Sydney and Diagnostic Services Laboratory,
Faculty of Veterinary Science,
The University of Sydney
Introduction
Mycobacteria are Gram-positive rods which have as their distinguishing feature a cell wall which has outer
layers rich in mycolic acids and mycosides. These lipids are considered responsible for most of the
characteristic features of the genus, including ‘acid fastness’, the ability to withstand drying as well as the
histological and immunological features of the disease process operating within the host.
The name Mycobacterium (fungus-bacterium) is a consequence of the hydrophobic nature of the lipid-rich
cell wall, which gives these organisms the tendency to grow as mould-like pellicles on the surface of liquid
medium and their ability to form stable aerosols when water is disturbed. Historically, mycobacteria have
been divided into the obligate pathogens such as M tuberculosis complex, which do not normally multiply
outside vertebrate hosts and saprophytic mycobacteria which have been divided into the facultative
pathogens such as M avium, M intracellulare (and related organisms grouped together as the MAC
complex) which normally exists as saprophytes in the environment, but sporadically cause disease and
those environmental saprophytes which almost never cause disease. Recently, however, the situation has
been made more complex. M leprae, for example, which was formerly considered an obligate pathogen of
humans and armadillos has recently been proposed to have some yet to be defined environmental niche,
while a number of formerly non-pathogenic species have been reported to cause significant disease in
HIV/AIDS patients.
Mycobacteria which give rise to human and animal disease entities include the tuberculosis complex (M
tuberculosis, M bovis, M africanum and M microti), M leprae (the cause of human leprosy) and M avium
subsp paratuberculosis (the cause of Johne’s disease of ruminants). Tuberculosis is acquired principally by
inhalation of aerosolised organisms directly or indirectly from an infected host although ingestion and
instillation are less common entry modalities. M leprae is probably acquired in a similar manner although
colonisation of the nasal passages is a common feature in infected individuals. As with many of the other
mycobacteria acquired from the environment, infection of the intestinal wall with M avium subsp
paratuberculosis is accomplished subsequent to successful passage of the lipid-rich organisms through the
stomach. However, due to the ability to form stable aerosols, most environmental mycobacteria may also
enter hosts via the respiratory tract or by direct instillation into wounds or abrasions.
Mycobacterium tuberculosis, M bovis and M microti have been isolated from tuberculosis in cats, but
these diseases will not be covered in great detail here as they are currently exotic to Australia. Classical
tuberculosis in cats is caused by M bovis and occasionally M tuberculosis. A ‘new’ feline tuberculosis
syndrome caused by a mycobacterium with characteristics intermediate between M bovis and M
tuberculosis has been the subject of considerable recent interest in Europe. It seems most likely to result
from infection with M microti strains inoculated following altercations with voles. In New Zealand, dogs
occasionally develop pulmonary tuberculosis due to M bovis.
In addition to these well known agents of disease, there are many different saprophytic mycobacterial
species that are capable of producing opportunistic infections in both immunocompetent and
immunoparetic cats and dogs, under certain circumstances. It is this group of organisms that are the
principal subject of this article. Although by no means a common cause of disease in small animal
practice, saprophytic mycobacteria are important ‘unusual’ pathogens. Recent developments in
antimicrobial chemotherapy and reconstructive surgery have enabled most mycobacterial infections
encountered in small animals to be potentially curable, given committed owners with sufficient financial
resources.
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When mycobacteria give rise to stereotyped clinical syndromes, diagnosis is usually straightforward,
providing the opportunity for a successful outcome. Unfortunately when mycobacteria give rise to
‘atypical’ disease, such as granulomatous pneumonia or disseminated disease with peripheral lymph node
and/or internal organ involvement, the clinical picture is suggestive of neoplasia such as lymphoma. Thus,
unless appropriate specimens are collected for laboratory examination, animals can be euthanased on the
basis of having an untreatable terminal disease. We have adopted a collaborative approach to the diagnosis
and management of cats, dogs and ferrets with mycobacteriosis. Our group consists of a group of
veterinarians interested in evaluating these cases, veterinary cytopathologists and microbiologists with a
special interest in diseases caused by unusual pathogens, a specialist surgeon interested in reconstructive
approaches to extensive cutaneous disease and interested medical colleagues and mycobacteriologists at
research institutes, reference laboratories and human hospitals.
Nontuberculous mycobacteria give rise to a number of different clinical syndromes in small animals,
although sometimes the distinction between the syndromes can become blurred. For purposes of
simplicity, the syndromes covered in this review include:
• mycobacterial panniculitis due to rapidly growing mycobacteria
• localised or disseminated cutaneous/subcutaneous disease (‘feline leprosy-like disease’ and ‘canine
leproid granuloma syndrome’)
• disseminated mycobacterial infection, with lymph node and/or internal organ involvement
• miscellaneous localised mycobacterial infections, eg mycobacterial pneumonia, keratitis
A feature of mycobacterial disease is the associated inflammatory response, which is generally
granulomatous or pyo-granulomatous, as might be expected for an infection in which antigen-specific cell
mediated immunity (CMI) is required to activate mononuclear phagocytes to deal effectively with bacteria
capable of intracellular survival. Pyogranulomatous inflammation is most usually characteristic of disease
associated with saprophytic mycobacterial infections from humans or animals whether associated with the
more slowly growing or poorly growing organisms such as MAC, M marinum, M xenopi, M malmoense,
M genavense or M scrofulaceum or the rapidly growing species such as M thermoresistibile, M fortuitum,
M smegmatis or M chelonae which have been described in a variety of animal and human diseases with
cutaneous manifestations. Similar pathology may be seen with diseases associated with other bacteria with
high lipid content in their cell walls (such as members of the genera Corynebacterium, Nocardia and
Rhodococcus) and with some fungal infections. The pathology, however, is clearly distinguishable from
that associated with tuberculous mycobacteria where granulomatous inflammation dominates the cellular
response in immunocompetent hosts.
Although many other disease processes and agents give rise to (pyo)granulomatous inflammation, for
example feline infectious peritonitis virus and the aforementioned microorganisms, mycobacteria should be
considered in the differential diagnosis when cytopathology demonstrates mixtures of mononuclear and
polymorphonuclear phagocytes and lymphoid cells. Under these circumstances, the diagnostic laboratory
should be requested to perform special stains to demonstrate mycobacteria, including both modified-acid
fast stains to demonstrate acid fast bacilli (AFB) directly and Romanowsky-type stains (such as Diff Quik)
which ‘negatively’ stain the bacilli. Furthermore, material should be collected for mycobacterial culture,
which, along with conventional solid nutrient media eg blood agar, involves using a variety of special
media, incubation conditions and preparatory techniques which maximise the likelihood of isolating causal
organisms. It is prudent also to freeze representative tissue from these suspect cases, in case molecular
analyses such as PCR or probe detection systems are contemplated at a later date. These molecular
techniques have had a big impact on the clinical management of mycobacterial infections in human
patients, as they have the potential to provide an etiological diagnosis more quickly and accurately than
culture, and in cases where routine mycobacterial culture is negative because of the fastidious nature of
disease producing strains.
In contrast to the situation in human patients, application of molecular tools for diagnosis of mycobacterial
infections of companion animals is still in its infancy. It is envisaged that molecular techniques will gain
more precedence for diagnosis of mycobacterial infections of cats and dogs in the future, particularly for
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species which are difficult or impossible to culture, such as the organisms that give rise to feline leprosy
and canine leproid granuloma syndrome cases.
Before discussing specific entities seen clinically in small animal practice, it is worth emphasizing that
non-tuberculous mycobacterial disease should be considered in two distinct conceptual categories.
1) Firstly, there are those immunocompetent patients in which some breach in the normal defense
mechanisms of the respiratory or alimentary tracts, or integument, allows mycobacteria to enter host
tissues and give rise to localized disease. In this type of scenario organisms may be inoculated into an
otherwise sterile site, eg following disruption of the skin by a penetrating injury or aspiration of abnormal
material into the tracheobronchial tree. Likewise, localized gastrointestinal involvement may occur
subsequent to ingestion of a large inoculum of organisms from an environmental source.
2) Secondly, there are cases where the host has some immunological defect, presumably affecting CMI,
which allows saprophytic mycobacteria to produce disseminated disease, often without an obvious breach
in integrity of the defense system to account for the site of primary infection. In these cases it is suspected
that organisms colonize some site, such as the alimentary or respiratory tract, and later spread
haematogenously to skin, lymph nodes, lungs, liver, brain, spleen and bone marrow, tissues which for
some reason favor multiplication of mycobacteria.
Some mycobacterial species, such as the rapid growers M fortuitum and M smegmatis, are strongly linked
with localized infections in immunocompetent hosts. Other species, such as M avium complex, can
produce either localized disease in an immunocompetent host, or disseminated infections in
immunodeficient hosts. This distinction is important clinically, as patients in the latter category may have
identifiable causes of immune compromise, such as inherited canine immune deficiency, FIV/FeLV
infection in cats or history of immunosupressive therapy, and may be at risk for developing other diseases
associated with immune dysfunction subsequent to successful treatment of their mycobacterial infection.
Mycobacterial panniculitis
Clinical features
Mycobacterial panniculitis refers to a clinical syndrome characterized by chronic infection of the subcutis
and skin of cats and dogs with ‘rapidly growing’ saprophytic mycobacteria. In Australia this condition is
quite common in cats, but rare in dogs. Rapidly growing mycobacteria (RGM) are a heterogenous group
of organisms that produce visible colonies on synthetic media within seven days when cultured at
temperatures ranging from 24oC to 45oC, depending on the species. They are distributed ubiquitously in
nature, and can be commonly isolated from soil and bodies of water. Bacteria in this group of potentially
pathogenic saprophytic mycobacteria include M fortuitum, M chelonae, M smegmatis, M phlei, and M
thermoresistibile.
If introduced through some breach in the integument, saprophytic mycobacteria are capable of replication
in mammalian tissues. The preference of certain saprophytic mycobacteria for fat results in their
propensity to produce disease in obese individuals, and in tissues rich in lipid, such as the inguinal fat pad
and infusions or injections with oil suspensions. The same phenomenon accounts for situations where these
organisms give rise to human infections, for example athletes that inject themselves with anabolic steroids
suspended in oily vehicles from contaminated multi-use vials, as a complication of lipoid pneumonia and
following augmentation mammoplasty and median sternotomy. Experimental and clinical observations
suggest that adipose tissue offers a favorable environment for survival and proliferation of saprophytic
mycobacteria, either by providing triglycerides for growth of organisms, or protecting them from the
phagocytic or immune responses of the host.
Initial reports suggested that mycobacterial panniculitis in cats was more common in warm humid tropical
and subtropical climates. This would appear to be the case in humans where for example, most reported
cases in the USA occur in Texas, Florida and Louisiana. However, cats from a variety of temperate
climates (including Finland) have subsequently been reported to develop these infections, and the causal
organisms have been readily cultivated in temperate soil samples collected from Japan.
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In cats, infections tend to start in the
inguinal region, usually following
environmental contamination of cat
fight injuries, typically raking wounds
inflicted with the hind claws. The
infection may spread subsequently to
contiguous subcutaneous tissues of the
ventral and lateral abdominal wall and
perineum. Penetrating injury by
sticks, metallic objects and vehicular
trauma may also give rise to these
infections, as can cat and dog bite
injuries contaminated with soil or
dirt. Sometimes infections start in the
axillae or flanks and spread into
adjacent tissues. We have encountered
RGM infection of the subcutis in only
one dog, a very obese Keeshond, with
abundant subcutaneous fat deposits. FIGURE 1: Indurated inguinal skin and subcutis in a cat with
Early in their clinical course, mycobacterial panniculitis. This case was caused by M smegmatis,
the commonest cause of this syndrome in eastern Australia.
infections can resemble conventional
cat fight abscesses, but without the
characteristic pungent odor and turbid pus. Instead, a circumscribed plaque or nodule is apparent at the
site of injury. Later there is progressive thickening of the nearby subcutis to which the overlying skin
becomes adherent. Affected areas become denuded of hair and numerous punctate fistulae appear,
discharging a watery exudate. Fistulae are intermingled with focal purple depressions which correspond to
thinning of the epidermis over accumulations of pus. The ‘lesion’ gradually increases in area and depth,
and may eventually involve the entire ventral abdomen, adjacent flanks or limbs. If cats are presented
promptly for veterinary attention and the lesion confused with an anaerobic cat bite abscess, standard
treatment consisting of surgical drainage and administration of a synthetic penicillin is typically followed
by wound breakdown and development of a large
non-healing sinus tract surrounded by indurated
suppurating granulation tissue (Figure 1).
Some affected cats with severe infections develop
constitutional signs. They become depressed, pyrexic,
inappetent, lose weight and are reluctant to move.
Surprisingly, other cats remain comparatively well
despite extensive pyogranulomatous disease. Usually
the problem remains localized to the cutaneous and
subcutaneous tissues, as might be expected from an
opportunistic infection in an immunocompetent host.
Although adjacent structures such as the abdominal
wall can be affected eventually, widespread
dissemination of the infection to the internal organs
and lymph nodes is unusual.
FIGURE 2: Another cat with mycobacterial

panniculitis. This case was unusual in that
involved the skin and subcutis over the dorsum of
this very obese cat., which was successfully
treated by Dr Linda Vogelnest
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Diagnosis
Cytology
A tentative diagnosis of mycobacteriosis can be confirmed by collection, usually under sedation or
anaesthesia, of aspirated pus or deep tissue samples. This material is used to confirm the diagnosis using
appropriately stained cytology specimens, histological sections and mycobacterial culture. A histological
diagnosis is unnecessary if appropriate samples for cytology and culture have been procured.
In our experience samples of pus obtained from needle aspirates of affected tissues provide the best
laboratory specimens. This material can be obtained from a palpably indurated portion of the subcutis,
following cleansing of the overlying skin with 70% ethanol. It is only necessary to obtain a small amount
of liquid material into the hub of the syringe; after replacing the needle with a sterile cover, the syringe
should be submitted to the laboratory for culture. Exudate from draining sinus tracts is too heavily
contaminated with Staph to be worthwhile submitting for culture. Finally, it is vital to give the laboratory
advanced warning that a mycobacterial aetiology is suspected.
Biopsies
Biopsies should be triturated in brain heart infusion broth using a sterile mortar and pestle. Smears
prepared from the resulting homogenate or from swabs or aspirates of the purulent exudate should be
stained using Diff Quik, Burke's modification of the Gram stain and a modified acid-fast procedure
(decolorizing with 5% sulphuric acid for three to five minutes). Cytology invariably demonstrates
pyogranulomatous inflammation and it is generally possible to visualize Gram positive and/or acid fast
rods in smears, although an exhaustive search of several smears is sometimes required. Many organisms
demonstrate beading.
Histologically
There is pyogranulomatous inflammation of subcutaneous adipose tissue, overlying dermis and underlying
abdominal fascia and musculature. AFB may be hard to find in Ziehl-Neelsen stained tissue sections and
are often located in lipid vacuoles.
Bacteriology
Tissue homogenates and pus should be streaked onto duplicate 5% sheep blood agar plates and a
mycobacterial medium such as Lowenstein-Jensen medium or 1% Ogawa egg yolk medium and incubated
aerobically at 37oC and 25oC. Moderate to heavy growth of pin-point, smooth or rough (depending on
species), non-hemolytic colonies is usually detected after two to three days on sheep blood agar at 37oC.
Where only contaminated specimens are available, tissue homogenates can be treated with 4% sodium
hydroxide followed by neutralization with dilute hydrochloric acid prior to inoculation onto media.
Another method which can be used to selectively differentiate RGM from contaminant flora is by primary
isolation around antibiotic sensitivity discs (first generation cephalosporins or isoxazolyl penicillins)
applied to the plate after inoculation; this has become our method of choice for dealing with skin and
subcutaneous samples contaminated with staphylococci.
Strain Identification: Species identification can be carried out in a well equipped veterinary bacteriology
laboratory although it if often more convenient to send the strain to a Mycobacteria Reference Laboratory
following primary isolation. Identification takes into account the following phenotypic features: organism
morphology in Ziehl-Neelsen stained smears of growth taken from Lowenstein-Jensen medium, colonial
morphology (rough or smooth), pigmentation in the dark and light, degree of acid fastness, rate of growth
at room temperature and 37oC, ability to grow at 42oC and 52oC, arylsulphatase activity, iron uptake, p-
amino salicylic acid (PAS) degradation, nitrate reduction, galactosidase activity, acid production from
carbohydrates (glucose, inositol, mannitol), utilization of compounds (glucose, fructose, inositol, mannitol,
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citrate) as the sole carbon source, tolerance to 5% sodium chloride in Lowenstein-Jensen medium and
susceptibility to polymyxin B and trimethoprim.
Isolates are identified as M fortuitum if they are acid-fast, grow in less than seven days at 28oC and 37oC,
produce rough, non-pigmented colonies, produce a positive three-day arylsulphatase reaction, are positive
for iron uptake, reduce nitrate and are susceptible to polymyxin B but not trimethoprim. M fortuitum
strains are divided into biovarieties according to their utilization of mannitol, inositol and citrate as sole
sources of carbon for growth. It is possible to differentiate M fortuitum strains from M chelonae strains
based on in vitro susceptibility to polymyxin B (M fortuitum sensitive, M chelonae resistant).
Isolates are identified as M smegmatis if they grow in less than seven days, grow well at 43oC but not at
52oC, are positive for iron uptake and have a negative three-day arylsulphatase reaction. Further, colonies
of M smegmatis from clinical material are typically smooth and not immediately pigmented, although a
late-developing yellow-to-orange pigmentation is seen in some isolates. Pigment develops in the dark but is
enhanced by light. M smegmatis strains have a relatively consistent susceptibility pattern, being resistant to
rifampicin and isoniazid, but susceptible to ethambutol, doxycycline, sulphamethoxazole, trimethoprim,
gentamicin and ciprofloxacin. It is possible to differentiate M smegmatis (sensitive) from M
fortuitum/chelonae (resistant) using a trimethoprim disc.
Antimicrobial susceptibility testing: Minimum inhibitory concentrations (MICs) for ciprofloxacin,
gentamicin, trimethoprim, clarithromycin and doxycycline can be determined easily using the Etest (AB
Biodisk, Solna, Sweden) method according to the manufacturer’s recommendations and using one Etest
strip per 90 mm plate containing 20 mL Brucella agar (Difco Laboratories, Detroit, USA). The MIC is
read after 72 hours from the scale on the strip and is determined as the place where the elliptical zone of
growth inhibition intersects the strip. This methodology is far less demanding than the gold standard of
broth microdilution. Antimicrobial susceptibility of clinical isolates can also be determined using disc
diffusion methodology. Typically, isolates are tested against discs containing representative antimicrobials
including doxycycline (30µg), gentamicin (10µg), ciprofloxacin (5µg), norfloxacin (10µg), trimethoprim
(5µg), polymyxin B (300µg), enrofloxacin (5µg) and clarithromycin (30µg). Some antibiotics are included
to determine a suitable agent for long-term oral therapy, while others (trimethoprim, polymyxin B) are
used to provide phenotypic information concerning strains. Suspensions of each organism in saline or
nutrient broth are sown onto sensitivity agar and incubated at 37oC. Results are recorded after incubation
for 48 and 72 hours.
Therapy
Our handling of these cases continues to evolve over time according to accumulating clinical experience
with a range of infected cats, the availability of new antimicrobial agents and development of new surgical
procedures. It is worth emphasizing that there is a great variation in the severity and extent of lesions in
individual patients. Our experience is based on cats presented to a referral centre or seen as a ‘second
opinion case’ after having been treated by other veterinarians on several occasions and subjected to at least
one attempt at surgical excision of lesions, usually followed by administration of an antibiotic such as
amoxycillin/clavulanic acid. Thus, the cases we have had experience with may be more severe than those
likely to be encountered as first opinion cases. The marked variation in disease severity makes it difficult
to compare different treatment options.
The strategy we currently use has evolved over the last 10 years at the University Veterinary Centre
Sydney. Treatment commences with either doxycycline (typically) or a flouroquinolone, often followed
some weeks later by radical surgical excision of all or nearly all infected tissues, with intra- and peri-
operative gentamicin followed subsequently by a long period (typically months) of follow-up antimicrobial
therapy. Severe cases benefit from the radical excision technique developed by one of the authors (GH)
where infected tissue is resected en bloc followed by rearrangement of nearby skin to fill the often
substantial tissue deficits created. This strategy has proved to be uniformly successful and all cases so
treated were cured on the basis of absence of disease recurrence for years after treatment. Gentamicin was
administered intra-operatively (2 mg/kg every eight hours intravenously or subcutaneously) and in the
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early post-operative period because it was bactericidal, available in a parenteral form, inexpensive and
displayed good in vitro activity against all mycobacterial isolates. Amikacin would be equally or more
effective in this setting, although it is substantially more expensive. Ciprofloxacin was used postoperatively
in many of the cases because enrofloxacin was not available commercially in Australia when this treatment
regimen was first instituted. Subsequent use of enrofloxacin has given comparable efficacy to ciprofloxacin
at a considerable cost advantage and with the convenience of once daily dosing. However current concerns
about the retinotoxic potential of enrofloxacin when given in doses exceeding 5 mg/kg daily may favor a
return to ciprofloxacin, which owners can purchase inexpensively from Pharmacy Direct.
Given the extent and severity of the pathology in many of these cats, it is understandable that adequate
levels of antimicrobial agent may not be achieved throughout all involved tissues and that in these cases the
best chance for a successful long-term outcome is to remove as much infected tissue as possible following
preliminary antimicrobial therapy. Residual foci of infection can then be targeted by the high
concentrations of antibiotics achieved during and after surgery. Obviously peri- and post-operative
antimicrobial therapy is vital to ensure primary intention healing of the extensive surgical wound. Similar
recommendations have been made for humans with deep-seated saprophytic mycobacterial infections,
where antibiotic therapy is used in addition to aggressive surgical debridement. In people, the current
recommendation is to pack the infected wound open following surgical intervention although our
preference in feline patients is a one-stage procedure including the judicious use of drains, because of
difficulties of maintaining wound toilet for a prolonged period in a fractious patient. It must be emphasised
that advanced cases with extensive subcutaneous involvement demand the skill of a very experienced
surgeon to adequately reconstruct the resulting tissue deficit without undue tension.
Interestingly, some cases treated in a preliminary fashion using doxycycline, enrofloxacin or ciprofloxacin
continue to improve to such an extent that surgery becomes unnecessary. These cases can thus be cured
using medical therapy alone, although treatment with oral antimicrobials for periods of up to six months is
required. As a generalisation, cases that resolve without the need for (further) surgical intervention involve
a lesser depth of tissues than those cases which eventually require surgery. Successful management of
mycobacterial panniculitis using enrofloxacin or clofazimine without surgery has been reported previously.
However, some cases are so severe that only limited improvement can be achieved with antimicrobial
therapy alone, and surgical intervention is required to effect a cure. Because it is not possible to predict
with certainty which cases will require operative debridement, our current recommendation is to start
empirical therapy using doxycycline or a quinolone, determine the in vitro susceptibility of the strain to
ensure the cat is on appropriate therapy, then re-assess the case every three to four weeks to decide if
continued improvement is occurring, or whether surgery is indicated.
Generally we have found that there is very good agreement between the in vitro susceptibility results and
in vivo effectiveness of antimicrobials administered at recommended dosages. For example, we have seen
cats infected with strains resistant to enrofloxacin in vitro that did not respond to oral dosing with that
drug, but were subsequently cured using doxycycline. Similarly, one cat infected with a strain resistant to
doxycycline in vitro did not respond to treatment using doxycycline, but was cured using clarithromycin
(1/4 to ½ 250mg tablet every 12 to 24 hours) to which the organism involved had recorded in vitro
susceptibility. The Etest method is easily applicable to this group of organisms and provides quantitative
information pertinent to selection of optimal drug therapy. However susceptibility testing using a disc
diffusion method provides very similar information and is cheaper to use in a general diagnostic
laboratory. As a group, M smegmatis strains are generally sensitive to a wide range of antimicrobial
agents well suited to treating chronic infections in cats, whereas M fortuitum strains generally demonstrate
resistance to one or several agents, and often have higher MICs than M smegmatis for agents to which
strains are sensitive. We have no first hand experience with M chelonae strains, but based on experience in
human patients, they tend to be resistant to all common agents available for oral dosing apart from
clarithromycin. Of the agents suitable for long term therapy in cats, we consider the fluoroquinolones
(ciprofloxacin and enrofloxacin) and doxycycline as the agents of choice for treating these infections, at
least in Australia where M smegmatis and M fortuitum strains predominate. Clarithromycin is a macrolide
derivative with an extended spectrum of activity and prolonged pharmacokinetics. It has proved to be an
extremely useful in treating mycobacterial infections in human patients, especially MAC infections, and we
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have likewise found it useful for certain mycobacterial infections in cats. Unfortunately a large proportion
of M smegmatis strains are not susceptible to this agent in vitro, and this unpredictable efficacy makes it
an unsuitable drug to recommend for empiric therapy.
It general, it is necessary to use as high doses as possible of antimicrobial agents when treating these
infections, because affected subcutaneous tissues are not well perfused and considerable diffusion barriers
can prevent blood levels of antibiotics reaching organisms in tissues. Doxycycline is the tetracycline of
choice for use in the cat, being well tolerated orally, present in a readily available form (Vibravet tablets;
Pfizer Australia) and having good lipid solubility. Doxycycline (25 to 50mg per cat orally every 8 to 12
hours), enrofloxacin (25 to 75 mg per cat orally once a day) and ciprofloxacin (62.5 mg to 125 mg per cat
orally every 12 hours) all have proved effective for monotherapy in our experience. Recent work has
indicated that high doses of enrofloxacin, in excess of 5 mg/kg every 24 hours, can produce retinal
degeneration and sometimes blindness in a proportion of cats, and this must be borne in mind when
selecting which agent is to be used. To the best of our knowledge, retinal toxicity has not been reported for
ciprofloxacin, marbofloxacin or orbofloxacin, although the authors have had no experience in using the
latter two drugs in the treatment of mycobacterial infections and no data on their use is available from the
literature. In general, treatment commences using the low end of the dose rates given above, and the
dosage slowly increased (over several weeks) until adverse side effects (inappetence, vomiting) suggest the
need for slight dose reduction.
Based on our Australian experience, one of the fluoroquinolones is recommended for empiric therapy
where microbiological data are lacking, or while susceptibility data is pending. These agents are
bactericidal, penetrate well into tissues including fat, and are concentrated in polymorphs and
macrophages. Doxycycline, on the other hand, has a cost advantage, and based on our experience has very
similar efficacy to enrofloxacin or ciprofloxacin, and is equally well suited to long term oral therapy. We
have had an interesting experience with two strains of M smegmatis which were resistant to enrofloxacin
and/or ciprofloxacin; both of these cats had been treated with fluoroquinolones prior to referral and
culture, and we suspect that resistance developed during a course of therapy as has been reported
previously for human patients. The propensity of mycobacteria species to develop resistance during
treatment is well known, especially for species such as M tuberculosis, M avium and M leprae, although
this phenomenon has been far less problematic for the RGM. It should be borne in mind that the
development of resistance is common for the quinolone agents, probably as a result of selection of pre-
existing mutants. There is probably insufficient information to recommend routine combination therapy in
these cases, especially in cats where administration of multiple agents can be problematic. However, the
possibility of resistance development during monotherapy, especially using fluoroquinolones, should be
considered in cases where the favorable response to therapy does not continue during a course of
treatment. The same dilemma is debated in the human literature, where some authorities recommend that
quinolones be given in combination with another agent, while others consider treatment with a single agent
to be appropriate. There has been no evidence of acquired resistance to doxycycline in our experience and
data from human patients indicates the likelihood of mutational resistance to doxycycline is low. Thus,
single-agent therapy with this agent may be employed with a lesser risk of the development of resistance
than for enrofloxacin or ciprofloxacin.
The total duration of therapy should be in the order of three to six months and typically agents should be
administered for at least one to two months after the affected tissues look and feel completely normal. We
have not evaluated trimethoprim or sulphamethoxazole/trimethoprim combinations even though in vitro
susceptibility data show many strains are sensitive to both components of the combination. Previous
reports, however, implied that this combination was inferior to doxycycline or a flouroquinolone for long
term therapy of these deep-seated infections in cats. In occasional refractory cases, clofazimine, cefoxitin
or amikacin may be used for monotherapy or in concert with other agents shown to be effective in vitro.
Cefoxitin and amikacin must be given by injection, however.
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Summary of Mycobacterial Panniculitis
To sum up, mycobacterial panniculitis is an eminently treatable disease in the cat. Diagnosis is
straightforward, especially for a practitioner familiar with the syndrome. The prognosis is excellent, even
in cases with severe, extensive and longstanding disease. Treatment involves long courses, typically three
to six months, of antimicrobial agents chosen on the basis of in vitro susceptibility testing, sometimes
combined with extensive surgical debridement and wound reconstruction. Even when surgical intervention
is not possible because of financial considerations, long term once daily therapy with doxycycline will
usually confine the infection sufficiently to enable the cat to lead a normal life. Finally, the routine
prophylactic use of doxycycline following treatment of penetrating injuries in obese cats may prevent the
development of this deep-seated infection.
Feline leprosy-like disease

Feline leprosy refers to a mycobacterial syndrome in which single or multiple granulomas form in the skin
or subcutis in association with large numbers of acid-fast bacilli (AFB) which are nonculturable using
standard mycobacteriological methods. The condition was first recorded in the literature by New Zealand
and Australian researchers, in 1962 and 1963, respectively, although Carne had encountered cases as early
as 1934. The disease has since been reported in Western Canada, the Netherlands, the UK and USA.
Although the causal organisms are likely to have a world-wide distribution, the infection seems especially
common in certain geographical locations such as the North Island of New Zealand, the Netherlands and
British Columbia. Based on available data, ‘feline leprosy’ would appear to be more common in temperate
coastal areas and port cities, rather than in inland or tropical habitats.
Historically, the causative agent of feline leprosy is purported to be Mycobacterium lepraemurium. This
bacterium causes murine leprosy, a systemic mycobacterial infection of rats. Cats are thought to contract
M lepraemurium following bite injuries from infected rodents. M lepraemurium is a fastidious, slow-
growing mycobacterial species which, with difficulty, can be cultured from large inoculae on Ogawa’s
enriched egg yolk medium under strictly controlled conditions of temperature (35oC) and atmosphere, or in
liquid medium enriched with cytochrome c and α-ketoglutarate. Although a few investigators have
successfully grown M lepraemurium from infected cats, the basis of ascribing this bacterium as the
aetiological agent of feline leprosy was historically dependent on transmission studies and the results of
delayed hypersensitivity reactions to intradermally administered tissue extracts. Several groups have been
able to show that material obtained from feline leprosy lesions can be used to transmit disease
experimentally to rats and mice, and subsequently back to cat. In such studies, the incubation period
varies from two months to one year or more. Interestingly, some cats appear much more susceptible to
infection than others.
According to the literature, cats with feline leprosy are typically young adults, less than 5 years-of-age,
perhaps with a preponderance of males among reported cases. Presumably these demographic
considerations reflect the need for the cat to interact with a rat to become infected. The initial lesion in
feline leprosy is said to be a focal granuloma of the subcutis and skin. Owners become aware of solitary,
or more commonly multiple, painless, raised, fleshy, tumour-like lesions, from a few millimetres up to 4
cm in diameter. These granulomas are freely movable over the underlying tissues. Lesions apparently
develop quite rapidly, and when large can ulcerate. Infection spreads to adjacent areas of the integument
and may invade adjacent and underlying local tissue such as muscle and fascia. There is often spread of the
infection to regional lymph nodes.
Lesions can occur anywhere on the body, but tend to be concentrated on the head and limbs. Small lesions
are occasionally found on the tongue, lips and nasal plane. Lesions, even if multiple, tend to be initially
concentrated in one region, and have the propensity to recur following attempted surgical excision.
Affected cats are usually in good general health, at least initially. In some cases, however, the condition
becomes generalised, with the development of large numbers of skin lesions of different sizes over the
286
entire body surface, presumably as a result of haematogenous or lymphatic spread. These cats suffer
malaise, depression, poor appetite and wasting and further investigation or necropsy reveals a systemic
infection with granulomatous lesions in the internal organs (especially liver, spleen or lungs), lymph nodes
and bone marrow.
Pathologically, feline leprosy has been subdivided into lepromatous or tuberculoid forms on the basis of
the number of AFB present, and the extent of the host immunological response. Because the causal
mycobacteria are slow-growing organisms capable of intracellular survival, the clinicohistological
manifestations of disease depend on the host’s innate CMI response to the parasite. When the immune
response is poor, lepromatous (or multibacillary) disease develops with infiltration of the dermis with large
numbers of ‘incompetent’ or ‘kamikaze’ macrophages loaded with AFB. If the host’s immune response is
more effective, multiplication of the organism is limited, with the formation of granulomatous lesions in
the dermis - the so called tuberculoid response. In the tuberculoid form, which appears to account for
perhaps two-thirds of cases in Western Canada and a substantial proportion of cases in New Zealand and
the Netherlands, epithelioid histiocytic cells are accompanied by moderate numbers of lymphoid cells and
plasma cells, and caseous necrosis is often evident. Other cases present with more pyogranulomatous
pathology. In the lepromatous form, granulomas are composed of large sheets of foamy macrophages that
contain enormous numbers of organisms. AFB are usually arranged in the cytoplasm of macrophages as
dense parallel accumulations known as globi, which displace the nucleus to an eccentric position within the
macrophage. The granulomas are not encapsulated and tend to spread into adjacent tissues. Invasion of
local nerves, a prominent feature of human leprosy, is rarely observed in patients with feline leprosy,
although a recent case report described a cat (without cutaneous lesions) which presented for mycobacterial
infiltration of one sciatic nerve.
AFB in smears and tissue sections appear as long slender rods, 3 to 6µm in length. In smears stained with
Romanowsky stains such as Diff Quik, organisms appear as negative-staining bacilli. In smears or sections
stained with modified acid-fast stains such as Ziehl-Neelsen (ZN) or Fite’s stain, organisms take up the
carbol fuschin, and are acid and alcohol fast. M lepraemurium is said to have a characteristic morphology
both in vitro and in vivo, with pleomorphic AFB including very long filamentous forms, beaded forms,
branching and swollen ends.
Recently, molecular-based methodologies have been applied to the investigation of presumptive feline
leprosy. Of eight cases of invasive or disseminated cutaneous mycobacterial disease investigated by
Hughes’ group in Belfast using material collected largely from New Zealand cats, four cases were shown
to have M lepraemurium infections. Of the remaining cases, one cat had a disseminated M avium
infection, the aetiology in one cat was undetermined and in two cases infection was attributable to a novel
mycobacterial species, which shared close nucleotide sequence identity with M malmoense. Given the
current state of knowledge, the term ‘feline leprosy’ is thus probably best considered to be a syndrome
rather than a specific infection. Similar situations exist in human mycobacteriology, where, for example,
the ‘Scrofula syndrome’ of localized cervical pyogranulomatous lymphadenitis in children can be caused
by a large variety of mycobacteria including M scrofulaceum, M avium complex (MAC), M genavense
and M interjectum.
Data from Hughes’ molecular-based studies encouraged us to re-appraise cases of feline leprosy
encountered by the Diagnostic Services Laboratory of the University Veterinary Centre Sydney (UVCS).
Our data has indicated that ‘cat leprosy’ actually refers to at least two different diseases, one caused by the
rat leprosy bacillus M lepraemurium and another by a single novel species of mycobacterium. The
molecular insights obtained in this study were clarifying, as we had been puzzled as to why most of the
‘feline leprosy’ cases referred to us did not conform with the expected textbook picture. Instead of a
localised lesion, or group of lesions, on the head or extremity of a young cat, the majority of cats we see
in Sydney are elderly and had typically developed widespread cutaneous disease by the time they are
referred for a second opinion or following diagnosis in practice.
287
FIGURE 3: A Californian cat with feline leprosy due to M lepraemurium
(photograph courtesy of Dr Peter Ihrke).
M lepraemurium infections
should have a number of
distinguishing features that
suggest this aetiology even
where molecular testing is
not practicable. In these
infections, a ‘tuberculoid’
pyogranulomatous response
with prominent involvement
of lymphoid cells and
neutrophils, in association
with regions of necrosis
containing sparse to
moderate numbers of AFB
is strongly suggestive of this
aetiology. In contrast, in
cases caused by the novel
species, caseous necrosis is
absent and the histological response is typically ‘lepromatous’, with enormous numbers of AFB within
epithelioid macrophages and giant cells.
Diagnosis of the ‘feline leprosy’ syndromes is usually straightforward, provided that the clinician has a
high index of suspicion for the condition. Needle aspirates, crush preparations of biopsy material and
histological sections stained with ZN or similar methods contain easily demonstrable AFB surrounded by
variable granulomatous to pyogranulomatous inflammation. In Diff Quik stained smears mycobacteria can
be recognized by their characteristic negative-staining appearance and location within macrophages and
giant cells. Material should be submitted also for culture, because occasionally slowly-growing species
such as MAC can produce an identical clinical presentation ,and in these cases optimal antimycobacterial
therapy can be selected more readily on the basis of in vitro susceptibility results and information available
in the literature. However, in the majority of cases conventional mycobacterial culture is negative due to
the fastidious nature of the causal organisms and a mycobacterial aetiology can only be proven using
molecular techniques such as PCR amplification and nucleotide sequence determination of gene fragments.
Fresh (frozen) tissue delivered to a mycobacteria reference laboratory with PCR facilities provides the
optimal sample, and we would be very happy to arrange this if cases can be referred to the UVCS.
FIGURE 4: Diff Quik stained

smear from a case of feline
leprosy in an elderly cat with the
novel species of mycobacteria.
The mycolic acid- rich cell wells
are not penetrated by the stain,
and thus show up as negative-
staining bacilli. These negatively
stained bacilli are very numerous,
and are present both within
macrophages and outside the
cells.
288
FIGURE 5: Subcutaneous nodule due
to infection with the novel mycobacterial
species. Similar but smaller lumps were
present over a large area of the cat’s
skin.
Too few cases with a documented aetiology have been reported to provide accurate guidelines for
treatment. Although M lepraemurium can be cultured in vitro, it is so fastidious and slow-growing that in
vitro susceptibility data is lacking to the best of our knowledge. There is enough information in the feline
literature to suggest that where a high index of suspicion for M lepraemurium exists and cases are
diagnosed early, while the disease is still localised, that wide surgical excision of infected tissues provides
the best chance to simply and rapidly effect a cure. Sufficient reconstructive surgical techniques applicable
to the head and limbs have been published to permit aggressive resection techniques to be adopted, with en
bloc resection of all lesions, and reconstruction of the resulting tissue deficits. Such an approach should be
combined with adjunct antimicrobial therapy, which should probably begin a few days prior to surgery
such that effective levels of antimicrobials are present in the blood and tissues at the time of surgery, and
continued into the post-operative period to ensure clean wound margins and primary intention healing.
The drug clofazimine (at a dose rate of up to 10 mg/kg once daily orally; typically 25 to 50 mg every 24 to
48 hours) has the best reported success rate, although it is likely that combination therapy using two or
more drugs will prove to be a superior approach. Drugs likely to have broad antimycobacterial activity
against slow-growing mycobacteria such as M lepraemurium include rifampicin, clofazimine and
clarithromycin although doxycycline, the fluoroquinolones and aminoglycosides may also prove to be
useful.
In cases caused by the novel mycobacterial species, we believe that combination therapy using two or three
of clofazimine (25 to 50 mg per cat orally every day or every other day), clarithromycin (62.5 to 125 mg
once or twice daily) or rifampicin (10 to 15 mg/kg per day) represents optimal therapy. Based on limited
experience, we are unsure, however, which component of therapy is the most efficacious. Clofazimine
capsules, which contain 50 mg of the dye, can be cut into halves using a scalpel blade while wearing
disposable latex gloves, and the two portions placed into gelatin capsules to facilitate dosing. Rifampicin is
made up extemporaneously by dividing the contents of a 150 mg capsule and reformulating the
approximate dose in a gelatin capsule. As clofazimine and rifampicin both can produce reversible
hepatotoxicity, biochemical monitoring of cats regularly during therapy is mandatory, while vomiting
and/or inappetence suggest the need for dosage reduction or temporary discontinuation of therapy. We
have encountered photosensitivity and pitting corneal lesions in cats, which we suspect are attributable to
clofazimine. Our experience is that of these different agents, clarithromycin is the least likely to cause
clinically relevant side effects. Monotherapy with this agent, however, is not recommended because of the
possibility of resistance developing during the course of treatment. Guidelines for duration of therapy are
hard to define, although generally speaking mycobacterial infections should be treated for several months
and typically therapy should be continued for at least 2 months (the life of a macrophage in the tissues)
after disappearance of lesions. Currently we recommend combination therapy using rifampicin and
clarithromycin.
289
Canine leproid granuloma syndrome (“canine leprosy”)
Canine leproid granuloma syndrome (CLGS) or canine leprosy is the most common mycobacterial disease
of dogs in Australia. Patients with this infection are not systemically ill and present with one or more
nodules in their subcutis or skin. The condition was first described in a Boxer and a Bullmastiff from
Zimbabwe in 1973, with similar reports from Australia appearing soon afterwards. Primary skin lesions
consist of single or multiple well circumscribed nodule(s). These lesions can appear anywhere on the dog,
although usually they are located on the head and typically on the dorsal fold of the ears (Figure 4). The
nodules are hard, painless, and vary in size from 2 mm up to 5 cm in diameter. Small nodules are detected
as hard subcutaneous lumps while larger nodules may show superficial hair loss. Very large lesions may
ulcerate. Leproid granulomas are confined to the subcutis and skin and do not involve regional lymph
nodes, nerves or internal organs. Consequently, affected dogs suffer no apparent systemic ill effects. This
suggests that the causal organism(s) have low pathogenicity or special prerequisites, such as a requirement
for low temperature, which permits them to survive and multiply in superficial tissues only.
CLGS has a wide
geographic distribution
in Australasia, with
cases recorded from
Sydney, coastal and
country New South
Wales, Western
Australia, Queensland,
Victoria, Tasmania and
New Zealand. The
causal organism(s) are
likely to have a world-
wide distribution based
on the fact that the
condition has also been
reported in Zimbabwe,
Brazil, California and
Florida.
FIGURE 6: Ulcerated leproid granuloma on the pinnae of a Boxer dog.
Interestingly, there is a
strong propensity for short-coated breeds to be affected, with Boxer and Boxer-cross dogs accounting for
nearly half the cases seen in Australia. Despite the fact that CLGS was first reported nearly 30 years ago,
its aetiopathogenesis has not been fully elucidated. The initial report of the disease by Richard Smith stated
‘lesions appear suddenly, and are usually seen on dogs pestered by biting flies’. This might suggest that
flies, or some other biting insect, inoculate mycobacteria from an environmental niche into susceptible
tissues. The predilection for lesions to develop in regions favored by biting insect vectors, such as the head
and particularly the ears, is consistent with this hypothesis, as is the overrepresentation of short-coated dog
breeds.
Diagnosis is usually straightforward as the distribution of lesions (especially the propensity for the dorsal
ear fold to be affected), coupled with the tendency for lesions to be multiple, particularly in an at-risk
breed, is strongly suggestive of CLGS. Diagnosis can be confirmed by obtaining specimens of
representative lesions for cytologic or histologic examination. Diff Quik stained smears from needle
aspirates typically demonstrate numerous macrophages with variable numbers of lymphocytes and plasma
cells and lower numbers of neutrophils. Usually few-to-moderate numbers of negatively stained, medium-
length bacilli can be detected within macrophages or extracellularly. Histologically, lesions within the
subcutis and dermis consist of pyogranulomas composed chiefly of epithelioid macrophages, Langerhans-
type giant cells with scattered neutrophils, plasma cells and small lymphocytes. The number and
290
morphology of AFB in Ziehl-Neelsen-stained section is highly variable from case to case. Currently, it is
impossible to confirm the diagnosis by culture, as the in vitro growth requirements for this fastidious
organism have not been determined. We are still interested in referral of cases with multiple lesions, as we
are still trying different methodologies in an attempt to culture the causal organism in vitro.
FIGURE 7: Leproid granuloma on the ear of a Mastiff-type dog. Similar lesions were also present on the
flank of this patient.
PCR methodologies using primers designed to amplify regions of the bacterial 16S rRNA gene have been
performed on leproid granuloma specimens from dogs. Using sequence capture PCR for paraffin-
embedded specimens and nested PCR on DNA from fresh tissue specimens, a novel PCR product has been
identified with identical sequence over a 350 bp region. In total, molecular methodologies identified this
proposed novel mycobacterial sequence in material from 16 cases of CLGS indicating that the species
represented by this sequence may be the principal causative agent of CLGS. Our continuing experience,
and those of colleagues in California, supports this contention. Interestingly, the species represented by
this sequence has never been recorded from mycobacterial granulomas affecting the skin or subcutis of
cats, horses, people, or other non-canine mammalian species.
Very little has been written concerning the treatment of CLGS. Many cases are self-limiting, with the
characteristic nodular skin lesions regressing spontaneously with time, typically within 3 to 4 weeks of
appearing. The stated time frame is based on our experiences consulting with veterinarians in relation to
cases diagnosed histologically; by the time the sections are submitted, processed, reported on and we
establish a dialogue with the clinician, lesions are often already starting to regress, either spontaneously, or
in response to antimicrobials (useful for secondary Staphylococcus intermedius, but with unlikely efficacy
for mycobacteria). This occurs presumably as a result of an effective host immune response.
In cases with a limited number of lesions, surgical excision can be curative and provides material with
which to confirm the diagnosis histologically. In other cases, however, the infection progresses to produce
chronic, disfiguring lesions that persist indefinitely. Limited information suggests that treatment with
conventional antimicrobial regimens using β-lactam drugs or doxycycline (as monotherapy) fails to have a
significant impact on the course of infection. One report concerning two dogs from Brazil suggested
topical antibacterial treatment and orally administered rifampicin may be effective.
Our evolving experience treating ‘canine leprosy’ suggests that this infection responds to therapy with
combinations of antimicrobial agents that are known to be effective against nontuberculous mycobacteria,
291
including rifampicin, clarithromycin, clofazimine and doxycycline. Based on our evolving experience, a
combination of rifampicin (10 to 15 mg/kg PO, every 24 h) and clarithromycin (15 to 25 mg/kg PO total
daily dose; divided and given every 8 to 12 h) is currently recommended for treating severe or refractory
cases of canine leproid granuloma syndrome. Unfortunately the clarithromycin component of therapy is
extremely expensive in large dogs. A far more affordable combination consists of rifampicin (at the same
dose) and doxycycline (5 mg/kg or higher every 12 hours), and further studies may prove this to have
similar efficacy to the former regimen. Treatment should be continued until lesions are substantially
reduced in size (typically for 4 to 8 weeks) and ideally until lesions have resolved completely. A topical
formulation containing clofazimine in petroleum jelly may be used as an adjunct to systemic drug therapy.
[This can be prepared by crushing (with a hammer) 40 50mg clofazimine ‘capsules’ within a plastic bag;
the extracted liquid dye is mixed into an ointment with 100 gm petroleum jelly]. Further work is required
to determine the most cost effective treatment regimen for this condition.
Disseminated mycobacterial disease with lymph node and/or

internal organ involvement
There have been numerous single case reports in the literature of disseminated non-tuberculous
mycobacterial infections in cats and dogs. Some of the recorded cases have been young-adult purebred
animals eg Siamese cats, although we have seen very similar disease in two young Abyssinian cats, and the
literature also records sporadic disease in mature adult domestic crossbred cats of various ages.
Apart from two ferrets with disseminated M genavense infections, our experience at the UCVS has been
exclusively with cats. Affected patients are presented for signs reflecting the particular tissues which are
involved, although typically they are gravely ill, reflecting widespread disseminated disease with
involvement of lymph nodes and organs in either or both body cavities. Sometimes nodular or ulcerative
skin lesions are present also in these cases. Rarely the diagnosis is made when negative staining bacilli are
detected in the cytoplasm of white blood cells in a smear of peripheral blood or bone marrow.
Occasionally a cat can present in good health with apparently localized lymph node involvement. Although
surgical excision of the infected node results in temporary resolution of the condition, affected cats may
subsequently succumb to disseminated disease some months later. Most reported cases have been
associated with MAC infections (usually M avium), which presumably develop as a result of some subtle
immunodeficiency that may be inherited in certain lines of feline and canine purebreds.
FIGURE 8: Ziehl-Neelsen
stained section of spleen
from an FIV-positive cat with
disseminated M genavense
infection. Note the
enormous number of acid
fast bacilli within the
macophages.
292
We recently reported a similar presentation in an FIV-positive cat that developed a severe disseminated M
genavense infection. Circumstantial evidence supported the notion that mycobacteriosis developed in this
cat as a consequence of an AIDS-like state.
Diagnosis in these cases is contingent on obtaining representative samples of tissue for laboratory
investigations, typically cytology and culture. The best diagnostic specimen varies from case to case, but
would include lymph node aspirates, bronchoalveolar lavage specimens and portions of abdominal organs
obtained via ultrasound guided biopsy or laparotomy. The presence of granulomatous or pyogranulomatous
inflammation should alert the cytopathologist to the possibility of a mycobacterial aetiology, and the need
for special stains and appropriate culture techniques. Organisms are usually not as numerous as in feline
leprosy-type specimens, although there are exceptions to this generalization. Because these infections can
sometimes be caused by very fastidious organisms such as M genavense, collection of appropriate fresh
specimens for PCR is indicated also.
We have had the opportunity of treating a small number of these cases, and have had gratifying results in
some (but not all) cats using similar drug combinations to those described earlier for the treatment of
‘feline leprosy-like disease’. Based on our limited experience and what has been described in the literature,
cats with localized lymph node involvement should be treated with two or three antimycobacterial agents
even if the affected lymph node has been removed surgically, for they are at risk of later developing
disseminated disease.
Miscellaneous localized mycobacterial infections in

immunocompetent hosts
Cats and dogs with no detectable immune deficiency can become infected by mycobacteria which gain
access to the body through a breach in normal defense mechanisms. Usually these infections remain
localized by a cellular response which is sufficient to prevent more widespread dissemination, but is
insufficiently effective to remove organism from the primary site of infection. Such infections are typically
treated most effectively using surgery and adjunctive antimycobacterial drug therapy, although in some
cases medical therapy alone will be successful.
Three infections the authors have consulted on illustrate the approach to this type of mycobacterial disease.
A cat with a localised corneal mycobacterial granuloma was treated by keratectomy following unsuccessful
attempts at topical and systemic therapy. M intracellulare was cultured from the resected lesion. Surgical
excision was successful, although the lesion recurred two years later, necessitating a second keratectomy.
Another cat had severe diffuse mycobacterial pneumonia due to M thermoresistibile, and this patient was
successfully cured following a long course of combination therapy using clarithromycin, rifampicin and
doxycycline. A similar case, but involving a dog successfully treated for M smegmatis lobar pneumonia
was reported recently. A third cat being treated with cytotoxic multi-agent chemotherapy for lymphoma
developed a focal mycobacterial lesion on its nasal bridge. This MAC infection was treated successfully
using cytoreductive surgery and follow up therapy with clofazimine.
Public Health Significance

Non-tuberculous mycobacteria are generally considered to pose no risk of contagion to other cats or human
beings. These organisms are saprophytes found in abundance in a variety of watery environments, and it is
extremely unlikely that infected feline patients would secrete enough organisms by any route to pose a
threat, even to immunosupressed individuals. In those countries in which tuberculosis is endemic, it is
important to differentiate infections attributable to saprophytic mycobacteria from those due to organisms
such as M tuberculosis and M bovis, which, in contrast, pose a significant threat to other household
members.
293
Further reading
Charles J, Martin P, Wigney DI et al. Pathology of canine leproid granuloma syndrome. Aust Vet J 1999; 77:
799-803.
Deykin AR, Wigney DI, Smith JS, Young BD: Corneal granuloma caused by Mycobacterium intracellulare in
a cat. Aust Vet Practit 1996; 26:23-26.
Foster SF, Martin P, Davis W et al. Chronic pneumonia caused by Mycobacterium thermoresistible in a cat. J
Small Anim Pract 1999; 40:433-438.
Hughes MS, Ball NW, Beck L-A, et al: Determination of the etiology of presumptive feline leprosy by 16S
rRNA Gene Analysis. J Clin Microbiol 1997; 35: 2464-2471.
Hughes MS, James G, Ball N et al. Identification by 16S rRNA gene analysis of a potential novel
mycobacterial species as an aetiological agent of canine leproid granuloma syndrome. J Clin Microbiol
2000;38:953-959.
Hughes MS, Ball NW, Love DN, Davis, Canfield PJ and Malik R: Disseminated Mycobacterium genavense
infection in an FIV-positive cat. Journal of Feline Medicine and Surgery 1999; 1: 23-30.
Hunt GB: Skin-Fold Advancement Flaps for Closing Large Sternal and Inguinal Wounds in Cats and Dogs.
Vet Surg 1995; 24: 172-175.
Lucas J, Lucas A, Furber H et al. Mycobacterium genavense infection in two aged ferrets with conjunctival
lesions. Aust Vet J 2000;78:685-689.
Michaud AJ: The use of clofazimine as treatment for Mycobacterium fortuitum in a cat. Fel Pract 1994;22: 7-
9.
Malik R, Gabor L, Martin P, et al: Subcutaneous granuloma caused by Mycobacterium avium complex
infection in a cat. Aust Vet J 1998; 76: 604-607. 1998.
Malik R, Hunt GB, Goldsmid SE, et al: Diagnosis and treatment of pyogranulomatous panniculitis due to
Mycobacterium smegmatis in cats. J Small Anim Pract 1994; 35: 524-530.
Malik R, Wigney DI, Dawson D, et al. Infection of the subcutis and skin of cats with rapidly-growing
mycobacteria: A review of microbiological and clinical findings. Journal of Feline Medicine and Surgery 2000;
2, 35-48.
Malik R, Love DN, Wigney DI et al. Mycobacterial nodular granulomas affecting the subcutis and skin of dogs
(canine leproid granuloma syndrome). Aust Vet J 1998; 76:403-407.
Mason KV, Wilkinson GT, Blacklock, Z. Some aspects of mycobacterial diseases of the dog and cat. In:
Proceedings of the Annual Meeting of AAVD and ACVD, Davis, California 1989;36.
Peters DH, Clissold SP. Clarithromycin. A review of its antimicrobial activity, pharmacokinetic properties and
therapeutic potential. Drugs 1992; 44:117-164.
Wilkinson GT, Mason KV: Chapter 19: Clinical Aspects of Mycobacterial Infections of the Skin. In: August,
JR, ed. In: Consultations in Feline Internal Medicine. WB Saunders, Philadelphia 1991, p 129-136.
White PD, Kowalski JJ: Enrofloxacin-responsive cutaneous atypical mycobacterial infection in two cats. In:
Proceedings of 7th Meeting of the American College of Veterinary Dermatology. 95, 1992.
ACKNOWLEDGEMENT
This article is devoted to the memory of Daria Love, who provided the inspiration
for the investigation of these various mycobacterial syndromes
294

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Textbook for

Veterinary Microbiology (VETS3040) and

Jennie Hodgson, Jacqui Norris, Katrina Bosward,

This book is copyright of the various source textbooks and the

The book is distributed to enrolled students on a non-profit cost

Commercial use of any material in the book is an infringement

SECTION 1: Gram Positive Cocci 1

SECTION 2: Gram Negative Rods 41

SECTION 3: Gram Positive Rods 173

FAMILY Actinomycetes 175

Gram Positive Cocci

SPECIES HOSTS Coagulase Disease

S. aureus++++ humans, horses, common pathogen (pyogenic) of

Staphylococci divide in several planes to form irregular CLUSTERS.

The distinction between coagulase positive and coagulase negative isolates

Where are they found?

 Staphylococci are found world-wide

KEY POINT Staphylococci are part of the NORMAL FLORA (commensals).

How do they enter the host?

How do they cause disease?

Staphylococci have many virulence factors and therefore are GOOD

Intracellular survival by some strains of Staphylococci allows development of

iii) Capsule and Pseudocapsule

iv) Peptidoglycan and Teichoic Acids

II) The Pathogenesis of Staphylococcal Infections

A BREAKDOWN in HOST DEFENCE MECHANISMS is required for staphylococcal infections

a. type IV hypersensitivity (delayed or cell mediated)

 the typical lesion is the abscess

FIGURE: Pus-filled Abscess in vertebral

I) Coagulase Positive Staphylococci

S. aureus and S. inermedius can cause a wide range of different diseases in

1. Pyogenic Infections ×Øb Õ

2. Arthritis and Osteomyelitis

FIGURE: A case of gangrenous

4. Urolithiasis and Cystitis ×

Diseases ONLY caused by Staphylococcus spp

10. Exudative Epidermitis = Greasy Pig Disease

11. Tick Pyemia

II) Coagulase Negative Staphylococci

Coagulase negative staphylococci rarely cause disease in

ii) Bovine Mastititis

 the role of coagulase-negative infections in bovine mastitis is contentious – mostly S. epidermidis, S.

How would you Diagnose an Infection?

If suppurative infections are observed, the likelihood of staphylococcal

 Inflammatory cells MUST be present in smears of collected samples to

Staphylococci are NOT difficult to ISOLATE, but their presence can be

Clumps of gram positive cocci

 clearance of staphylococci depends chiefly on phagocytosis by macrophages and neutrophils - therefore

How would you Treat an Infection?

General Approach to Therapy

Staphylococci cause opportunistic infections, therefore ALWAYS look for

In general, antibiotics penetrate poorly into abscess. Therefore drainage of

 topical administration of mild antiseptics (e.g. bathing in antibacterial shampoos such as

Specific Treatments – Antibiotics

 antibiotics may be given either topically or systemically

Antibiotic Sensitivity Testing should be performed on isolated of S. areus

Antimicrobial therapy may be ineffective as Staphylococci can survive in

How would you control disease?

What are the Public Health Considerations?

SPECIES 1 2 HOSTS DISEASES

tonsillitis, pharyngitis, otitis, pyoderma,

 Streptococci are gram positive cocci

 their morphology also can vary from cocci to short rods

Staphylococci are found world-wide

the typical lesion is the abscess

the role of coagulase-negative infections in bovine mastitis is contentious – mostly S. epidermidis, S.

Inflammatory cells MUST be present in smears of collected samples to

clearance of staphylococci depends chiefly on phagocytosis by macrophages and neutrophils - therefore

topical administration of mild antiseptics (e.g. bathing in antibacterial shampoos such as

antibiotics may be given either topically or systemically

Streptococci are gram positive cocci

their morphology also can vary from cocci to short rods

the basic pathological process resembles that of Staphylococcal infections

a number of very important and common diseases are caused by streptococci

Streptococci will also frequently grow better in 5% CO2

all members of the Enterobacteriaceae are large, gram negative rods

they are all facultatively anaerobic