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KOALA DISEASE - AN OVERVIEW

Mark Krockenberger
Associate Professor of Veterinary Pathology,
Faculty of Veterinary Science, University of Sydney, NSW. Australia
mark.krockenberger@sydney.edu.au

Acknowledgements
Begun in the early 1980s, the database of koala disease at the Faculty of Veterinary Science was an
initiative of Professor Emeritus Paul Canfield in partnership with the Koala Preservation Society of
NSW. Over a period of 30+ years assisted by a number of people in the Faculty including Alison
Spencer, Susan Hemsley, David Gee, Joanne Connolly, Mark Krockenberger, Damien Higgins,
Kathryn Stalder, Joanna Griffith, Alison Stuart, Lisa Black and large numbers of volunteers in the
Koala Preservation Society of NSW at Port Macquarie.
Introduction
Disease becomes a more important impact on any population as the individual animal becomes a
larger proportion of the population. This is particularly the case in fragmented populations. Disease
prevalence in any wildlife species is a difficult thing to measure. It is unusual for wildlife populations
to be closely monitored prospectively for disease in situ. Apart from anecdote, the published literature
and retrospective mortality studies are the only tools available to attempt the measurement of disease
prevalence. A number of mortality studies have been reported for the koala. This paper presents an
overview of important koala diseases by presenting a summary of a mortality database and reference
to published literature.
Summary of Koala Disease Patterns in Eastern NSW
The necropsy population was predominantly drawn from the north coast of NSW, although a small
number of captive koalas from various collections mainly in Sydney and coastal NSW, and koalas
from other places are included.
Urogenital tract disease is the most important disease category in koalas. This is predominantly
attributable to chlamydiosis. Half of the koalas in this category had cystitis, half had paraovarian
cysts, a quarter had uterine inflammation and about 12% had renal disease (including 3.7% with
oxalate nephrosis). One-third of males in this category had prostatitis. Notably, about 20% of koalas
with urogenital tract disease also had conjunctivitis. Overall, 12% of koalas presented with
conjunctivitis/keratitis. Of these, approx 90% also had urogenital tract disease. There was no sex
predilection but animals over 4 years were over-represented.
The high prevalence of urogenital tract disease is consistent with previous findings (Weigler, Booth et
al. 1987; Degabriele 1989) (30% prevalence of urogenital disease in koalas from Victoria and
southeast Queensland, respectively). Overrepresentation of females with urogenital disease is
supported by the findings of other studies (Obendorf 1983; Canfield 1987). The finding that urogenital
disease is more commonly observed in free-living, rather than captive koalas is supported by previous
evidence (Canfield 1989; Canfield 1990) likely because Chlamydophila positive animals are actively
excluded from captive populations. Over-crowding of free-living koalas due to habitat destruction is
suspected of enhancing the impact of Chlamydophila through increased competition for food and
shelter leading to stress and resultant reduction in host immunity, and enhanced rates of spread
because of increased or closer contact. The prevalence of conjunctivitis/keratoconjunctivitis is
consistent with previous reports, in which prevalence ranged between 8-15% (McKenzie 1981;
Obendorf 1983; Canfield 1987; Weigler, Booth et al. 1987; Connolly 1999). Chronic inflammatory
changes were more commonly recorded, especially in cases where this was the principal finding
(8.8%); but this was to be expected considering that acute cases may resolve spontaneously
(Kempster, Hall et al. 1996). Frequently, koalas with conjunctivitis had concurrent systemic disease ,
with 67.2% having concurrent urogenital tract disease, perhaps indicating that
conjunctivitis/keratoconjunctivitis was not likely to be the primary cause of death (Hirst, Brown et al.
1992). However, it is possible that the progressive body wasting of koalas with chronic ocular
inflammation, as reported by Canfield (Canfield 1990), may have predisposed the animal to the
development of other complicating diseases, which in turn led to death.

Trauma is the next most important category of disease in koalas, largely related to urban expansion.
Almost one-third of koalas presented for trauma, with males and young adults over-represented,
predominantly in winter and spring. Over half of trauma cases were as a result of motor vehicles,
approx 20% due to dog attacks, 4% drowning, 3% falls and only 3% due to bush fires. Bush fire
deaths are likely significantly under-represented.
Trauma has been consistently reported in many mortality surveys, accounting for 35% (Obendorf
1983), 60% (Weigler, Booth et al. 1987), 38% (Canfield 1987), and 42% (Canfield 1990) of cases
seen, respectively. This can be directly attributed to habitat loss and fragmentation and is identified
as a major wild population management issue. Young adult males were over-represented in both
trauma and motor vehicle accident categories, and tended to present during Summer and Spring.
This has been previously reported by a study, which noted that the seasonal trend for deaths due to
trauma correlated with the koala breeding season (Weigler, Booth et al. 1987). This was in contrast
to predation where no significant difference between the sexes was detected. Previous studies have
found that motor vehicle victims were often in good body condition, whereas koalas that had been
attacked by a carnivore tended to be in poor body condition (Canfield 1987; Canfield 1990). It was
suggested that koalas in poor condition were more likely to move to lower parts of the tree, or sit at
the base and therefore be easy targets for predators.
Respiratory tract disease is an important cause of mortality in koalas. This includes aspiration
pneumonia and disease caused by a variety of infectious agents of disease but the most common
specific disease was cryptococcosis (approx 30% of respiratory cases).
Respiratory disease in koalas has been attributed to a range of aetiological agents (Canfield,
Oxenford et al. 1986; Letcher, Weisenberg et al. 1993) (Wigney, Gee et al. 1989). Captive koalas
were over-represented, as reported previously (McKenzie, Wood et al. 1979). The most common
specific aetiology was cryptococcosis, consistent with findings from a recent retrospective study of 43
cases of cryptococcosis (Krockenberger, Canfield et al. 2003). As previously reported, no sex or age
predilections were observed and seasonality was not a feature of cryptococcosis.
Alimentary tract disease is important. 40% of koalas with alimentary tract disease had liver disease
and 35% had intestinal disease, of which the majority were inflammation/necrosis of the
caecum/colon, including a significant number of koalas with haemorrhagic typhlitis.
Neoplasia is an important category of disease in koalas. By far the most common were lymphoid
neoplasias, accounting for approximately half this group (i.e. approx 6% of necropsies), of which
approximately half were lymphoid leukaemias. Mesothelioma accounted for 12% of this group,
cranio-facial tumours of mixed cartilage and bone for 5% of this group, adenocarcinomas of various
organs for 6% of this group (mammary, biliary, hepatic, adnexal), squamous cell carcinomas and
osteosarcoma were each seen in two animals. A single fibrosarcoma and a teratoma were observed.
Neoplasia was observed in 12% of koalas, particularly adults and aged, presenting for necropsy. This
is slightly higher than others have observed (3-11.5%) (McKenzie 1981; Canfield 1987; Weigler,
Booth et al. 1987; Canfield 1990 respectively).
Tumour types commonly seen include
lymphosarcoma; mesothelioma (earlier recorded as nodular peritonitis) and craniofacial tumours, and
have all been described previously (Backhouse and Bolliger 1961; McKenzie 1981; Sutton 1986;
Canfield, Perry et al. 1987; Canfield, Sabine et al. 1988; Canfield, Hartley et al. 1990; Canfield,
Hartley et al. 1990; Canfield and Hemsley 1996). Lymphosarcoma was first observed in two koalas in
1961 (Backhouse and Bolliger 1961). Since then, several case series have been reported (Finnie
1978; McKenzie 1981; Canfield, Sabine et al. 1988; Canfield and Hemsley 1996), and lymphoid
neoplasia is now considered the most common neoplasm of the koala (Canfield 1990). Koalas are
commonly leukemic, and lesions may be present in any of a number of organs, although found most
commonly in liver and spleen (Connolly, Canfield et al. 1998). While early work identified viral
particles (Canfield, Sabine et al. 1988), it is not until recently that an association with Koala Retrovirus
(KoRV) has been established (Tarlinton, Meers et al. 2005).

Table 1: Summary of Retrospective Mortality Survey of >1100 Koalas from eastern NSW
including sex predilection and age predilection
Disease
% of Necropsy
Sex
Age Predilection
Population

Predilection

No Significant Lesions

8.4%

Joeys and Juveniles

Urogenital

40%

Female

Aged

Cystitis

20%

Female

Adult and Aged

12.5% Females

N/A

Adult and Aged

Paraovarian Cysts

25% Females

N/A

Aged

Oxalate Nephrosis

1.5%

Joeys,

Uterine Inflammation

Juveniles

and

Young Adults
Conjunctivitis/keratitis

12%

Adult and Aged

Trauma

28.5%

Male

Young Adult

Motor Vehicle Trauma

15.8%

Male

Young Adult

Attacked By Dog

5.7%

Joeys,

Juveniles

and

Young Adult
Respiratory

15%

Cryptococcosis

4%

Gastrointestinal

15%

Neoplasia

12%

Adult and Aged

Lymphosarcoma

6%

Adult

Mesothelioma

1.4%

Craniofacial tumours

0.6%

Bones/Joints

3.0%

Aged

Skin

3.1%

Limitations
Mortality studies have their limitations. The most important ones are the selection bias at several
levels and the limitations of incomplete records. Selection bias occurs naturally with an unknown
proportion of sick and injured koalas being detected by people. Further bias is transmitted into the
study due to case selection for necropsy examination. This is controlled at the carer group level and
varies with the person at the decision making level. An obvious instance of both these issues is the
low representation of bushfire cases in the database. Logistical issues also interfere at times with
cadavers sometimes having to be frozen, often precluding histopathological diagnosis.
Additional Koala Disease Case Reports from the Literature
Inflammatory Conditions
- Parasitic Diseases
- Sarcoptic mange (Barker 1974; Brown, Seawright et al. 1982; Obendorf 1983)
- Demodectic mange (Vogelnest, Vogelnest et al. 2000)

Toxoplasmosis (Canfield, Hartley et al. 1990; Hartley, Dubey et al. 1990)


Cryptosporidium (Morgan, Deplazes et al. 1999)
Encephalitizoonosis (Nimmo, Snowden et al. 2007)
Cat fleas (Griffin, Canfield et al. 1983)
Bartonella (tick borne diseases) (Vilcins, Old et al. 2008; Vilcins, Kosoy et al. 2009; Vilcins,
Old et al. 2009; Kaewmongkol, Kaewmongkol et al. 2011)
Metastrongylus spp. parasitic pneumonia (McColl and Spratt 1982)
Trypanosomiasis (McInnes, Gillett et al. 2009; McInnes, Hanger et al. 2011)

Viral Diseases
- Papillomavirus has been isolated from koalas and has the potential to cause disease
(Antonsson and McMillan 2006).
- GammaHerpesvirus (Vaz, Whiteley et al. 2011)
Bacterial Disease
- Chlamydiosis (see above)
- Tyzzers disease (Bacillus piliformis) (Canfield and Hartley 1991)
- Melioidosis (Ladds, Thomas et al. 1990)
- Mycobacteriosis (Mycobacterium ulcerans) (Mitchell, Jerrett et al. 1984; McOrist, Jerrett et al.
1985; Mitchell, McOrist et al. 1987; Portaels, Chemlal et al. 2001)
Idiopathic
- Pancreatitis (Canfield 1986; Higgins and Canfield 2009),
Degenerative Disease
- Diabetes mellitus (Shimizu, Yasuda et al. 1989; Hemsley, Govendir et al. 1998)
Disorders of Growth
- Various congenital defects have been reported including an atrial septal defect (Atwell and
Booth 1990), cerebellar abiotrophy (Kuwamura, Murai et al. 2009), shoulder and hip dysplasia
(Pye, Hamlin-Andrus et al. 2008; Pye 2009)

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CRYPTOCOCCOSIS IN KOALAS
Dr Mark Krockenberger
Associate Professor of Veterinary Pathology
Koala Infectious Diseases Research Group
Faculty of Veterinary Science, University of Sydney, NSW, Australia.
mark.krockenberger@sydney.edu.au
Agent Nomenclature
Cryptococcosis is an important systemic mycosis of animals and people, with a worldwide distribution.
It is the second most common specific infectious disease of koalas, although is not nearly as common
as chlamydiosis overall, is much more common than chlamydiosis in captive populations.
Cryptococcosis is caused by two species of fungus; Cryptococcus neoformans and Cryptococcus
gattii. These are closely related and are regarded as part of a species complex but have different
epidemiologies.
Unfortunately, the two species of fungus used to be regarded as varieties of the same species and
were named C. neoformans var. neoformans and C. neoformans var. gattii, respectively (See Table
1).
Table 1: Nomenclature of the Cryptococcus species complex
Kwon-Chung,
Franzot, Salkin Boekhout,
Kwon-Chung,
Bennett et al.
et al. 1999 [4]
Theelen et al.
Boekhut et al.
1982 [3]
2001 [5]
2002 [6]
C. neoformans

C. neoformans

- var. neoformans
(serotypes A, D,
AD)

- var. grubii
(serotype A)

C. neoformans
(serotypes A,
D, AD)

C. neoformans

C. neoformans

- var. grubii
(serotype A)

- var. grubii
(serotype A)
(molecular types VNI,
VNII)

- var.
neoformans
(serotype D)

- hybrid
(serotype AD)
(molecular type VNIII)
- var.
neoformans
(serotype D)

- var. gattii
(serotypes B, C)

- var. gattii
(serotypes B,
C)

Current Acccepted
Nomenclature

C.
bacillisporus

C. gattii

- var. neoformans
(serotype D) (molecular
type VNIV)

C. gattii

(serotypes B,
(serotypes B, C)
(serotypes B,
C)
(molecular types VGI,
C)
VGII, VGIII, VGIV)
NB: The current C. neoformans-C.gattii species complex is considered to contain 4 molecular types of
C. neoformans (VNI-IV) and 4 molecular types of C. gattii (VGI-IV). Two molecular types (VNII and
VGII) appear to be globally expanding.
Epidemiology of Cryptococcosis An Environmentally Acquired Disease
Broadly, cryptococcosis in immunocompromised people is caused by C. neoformans and in
immunocompetent people is caused by either C. neoformans or C. gattii. In animal species, an
association with prior immunosuppression has not been demonstrated.
Both species of fungus are acquired from the environment and are not contagious and are not
regarded as zoonoses with regards to direct spread of disease from one sick animal to another
animal or person. Exposure to the same highly contaminated environmental source has certainly
been associated with outbreaks of disease in groups of animals (both single species groups and

mixed species groups). In fact, in Vancouver Island there are documented cases of clinical disease in
dogs, cats, horses and people in the same household. All apparently exposed to the same
environmental source of the fungus [8]. With cryptococcosis in captive groups of koalas, cases of
clinical cryptococcosis are generally associated with high environmental loads [personal observation].
There is a single case report of cryptococcosis as a zoonosis. In that case, the isolate of C.
neoformans var. grubii from an immunosuppressed person with cryptococcosis was found to be
identical to that found cultured from the cage of the persons pet bird. The bird was not found to be
suffering cryptococcosis [7], but obviously provided the dried bird guano substrate that is an excellent
environmental source of C. neoformans. Good hygiene, including minimizing aerosolisation, is
recommended when in contact with environmental substrates likely to be contaminated with fungi.
Cryptococcosis in koalas can be predominantly attributed to C. gattii. Very rarely C. neoformans and
indeed other Cryptococcus spp. may cause disease in koalas, however, because of the vast
predominance of C. gattii as the causative agent it is worth checking with the diagnostic laboratory
about identification methods used in any cases suspected to be caused by any other cryptococcal
species. See comments about laboratory diagnosis.
What the fungus actually does in the environment is of great importance, for a number of reasons. It
influences the morphological form of the fungus and therefore the nature of its interactions with the
hosts innate defences but it is also postulated that environmental interactions/pressures have led to
the acquisition of a number of virulence factors including the capacity to survive intracellularly [9],
formation of the large polysaccharide capsule and capacity to utilise phenolic substrates found in the
brain. C. neoformans and C. gattii are both dimorphic, and can exist in either a yeast form or in a
hyphal or mycelial form. In the yeast form, both species usually have a thick extracellular capsule
(thicker in C. gattii) and are large (5-50 microns in diameter), depending on the thickness of the
capsule. The yeast form is not easily aerosolised and is larger than the size of particles that can
evade normal innate defences of the respiratory tract and therefore is not in a highly infective state.
In diseased tissues and on standard laboratory culture media, the fungus is in the yeast form.
The hyphal or mycelial form of the fungus may produce very small basidiospores by sexual
reproduction, given the right environmental conditions. These spores are 1-2 microns in diameter and
can easily evade host respiratory tract defences and penetrate to the alveolar spaces of the lungs. It
is thought that the basidiospore is the most likely infectious particle resulting in disease.
It appears that VGII and VNII strains are particularly good at forming basidiospores, which is likely
important in the emergence of these two strains globally over the past 10 years.
How do we detect the morphological form of the fungus in the environment? As soon as we
culture it on laboratory media it immediately assumes a yeast form, irrespective of its prior
morphology. So, with selective media (birdseed agar) we can easily detect the presence of the
organism but we cant determine the environmental morphological form. From indirect evidence
(evidence of genetic recombination) we know that C. gattii VGI and VGII exist in the environment in
the hyphal form [10]. Molecular epidemiological studies continue to elucidate the cryptic life of C.
neoformans species complex in the environment.
What is the distribution of C. gattii in the environment? Cryptococcosis caused by C. gattii has a
relatively restricted global distribution, including Australia and mainly subtropical and tropical regions,
which has been attributed to the natural distribution and human-aided dispersal of eucalypts with
which it has been associated [11, 12]. It has always been present in some temperate regions of
Australia. In fact, the most reliable and consistent environmental sources of this fungus are trees of
the Murray Darling river system in Mediterranean climate and temperate regions of south east
Australia. This distribution reflects the distribution of C. gattii VGI (the most likely cause of koala
cryptococcosis).
Relatively recently (past 10-15 years), C. gattii VGII has dramatically expanded into new temperate
climates in the world including western Canada and again more recently into north-western United
States of America (Washington State) [8]. Even more recently, there is increased detection of VGIII
isolates, particularly in California and western USA.

C. gattii is associated with disease in individuals with apparently normal prior immune
function and is only rarely associated with disease in severely immunocompromised
individuals. In people, this has been attributed in the past to lack of exposure of this population of
patients to highly contaminated environments containing C. gattii. There is no data to support this
premise and in fact it appears to be quite implausible considering the size and distribution of
immunocompromised population of people; and the ability to isolate C. gattii from large geographical
regions of Australia, including within urban areas. It is more likely to be related to features of the hostpathogen interaction at a tissue, cellular and subcellular level. A greater understanding of how C.
gattii causes disease at a cellular and tissue level is beginning to emerge [19].
What is the distribution of C. neoformans? C. neoformans is associated environmentally with bird
guano predominantly but also some trees, predominantly rainforest trees from South America and
some species found in Australia. It is associated with high prevalence in immunosuppressed patients
and was for many years an AIDS-defining illness in many parts of the world. However, if you remove
the severely immunocompromised human patients from the mix, it is possible to detect approximately
equal prevalence of cryptococcosis caused by C. neoformans and that caused by C. gattii in people in
Australia [13]. Therefore, it is important to remember that even in humans C. neoformans can affect
immunocompetent people. In dogs and cats and other animals, we certainly see C. neoformans in
apparently immunocompetent individuals.
Epidemiology of Koala Cryptococcosis [14-17]
The vast predominance of cases of cryptococcosis in koalas has been attributed to C. gattii with
extremely rare isolation of other cryptococcal species. This contrasts with approximately 1/3 of
canine cases and 1/5 of feline cases of cryptococcosis attributed to C. gattii, and approximately of
human cases attributed to C. gattii (in people of apparently normal immune function), depending on
geographical region.
The host-pathogen-environment interaction has three distinct possible outcomes that are particularly
prominent in the koala (these possible outcomes are a feature of the Host-Pathogen-Environment
Interactions (HPEI) of cryptococcosis in other animals and people also). The fourth possible outcome
is elimination of the fungus by the host upon exposure. This is likely occurring all the time but cannot
be detected.
1.
colonisation of mucosal surfaces (upper respiratory tract and skin)
In this outcome, the fungus can be cultured from the nasal cavity mucosal surface or the skin,
in the absence of any clinical or subclinical evidence of tissue invasion or disease. There is no
evidence of inflammation. There is no structural evidence of tissue invasion. There is no
laboratory evidence of tissue invasion.
2.
subclinical disease (respiratory tract, lymph nodes)
There are no clinically obvious manifestations of disease, however, limited structural evidence
of tissue invasion can be detected microscopically and there is laboratory evidence of tissue
invasion.
3.
clinical disease (respiratory tract, tissues of the head, CNS, skin, disseminated disease)
Clinical manifestations of the disease do vary a little but include predominantly signs reflecting
respiratory tract involvement or central nervous system involvement. There is little doubt that
the respiratory tract is the primary focus of disease in koalas, apart from a small minority of
direct cutaneous inoculation. The incidence of involvement of the CNS in dogs and koalas is
similar and much greater than that seen in cats, and that the local involvement of structures
contiguous with the nasal cavity is greater in cats than dogs or koalas. The upper and lower
respiratory tract are important sites of disease in the koala, whereas in dogs, cats and birds the
lower respiratory tract is affected less frequently.
Our general findings over the past 15 years can be summarised in the following way. ( For only a
small percentage of exposed animals is the HPEI outcome clinical disease, but that for a variable but
much greater percentage of koalas the outcome is subclinical disease, and for a very high proportion
of koalas mucosal colonisation is the outcome of the HPEI.)
Furthermore, we have documented progression from colonisation to subclinical disease and from
subclinical disease to clinical disease in a number of individuals and we have formulated a general set
of guidelines determining intervention at various stages.

A predictive conclusion from our work has been that in areas of high environmental contamination, the
HPEI outcome is skewed towards colonisation, subclinical disease and clinical disease. Alternately, it
can also be shown that clinical disease correlates strongly with prevalence of subclinical disease and
degree of environmental contamination by this fungus.
There has been no apparent indication of general immunodeficiency in koalas that contract
cryptococcosis compared with those that dont at a very broad level. In other words there is no
general evidence of predisposition to disease caused by infectious agents of disease in animals that
later contract cryptococcosis. A potential role for Koala retrovirus (KoRV) and koala immune
genotype in association with cryptococcosis is still to be elucidated and the current work in this area
will be of great interest.
At present the genotype information that we have formulated for parts of the MHCII locus of the koala
hasnt promised an easy answer to the differing susceptibilities to cryptococcosis.
Clinical Signs
The most frequent clinical signs seen in koalas are upper respiratory tract signs, intracranial
neurological signs and skin signs.
Upper respiratory tract signs range from massive epistaxis to persistent mucopurulent nasal
discharge (unilateral or bilateral) to dyspnoea and marked stertor; depending on the primary site
involved, chronicity and severity of disease. When the rostral nasal cavity is involved extension to the
oral cavity has been seen in some cases.
Central nervous system involvement usually is generalised with seizures, with localising signs
unusual.
Skin disease is manifested by dermal and subcutaneous mass lesions primarily of the skin of the face
(nasal bridge, lip, lateral face), although we have seen skin lesions of the pinnae of the ears and also
the feet. When involving the skin of the face it is usually an extension of nasal cavity or paranasal
sinus disease.
At all sites, the mass lesions produced may vary from a reasonably firm consistency through to a
fluctuant fluid consistency on palpation, with the majority having a gelatinous to fluctuant consistency
on palpation. The consistency of the mass is very dependent on the degree of host response, with the
gelatinous to fluctuant swellings occasioning little host response and being comprised simply of
masses of fungus; while the more firm swellings will have a considerable granulomatous host
inflammatory response.
Diagnosis
Diagnosis is simple when there is a mass lesion that can be aspirated or biopsied. Every clinician
should be able to do this in their practice. All that is needed is a Romanowsky type stain (DiffQuik )
and a microscope.
Diagnosis is established by observation of heavily encapsulated yeasts forming mass lesions
microscopically by cytology or histopathology. The yeasts should display narrow-necked
budding and be approximately 10-50 m in diameter (including capsule) with a yeast cell body approx
5-25 m in diameter. An inflammatory host response is evident to varying degrees from the presence
of neutrophils and macrophages to granulomatous inflammation with epithelioid and multinucleate
giant cell macrophages. In Australia, there are few differentials on microscopic examination. In other
parts of the world, agents such as Histoplasma capsulatum, Blastomyces dermatiidis, Coccidioides
immitis, Paracoccidioides brasiliensis need to be considered as alternate possibilities.
Confirmation of the identity of the fungus should be performed by culture from the lesion
(preferably a normally sterile site with specimens taken appropriately). Note that different laboratories
identify Cryptococcus species in different ways and it is always worth clarifying the result with the lab
if they have not made it clear to what level they have identified the organism. Some labs still report
both C. neoformans and C. gattii as C. neoformans.

If culture is not available, immunohistochemistry can be performed to confirm species. This method
has some limitations with one molecular sub-type of C. gattii (VGII).
PCR detection and speciation is increasingly successful, based on sequencing of the ITS region
(internal transcriber sequence) of the fungal genome. It is not always possible to extract sufficient
quality DNA from formalin fixed tissues in order to get a result by this method.
Serology (detection of circulating cryptococcal antigen)
Most clinicians would follow up diagnosis with an LCAT (latex agglutination test for circulating
cryptococcal antigen). This can be used to confirm tissue invasion if samples cannot be obtained for
microscopy and the titre may be used serially to monitor response to treatment. While the serological
titre does not really provide a good handle on prognosis, what we have noticed is that subclinical
disease can be seen with titres of up to 64, whereas clinical cryptococcosis in koalas usually has a
serological titre of cryptococcal antigen in excess of 128. These are generalisations and clinical
disease has been observed with a positive titre of only 16. A positive titre in excess of 2 is evidence
of tissue invasion. Depending on the circumstance of the animal different strategies will be employed
(see below in Treatment section). It is worth remembering that titres cannot be accurately compared
amongst laboratories or amongst detection methods, however, within a laboratory titres are
comparable.
NB: we are currently validating a new immunochromatographic method of detecting circulating
cryptococcal capsule antigen against existing latex agglutination methods.
It is important to remember that normal koalas will display nasal colonisation by C. gattii (and
infrequently C. neoformans) without any tissue invasion or clinical signs of disease. Therefore culture
of the nasal cavity alone is insufficient to diagnose cryptococcosis. Evidence of tissue invasion and/or
host response must be documented either microscopically or serologically.
Pathology
Location. It is very common to see nasal cavity mass lesions that extend into surrounding tissues
including the paranasal sinuses; punching through bone to the subcutaneous tissues of the face;
extension down the incisive duct to the rostral oral cavity and the lips; punching through the hard
palate to the oral cavity; extension through the cribriform ethmoidal turbinates to the rostral cranial
cavity; nasopharyngeal masses that may obstruct the whole nasopharynx by time of diagnosis.
Size & Shape. The lesions observed may be very small (2-5mm diameter) through to involving the
whole nasal cavity (>40mm diameter). The lesions are usually well circumscribed focal or multifocal
mass lesions, however, more diffuse lesions are also possible (especially in the meninges).
Consistency. The lesions are generally gelatinous in consistency but may be firmer if there is a
significant host inflammatory response developed. We sometimes see this in the treatment phase
more than prior to diagnosis.
Colour. This will vary with the degree of host inflammatory response. When there is minimal host
response, the lesions are often a yellow tinged relatively clear colour. When there is a significant
granulomatous response, the lesions will be yellow-white and firm on cut surface.
Microscopic Pathology. Presence of abundant, usually budding, encapsulated yeasts with a variable
host response usually skewed towards granulomatous inflammation with the presence of
macrophages and multinucleated giant cell macrophages. Neutrophils, eosinophils, lymphocytes and
plasma cells may also be seen in the inflammatory exudate. Neutrophils predominate in the very
early lesions, however, this stage of disease is usually not seen diagnostically. Organisms may be
seen intracellulary in macrophages or (predominantly) extracellularly. A common occurrence is to see
large numbers of organisms with very little host response. With therapeutic intervention, the
inflammatory response becomes more marked.
Treatment
Antifungal agents. Antifungal therapy revolves around 3 possible agents amphotericin B,
fluconazole and itraconazole. Ketoconazole can be used but is probably the least favoured choice
due to limited efficacy against Cryptococcus and side-effects. Successful therapy of significant

clinical disease requires the use of amphotericin B in addition to the long term therapy with
one of the azoles.
Amphotericin B can be successfully delivered to koalas using a modification of the feline protocol
developed by Professor Richard Malik [18]. Generally, a cumulative dose of at least 20mg/kg over a
period of weeks to months seems to be important. Sometimes the cumulative dose may need to be
40mg/kg.
We always have used either itraconazole or fluconazole in conjunction with the amphotericin therapy
and for long periods after cessation of amphotericin.
Itraconazole or fluconazole can be used as a sole agent of therapy but care should be taken to
measure for effective serum levels especially if using as sole therapeutic agent. Treatment failure
with the azoles appears likely to be related to absorption or metabolism factors resulting in suboptimal
serum levels (both duration of detectable levels and peak levels)[pers. obs]. For fluconazole, twice
daily 15mg/kg is currently recommended.
Nursing. Nursing of these cases is of utmost importance. These animals undergo long periods of
therapy (months to years) and close attention needs to be paid to nutritional status (food intake and
energy intake), body weight fluctuations, stresses like breeding stress, transport stress,
treatment/handling stress. A dedicated keeper/nurse seems to be very important.
Environmental treatment. It is appearing to be more and more important in the treatment of
subclinical and clinical cryptococcosis to reduce the environmental load of the fungus. Failure to do
this is most important in the final eradication of small foci of disease and prevention of re-infection.
We currently advise an initial environmental treatment of replacement of static climbing branches
where possible and topical treatment with a suitable disinfectant at the appropriate concentration on a
weekly basis. Initially, using a scrubbing brush where possible and a spray otherwise. Monitor the
koalas for signs of skin irritation of the paws.
Topical treatment of the environment can then continue fortnightly for a number of months and may
even continue indefinitely depending on the scope of the problem. Currently we monitor the effects of
the environmental treatment on environmental load where we can.
This approach has lowered subclinical disease substantially in captive groups of koalas that had high
prevalence of subclinical disease.

What to do with a low positive LCAT in the absence of clinical signs of disease.
In the absence of clinical signs, cryptococcal antigen serology titres of 2-16 may be monitored
monthly and a treatment regime put into place if there is a gradual or rapid elevation in the titre. That
is my usual approach. Additionally, as mentioned above, it may be worth implementing an
environmental treatment protocol.
Alternatively, the koala may be placed onto either daily itraconazole therapy or twice daily fluconazole
therapy and monitored. At least the cryptococcal antigen serology should be monitored but ideally
drug level should also be monitored.
A cryptococcal antigen serology titre in excess of 32 should be investigated further for evidence of
detectable disease and monitored very closely. I now almost always institute therapy in these koalas
(even without any evidence of detectable disease) because monthly serological monitoring may miss
a sudden elevation prior to subsequent severe clinical disease and death of the koala.
Koalas with any evidence of subclinical disease should not be transported or traded. Any subclinical
disease should be resolved prior to the stresses of handling involved in these sorts of transactions.
ACKNOWLEDGEMENTS
The Koala Infectious Diseases Research Group has had research grant support from the Australian
Research Council (with Linkage partners The Australian Koala Foundation, The Koala Preservation
Society of NSW, Symbion Vetnostics, Pfizer, and WIRES) and the Hermon Slade Foundation. The
Koala Infectious Diseases Research Group includes Mark Krockenberger, Paul Canfield, Damien
Higgins, Merran Govendir, Susan Hemsley, Joanna Griffith, Sarah Jobbins, Nathan Saul, Quintin Lau,

Ben Kimble, Lisa Black, Tiziana Beninati. A number of other people have contributed to research into
koala disease at Sydney University over the years.

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CHLAMYDIAL DISEASE IN KOALAS


Dr Damien P Higgins
Koala Infectious Diseases Research Group
Faculty of Veterinary Science, University of Sydney, NSW, Australia.
damien.higgins@sydney.edu.au
AGENT
Chlamydiosis is the most commonly occurring known disease of wild koalas and is accredited with
causing proliferative conjunctivitis, rhinitis/ pneumonia complex, and disease of the urinary and
reproductive tract (Brown and Grice, 1986; Obendorf and Handasyde, 1990). Chlamydial organisms
isolated from koalas were initially classified as Chlamydia psittaci (Brown and Grice, 1984), and then
reclassified as Chlamydophila pecorum and Chlamydophila pneumoniae koala biovar (Glassick et al.,
1996; Everett et al., 1999), though strong arguments have been made for a return to the single genus
Chlamydia (Schachter et al. 2001; Stephens et al. 2009). Both species can be isolated at ocular and
urogenital sites, and equally in males and females, though disease associated with Ch. pecorum is
more commonly severe (Jackson et al., 1999). Several Chlamydia-like organisms of unknown
significance have also been isolated from koalas (Devereaux et al., 2003). The burning question of
how and when Ch. pecorum and Ch. pneumoniae arrived in Australia remains to be answered
definitively, but the strongest data to date, obtained from multi-locus studies and contrary to previous
partial ompA locus-based analysis (Jackson et al. 1997), indicates that koala Ch. pecorum has most
likely evolved as a discrete lineage following a limited number of introduction events (Marsh et al,
2011).
The Chlamydiaceae are weakly gram-negative, coccoid, obligate intracytoplasmic parasites sized
between bacteria and viruses. There are two morphologic forms of these organisms within cells; the
elementary body (0.3- 0.35 m) and the reticulate body (0.5-1.3 m). The life cycle of the
Chlamydiaceae is characterised by: uptake of the infectious elementary body (EB) into a phagosome;
reorganisation of the infecting EB into a reticulate body (RB), which multiplies by binary division;
secondary reorganisation of the RB into infectious EB; and finally, release of infectious EB from host
cells by cell lysis (Storz and Kaltenboeck, 1993). Several factors, such as exposure to interferon
gamma (IFNg), or some antibiotics, can induce formation of persistent aberrant forms (Beatty et al.,
1995). Aberrant bodies represent an arrested stage of development, showing characteristics
consistent with incomplete or inappropriate division of RB, and play a key role in persistence of the
organism in tissues. The induction of these more cryptic forms is mediated via tryptophan depletion
and, at least in C. trachomatis of humans, is therefore influenced by tryptophan-regulating genotype
of the chlamydial strain (Perry et al., 1999; Nelson et al., 2005) as well as the type and intensity of the
host-response which, in turn, is likely to be affected by a range of environmental and host factors.
Persistence, and the antigenic stimulation produced by these forms, are key elements of the
pathogenesis of this disease.
Pathogenesis
In koalas, Ch. pecorum is transmitted venereally (Obendorf, 1983; Handasyde, 1986), and possibly by
non-venereal contact between fighting animals during the mating season (Brown and Woolcock,
1988; Weigler et al., 1988; Jackson et al., 1999). Jackson et al. (1999) commonly found Ch. pecorum
and Ch. pneumoniae in koalas less than one year of age, suggesting that vertical transmission,
perhaps during birth, via monocytes in milk, or during pap-feeding (establishment of intestinal flora of
young by ingestion of maternal caecal faeces), may be significant. Transmission between anatomical
sites can occur (Brown and Grice, 1986). This might be external, but systemic spread also appears
possible as Ch. pneumoniae circulates in blood mononuclear cells (Bodetti and Timms, 2000) and
systemic infection has also been described for Ch. pecorum in cattle and sheep (Storz and
Kaltenboeck, 1993), Ch. felis in cats (TerWee et al., 1998), Ch. psittaci in birds and Ch. suis in pigs
(Storz and Kaltenboeck, 1993).
The majority of tissue damage in chlamydiosis is caused by the hosts response rather than by cell
lysis associated with chlamydial replication. However, it is unclear whether it is persistent local proinflammatory responses of Chlamydia-infected cells (Stephens, 2003), or delayed-type
hypersensitivity to chlamydial heat shock antigens (hsp60), developed during reinfection or

recrudescence (Grayston et al., 1985), that induces chronic inflammation and disease. Heat shock
proteins, which are up-regulated in persistent life stages of Chlamydia, have some direct effects on
the immune system and also contain epitopes conserved across chlamydial species, other microbes
and, in some cases, across mammals. As a result, they can stimulate deleterious non-specific (Kol et
al., 2000), pathogen-specific, trans-species, trans-genera and even host auto-immune cell mediated
and humoral immune responses (Morrison, 1991). Some evidence exists that these mechanisms,
elucidated for C. trachomatis in humans, apply to koalas; histological lesions are similar between the
species (Hemsley and Canfield, 1997) and, like women, koalas with tubal fibrosis and infertility display
elevated anti-hsp60 antibodies (Higgins et al, 2005a).
The immune evasion necessary to allow repeated infection by C. trachomatis in humans is achieved
by variation at a dominant surface antigen, the Major Outer Membrane Protein (MOMP), coded by the
ompA locus. Lack of ompA diversity within a single large koala population where disease is common
(Higgins et al 2011) indicates that, as for koalas infected with larger experimental doses (Kempster et
al, 1996), repeated infection by multiple MOMP serotypes is not necessary to induce disease in
naturally infected koalas. Nevertheless, due to the wide conservation of hsp antigens, a pathogenic
role for infection by multiple Ch pecorum serotypes, concurrent infection by Ch. pecorum and Ch.
pneumoniae or other Chlamydia-like organisms should still be considered possible. In addition the
potential impact of novel or virulent Ch. pecorum strains should be considered. Antigenic diversity of
Ch pecorum exists among koala populations (Jackson et al. 1997; Higgins et al, 2011), and marked
diversity at a proposed virulence locus, ORF663 (Yousef Mohamed et al 2008), also exists, even
within a single MOMP serotype within one population (Higgins et al 2011).
SIGNIFICANCE
Chlamydiosis is one of the most commonly treated wildlife diseases in Australia, with significant
dedication of resources. Nevertheless, the more we learn about this disease and its treatment, the
more questions are raised. Anecdotal evidence and extrapolation have formed the basis of many of
our conceptions about this disease; pathology research has been hamstrung by a lack of koalaspecific reagents, and as with most wildlife species it is difficult to perform controlled studies with
small sample sizes and limits on invasiveness of procedures.
Modelling of the effect of chlamydiosis on koala populations by Augustine (1998) indicated that
chlamydiosis alone is unlikely to result in the extinction of koala populations. However, this prediction
came with several provisos: that the populations are not subject to other pressures; and that the
transmission rate is not increased due to previously unrecognised transmission pathways, or
increased mating frequency. These predictions need to be reviewed as new knowledge of the
pathogenesis and epidemiology of chlamydiosis emerges and as new environmental scenarios
develop. For example, one might expect transmission rate to increase in populations where
increasing population densities affect normal social structures; if environmental factors affect
shedding of the organism by altering the host response; or where a high proportion of females have
uterine or salpingeal fibrosis but intact ovaries, thereby failing to conceive and returning to oestrus
several times throughout the breeding season. In addition, we currently know virtually nothing of the
impact of preventing mortality of carriers of Chlamydiae in koala populations as a result of treatment
and re-release. Recent work suggests strongly that, although hospitalisation and treatment improve
the clinical status and reduce chlamydial shedding, long term elimination of the organism is
questionable (Griffith, 2010) due to the chronic status of most infections encountered and the low
blood drug concentrations achieved following administration of enrofloxacin (Griffith et al 2010) and
chloramphenicol (Govendir et al 2011), the most commonly applied antibiotics, to koalas.
Prevalence of infection and disease vary among populations. A wide range of host, pathogen and
environmental factors have been suggested to affect disease outcomes from chlamydial infection and
are under investigation. These include: genetic diversity (Jobbins 2010; Lau, unpublished data);
territorial stress; poor nutrition; exposure to toxins; infection by the koala retrovirus (Simmons, 2011);
chlamydial strain virulence (Higgins, unpublished data) and multiple strain infection. Consideration of
these should inform management of koalas and their habitat and, in the case of chlamydial strain
diversity, relocation and corridor construction programs.

CLINICAL SIGNS
External examination
Chlamydial infection can be clinical or subclinical. Koalas with cystitis, urethritis or bladder
dysfunction (oedema, fibrosis or smooth muscle hyperplasia) exhibit frequent and occasionally painful
urination, and staining and/ or ulceration of the rump. If lower urinary tract disease is absent,
advanced structural reproductive tract and renal disease can exist without external signs. Weight loss
is often a feature, though females with infertility from advanced structural reproductive tract disease
can be in very good body condition.
Ultrasonography and palpation
With some practice, spherical ovarian bursal cysts of approx 15mm diameter or greater can be
reliably palpated, adjacent to the dorsal internal abdominal wall. The animal is placed in dorsal
recumbency and fingers pressed into the space lateral to the epipubic bones, and moved across the
dorsal abdominal wall. Large cysts (> 4cm diameter) can often be palpated in relaxed conscious
animals; they often extend to the ventral abdominal wall beside the epipubic bones, or even anterior
to the epipubic bones when very large (up to 15cm diameter). As in other species, the kidneys can be
palpated for irregularity in shape or size.
Ultrasonography of the koala has been partially described (Stalder, 2003). In that study and others
(Griffith, 2010), the following could be visualized readily by ultrasonography: thickening of the bladder
wall, pyometron, ovarian bursal cysts (single or multiloculated), hydroureter and gross renal changes
such as hydronephrosis and renal fibrosis. Bladder lesions and ovarian bursal cysts have been seen
to progress during hospitalisation. Bladder thickening sometimes partially resolves (presumably the
component related to oedema) but in almost all cases ovarian bursal cysts do not.
Presence of ovarian bursal cysts is the most commonly used indicator of infertility but this is not
infallible. Gross and histopathological study of 34 female koalas revealed that, of 21 koalas with
occlusion of the reproductive tract lumen, five had no ovarian bursal cysts. Also, five of 21 koalas
with ovarian bursal cysts had no histological evidence of reproductive tract occlusion, and one of
these had no evidence of chlamydial infection or reproductive tract pathology (Higgins et al., 2005b).
Depending upon the extent of uterine fibrosis, it is reasonable to assume that some koalas with
unilateral cysts should be able to breed, but these appear likely to be exceptions and presence of
cysts, especially if found in association with a small and undeveloped pouch during breeding season,
is the most reliable indicator of infertility of koalas to date.
Haematological and biochemical analysis, including urinalysis
There are no clinical pathological findings that are typical of chlamydiosis. Leukocytes are usually
within reference values. Anaemia (typically non-regenerative), hypoproteinaemia, and dehydration are
common in debilitated koalas, but are not specific for chlamydiosis. Renal disease is not uncommon
in chlamydiosis and should be evaluated similarly to other species, by examination of blood urea,
creatinine, water intake, urinalysis and clinical signs and history.
Ocular disease
In an infection trial of five koalas with a wild koala ocular strain of C. psittaci, Kempster et al. (1996)
described the course of disease in three phases: the first begins two to six days post inoculation, with
a mild serous discharge, blepharospasm and very mild palpebral and conjunctival oedema. The
second phase, beginning at 8-11 days post inoculation and progressing to maximum severity over 3-5
weeks, consists of mucopurulent discharge, hyperaemia, severe chemosis of the conjunctiva and
nictitating membrane, papillary hypertrophy of the nictitating membrane, corneal oedema and
vascularisation, epithelial punctate stippling, rhinitis, and peri-ocular alopecia. The third phase,
resolution, begins at 5-7 weeks post inoculation and coincided with the resolution of a naturally
transferred infection, which had been detected 3-5 weeks earlier in the non-inoculated eye.
Resolution of papillary conjunctival hypertrophy and residual corneal vascularisation was complete 14
weeks post infection. Mild scarring persisted.
The subconjunctival infection of a single koala with koala isolates of Chlamydia psittaci by Brown
and Grice (1986) induced persistent infection and conjunctival proliferation at 9 weeks. Keratitis was
observed at 16 weeks and remained beyond 13 months.

GROSS PATHOLOGY
Urogenital tract
Gross findings are typical of pyometron, metritis, cystitis, or occlusive fibrosis of any part of the
reproductive tract or bladder. Obendorf (1988) observed that the peritoneal opening to the bursa
(peritoneal ostium) was patent in undistended ovarian bursae but not distended bursae (ovarian
bursal cysts or peri-ovarian cysts). Typically, the bursa was adhered to the ovary with fibrinous
strands, there was damage to associated uterus and uterine tube and the ovaries were small and
flattened against the bursal wall (Obendorf and Handasyde, 1990). Hydroureter and hydronephron
are common sequelae of fibrotic cystitis and renal changes are typical of hydronephrosis or
pyelonephritis in other species, with or without resulting fibrosis (Higgins et al, 2005b). Severe cases
of urogenital disease can also show fibrotic adhesion of any of the pelvic viscera (Obendorf, 1988).
MICROSCOPIC PATHOLOGY
Ocular
Hemsley and Canfield (1997) described the histological appearance of chronic lesions. They reported
villous epithelial hypertrophy and hyperplasia, and occasional granulation, fibrosis and focal
squamous metaplasia. Leukocyte infiltrates were heaviest in the lamina propria-superficial
submucosa and consisted of neutrophils (most commonly epithelial), lymphocytes (throughout, but
most common in lamina propria and submucosa), and occasional macrophages (most common in
lamina propria and submucosa). In addition, plasma cells, which were mostly distributed immediately
beneath the basal epithelium, became predominant and extended deeper into the submucosa in more
severe cases. These findings were consistent with descriptions of trachomal conjunctivitis in humans
(el-Asrar et al., 1989).
Pathological processes in the female reproductive tract can be classified as any combination of
occluded or patent, and active or inactive (Higgins et al., 2005b): Active components are
characterised by oedema, hyperaemia, and submucosal and epithelial infiltrates with a prominent
neutrophil component and a variable, but usually prominent, lymphocyte component. Generally,
these also have intense, plasmacytic submucosal infiltrates. In inactive tracts, only mild plasmacytic
submucosal infiltrates and scattered macrophages remain. Occluded tracts show one or more
areas where the lumen and epithelium is replaced by fibrous tissue that occasionally contains
remnants of epithelial cells and uterine glands. Chlamydial inclusions have been demonstrated in
epithelial cells of all parts of the reproductive tract, their presence being associated with lesions
containing an active inflammatory component. Chlamydial inclusions have also been detected in
submucosal macrophages in some sections, scattered immediately under the infected epithelium and
in aggregates deep in the submucosa.
Canfield (1989) also described a range of histological changes in the bladders of koalas, similar to
those described in the reproductive tract. In addition, he described epithelial hyperplasia and
hydropic change resulting in thickening and folding of the mucosa, oedema and fibrosis of the
submucosa, and hyperplasia of the muscularis. Using immunohistochemistry, Higgins et al., (2005b)
and Hemsley and Canfield (1997) demonstrated chlamydial inclusions in association with lesions in
the kidney, bladder and urogenital sinus.
Renal histological abnormalities range from pyogranulomatous pyelonephritis, to focal mononuclear
interstitial nephritis, and interstitial fibrosis without evident inflammation (Canfield and Spencer, 1993;
Higgins et al., 2005b).
Canfield (1989) described prostatitis of koalas. It was characterised by large accumulations of
neutrophils in the ducts and glands, and fibrosis and mononuclear infiltration of the lamina propria of
the urethral mucosa. Chlamydial inclusions have been identified in situ by immunohistochemistry
(Hemsley and Canfield, 1997). More recently, orchitis and epididymitis of koalas, associated with Ch.
pecorum has been described (Dief et al, 2010).
DIFFERENTIAL DIAGNOSIS
It is possible that other, non-chlamydial bacteria cause reproductive or urinary tract disease, either in
combination with, or instead of Chlamydophila spp. Non-chlamydial bacteria cultured from the
reproductive tract of female koalas include: alpha haemolytic Streptococci (pyometra), Staphylococci,
Corynebacterium spp., and non-haemolytic E. coli (uterine tubes and bursae and pyometra)

(Obendorf, 1981; Canfield et al., 1983; Obendorf, 1988). In addition, Weigler et al. (1988) isolated
diptheroid rods, Bacillus spp., Pasteurella spp., Aspergillus spp., and Klebsiella oxytoca. The
significance of these in disease is currently unknown.
DIAGNOSTIC PATHWAY
Methods to detect chlamydial infection reflect the development of technologies over 30 years.
Culture, immunofluorescence, latex agglutination, antigen and antibody ELISAs and PCR have all
been used historically. Each has its attributes in terms of sensitivity, specificity, availability and utility,
but it is the complexities in interpreting these tests that has probably led to the most confusion for
practitioners, both when assessing for treatment and when screening animals for translocation or
quarantine.
Swab collection: Immunohistochemical studies tell us that even in some advanced lesions, epithelial
chlamydial inclusions range from frequent to extremely rare (Hemsley et al., 1997). These studies
also indicate that significant numbers of inclusions can be present in circulating monocytes (Bodetti et
al., 2000), in submucosal macrophages, and in histiocytes of the spleen, liver, lung and inflammatory
sites (Higgins et al., 2005b). It is important to remember that a negative swab indicates that epithelial
inclusions are not present at that site at that time; the host is not necessarily free of the organism.
Swab technique is also important. Swabs should be drawn across the mucosa firmly to ensure a
representative sample of epithelial cells is removed. Viable organisms are more reliably obtained on
a second swab (Wood et al., 1992), the first having removed predominantly exudate and surface
debris, the second removing viable epithelial cells. Higher antigen yield on the first swab is
associated with more severe clinical disease (Wood et al., 1992); yield on the first swab probably
reflects active replication and shedding, the second swab probably reflects presence of replicating
and/or inactive persistent forms in the epithelial cells.
TM

Antigen ELISA: The Clearview test kit is the most widely accessible test kit for diagnosis of
chlamydiosis in koalas. It is a point-of-care membrane-based ELISA for C. trachomatis antigen. Its
sensitivity for koala Chlamydophila is 130-1300 EBs / ml and when compared to a gold standard of
culture and long-term clinical observation of a group of 18 koalas, its sensitivity and specificity were
91% and 100%, respectively (Wood et al., 1992). False positives occurred in this study when non8
chlamydial bacteria were present at 10 / ml, so faecal contamination should be avoided.
TM

In our experience, Clearview is reliable for detecting Chlamydophila in koalas with active clinical or
subclinical disease, but is less reliable in koalas with old, inactive structural lesions (Griffith 2010).
Based in immunohistochemical observations on these types of cases (Higgins et al 2005b), this is
likely to be related to the lesser number of epithelial inclusions in these cases.
Immunohistochemistry: Immunohistochemistry for chlamydial organisms (Higgins et al 2005b) is now
available commercially for use on formalin-fixed necropsy material (Veterinary Pathology Diagnostic
Services, University of Sydney). As Chlamydiae are primarily within the epithelial cells, tissues must
be freshly fixed and selected to maximise the quality and quantity of epithelium on the slide.
PCR: The sensitivity of PCR, particularly real-time PCR has revolutionized the way we look at
chlamydial testing. All genes have conserved and variable regions, but there is a tendency to
increasing specificity going from PCRs based on conserved 16s rRNA regions, to GroEL, and to
ompB, to ompA (MOMP) genes, which have regions that are species, or even strain, specific (Bush et
al., 2001). Liaison with those performing the PCR is essential to ensure results are interpreted
correctly. For example, a PCR using 16s regions may detect the recognised pathogens Ch. pecorum
and Ch. pneumoniae, and a range of novel chlamydia-like organisms whose clinical relevance
remains unknown (Devereaux et al. 2003). PCR of a more variable region, such as ompA or ompB is
more likely to detect only the currently recognised pathogens. Species-specific real-time PCR for Ch.
pecorum and Ch. pneumoniae (Griffith 2010) is now available commercially (Veterinary Pathology
Diagnostic Services, University of Sydney).
Owing to the sensitivity of PCR, false negatives are most likely to occur in cases where infection is not
actively progressing at the swab site. In such cases, systemic tests may be of greater use.

Systemic tests
Serology: Serology has been used widely in field studies of prevalence of infection, with results that
are difficult to interpret. This is partly due to the different tests used, and partly due to our limited
understanding of the marsupial immune response. We found that Chlamydial hsp60 and hsp10
seropositivity is as strongly influenced by the disease status of the koala as it is by infection status
(Higgins et al., 2005a): low titres were associated with more chlamydial inclusions, and intense
submucosal and epithelial infiltrates of neutrophils and lymphocytes, with or without a background of
fibrosis and plasma cell infiltration; whereas high titres were associated with advanced reproductive
tract fibrosis, but few chlamydial inclusions and either no inflammatory infiltrate, or mild infiltrates of
macrophages and plasma cells only. It appears that antibody testing is not likely to be useful in
detecting infected animals during active disease but is likely to detect chronic, inactive cases where
swab-based PCR or antigen ELISA are more likely to be falsely negative. There is currently no
commercially available serological test for Chlamydia in koalas, largely due to the transient availability
of marsupial-specific immunological reagents.

ACKNOWLEDGEMENTS
Much of the research performed by the Koala Infections Diseases Research Group and described in
this paper was supported by an Australian Research Council Linkage Grant with partners: The
Australian Koala Foundation, The Koala Preservation Society of NSW, Symbion Vetnostics, Pfizer,
and WIRES; and by the Hermon Slade Foundation. More information on koalas and the Australian
Koala Foundation can be found at www.savethekoala.com
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KOALA LYMPHOSARCOMA AND OTHER HAEMATOLOGICAL OR IMMUNE DISORDERS


Dr Damien P Higgins
Koala Infectious Diseases Research Group
Faculty of Veterinary Science, University of Sydney, NSW, Australia.
damienh@vetp.usyd.edu.au
AETIOLOGICAL AGENT
This is currently unknown, but the koala retrovirus (KoRV) has been considered as a potential
aetiological agent of lymphosarcoma, myeloid leukaemia, aplastic anaemia, myelodysplasia and
immune dysfunction (Worley et al., 1993; OBrien et al., 1997; Hanger et al 2003). Tarlinton et al.
(2005) found an association between plasma koala retroviral load and lymphoid disorders, and a
weaker, non-significant association between KoRV plasma retroviral load (viraemia) and
chlamydiosis. However, a causative role for KoRV in disease is not yet established; in other species
retroviral titers can increase as a result of lymphoproliferative disorders or chronic inflammation. In
addition, a subsequent, larger study, with more rigorous investigation of disease status, failed to find
similar associations (Simmons 2011). There is, however, some preliminary supportive evidence for a
role for KoRV in immunomodulation, in that a conserved retroviral protein, which is present in KoRV,
inhibits lymphocyte proliferation and alters cytokine expression in-vitro in other species (Fiebig et al.,
2006). This remains to be tested in the koala and verified by in-vivo observations.
The latest advances in the study of KoRV come from the thesis of Simmons (2011). Key findings
include: that KoRV is widespread in Australia, with 100% prevalence in Qld and NSW, 73% in
mainland Victoria, 28% on Victorian Islands, and 15% on Kangaroo Island, South Australia; it appears
that the virus may be in the process of endogenising, showing more exogenous behaviour in
southern populations; there is a very similar virus in the native Australian rat (Melomys burtoni),
strongly suggesting that it is the original source of KoRV; and, though transmission has not yet been
confirmed, arthropod transmission appears possible.
SIGNIFICANCE
Lymphosarcoma is a widely recognised disease of koalas. No formal studies of its prevalence have
been performed, but it has been reported to occur in approximately 4% of animals necropsied at a
NSW koala hospital over a 5 year period (Canfield et al., 1987). Hanger et al. (2000) suggests that up
to 80% of mortalities in captive koalas might be attributed to lymphoma and a variety of leukaemias.
CLINICAL
Common clinical presentations of lymphosarcoma include lymphadenomegaly, weight loss, poor coat
condition, or any clinical sign that might be caused by metastasis-induced organ dysfunction. It most
commonly occurs in middle-aged koalas, without sex predilection and progression of disease can be
rapid. Connolly et al. (1998), report that 63% of affected animals are leukaemic but suggest that this
high percentage might be associated with the late presentation of koalas with this disorder, in
comparison to cats (25%) or dogs (33%). Due to the high prevalence of leukaemia in koalas with
lymphosarcoma, haematological examination is a useful tool. Abnormal lymphocytes or
lymphocytosis are commonly seen, and flow cytometric and immunohistochemical methods have
been developed to discriminate between T and B lymphocytes in blood or in fine-needle aspirate or
biopsy of enlarged lymph nodes, or bone marrow.
Hanger et al (2003) describe aplastic anaemia, myelodysplasia and immune dysfunction in koalas,
presenting with some or all of the following: chronic ill thrift, lymphopaenia, marginal anaemia, atypical
lymphocytes, small, gelatinous and poorly cellular lymph nodes, intractable or chronic infection by
ubiquitous organisms, stomatitis and glossitis, typhlocolitis or caeco-colic dysbiosis syndrome,
cryptococcosis, severe chlamydiosis. The non-specific nature of many of these signs make difficult
interpretation of their cause, but diagnosis of aplastic anaemia and myelodysplasia are based on
changes in blood and bone marrow, while remaining cases are classified tentatively as KIDS, or koala
immune deficiency syndrome. The aetiology of these, and the degree of overlap among these
conditions and with general debilitation due to displacement and starvation, remains to be elucidated
and pose an important area for research.

GROSS PATHOLOGY
In koalas, lymphosarcomas have been described in most tissues. Most common are: lymph nodes
and abdominal tissues (predominantly liver and spleen, but also gastrointestinal and urogenital tracts
and kidney), and cervico-mediastinal tissues such as thymus or cervical lymph nodes (Connolly et al.,
1998).
HISTOPATHOLOGY
Lesions are typical of lymphoid tumours of other species, consisting of diffuse sheets of neoplastic
cells infiltrating or replacing normal parenchyma. Sheets of lymphocytes commonly exhibit a starry
sky appearance due to the presence of histiocytic cells (macrophages) scattered throughout.
Lymphocyte types can be small, medium or large; with more than half being medium. Nucleoli are
prominent only in larger cell types (Connolly et al., 1998). In many cases neoplastic lymphocytes are
difficult to differentiate from normal lymphocytes and classification as neoplasia must rest on clinical
history, the behaviour of the cells (sheeting, invasion and exclusion of other cell types), and
demonstration of clonality by immunohistochemistry.
Koala lymphosarcomas examined by Connolly et al. 1998 were of T cell (51%), B cell (24%) and
unlabelled (25%) immunophenotypes. As discussed by these authors, unlabelled lymphosarcomas
might be either not expressing surface markers due to their anaplastic state, might have originated
from CD3-/CD79b- cells, e.g. NK cells, or might have failed to react due to poor fixation or autolysis.
Abdominal metastasis is common and immunophenotype prevalence in this region approximates that
observed overall, indicating no immunophenotypic predilection for this area (Connolly et al. 1998).

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