WDA Conference 2003 Papers Final

WILDLIFE DISEASE ASSOCIATION AUSTRALASIAN SECTION

2003 Annual Conference Healesville, Victoria 1-5 December 2003

Barry Laing Munday 1932 - 2003

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Barry Laing Munday, died on the 10 of May, 2003 in Launceston. Barry was born in Hawthorne Victoria in 1932 but moved with his family to Hobart when still a youngster. He commenced his veterinary science degree in 1951 undertaking a first year general science course at the University of Tasmania and then 4 years study at the University of Sydney Veterinary School, supported by a Tasmanian Department of Agriculture studentship. He graduated with Honours in 1955 and with distinctions in bacteriology, protozoology and parasitology. In 1956 he became Government Veterinary Officer (GVO) in Launceston and Burnie. In late 1957, having recently married Faye Gluskie, Barry took up the GVO position on King Island providing both a clinical service and disease control work for production livestock and companion animals under the Department of Agriculture’s Veterinary Nationalisation Scheme. As Veterinary Officerin-Charge of disease control his responsibilities included formulation of State guidelines and policy for the Bovine and Ovine Brucellosis Eradication program and the Ovine Footrot Campaign. A field vet on King Island was on call virtually 24 hours a day, 7 days a week but Barry also conducted collaborative work with K.D. Skerman and A.K. Sutherland on micronutrient deficiencies, specifically copper and cobalt deficiencies affecting livestock production. In December 1960, Barry moved back to Launceston to commence his career in pathology in the Mt. Pleasant Laboratories. th My acquaintance with Barry goes back to 28 November 1967 when I identified some filarioid nematodes from Tasmanian possums which he had sent to John Sprent at the University of Queensland. Thus commenced decades of correspondence, some collaborative research on Sarcocystis spp. in native rodents and snakes in southeastern New South Wales and the uninterrupted movement of wildlife parasite specimens from Launceston to Brisbane and later to Canberra. Barry was awarded a Wool Research Trust Post Graduate Scholarship to the University of Melbourne Veterinary School in 1968 where he obtained a Master of Veterinary Science degree for his study of the

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epidemiology of toxoplasmosis, a common cause of ovine abortion. In 1971 he was invited to become a Foundation Member of the Australian College of Veterinary Science. In 1972 Barry commenced to drum up support for an Australasian Section of the international Wildlife Disease Association (WDA). By April 1973 he had garnered the required 10 signatures of supporters, myself included, and was able to forward a petition to the parent body in the USA requesting formation of an Australasian Section of the WDA. He was successful and, at the request of the Parent Body, undertook the role of inaugural Chairman until the first scientific and annual general meeting was held in conjunction with an ANZAAS meeting at the Australian National University in Canberra in January 1975. To his credit, the Australasian Section of the WDA has never looked back and remains a strong and forceful body of interested and active scientists and practitioners to this day. For that effort, and for his enormous contribution to wildlife disease studies and education in Australia, Barry was awarded a trip to Uppsala, presented a keynote address on wildlife disease in Australia and accepted the Distinguished Service Award of the WDA parent body in July1985. Or, as Barry put it to me in a letter seeking some slides for his talk, “As you well know, some very convincing liars have managed to organise a trip to Sweden for me.” This was followed by life membership in the Australasian Section. Barry’s research interests centered on the comparative disease processes of wild and domestic animals, particularly the zoonotic diseases toxoplasmosis, leptospirosis and brucellosis. He also played a major role in sarcosporidiosis research demonstrating that several species could infect individual intermediate host species but that each species of Sarcocystis had a specific definitive host. He tested numerous predator-prey associations in his life cycle studies and demonstrated species of Sarcocystis cycling between owls and rodents, between snakes and rodents, between rabbits and cats and between rodents and cats. Much of his parasitological and wildlife disease work was conducted as a sideline to his large workload and duties as a diagnostic veterinary pathologist. Barry was Chief Veterinary Pathologist in the Tasmanian Department of Primary Industry (DPI) and Officer-in-Charge of the Mt. Pleasant Laboratories during the period 1978-1988. He was involved with the Thyroid Advisory Committee, cetacean stranding investigations, the establishment of a fish pathology capability at the Mt. Pleasant Labs, an important adjunct to development of the farmed trout industry and a participant in the medical science body - The Haematology, Immunology and Neoplasia Group. He became an Honorary Research Associate of the Medical School, University of Tasmania in 1982. Following resignation from the DPI, Barry entered academia as Reader in Aquaculture at the Launceston Campus of the University of Tasmania and was invited to join the editorial board of the Journal of Fish Diseases. In the last few years Barry returned to his life-long interest in wildlife disease and parasitology in the School of Human Life Sciences at the Launceston Campus, with involvement in further studies of Sarcocystis spp. infections in native animals and of the frequently fatal infection of platypus with the fungus Mucor amphibiorum. Barry’s untiring efforts at liason and co-operation with, and education of a diverse spectrum of people interested in the many aspects animal health over more than four decades, is to be applauded. My pile of correspondence, almost all handwritten, is more than 30 mm deep, many of the Dept of Agriculture, Mt Pleasant Laboratories pages having been splashed with formalin during the writing and th still bearing hints of that malodorous chemical. On 30 July 1973, still having never met each other, he wrote to me, “As you say, once again we seem fated not to meet. I sincerely hope our first meeting is not in a geriatric ward!” We finally came face to face 7 years after our first correspondence, when Barry came to Canberra in January 1975 for the inaugural meeting of the Australasian Section of the WDA. Barry would often stay with my wife and I during his one or two day trips to Canberra on “Veterinary/Quarantine/Vertebrate Pest Control” business. He always arrived with something in hand, a good South Australian wine or a coloured fleece for spinning, although on one occasion we missed out on a forequarter of venison because the family was to be away on holidays during his visit. He was also a generous host and who could ever forget Barry’s slow roasted Tasmanian potoroo garnished with bacon! Barry Munday’s name shall remain associated with the study of wildlife parasitology and disease in Australia. The news of his death wasn’t helped by the stark realisation that I still owed him a beer for being unable to meet with him the last time he was in Canberra. He is survived by his wife Faye and their three children, Phillip, Paul and Louise. Dave Spratt 1 July 2003

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Contents: PAPERS AND ABSTRACTS HAEMATOLOGY AND SERUM BIOCHEMISTRY IN THE LEOPARD SEAL (Hydrurga leptonynx) AND WEDDELL SEAL (Leptonychotes weddellii) IN EASTERN ANTARCTICA – R.B. Gray, P.J. Canfield, and T.L. Rogers………………………………………………………………………………………………………….. 7 HAEMATOLOGICAL CHARACTERISTICS OF COMMON RINGTAIL POSSUMS (Pseudocheirus peregrinus) WITH BITE INJURIES - P. Clark and P. Holz………………………………………………………8

DEVELOPMENT AND INVESTIGATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF SERUM ANTIBODIES TO HENDRA VIRUS IN FLYING FOXES - Andrew Breed, Hume Field, John Molloy, Kim Halpin and Peter Daszak…………………………………………….. 11

LYSSAVIRUSES AND WILDLIFE IN THE AUSTRALIAN CONTEXT - John Bingham…………………….12

A REVIEW OF AVIAN INFLUENZA IN AUSTRALIA - Chris Bunn……………………………………………15 ANIMAL DISEASE INVESTIGATION AT AAHL - Maria Cardoso…………………………………………….20 PACHECO’S DISEASE – AN AUSTRALIAN EXPERIENCE - Ian Hough………………………………..…22 TASMANIAN DEVIL FACIAL TUMOURS - Tim McManus……………………………………………………24 POWERFUL OWL – Ed McNabb…………………………………………………………………………………26 RADIAL NERVE DAMAGE IN RAPTORS - Franciscus Scheelings and Paul Ramos……………………..27 MANAGEMENT OF CHRONIC CORNEAL DISEASE IN A MALAYAN TAPIR (Tapirus indicus) AT MELBOURNE ZOO - Kate Bodley and Chloë Hardman……………………………………………………….29 THE TALE OF TWO OCEAN SUNFISH (MOLA MOLA) – AN UNUSUAL MORTALITY IN AN UNUSUAL SPECIES - Larry Vogelnest……………………………………………………………………………………….30 SARCOPTIC MANGE IN URBAN COMMON BRUSHTAIL POSSUMS (Trichosurus vulpecula) - Anne Fowler………………………………………………………………………………………………………………..33 SARCOPTIC MANGE IN AGILE WALLABIES - David McLelland and Jennifer Youl………………………35

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MOLECULAR CHARACTERIZATION OF A HERPESVIRUS ISOLATED FROM INDIAN GYPS VULTURES - Maria Cardoso……………………………………………………………………………………...36

DISEASES OF CROCODILE HATCHLINGS IN CAPTIVITY - Philip Ladds…………………………………37 ATYPICAL MYCOBACTERIOSIS IN A GROUP OF BARBARY SHEEP - Tim Portas…………………….40 THE CAPE OTWAY CENTRE FOR CONSERVATION ECOLOGY - Lizzie Corke and Shayne Neal……41

KOOKABURRAS - A MODEL SPECIES FOR DETERMINING REHABILITATIVE SUCCESS - Phillipa Mason………………………………………………………………………………………………………………..42

HOP, SKIP and JUMP!....Ogden Nash re-visited on Oz Small Mammals OR Handling specimens, data and analyses in long-term studies in parasite ecology - a case history - David M. Spratt and E. MargaretCawsey……………………………………………………………………………………………………43 HEALTH MONITORING OF THE JENOLAN CAVES BRUSH-TAILED ROCK WALLABY (Petrogale penicillata) COLONY - Larry Vogelnest and Mikaylie Wilson………………………………………………….51

CROSS REACTIVE ANTIBODIES WITH POTENTIAL TO IMMUNOPHENOTYPE MARSUPIAL TUMOURS S Hemsley, M Jones and J Cordell………………………………………………………………………………52 GAMMA INTERFERON ENZYME IMMUNOASSAYS AND THEIR USE IN THE INVESTIGATION OF TUBERCULOSIS IN A WESTERN LOWLAND GORILLA - Helen E. McCracken………………………….55 SURVEILLANCE OF CHYTRID IN TADPOLE POPULATIONS IN SE QLD - Pearl Symonds …………..62 ADVENTURES IN AVIAN ORTHOPAEDICS AND BEAKISTRY - Jennifer Youl and David Mclelland…..63

SHELL FRACTURE REPAIR USING GLASS IONOMER CEMENT IN THE LONG-NECK TORTOISE (Chelodina longicollis) - Anne Fowler and Nick Magelakis…………………………………………………….66

FURTHER INVESTIGATIONS INTO DISEASES OF THE KOALA (Phascolarctos cinereus) - K Stalder, G Allen and M Krockenberger………………………………………………………………………………………..68 ABDOMINAL LUMPS IN THREE PYTHONS - Teri Bellamy ………………………………………………….70 FLUOROSIS AS A PROBABLE CAUSE OF CHRONIC LAMENESS IN EASTERN GREY KANGAROOS Emily Clarke ………………………………………………………………………………………………………..71

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POSTERS ROSS RIVER VIRUS INFECTION IN CAPTIVE MACROPOD POPULATIONS IN URBAN NSW – J M Old, O Chan and E M Deane……………………………………………………………………………………...87 IMMUNOPATHOLOGY OF LYMPHOID TISSUES FROM LONG-NOSED AND LONG-FOOTED POTOROOS – L.J. Young, P.H. Holz, and E.M. Deane……………………………………………………….88 IDENTIFICATION OF IMMUNOREGULATORY MOLECULES IN SMALL WALLABY SPECIES – L.J. Young, G.A. Harrison, P.H. Holz, and E.M. Deane…………………………………………………………….90

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HAEMATOLOGY AND SERUM BIOCHEMISTRY IN THE LEOPARD SEAL (Hydrurga leptonynx) AND WEDDELL SEAL (Leptonychotes weddellii) IN EASTERN ANTARCTICA RB Gray, PJ Canfield, and TL Rogers Australian Marine Mammal Research Centre PO Box 20 Mosman NSW 2088 Faculty of Veterinary Science University of Sydney NSW 20062 Email: [email protected]

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As upper trophic species, leopard seals (Hydrurga leptonynx) and Weddell seals (Leptonychotes weddellii) may be useful indicator species of change within the Antarctic ecosystem. To enable monitoring of the health status of these species, reference ranges for parameters of health, including haematology and serum biochemistry, need to be determined. The objectives of the present study were to: 1. Establish reference ranges for haematology and serum biochemistry in these species, 2. Ascertain the effect of seal sex, moult status and year of sampling on these analytes in the leopard seal, 3. Compare haematology and biochemistry in these species, and in leopard seals hauling out in NSW and in Antarctica. Blood was collected from adult leopard and Weddell seals during the Austral Summer seasons of 1999-2002 near Davis Station, Eastern Antarctica and from leopard seals hauled out along the NSW coast between 1972-2003. Manual haematology and commercial biochemistry were performed. Reference ranges were developed for haematology and biochemistry in the leopard and Weddell seal. Significant differences between species were seen in haematology and serum biochemistry values, however, for the majority of the analytes, the levels were generally within the normal ranges reported for domestic species and other seal species. No significant differences in haematology and biochemistry were seen with seal sex, season of sampling, or moult status in the leopard seal, except for differences in anion gap and fibrinogen between seasons. Significant differences in haematology and biochemistry values were determined for leopard seals in Antarctica compared to those in NSW waters, reflecting differing health status, environmental and physiological influences. The development of reference ranges for these indicators of health in the leopard and Weddell seal are an essential prerequisite for the future monitoring of the health status of these populations and the ecosystem they occupy.

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HAEMATOLOGICAL CHARACTERISTICS OF COMMON RINGTAIL POSSUMS (Pseudocheirus peregrinus) WITH BITE INJURIES 1

P. Clark and P. Holz

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School of Clinical Sciences, Division of Veterinary and Biomedical Sciences, Murdoch University, South St, Murdoch, Western Australia, 6150. 2 Healesville Sanctuary, P.O. Box 248, Healesville, VIC, 3777.

Introduction Common ringtail possums (Pseudocheirus peregrinus) are widely distributed over eastern Australia, including suburban areas. These possums may encounter several non-native predators including cats, dogs and foxes and consequently sustain bite injuries. The resulting tissue damage and inoculation of bacteria may cause significant inflammation. Haematological alterations, notably changes to leukocyte concentration and morphology, may allow recognition of an inflammatory disorder and aid in determining the likelihood of survival of injured possums. To the authors’ knowledge, no previous studies have characterised the leukocyte response of common ringtail possums with clinical inflammation. We examined the leukocyte response in six injured, free-living, common ringtail possums that were presented to the veterinary service of Healesville Sanctuary. Case Histories and Methods Case 1, an adult male, was presented in a poorly responsive state with multiple bite wounds evident. Case 2, an adult, was moribund and had multiple bite wounds evident. Case 3, a sub-adult female, presented unable to use its hind legs and with multiple puncture wounds around the stifle, abdomen and lumbar region. Case 4, an adult female, was moribund and had multiple bite wounds evident. All of these cases were euthenased after examination, due to the poor likelihood of survival. Case 5, a sub-adult male, presented after having been extracted from a cat's mouth and had a single puncture wound evident. The possum was alert and was treated with an oral antibiotic solution (0.1 ml/kg Noroclav: 140 mg/ml amoxycillin and 35 mg/ml clavulanic acid, Norbrook Laboratories Australia Pty Ltd, New Gisborne, VIC) but deteriorated and died five days later. Case 6, an adult female, was presented moribund with bite wounds and was euthenased after examination. For each of the cases, a sample of blood was collected by venepuncture of the ventral caudal vein. These were mixed with EDTA and analysed by a commercial veterinary laboratory using an automated analyser (Cell Dyn 3500, Abbott Diagnostics). Manual differential leukocyte counts were performed on blood films stained with Wright’s and Giemsa stains. The morphology of haematological cells was assessed by light microscopic examination of stained blood films. Results The haematological data for all of the possums is given in Table. 1. Compared to published haematological values for common ringtail possums, five possums (cases 1-4 and 6) had a leukopenia due to both neutropenia and lymphopenia and one possum (case 5) had a total leukocyte concentration that was within reference values but had a mild neutrophilia and concurrent lymphopenia. Neutrophils with morphological atypia, such as Döhle bodies, increased cytoplasmic basophilia and vacuolation were present but difficult to objectively assess due to the paucity of cells. A mild anaemia was evident in three possums (cases 1, 3 and 5) and a mild polycythaemia in one (case 4). Discussion Common ringtail possums in a suburban environment may encounter several non-native predators including cats, dogs and foxes. Cat bites usually result in small puncture wounds, whereas bite wounds from dogs and foxes cause crush/tear injuries (Rose 1999). Cat bites may also result in the inoculation of a range of bacteria, from the oral cavity, which may subsequently result in localised or systemic inflammation (Love et al. 2000).

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In the current study all possums exhibited an inflammatory leukogram. Four possums displayed a neutropenia consistent with acute or severe inflammation (Jain, 1986). This is consistent with acute inflammation inciting neutrophils to migrate from the blood to the site of tissue injury, which have not been replaced by release of cells from the bone marrow. As these were free-living animals, the time from the bite and presentation could not be determined (with the exception of case 5). One case exhibited a mild neutrophilia and atypical neutrophil morphology (Döhle bodies, cytoplasmic basophilia). All cases showed a lymphopenia that was consistent with the effects of increased secretion of glucocorticoids stimulated by pro-inflammatory cytokines (Turnbull & Rivier, 1999). The ‘eosinopenia’ present in all cases is also consistent with glucocorticoid-mediated effects. The ‘monocytopenia’ seen in five animals is of undetermined significance. Neutrophils from clinically healthy common ringtail possums typically have 3-6 nuclear lobes of coarsely clumped chromatin and a moderate amount of colourless to slightly eosinophilic cytoplasm (Clark 2003). In the current study, atypical neutrophil morphology encountered included Döhle bodies, increased basophilia of the cytoplasm, ‘foamy’ or vacuolated cytoplasm and a hypo-segmented nucleus. To the authors’ knowledge, there have been no clinical or experimental studies published on the haematological response to inflammation by common ringtail possums. The only species of Australian possum for which experimental studies have assessed the haematological characteristics of an inflammatory response is the common brushtail possum (Trichosurus vulpecula). Intravenous administration of recombinant common brushtail possum IL-1•resulted in a decrease in total leukocyte, neutrophil and lymphocyte concentrations from 2 to 6 hours post injection then a four to six fold increase in neutrophil concentration and two to three fold increase in lymphocyte concentration (compared to baseline values) at 24h post injection (Wedlock et al, 1999a). Similarly, administration of rpTNF-• resulted in decreased neutrophil concentrations at 2h post injection and a three to four fold increase in neutrophil concentration at 24h. Lymphocytes were decreased compared to control groups at 2 and 24h (Wedlock et al, 1999b). Clinically healthy common brushtail possums typically have a slightly greater number of neutrophils than clinically healthy common ringtail possums and a difference in the magnitude of the cellular response to inflammatory challenge may also exist between the two species. Four of the possums were mildly anaemic compared to the published reference interval for the erythrocytic values of common ringtail possums. This may reflect a statistical difference to the reference values, or a pre-existing condition and is unlikely to be caused by the acute inflammation. One possum exhibited a mild polycythaemia that was probably caused by dehydration. In the current study, all the possums had an ineffective haematological response to the inflammatory demand incited by the bite wounds. Further work to determine the time period between the bite (and inoculation of bacteria) and the onset of clinical signs, identification of bacteria that may be inciting the inflammatory response and response to antimicrobial therapy needs to be undertaken. Acknowledgements We thank Gribbles Veterinary Pathology for analysis of blood samples. References Clark P (2003) Haematology of Australian Mammals. CSIRO Publishing, Collingwood. In Press th

Jain NC (1986). The neutrophils. In Schalm’s Veterinary Hematology 4 Edition (Ed NC Jain) pp676–730 (Lippincott, Williams & Wilkins: Philadelphia). Love DN, Malik R, Norris JM (2000) Bacteriological warfare amongst cats: what have we learned about cat bite infections? Vet Microbiol 74:179-93 Presidente PJA (1979) Common ringtail possum Pseudocherius peregrinus: maintenance in captivity, blood values and diseases. Proceedings of the Scientific Meeting of the Australian Mammal Society, Healesville, Victoria, February 1979. The Management of Australian Mammals in Captivity, Editor: D.D. Evans, 75-81

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Presidente PJA, Correa J (1981) Haematology, plasma electrolytes and serum biochemical values of Trichosurus vulpecula (Kerr) (Marsupialia: Phalangeridae). Aust J Zool 29:507–517 Rose K (1999) Common diseases of urban wildlife. In: Wildlife in Australia: Healthcare and Management, Proceedings 327 of the Post-Graduate Foundation in Veterinary Science, University of Sydney, NSW, 365-427 Turnbull AV, Rivier CL (1999) Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action. Physiol Rev 79:1-71 Wedlock DN, Goh LP, Parlane NA, Buddle BM(1999a) Molecular cloning and physiological effects of brushtail possum interleukin–1β. Vet Immunol Pathol 67: 359–372 Wedlock DN, Goh LP, McCarthy AR, Midwinter RG, Parlane NA, Buddle BM (1999b) Physiological effects and adjuvanticity of recombinant brushtail possum TNF–α. Immunol Cell Biol 77:28–33 Table 1 Haematological data for Common Ringtail Possums (Pseudocheirus peregrinus) with bite injuries Analytes

Case 1

Case 2

Case 3

Case 4

Case 5

Case 6

Reference (President e 1979, n=6)

0.38

0.43

0.36

0.50

0.36

0.46

0.40-0.48

RCC (x10 /L)

4.69

5.28

4.10

5.62

4.65

5.46

4.5-6.6

Hb (g/L)

123

134

124

158

125

145

134-150

MCV (fL)

81

82

88

89

78

85

68-89

326

310

341

318

341

315

312-335

0.8

0.2

0.2

0.3

5.3

1.4

4.0-9.6

0

0

0

0

0

0.1

0

Neutrophils (x10 /L)

0.5

0.1

0.1

0.2

3.6

0.3

0.7-2.7

Lymphocytes 9 (x10 /L)

0.3

0.1

0.1

0.1

1.4

0.9

1.5-6.6

0

0

0

0

0.3

0.1

0.2-1.0

0

0

0

0

0

0

0.2-0.6

0

0

0

0

0

0

0

PCV (L/L) 12

MCHC (g/L) 9

WBC (x10 /L) 9

Bands (x10 /L) 9

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Monocytes (x10 /L) 9

Eosinophils (x10 /L) 9

Basophils (x10 /L)

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DEVELOPMENT AND INVESTIGATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF SERUM ANTIBODIES TO HENDRA VIRUS IN FLYING FOXES 1

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Andrew Breed , Hume Field , John Molloy , Kim Halpin and Peter Daszak . ¹ Taronga Zoo, Mosman, Sydney, New South Wales. Email [email protected] ² Animal Research Institute, Queensland Department of Primary Industries, Queensland. ³ Centres for Disease Control and Prevention, Atlanta, Georgia, USA. 4 The Consortium for Conservation Medicine, Palisades, New York, USA. Abstract Hendra virus is a paramyxovirus known to infect flying foxes, horses and humans. Flying foxes appear to be the natural host of the virus and research is currently in progress to determine the prevalence of antibodies to Hendra virus in flying foxes throughout Australia and neighbouring countries. The currently used serum neutralisation test (SNT) is not ideal for this purpose. This study developed an indirect enzyme linked immunosorbent assay (ELISA) to Hendra virus antibodies using flying fox specific antiserum. SNT results were used as the gold standard for development of the ELISA. The ELISA cut-off (positivenegative threshold) was determined by receiver operating characteristic (ROC) analysis and inspection of frequency distribution histograms. ELISA results were compared with SNT results of bats from three different populations; (i) a colony of captive flying foxes (n = 46); (ii) wild flying foxes from the Brisbane area (n = 103); (iii) wild flying foxes from the Ross River area (n = 67). Agreement between the ELISA and SNT was excellent for the captive population (kappa = 0.76), good for the Brisbane population (kappa = 0.6), and poor for the Ross River population (kappa = 0.22). Possible reasons for variation in level of agreement of the two tests between populations are discussed. The ELISA has potential to be a useful diagnostic tool in monitoring Hendra virus seroprevalence in flying foxes. With further work the ELISA could also be a useful research tool to help determine if other currently unidentified paramyxoviruses are present in flying fox populations.

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LYSSAVIRUSES AND WILDLIFE IN THE AUSTRALIAN CONTEXT John Bingham CSIRO Australian Animal Health Laboratory, Private Bag 24, Geelong, Victoria 3220, Australia, Email: [email protected] Australia has always been considered free of rabies apart from isolated introductions of the disease, none of which led to establishment of a rabies cycle (McColl et al. 1993). Freedom from infection has been maintained by strict quarantine regulations applied to the importation of dogs, cats and other rabies vectors. In 1996 an indigenous lyssavirus, subsequently named Australian bat lyssavirus (ABL) was detected in Australian bats suffering from neurological disease (Hooper et al. 1997). Further cases were subsequently diagnosed, mostly in the native flying foxes (Pteropus spp. Megachiroptera; Pteropodidae), indicating that some or all of these species are maintenance hosts of ABL. A small number of isolates were made from an insectivorous bat species, the yellow-bellied sheathtail bat (Saccolaimus flaviventris, Microchiroptera; Emballonuridae), indicating that this species may be a maintenance host. Gene sequence analysis indicates that isolates from the pteropid and insectivorous hosts are different (Gould et al. 2002), indicating the existence of two separate lyssavirus cycles. Lyssaviruses infect all mammalian species, including humans, and therefore pose a risk to the public and to domestic and wild animals. This paper discusses these risks and ideas on their management, with particular regard to the Australian context. At present the major perceived risk from ABL is that of human infection caused by contact (biting and scratching) with infected bats. This risk can be managed by public awareness and provision of pre- and post-exposure vaccination where necessary (guidelines are available at http://www.cda.gov.au/pubs/other/bat_lyssa.htm). Preliminary trials have indicated that conventional rabies vaccines provide a reasonably high degree of protection against ABL infection (CDC, unpublished data), although this needs further assessment. As contact of bats with humans is generally minimal, the risks that bats pose as a source of ABL infection is small, except to people who have regular contact with bats. Lyssaviruses in Australia are presently confined to bats. However, if rabies or other lyssaviruses should establish in terrestrial animal species the potential threat to public health could increase considerably. There are two possible pathways for the appearance of terrestrial animal lyssavirus cycles in Australia: 1) the introduction of foreign lyssavirus variants, particularly rabies, via infected animals and 2) the spread of ABL into terrestrial hosts to establish new cycles of virus transmission. To allow assessment of these risks certain important biological concepts should be defined. Aspects of the biology of lyssaviruses -

-

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Lyssavirus variants are adapted to one species (or species guild, such as the pteropid bats) alone, which maintains the virus cycle. The term “cycle” is used to refer to a self-sustaining virus maintenance system independent of other host species; the species that supports the cycle is referred to as the “maintenance host”. However, lyssaviruses can infect any other mammalian host species, a process known as “spillover”. Spill-over hosts are usually (but not always) dead-end hosts. The spill-over host may begin supporting a new virus cycle if the virus is able to undergo genetic adaptation to the new host and if the host is ecologically able to support transmission ratios above one, where each infected host transmits the infection to an average of at least one other host. To our knowledge, lyssavirus-infected hosts do not have a significant sub-clinical shedding phase and infective animals survive for a limited number of days. Although shedding without clinical

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signs has been reported (Aubert et al. 1991; Fekadu et al. 1981), it is not known whether this is significant in the maintenance of the virus cycle under natural conditions. Introduction of foreign lyssaviruses into Australia Classical rabies virus could become established after introduction by an infected animal. There are several theoretical mechanisms of entry of infected or incubating animals: - Long incubators that are released from quarantine - Introduction by undeclared importations of improperly vaccinated pets - Deliberate introduction of infected animals or virus (‘bioterrorism’) The first route is extremely unlikely as Australia has very stringent quarantine requirements, including comprehensive vaccination and antibody-testing regimens for animals in quarantine. To my knowledge there have been no breakthroughs in island countries that have implemented similar regimens. Once introduced, the probability of rabies establishing a cycle in the introduced species depends on the correct ecological conditions to maintain the basic reproductive ratio above one (R0 >1), that is, where each infected host transmits the infection to an average of at least one other host. Any condition that enhances contact of susceptible and infected animals will favour the emergence of a new cycle. These include dense populations and factors that increase the movement and social contact of hosts. It is probable that the red fox, which occurs over much of temperate Australia, is able to support cycles as the population density in many areas is comparable to that of European populations of foxes that support rabies (Marks & Bloomfield 1999). Domestic dogs are unlikely to support rabies as although urban populations are dense, dogs are usually kept under a high degree of confinement. Feral dogs and dingos may transmit rabies effectively, but where they live in pack social structures they may be precluded from becoming long-term maintenance hosts (no pack-forming canids are maintenance hosts of rabies). Feral cats may act as vectors, although their ability to support long-term cycles is unknown. Spread of ABL into new hosts Evaluation of the risks of cycle establishment in new hosts must take into account the infectivity of ABL in new hosts, the subsequent transmissibility of the virus into conspecifics of the infected host and the ecological ability of the new species to maintain the cycle. Current understanding of these stages is very limited. The first two can be investigated through carefully designed pathogenesis trials in the appropriate species, the third through ecological studies of potential maintenance hosts. Our lack of understanding of the risks posed by ABL is aggravated by ignorance of the mechanism of persistence and transmission in the maintenance hosts. Pathogenesis studies in bat species (e.g. McColl et al. 2002) will further our knowledge on these issues, but the foundation of our knowledge – on which we must base informed decisions on policy and scientific direction – is our understanding of the disease in nature, which is dependant on comprehensive national surveillance. As ABL has caused the death of two humans (Samaratunga et al. 1998; Hanna et al. 2000), it has to be assumed that at least under some conditions it can cause spill-over into other mammals, as with all other lyssavirus genotypes. With (probably relatively minor) genetic adaptations to the spill-over host, ABL could become efficiently transmitted to conspecific hosts. Thereafter, the successful establishment of new cycles is dependent only on ecological conditions that favour the continuous transmission to new hosts. Establishment of lyssavirus cycles in new hosts is most likely to occur in those host species where potential transmission dynamics are favourable. As discussed above with regard to rabies epidemics, the red fox is probably the terrestrial species most ecologically suitable for virus maintenance in Australia. Lyssaviruses must be regarded as dynamic organisms that will opportunistically colonise any host population that is capable of maintaining a virus cycle. During the last 80 years a large number of new lyssavirus cycles have emerged in wildlife species throughout the world (Bingham 2003). The dynamic nature of ABL may be inferred from examination of phylogenetic trees, which show narrow genetic diversity of pteropid ABL isolates (Gould et al. 2002; Guyatt et al. 2003). This implies that the isolates have recently radiated from a common ancestral virus and therefore that this lineage of pteropid ABL is a

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new phenomenon. Pteropid ABL may have originated from a microchiropterid cycle or alternatively may have recently entered Australian pteropid populations from an external pteropid source. Spill-over cases from infected bats will almost certainly be reported in future. Such cases – apart from human cases, which can be avoided – do not necessarily need to be treated with alarm. However, they will need to be differentiated from cases originating from new terrestrial lyssavirus cycles. It is necessary to monitor the prevalence of lyssaviruses in domestic and wild animals, and to be able to recognise cycles when they appear. This is not to say that the establishment of bat lyssavirus cycles in terrestrial Australian wildlife will occur, or is a likely event. However, it must be borne in mind that such an event is possible, with serious repercussions should an epidemic become widespread. Minimising the impact of such an epidemic would depend on early detection through appropriate surveillance. References Aubert, MFA, Blancou, J, Barrat, J, Artois, M & Barrat, M-J (1991) Transmission et pathogenie chez le renard roux de doux isolats a dix ans d'intervalle du virus de la rage vulpine. Annals de Recherche Veterinaire 22: 77-93. th

Bingham, J. (2003) Ecological change and the global emergence of lyssaviruses. Proceedings of the 10 conference of the International Society for Veterinary Epidemiology and Economics, Viña del Mar, Chile, 17-21 November 2003. Fekadu, M, Shaddock, JH, & Baer, GM (1981) Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from experimental rabies. American Journal of Tropical Medicine and Hygiene 30:1113-1115. Gould, AR, Kattenbelt, JA, Gumley, SG & Lunt, RA (2002) Characterisation of an Australian bat lyssavirus variant isolated from an insectivorous bat. Virus Research 89: 1-28. Guyatt, KJ, Twin, J, Davis, P, Holmes, EC, Smith, GA, Smith, IL, Mackenzie, JS & Young, PL (2003) A molecular epidemiological study of Australian bat lyssavirus. Journal of General Virology 84: 485-496. Hanna, JN, Carney, IK, Smith, GA, Tannenberg, AEG, Deverill, JE, Botha, JA, Serfin, IA, Harrower, BJ, Fitzpatrick, PF & Searle, JW (2000) Australian bat lyssavirus infection: a second human case, with a long incubation period. Medical Journal of Australia 172: 597-599. Hooper, PT, Lunt, RA, Gould, AR, Samaratunga, H, Hyatt, AD, Gleeson, LJ, Rodwell, BJ, Rupprecht, CE, Smith, JS & Murray, PK (1997) A new lyssavirus - the first endemic rabies-related virus recognized in Australia. Bulletin Institute Pasteur 95: 209-218. Marks, CA & Bloomfield, TE (1999) Distribution and density estimates for urban foxes (Vulpes vulpes) in Melbourne: implications for rabies control. Wildlife Research 26: 763-775. McColl, KA, Gould, AR, Selleck, PW, Hooper, PT, Westbury, HA & Smith, JS (1993) Polymerase chain reaction and other laboratory techniques in the diagnosis of long incubation rabies in Australia. Australian Veterinary Journal 70: 84-89. McColl, KA, Chamberlain, T, Lunt, RA, Newberry, KM, Middleton, D & Westbury, HA (2002) Pathogenesis studies with Australian bat lyssaviruses in grey-headed flying foxes (Pteropus poliocephalus). Australian Veterinary Journal 80: 636-641. Samaratunga, H, Searle, JW & Hudson, N (1998) Non-rabies lyssavirus human encephalitis from fruit bats: Australian bat lyssavirus (pteropid lyssavirus) infection. Neuropathology and Applied Neurobiology 24: 331-335.

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A REVIEW OF AVIAN INFLUENZA IN AUSTRALIA Chris Bunn Agriculture, Fisheries and Forestry- Australia The virus

• • •

influenza A viruses occur worldwide all highly pathogenic isolates have been influenza A viruses of subtypes H5 and H7 Remains viable for long periods in tissues, faeces and also in water

The disease

• • • • •

Severe depression, inappetence Drastic decline in egg production Facial oedema with swollen and cyanotic combs and wattles Petechial haemorrhages on internal membrane surfaces Sudden deaths (mortality can reach 100%)

Ducks infected with HPAI and excreting the virus may not show any clinical signs or lesions History of HPAI in Australia Keysborough 1976 H7N7 $0.2 million

• • • • •

Three farms involved Management poor with intercurrent disease Ducks delivered to one of the two farms 4 days before the outbreak – later negative Further virus isolation made from one duck on a duck farm in the control area Rising level of titres detected on the duck farm therefore slaughtered out

Keysborough (Surveillance)

•

Widespread Surveillance Instituted - 3,400 swabs (sick/dead bird pickup; free flying; lab submissions; other investigations)

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- 75,000 sera from throughout the State - 31,000 sera from other States -Transmission tests demonstrated fairly lethal for chickens and turkeys; spread easily in ducks without causing clinical signs; rapid seroconversion Bendigo 1985 H7N7 $2.2 million

• • • •

Major deficiencies in biosecurity Very poor hygiene Wild birds present in large numbers Two dams supplied untreated water

Bendigo 1985 (Surveillance)

• • •

20 km testing of all poultry flocks (including one duck farm) at a 95% level to detect 1% seropositive (73 flocks 13,000) plus dead bird pickup One third of all commercial flocks in the State at a prevalence of 2-5% (247 flocks 25000 samples 208 wild birds surveyed with one starling positive (7 days after stamping out had commenced )

Bendigo 1992 H7N3 $1.2 million

• • •

Two farms one a broiler breeder; the other a duck farm only 50 metres apart Poor hygiene practices on both farms Free range and wild ducks intermingled in a dam/swamp situation

Bendigo 1992 (Surveillance)

• • • •

Monitoring of farms at highest risk with - dead bird pickup - serological testing (1% at 95% confidence) After 3 weeks serological testing of all commercial flocks (and a few backyard) in the control area (5% level with 95% confidence) Retested after a 2 week interval Surveillance on 24 farms with a contact history by telephone twice a week Minor wild bird surveillance (< 20 birds)

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• • • •

Surveillance done by spreading out from the infected premises No State surveillance testing undertaken (estimated cost $385 000) No of farms visited 155 on 528 occasions involving almost 6 million birds. Control area quickly reduced from State to a 25km radius

Lowood 1994 H7N3 $0.4 million

• • • •

Isolated egg production farm (nearest commercial farm 12 kms away) A number of deficiencies in biosecurity Untreated water supplied from a watercourse (at low level due to drought) Large numbers of waterbirds

Lowood 1994 (Surveillance)

• •

A serological survey on 17 surrounding properties in a 1km radius one month after stamping out completed

Aimed at 5% level with 95% confidence Involved 222 chickens 26 ducks 26 turkeys 8 geese All with negative results

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Tamworth 1997 H7N4 $4.0 million

•

Occurred in broiler–breeder flocks in modern facilities with climate-controlled sheds, which had staff amenities, including showers.

•

However biosecurity deficiencies were identified: lack of quality control in staff movement and use of facilities

• •

Lack of maintenance of a water supply treatment plant

Free-ranging emus, which were found to be excreting AI virus, in open pens adjacent to chicken sheds

•

A contract dead-bird pickup service moving from farm to farm

Tamworth 1997 (Surveillance)

• • •

A declaration of a restricted zone of 3 kilometres and a surveillance zone of 10 kms was established. Concentrated on clinical and post mortem examinations and serological sampling in the restricted and control areas. (5% at 95% level and 60 birds per farm) Opportunistic sampling of wild waterfowl related to any significant mortalities

Wild bird Surveillance Influenza viruses have been isolated from a diverse range of apparently healthy wild birds over a wide geographical area. In a major study 0.65% of 7193 samples from 116 species demonstrated haemagglutinating agents and 6500 cloacal swabs resulted in isolation of 45 influenza viruses In Victoria 16 avian influenza virusess have been isolated from wild ducks H5 or H7 strains have only been isolated once (1985 Bendigo) Recent Developments

• •

Human health concerns (including planning for a human pandemic in Australia) Emergence of HPAI from mutation of LPAI strains

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• • • •

Increasing international surveillance programs Definition of AI Diagnostic and vaccine advances Efficiency auditing under the EAD response agreement

Conclusions We will come under more and more pressure to increase our active surveillance

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ANIMAL DISEASE INVESTIGATION AT AAHL Maria Cardoso CSIRO Livestock Industries, Australian Animal Health Laboratories, Geelong, Victoria The Australian Animal Health laboratories in Geelong are a sector of CSIRO Livestock Industries that specializes in disease diagnosis, research and policy advice in animal health. AAHL plays a major role in maintaining Australia free from exotic diseases of livestock as well as aquatic animals and wildlife. The facility is able to function at level 4 biocontainment, allowing research into zoonotic diseases such as Hendra and Nipah viruses, rabies and Newcastle disease virus to be performed. The Large animal facility is used to train veterinarians in the detection and identification of diseases exotic to Australia. There are at least seven major areas in the Diagnostic and Epidemiology project currently set up at AAHL: • • • • • • •

Virology Bacteriology Serology Electron microscopy Histology Pathology Molecular biology

The Molecular Diagnosis group supports the laboratory’s emergency disease preparedness capability by providing a 24 hour diagnostic service on a variety of samples from terrestrial and aquatic animals. AAHL is the national OIE reference laboratory for rabies, brucella, bluetongue, Newcastle disease virus and virulent avian influenza. The laboratory’s diagnostic cababilities have recently been enhanced by the implementation of automated high sample throughput technologies: • • • • •

DNA sequencing and pathotyping Real Time PCR for rapid pathogen detection Automated nucleic acid extraction Automated serum dilution and ELISA setup Multiplexing detection systems (Luminex under development)

Martyn Jeggo, the newly appointed director of AAHL outlined to Animal Health Australian (AHA) the importance of an effective system of surveillance to deal with emerging infectious diseases that may pose a threat to Australia’s livestock and wildlife populations. The link between human, livestock and wildlife diseases is now better recognized and steps must be taken to ensure that diseases such as SARS, Hendra and Nipah are efficiently managed if they are to emerge in Australia. The Australian Biosecurity CRC (AB-CRC) is a venture set up to utilize new technologies to improve the surveillance and diagnosis of emerging infectious diseases. Funding is estimated to be $60 million over 7 years to undertake research in 3 major areas: • • •

Molecular detection systems Disease ecology in wildlife reservoirs Spatial modelling systems to map animal-disease-environment interactions

These areas will be able to come together to generate an improved national disease surveillance of livestock and wildlife populations. The projects being undertaken at AAHL, with PhD and postdoctoral opportunities are:

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• • •

Development of DNA chips and hand-held devices for rapid disease detection Development of diagnostic tests to differentiate between WNV and Kunjin. Pathogenesis and transmission studies of Nipah virus in Australian flying foxes.

Other systems being implemented include a national information management system database (LIMS) for the reporting of all results associated with particular samples. The information gathered can then be shared between different animal health agencies. Sharing of information will also be made possible by communication between experts in real time using a digital camera and laptop. Expertise can now be rapidly sought from different parts of the world to enable the rapid diagnosis of unknown agents. In order to gain better knowledge on animal diseases, there is a major need for better collaboration between veterinarians, field biologists and laboratory scientists. New technologies are now making communication between different parties much easier. Priorities and opportunities for collaboration should be identified to make use of existing data, expertise and new advanced technologies that will enhance our knowledge into those emerging diseases that may threaten the wellbeing of humans, livestock and wildlife populations in Australia.

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PACHECO’S DISEASE – AN AUSTRALIAN EXPERIENCE Ian Hough Lower Mitcham Veterinary Clinic, 22A Chasewater Rd., Lower Mitcham, SA 5062 Cause: Herpes Virus Clinical Signs: Usually none, may demonstrate classical sick bird look. Susceptible: Patagonian conures, Nanday conures, Aratinga sp. and any psittacine. Diagnosis: Acute death. Possible splenomegaly and hepatomegaly. Histopathology: Necrotising hepatitis and splenitis with eosinophilic intranuclear inclusion bodies. Treatment: High doses of acyclovir (Zovirax) have been reported successful, but unlikely to be in most cases. Prevention: Vaccination has been used in the US. The Scene: A small animal practice, Adelaide, not computerised. A new client form the South East. The Problem: Deaths in aviaries since new introductions. The Sample: Dead bird for post mortem, sent via courier from the South East. The Findings: Good body condition, broken neck (owner’s method of euthanasia), mottled liver, splenic haemorrhage and enlarged bursa. Organisation: Interesting samples sit in pot on typical veterinary desk in typical office. Samples submitted to Australian Registry of Wildlife Pathology in December 2002. Trouble Begins: Karrie finds intranuclear inclusion bodies and magenta intracytoplasmic inclusion bodies in March 2003. Alarm Bells: Karrie reports findings to authorities, and the submitting veterinarian. Departmental Action: SA Department of Agriculture Officers away on training exercise, including CVO. Response: Submitting veterinarian receives phone calls from 4 different PISA Officers, all ask the same questions, especially “What is Pacheco’s?” Dilemma: No fresh (frozen) tissue, DNA testing not possible if EM work done. Solution: Tissues to the US for DNA hybridisation testing. Results: Negative Circovirus, Adenovirus, Pacheco’s, and Polyomavirus. Next Step: RVL Menangle for transmission electron microscopy. Further Complications: Wax blocks melted on return from US lab. Results: Consistent with adenovirus BUT cell and virus morphology distorted due to transport problems.

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PIRSA Response: Very keen to go to the owner’s property to carry out testing. Do not know what, but still want to do it. Owner Problems: Owner had heart attack soon after original problems. Most of his birds were sold through dealers in Adelaide and Melbourne. Veterinary Problems: Submitting veterinarian has no record of the owner except a phone number in post mortem book. Luckily veterinarian had told owner that tissue was going to Taronga initially. Discussion: Time lag in diagnosis, lost owner records, could not identify in Australia meaning further delay, inconclusive results from US, damaged tissue on return, and lack of information in Departments.

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TASMANIAN DEVIL FACIAL TUMOURS Tim McManus Consultant – Zoonotic Diseases, 35 Hammond St., Falmouth, Tasmania 7215 The Tasmanian devil (Sarcophilus harrisii) is the largest of the three carnivorous marsupials extant in Tasmania. It is believed to have become extinct on mainland Australia approximately 600 years ago. It stands about 30 cm at the shoulder. Males weight up to 12 kg and females up to 8 kg. They are aggressive and fight each other especially over food. Their favoured habitat is open, dry and wet sclerophyll bushland. They are mainly carrion eaters and scavengers on anything of animal origin. Cannibalism is also common. Devils eat the lot – skin, bones, feathers and all, making identification of faeces relatively easy. They will also hunt live prey such as possums and small marsupials. They are essentially nocturnal, hiding by day in a den or hollow log. The female has a rearward opening pouch with 4 nipples. They usually mate in March. The gestation period is 21 days. They have up to 4 young per litter. The young remain in the pouch for 15 weeks and are completely weaned by 40 weeks. They sometimes ride on their mother’s back. Young devils are more agile than adults and can readily climb trees. th

Devil numbers in Tasmania were low right through the first half of the 20 century. University of Tasmania zoologist, Dr. Eric Guiler, attributed this to an “epidemic disease”. See R.H. Green, QVM&AG Records No. 27. “Old-timer” trappers recall seeing devils with sores on their faces and necks, but only from fighting and from wire snares. No investigative work was done before the 1960s because the animal was considered a pest. They were trapped, shot and poisoned with strychnine for over 100 years. Devils ate wallabies in snares, all the facial muscles of cast sheep and, allegedly, killed new-born lambs. The Van Diemen’s Land Co. (VDL) in the far north-west of the state actually introduced a bounty of 2/6 (25 cents) for male devils and 3/6 (35 cents) for females. It was not until 1941 that devils were protected by law, although “Permits to Cull” were issued even as late as the 1960s. An enlightened attitude was heralded with the institution of the Tasmanian Fauna Board, which pre-dated the Parks and Wildlife Service. Devil numbers have spectacularly escalated since, especially in the north. In 1964 Bob Green trapped 33 near Gladstone in just 10 nights. He never saw any then or since with cancers (QVMAG No. 27). Facial tumours were first observed on devils in the north-east in the late 1990s in areas of former high population density. The incidence has since spread south and west to the edge of the Central Highlands. Recent surveys indicate major population crashes. Facial lesions are severe, caused by slow growing malignant lymphoma-like tumours. They always start on the face before subsequently spreading throughout the body via the lymphatic system. Devils take several months to die. Males are affected first, then females. Animals are obviously sick and become disorientated. A virus is suspected in the epidemiology, spread by individuals biting each other especially when competing for food at a carcase site. It could be a retrovirus. A retrovirus, named the Human T-cell Lymphotropic Virus – HTLV 1 – is known to be linked to T cell lymphomas in humans. Similarly, the AIDs retroviruses, HIVs I and II, can produce malignant lymphomas in their victims. They also compromise immunity, which is thought to occur in the tumour-affected devils. Another possible virus source is a herpes virus such Epstein Barr (EBV), which is responsible for Burkitt’s Lymphoma, particularly prevalent among Africans.

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Three Tasmanian Veterinary pathologists, Drs. Steve Pyecroft, Richmond Loh and Brad Chadwick, are investigating the disease. Histopathology is confused. It looks to be lymphohistiocytic, which could be so because, in humans, Class III histiocytosis syndromes cause, among other things, malignant lymphomas. If a retrovirus is the initiating factor, from whence has it come? Is it a low grade, constantly circulating pathogen, capitalising on enhanced infectivity afforded by high local devil population densities? There is no past evidence of any similar outbreak. Or might a retrovirus have crossed a species barrier from cats, humans or even goats? Feral cats abound in the Tasmanian bush and can’t be ignored as a possible retrovirus source if such pathogens can cross the species barrier via vector or other unknown pathways. The State government has allocated $1.8 million to the DPIW&E and the NP&WLS over the next 4 years to fund a detailed investigative program. Other strategies being mooted are isolation of unaffected animals, although this will be risky if a slow retrovirus is involved. Also nearly all adult devils have a degree of facial scarring from constant brawling, so it is hard to ensure they are clean. Establishing safe populations on offshore islands is another suggestion. To date there have been no reported cases of facial tumours on devils in managed wildlife parks or enclosed reserves. Is the problem a storm in a teacup which will resolve itself as might have happened before with a different infectious epidemic? Or has the spread of a modern or mutated pathogen created an insurmountable problem likely to lead to yet another extinction of a Tasmanian marsupial carnivore?

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POWERFUL OWL (Ninox strenua) Ed McNabb Arthur Rylah Institute for Environmental Research Department of Sustainability and Environment Vulnerable in Victoria. Home range: ~300 ha in rich habitat, and greater than 3000 ha in poor habitat. Powerful Owl Prey (Total individuals 200):

Common Ring-tail Possum 88% Brush-tail Possum Mountain/Common 5% Crimson Rosella 1% Unidentified 6%

Important points for rehabilitation into the wild If healthy, return to collection site ASAP If held captive, fatten the bird up, but fast for 24hr prior to release Avoid imprinting Daytime: Locate adult or sibling roost, and place in the same or nearby tree Evening: Release just prior to dusk Release in view of adults or at least within close audible range

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RADIAL NERVE DAMAGE IN RAPTORS Franciscus Scheelings and Paul Ramos University of Melbourne, School of Veterinary Science, Werribee The radial nerve originates from the dorsal fascicle of the brachial plexus. The dorsal fascicle divides into the axillary nerve (innervates deltoid and triceps muscles), and the radial nerve (innervates the extensors of the carpus and digits and sensory supply to dorsal propatagial area). Muscles innervated by radial n.:

Extensor metacarpi radialis extends the carpus and metacarpus. Supinator flexes the elbow and elevates the cranial edge of the wing. Extensor metacarpi ulnaris flexes both the forearm and the manus. Extensor digitorum communis extends the manus as well as flattens the alula against the metacarpus to decrease lift, or stabilise the position of the alula.

Differential diagnosis of radial nerve damage includes: Infectious e.g. aspergillosis Inflammatory e.g. neuritis Metabolic e.g.hypocalcemia Neoplastic Nutritional e.g.calcium deficiency Toxic e.g. lead poisoning Trauma Birds with radial nerve damage are unable to: Extend the carpus and metacarpus i.e. extend the wing distal to the shoulder or flex the elbow (extensor carpi radialis). Extend the manus or flatten and stabilise the alula against the metacarpus (extensor digitorum communis). Elevate the cranial edge of the wing (supinator). Flex the forearm and manus (extensor metacarpi ulnaris). Other signs may include analgesia distal to the mid-humeral area. Clinical Case Flightless powerful owl found by the roadside 15/04/03. On physical examination the bird was bright, alert, responsive and in good body condition. On examination under general anaesthesia a small penetrating wound was found on the left dorsal wing surface. There was no exit wound, no palpable fractures, and mild, bilateral, superficial abrasions on the dorsal metatarsals. The wound was explored. The distal portion of the severed radial nerve was visible. Treatment: 1) Muscle bellies were dissected to retrieve the proximal portion of the nerve. Mattress sutures were placed using 4/0 vicryl to appose nerve ends. The site was infiltrated with 0.2ml dexamethasone, 1ml benzylpenicillin. 2) 2.5mg dexamethasone IM, 0.25ml multivitamins IM, 10ml warmed dextrose saline IV, 40ml warmed dextrose saline SC, 25mg itraconazole liquid & 10ml warmed dextrose saline PO were administered on recovery.

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Continued Treatment:

Prednisolone 2.5mg in food SID for 7 days.

Response to treatment: Sent to carer and test flown 1 month later. 26/07/03 transmitter attached. 10/08/03 bird released. 14/09/03 bird found dead. Necropsy:

Extensor muscles of the affected wing were grossly atrophic and pale. Muscles innervated by the radial nerve were bilaterally dissected out with the following results: Extensor carpi radialis: left 1.2 g right 2.4 g. Extensor digitorum communis: left 0.5 g right 0.8 g. Supinator: left 0.3 g right 0.45 g. Extensor metacarpi ulnaris: left 0.8 g right 1.2 g.

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MANAGEMENT OF CHRONIC CORNEAL DISEASE IN A MALAYAN TAPIR (Tapirus indicus) AT MELBOURNE ZOO 1

2

Kate Bodley and Chloë Hardman

1

Associate Veterinarian, Melbourne Zoo Veterinary Ophthalmologist, Animal Eye Care 2 A female Malayan tapir, maintained in an outdoor exhibit at Melbourne Zoo, developed signs of severe bilateral keratopathy during January 2003. Despite intensive anti-inflammatory and antimicrobial therapy, bilateral keratitis persisted for approximately eight weeks. Chronic keratopathy resulted in development of a deep corneal ulcer and descemetocoele in the left eye. The ulcer was managed using a conjunctival pedicle graft. Bilateral canthal closures were also performed. Topical cyclosporin and hypertonic saline and systemic doxycycline and carprofen were used to reduce inflammation/oedema and encourage healing during the post-operative period. The left corneal lesion healed well post-operatively. The tapir continues to demonstrate clinical signs of mild to moderate bilateral keratitis. Malayan tapirs frequently develop corneal disease in captivity (Dockerill R, 2000 (unpublished); Janssen, Rideout and Edwards, 1996). A number of management practices and enclosure features have been modified at Melbourne Zoo so that the incidence and severity of corneal disease in this species is reduced in future. References Janssen DL, Rideout BA and Edwards ME (1996) Medical management of captive tapirs (Tapirus spp.) Proceedings of the American Association of Zoo Veterinarians, 1996. Pp: 1-11. Dockerill R (2000) Eye problems in tapir – analysis of survey data (unpublished, Taronga Zoo).

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THE TALE OF TWO OCEAN SUNFISH (MOLA MOLA) – AN UNUSUAL MORTALITY IN AN UNUSUAL SPECIES Larry Vogelnest Senior Veterinarian, Taronga Zoo There are four species of sunfish; the Ocean sunfish or mola (Mola mola), the Southern Ocean Sunfish (Mola ramsayi), the Slender Sunfish (Ranzania laevis) and the Sharptail Sunfish (Masturus lanceolatus). They are in the Class Osteichthyses (the bony fishes), Order Tetraodontiformes (trigger fish, boxfish, porcupine fish, and puffers) and Family Molidae. The common name "sunfish" is used to describe the marine family, Molidae, as well as the freshwater family, Centrarchidae. The common name "ocean sunfish" comes from the mola’s habit of lying atop the surface of the ocean appearing to sunbathe. While most sightings are at the surface, molas have been sighted in deeper waters with submersibles and remotely operated vehicles down to 500m. The mola is found world wide in tropical and temperate oceans and is the most common of the sunfish. It is the largest teleost fish. They are pelagic but occasionally come in-shore. Apart from their size they have a number of other unusual features. They do not have a caudal fin. Instead there is a clavus which is formed by extensions of the dorsal and anal fin rays. Mola feed on gelatinous zooplankton like jellyfish, Portuguese man-o-war, ctenophores and salps. Squid, sponges, serpent star bits, eel grass, crustaceans, small fishes and deepwater eel larvae have also been found in mola indicating that they forage both at the surface, among floating weeds, on the seafloor and into deep water. The teeth in each jaw are fused to form a beak like structure. The beak is internal and covered by lips. The mouth is small compared to the body size. Long claw-like “teeth” in the pharynx assist in prehension of prey items. The skin is thick with rough denticles and produces copious amounts of mucus. The skin is a silvery-grey colour with a slight opalescent sheen that can exhibit strikingly changeable spotted patterns. They produce up to 300 million eggs per spawning – the largest number of eggs recorded in any vertebrate at one time. Mola larvae after hatching are about 1.8mm long and look more like their puffer fish and boxfish relatives. The body is broader and they have a primordial tail fin, a large pectoral fin and body spines. By about 40mm the body spines are much less prominent, the body is deeper and more compressed, and the beak and clavus have developed. The adult mola has no spines. No data exist on how fast mola grow in the wild but one individual in captivity at the Monterey Bay Aquarium gained 364 kg in 14 months. There is also no data on longevity however one captive animal lived 10 years. Mola have few predators. They have been known to be taken by orca, sharks and sea lions. They are unfortunately common bycatch in the fishing industry (particularly driftnets). While unpopular in Europe and the United States, mola are eaten throughout Asia. Taiwan and Japan are the largest markets. All parts of the mola are eaten including the skin, fin muscles, backbone, testes, and gut which is viewed as a delicacy. In Taiwan, the gut is served as "Dragon Intestines." Unlike their puffer fish relatives, mola do not carry the deadly neurotoxin, tetratrodotoxin The average size of an adult mola is 1.8 m horizontal length and 2.4 m between the tips of the dorsal and anal fins. The average weight is up to 1000kg. The current record for the heaviest mola in The Guinness Book of Records belongs to a mola that was struck on 18 September, 1908 by the Australian steamship SS Fiona about 65km from Sydney. The fish measured 3.1m in horizontal length, 4.26m in vertical length and weighed 2235kg (probably an estimate). A mola caught off Japan in 1996 however weighed in at 2300kg but was only 2.7m long. Another also caught off Japan in August 1999 measured 3.25m but was only 1800kg (may not have been an accurate weight). In Australia, mola have been reported from Sydney to Tasmania and west to Mandurah in Western Australia. On the 13 October 1998 a mola was found stuck on the bulbous bow of the cement carrier MV Goliath. The fish became struck on the bow off Jervis Bay and slowed the ship from 14 to 11 knots. The rough skin of the fish wore the paint work back to bare metal. The fish measured 3.1m from the tip of the dorsal fin to the tip of the anal fin, and 2.5m from the tip of the snout to the end of the clavus and weighed approximately 1400kg.

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In September 2002 two mola were found in in-shore waters in Sydney. The last mola to be seen in Sydney waters was on 13 October 1998. The first animal was sighted in Narrabeen Lake on 10 September 2002. It was then found the following day floundering under a bridge further in-shore. The animal was in poor condition, had multiple skin abrasions and appeared disorientated. Euthanasia was elected. An incision was made in the thick skin with a knife where the heart was estimated to be and a 6g needle inserted into the animal. After numerus attempts the heart could not be found. Approximately 200ml of sodium pentobarbitone was injected into the coelomic cavity and the animal died within 20 minutes. The animal was taken to Taronga Zoo for necropsy. The animal weighed 211kg had a total body length of 178cm and was 215cm from the tip of the dorsal fin to the tip of the anal fin. There were 12 fin rays and 8 ossicles on the clavus confirming the species as M. mola (M. ramsayi has 16 fin rays and 12 ossicles). The animal had numerus monogenean parasites (Capsala martinieri – Ian Whittington pers com) around the head. Numerous different but, possibly also monogenean parasites were found in the mouth. The skin was extremely abrasive, 1-2mm thick and very tough. A dramatic and interesting finding was that despite being a teleost fish the entire skeleton (including the skull) was completely cartilaginous and very easy to cut. There were no obvious abnormalities of the internal organs apart from parasitic tracts in the liver. The animal was a female although the ovary did not look particularly active. Based on this and the relatively small size of the animal it was probably a sub-adult. Multiple different parasites were present from the pharynx and throughout the rest of the gastrointestinal tract. One the parasites were identified as an Accaocoeliid digenean trematode (Tom Cribb pers com) of which many species have been identified in mola. The stomach was empty. The tiny brain sat within a fluid filled cavity in the cartilaginous head. Numerus helminth parasites were present within this cavity. Histologically there were multifocal hepatic and renal granulomas. The meninges contained marked numbers of cestode parasites, probably plerocercoids. There was very mild perivascular microgliosis. Despite this mola’s condition no cause was evident on necropsy or histologically. The granulomas in the liver and kidney may have represented previous parasite migration however no parasites were evident in the lesions. The larval cestodes in the meninges were not associated with significant lesions, and they were most likely an incidental finding. A second animal was seen in Botany Bay on 16 September 2003. It was towed out to sea but was found in the bay again the following day. The fish appeared disorientated and did not swim upright. Seas were very rough and the animal was not seen again until the following day when it was found washed up dead on Lady Robinson Beach in Botany Bay. The animal weighed 300kg had a total body length of 197cm and was 284cm from the tip of the dorsal fin to the tip of the anal fin. The animal was in reasonable body condition, with minor abrasions on the trailing edge of the clavus but no other significant wounds or abrasions. The skin was much smoother than the previous animal and there were no external parasites. The liver had numerous parasitic tracts and parasites present. Parasites were present within the cavity surrounding the brain. The gastrointestinal tract was empty and several parasites were found within the colon. The animal was a male. The most significant histological findings were multifocal hepatic cestodiasis, focal intestinal nematodiasis and marked cerebellar malacia. The malacic lesions were sufficient to account for the animal’s apparent ataxia. Aetiologic agents were not evident within the lesion, but a vasculopathy was present in the lesion and may have been the mechanism of the tissue injury. The larval cestodes within the biliary ducts were not associated with significant lesions. There did not appear to be any link between these two cases. The large number and range of parasites found in these two animals is probably normal. Mola are infamous for their impressive parasite load. Some 40 different genera of parasites have been recorded in this species alone. Mola act as intermediate hosts for parasites of other species. For example one mola parasite is the plerocercoid of a shark tapeworm indicating that mola do fall pray to sharks thus completing the life cycle of this parasite. While

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at the surface of the ocean mola are often accompanied by seagulls who presumably feed on the mola skin parasites. Acknowledgements I would like to thank Mikaylie Wilson and Richard Buzas for their assistance with the necropsies, Kerryn Parkinson for assistance with the identification of the animals, Karrie Rose for the histopathology, and Ian Beveridge for assistance with identification of some of the parasites. References 1. www.amonline.net.au/fishes/fishfacts/fish/mola.htm 2. www.oceansunfish.org

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SARCOPTIC MANGE IN URBAN COMMON BRUSHTAIL POSSUMS (Trichosurus vulpecula) Anne Fowler BSc(Vet)(Hons) BVSc Melrose Animal Hospital Email: [email protected] Introduction Common Brushtail possums (Trichosurus vulpecula) have successfully colonized the urban environment. This is a result of their omnivorous appetites and ability to nest in less than perfect hollows such as roofs. In the Albury-Wodonga area, most possums that present the veterinary clinic are from the central Albury area. This area has been urbanized for a long time and has many large, established trees and older houses. The most common presentation is due to vehicular trauma causing facial damage and concussion. CASE 1 An adult female possum without pouch young was presented to the clinic having been found on the ground. Upon examination, she was depressed and pale with fur loss and abrasions over her trunk. Small nodules were noted on her ears. Internal bleeding from blunt trauma was suspected. She deteriorated over the next few hours despite warmth and subcutaneous fluids and was euthanased. An examination of the skin nodules postmortem was performed. The lesions were distributed over the edges of the ears, on the underside of the tail where the furless area meets the dense fur and on the junction between the hair on her feet and the smooth skin of the palms. No further investigation was performed.

Nodules on the ears of case 1 and nodules on the tail of case 2 CASE 2 An adult female possum without pouch young was presented to the clinic in a depressed state. Dull mentation and trauma to the face suggested vehicular trauma had caused concussion. Similar skin lesions were observed. The possum became more depressed and recumbent and was euthanased. The distribution of the skin lesions was similar to case 1. The lesions were present on the ears, underside of tail and around the paws. There was no fur loss on the body, nor signs of irritation in the affected areas. A skin scrape was performed and many Sarcoptes mites were seen. Histopathology of an ear lesion revealed a chronic hyperplastic dermatitis associated with Sarcoptes mite. It caused a hyperplasia of the epidermis and possibly hyperplasia of the pilosebaceous units. It was observed that the parasite had not stimulated much of a cellular reaction. Yeasts and pigmented fungal spores were also observed in the keratin and their significance was not determined. Discussion This paper documents case reports of nodular hyperplastic dermatitis associated with Sarcoptes mite in brushtail possums. Unlike the condition in dogs, where Sarcoptes parasitise all of the skin, with preference for ears, elbows and hocks, the lesions in brushtail possums appear to be very discrete and

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limited to ear tips, paws and the tail. In wombats Sarcoptes is often associated with erythema, parakeratosis and hairloss. Neither redness or hairloss was observed in the possums. For the veterinary clinic, the discovery of sarcoptes in a free-ranging urban population of possums has highlighted that Sarcoptes is not only a rural disease, as previously thought in the area, but may exist in pockets in the urban environment. Sarcoptes mite infestation is now considered as a differential cause of itchy skin disease in dogs in the central Albury area. Acknowledgements Thanks to CVDL for providing recuts of the histopathology. References Scott DW, Mueller GH: Small Animal Dermatology, pub: WB Saunders, 1989, p 396 -404. Skerratt L: Management of Sarcoptic Mange in Wombat Populations. In Proceedings: Veterinary Conservation Biology Wildlife health and Management in Australasia, 2001. p271 - 278.

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SARCOPTIC MANGE IN AGILE WALLABIES David McLelland and Jennifer Youl Litchfield Veterinary Clinic, PO Box 1614, Palmerston, NT 0831 Sarcoptic mange, caused by the mite Sarcoptes scabiei, is a disease typified by pruritic dermatitis in a number of mammalian species. In Australia, sarcoptic mange is an important disease in populations of humans and dogs, and is considered the most significant infectious disease of common wombats. The common wombat, southern hairy-nosed wombat, koala, common ringtail possum, Tasmanian devil and quoll are the Australian native mammals in which sarcoptic mange has previously been reported. A number of cases of sarcoptic mange in free-living and captive agile wallabies seen recently in the Northern Territory will be discussed.

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MOLECULAR CHARACTERIZATION OF A HERPESVIRUS ISOLATED FROM INDIAN GYPS VULTURES Maria Cardoso Bsc (Hons) CSIRO Australian Animal Health Laboratories, Geelong, Victoria A herpesvirus isolated from tissue of a sick vulture may be linked to the recent declines observed in the populations of Gyps vultures in the Asian subcontinent. The main aim of this project was to identify vulture herpesvirus (VHV) genes and in particular, to characterize the DNA polymerase gene as proof of evidence that this virus does in fact belong to the family Herpesviridae and that it is closely related to other avian herpesviruses of the subfamily Alphaherpesvirinae. This aim was achieved by cloning Hind III fragments of VHV DNA into a plasmid vector for sequence analysis. Primer walking and transposon insertion strategies led to the identification of VHV genes involved in virus structure and replication of the herpesvirus genome. The UL9 gene is especially interesting as, with the exception of one genus of the subfamily Betaherpesvirinae, it is only found in other members of the subfamily Alphaherpesvirinae. The complete sequence of the VHV DNA polymerase gene (UL30) that encodes the catalytic subunit of the enzyme, contained a single open reading frame (ORF) of 3660 nucleotides able to encode 1219 amino acids. Nine motifs were identified that are conserved amongst all known herpesviruses and are found within the 3’-5’ exonuclease and DNA binding domains of the DNA polymerase enzyme. The 5’-3’ exonuclease region thought to be responsible for the RNase H activity of DNA polymerase was also identified. In order to compare VHV to other members of the family Herpesviridae, the conserved domains were aligned by ClustalW with those of 21 herpesviruses from different subfamilies and also used to construct an unrooted phylogenetic tree by the neighbour-joining method. VHV was more similar in these regions to the alphaherpesviruses compared to members of the β and γ subfamilies and clustered closer to other avian herpesviruses, except for ILTV. A further aim of this study was to develop a diagnostic real time PCR assay for use in the investigation into the cause of Gyps vulture declines. A Taqman assay was optimized for the detection and quantitation of VHV genomic material in clinical samples. This assay must be further validated for testing future fixed samples from sick vultures submitted to the laboratory.

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DISEASES OF CROCODILE HATCHLINGS IN CAPTIVITY Philip Ladds Graduate Research College, Southern Cross University, PO Box 157, Lismore, NSW 2480 Disease with high mortality of hatchlings has been a major impediment to successful commercial crocodile farming in tropical Australia and elsewhere. Perhaps ironically, these mortalities during the “early days” of crocodile farming were in large measure due to inappropriate husbandry and management procedures, as farmers tried to replicate free-range micro-habitat on farms. Viral diseases include poxvirus infection with focal proliferative lesions especially over the head of both Crocodylus porosus and Crocodylus johnstoni, and, in Africa, an adenoviral hepatitis with typical intranuclear inclusions (Huchzermeyer 2002). Inclusions characteristic of herpesvirus infection were recently observed as an incidental finding in the skin of C porosus hatchlings that had no lesions apparent grossly (McCowan, pers. com.). A non-suppurative meningitis with pronounced peri-vascular cuffing was also considered likely to be of viral, perhaps arboviral, cause. Overwhelmingly, the fatal diseases of farmed hatchlings are those caused by opportunistic infections with ubiquitous bacteria or fungi, or by inappropriate nutrition. Most bacterial isolates from dead crocodiles are Gram negative and infections may be acute or chronic: multiple foci of necrosis are common in the liver and spleen of hatchlings with septicaemia. Aeromonas hydrophila, Providentia rettgeri and Salmonella spp. are frequent isolates. Sudden death of fat hatchlings and the finding of “wet” oedematous tissues and haemorrhage at necropsy suggest an enterotoxaemia-like syndrome (Ladds and Sims 1990). In mycobacteriosis in C johnstoni hatchlings, multiple pale protruding nodules up to 4 mm in diameter were in several organs including lung, liver, spleen and kidney (Ariel et al 1997b). Chlamydiosis, which in Zimbabwe causes an acute hepatitis (Huchzermeyer 2002) or a chronic conjunctivitis, appears not to have been diagnosed in hatchlings in Australia. Dermatophilosis (so-called “brown spot”) is a common and important skin disease in young crocodiles. Grossly, lesions are multi-focal, and as in domestic species typical branching filaments of Dermatophilus sp. can be seen microscopically within superficial debris (Buenviaje et al 1998). Fungal infections are mostly precipitated by sub-optimal water and or pen temperatures. The skin is especially involved and pale gelatinous areas or discrete white foci may develop on any part of the body. Ulcers may occur if this material is sloughed. Fungal growth in primary poxvirus lesions may complicate histological diagnosis. In deep mycosis pale foci may be present in any organ but especially the liver and lungs. Common fungal isolates include Fusarium sp., Penicillium spp. and Aspergillus spp. (Hibberd et al 1996). In regard to protozoan diseases, although Haemogregarines are often seen in blood smears, they seem unrelated causally to disease. Coccidiosis caused by Goussisa –like organisms does however appear to sometimes be an important cause of ill-thrift and mortality. In the intestine there is loss and “fusion” of villi associated with organisms, but in many other tissues sporulated oocysts and sporozoites are not associated with an overt host response. A protozoan also appeared to be the cause of a severe enterohepatitis on several farms. The proximal intestine was markedly thickened, and multinucleate giant cells containing organisms replaced normal intestinal lamina propria. This infiltrate sometimes extended deep into the muscularis and beyond; malabsorbtion was considered the main cause of emaciation in animals so affected (Ladds et al 1994). Helminth infections are no longer a problem on farms in which replacement stock are derived from artificial incubation of eggs collected on the farm or from the wild. They include ascariasis and capillariasis (with worms and their ova present in the stomach), the filarid nematode Micropleura sp. free

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in the abdominal cavity, and trematodes in the gut, kidney or blood (Ladds and Sims 1990; Ladds et al 1995). The number of ascarids (Gedoelstascaris spp.) present seems unrelated to body condition, the fattest hatchlings often having the most worms! Sometimes, however, the adult worms are clearly associated with quite severe gastric ulceration. Microfilaria that are occasionally seen microscopically in tissues are presumably those of filarid nematodes such as Micropleura sp. that usually do not seem to elicit a host response. Whereas the minute blood flukes likewise do not appear to induce a host reaction other than micro-granulomas, quite severe microscopic lesions with loss of intestinal villi, and pyelonephrtis, respectively, may be seen in association with intestinal flukes (probably Cyathocotyle crocodili) and renal flukes (probably Deurithitrema sp.). Depending on husbandry practices, and food offered, parasitic pneumonia caused by the pentastome, Sebekia sp. may be extensive. Grossly the lungs are irregular in colour (sometimes with black sub-pleural foci) and consistency, and histologically various stages of the parasites and their ova are associated with bronchiectasis and inflammation. Diseases of hatchlings resulting from inappropriate nutrition include gout, osteomalacia, a nervous disorder due to deficiency of thiamine, squamous metaplasia in hypovitaminosis A, and perhaps fat necrosis with steatitis due to inadequate vitamin E – although this latter change is more common in older animals. Osteomalacia is characterized by flexible “rubber” jaws and glassy teeth. In gout, white crystalline precipitates are around joints or in deeper organs, particularly the kidneys. Overfeeding for maximum growth with high protein diets appears to be the underlying cause. Thiamine deficiency may be due to thiaminase in frozen fish, and perhaps sulphides in preserved meat. Signs include loss of righting reflex so hatchlings are found floating or lying on their sides with their jaws open (Jubb 1992). Microscopically there is malacia with vacuolation apparent. In deficiency of Vitamin A, multiple pale brown nodules up to 5 mm in diameter were confined to the dorsum of the tongue. Microscopically these nodules consisted of much keratin trapped in affected glands (Ariel et al 1997a). An interesting hatchling disease encountered on several Australian crocodile farms was interdigital subcutaneous emphysema – so-called “bubble foot”. Clinically, gaseous interdigital swellings involve one or more limbs and in severe cases may extend to the base of the tail. These lesions may prevent normal swimming and hence compromise the hatchling’s ability to compete for food. Loss of digits may ultimately occur (Turton et al 1996). Cause of the disease is unknown but may be comparable to gas bubble disease in frogs in which the water source was low in dissolved oxygen but supersaturated with nitrogen and argon gas (Colt et al 1984). In all surveys of hatchling ill-thrift and mortality, the problem of “maladaption” and or “runt” animals has been revealed as significant. Essentially these crocodiles simply refuse to eat normally and become emaciated. Unfortunately, however, detailed necropsy and follow-up laboratory examination in such cases fails to reveal any infectious cause. References Ariel, E., Ladds, P.W., and Buenviaje, G.N. (1997a). Concurrent gout and suspected hypovitaminosis A in crocodile hatchlings. Aust vet J 75, 247- 249. Ariel, E., Ladds, P.W., and Roberts, B.L. (1997b). Mycobacteriosis in young fresh-water crocodiles (Crocodylus johnstoni). Aust vet J, 75, 831 - 832. Buenviaje, G.N., Ladds, P.W. and Martin, Y. (1998). Pathology of skin diseases in crocodiles. Aust vet J, 76, 357 – 363. Colt, J., Orwicz, K. and Brooks, D. (1984). Gas bubble disease in the African Clawed frog (Xenopus laevis). J Herpetology, 18, 131 – 137.

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Hibberd, E.M.A., Pierce, R.J., Hill, B.D. and Kelly, M.A. (1996). Diseases of juvenile farmed estuarine th crocodiles (Crocodylus porosus). Proc 13 Working Meeting of the IUCN Crocodile Specialist Group, Sante Fe, Argentina, 13-17 May 1996. Huchzermeyer, F.W. (2002). Diseases of farmed crocodiles and ostriches. Rev sci tech Off int Epiz, 21, 265 – 278. Jubb, T.F. (1992). A thiamine responsive nervous disease in saltwater crocodiles (Crocodylus porosus). Vet Rec, 131, 347 – 348. Ladds, P.W. and Sims, L.D. (1990). Diseases of young captive crocodiles in Papua New Guinea. Aus vet J, 67, 323 – 330. Ladds, P.W., Donovan, J.A., Reynolds, A. and Turton, J.A. (1994). Giant cell enteritis in young crocodiles. Aus vet J, 71, 300 – 301. Ladds, P.W., Mangunwirjo, H., Sebayang D. and Daniels, P.W. (1995). Diseases of young farmed crocodiles in Irian Jaya. Vet Rec, 136, 121 – 124. Turton, J.A., Ladds, P.W. and Melville, L.F. (1996). Interdigital subcutaneous emphysema (‘bubble foot’) in Crocodylus porosus hatchlings. Aust vet J, 74, 395 – 396.

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ATYPICAL MYCOBACTERIOSIS IN A CAPTIVE HERD OF BARBARY SHEEP (Ammotragus lervia) 1

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TJ Portas , BR Bryant , KR Rose , SL Jones and K Humphries 1

Western Plains Zoo, PO Box 831, Dubbo, NSW 2830 Taronga Zoo, PO Box 20, Mosman, NSW 2088

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Abstract Gross and histopathological lesions consistent with pulmonary mycobacteriosis were identified in two, adult, male Barbary sheep from a captive herd maintained at Western Plains Zoo. A third animal, an adult female was euthanased subsequent to an illness characterised by respiratory signs and was found to have histopathological lesions consistent with pulmonary and mesenteric lymph node mycobacteriosis. In an attempt to characterise the organism and rule out tuberculosis associated with Mycobacterium bovis or M. avium, Ziehl-Neelsen acid fast stains were performed on formalin fixed tissue, impression smears from frozen tissue and on smears made utilizing a concentration technique. Bacteriological samples were set up for aerobic and anaerobic culture and inoculated into Lowenstein-Jensen medium and frozen tissue was submitted for PCR. Acid fast bacilli were evident in pulmonary tissue from both males and from pulmonary tissue and a mesenteric lymph node in the female sheep. M. avium was cultured from the lung parenchyma of the female sheep and a rapid growing Mycobacterium species, later identified as M. parafortuitum by 16s rDNA sequencing, was cultured from a mesenteric lymph node. Diagnostic testing of the herd included collection of blood for a gamma interferon assay, intradermal skin testing and faecal culture for Mycobacterium avium subsp. parartuberculsosis. On the basis of these investigations atypical mycobacteriosis was confirmed in this herd. Immunocompromise secondary to retroviral infection is suspected as the underlying cause but serological investigations to date have been unrewarding.

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THE CAPE OTWAY CENTRE FOR CONSERVATION ECOLOGY Pty Ltd Lizzie Corke and Shayne Neal The Cape Otway Centre for Conservation Ecology Pty Ltd, 635 Lighthouse Rd, Cape Otway, PO Box 296, Apollo Bay, Victoria 3233, t: (03) 5237 9297, f: (03) 52376534, e: [email protected], w: www.capeotwaycentre.com.au Abstract “In the end we will conserve only what we love and we will love only what we understand” BABA DIOUM, African Ecologist. At The Cape Otway Centre for Conservation Ecology we strive to improve our understanding of Australian ecology, and through this, to encourage a love for its beauty and diversity, ensuring its conservation in the future. Our mission statement is …to create and preserve a safe and natural environment for the indigenous flora and fauna, and to share these with people in order that understanding be gained, the animals and vegetation always protected, and the admiration for them never lost. Through our presentation we will discuss how we go about achieving the aims of our mission statement. The Centre will be opening in early 2004, and we look forward to sharing it with you then as we all strive together for the future health of Australia’s wildlife.

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KOOKABURRAS AS A MODEL SPECIES FOR DETERMINING REHABILITATIVE SUCCESS PRELIMINARY RESULTS – MASTERS OF VETERINARY SCIENCE Phillipa Mason Healesville Sanctuary and Melbourne University,, P.O. Box 248, Healesville, Vic. 3777, [email protected] Abstract Over an 18 month period, from May 2002 to December 2003, 21 kookaburras, Dacelo novaeguineae, were released with radiotransmitters in order to determine if rehabilitation had been successful by whether or not they survived. They initially presented to Healesville Sanctuary suffering various traumatic injuries. The majority were released at their point of rescue, but three were released away from their familiar territory. These three moved greater distances, spent less time finding food and were never found at the same place twice. Birds released at their point of rescue developed habitual routines within a week and could be found at set spots at certain times of the day. With the limited data available so far it seems that kookaburras should be released as close to their territory as possible. Of the 10 kookaburras monitored for greater than 14 days, the following was observed. The majority of rehabilitated kookaburras survived once released. 2 were found dead after further misadventure, but neither had lost weight. One of these kookaburras was found 200 meters from the initial rescue point, presumably hit by a car again. 4 birds reintegrated with their family groups within 24 hours, despite being in captivity from 25 to 60 days. The time spent in hospital did not appear to affect the ability of the bird to survive, but all birds were fit at release. Research is continuing and statistical analysis is yet to be done. ACKNOWLEDGMENTS This research could not have come so far without the help of the following: David Middleton,, Peter Holz, Brett Chester, Ian Beveridge,, Ron Slocombe,, Bruce Parry,, Janine Ferguson,, Sam Young,, Vonika Linley,, Karen Moore, Tiffany Eastley, Paul Slinger, Jane Colbert, Anne Cowden and Cybec Trust, nor could it continue without their generous support and encouragement.

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HOP, SKIP and JUMP!....Ogden Nash re-visited on Oz Small Mammals Big fleas have little fleas Upon their backs to bite ‘em Little fleas have lesser fleas And so ad infinitum OR Handling specimens, data and analyses in long-term studies in parasite ecology - a case history. David M. Spratt* and E. Margaret Cawsey CSIRO Sustainable Ecosystems, GPO Box 284, Canberra ACT 2601. Introduction Long-term research in three related areas produced a volume of data to be interrogated, analysed and interpreted efficiently and effectively, and to serve in the future as an accessible repository of parasitological knowledge. Area 1: a 15 year study of the succession of small mammal species following wildfire in eucalypt forests in south-eastern NSW, involving trap, mark, release, re-trap and sub-sample of animals from 6 habitat types 4 times annually to examine quantitatively the re-colonisation and re-structuring of their ecto- and endoparasite communities. Area 2: 30 years of postmortem data from Australian reptiles, birds and mammals including those from the succession study. Area 3: 30 years of data in the form of accession records supporting the CSIRO Wildlife Parasite and Pathology Collections. Collating all of this data from field note books, post mortem sheets and the catalogue of parasitological and pathological specimens was a nightmarish exercise designed to frustrate and infuriate. The solution was the development of a relational database which we call Parabase. This research and analysis engine permits queries into all aspects of these studies including relational information on parasite identification, prevalence, intensity etc. derived from individual hosts or individual host species in relation to season, sex, age, time after perturbation, specific habitat and linked to voucher parasite specimens held in the CSIRO Wildlife Parasite and Pathology Collections. Using that engine, we present a first exploratory interrogation, rather than rigorous analyses, of the flea communities associated with small mammal communities pre- and post-wildfire. Although there has been a considerable amount of work on the fleas of Australia, most research has focused on description of species, their distribution and their general host associations, culminating in the monograph, almost 30 years ago, of Australian fleas by Dunnet and Mardon (1974). There have been no field studies where the structure and dynamics of a flea community have been examined nor where a flea community has been studied after major environmental disturbance, including fire. Fleas have not been considered as indicators of disturbance, although it is possible that some species which are primarily nest parasites may be more severely impacted by disturbances such as fire, where their hosts are eliminated and consequently the flea’s propensity to disperse with new colonisers is severely compromised. Materials and Methods Small mammals were examined at six sites in Nadgee and Timbillica State Forests and the adjoining Nadgee Nature Reserve in coastal southeastern New South Wales near the Victorian border. LU = Ludwigs Swamp - moist open forest, herbaceous understory, adjacent to Ludwigs Ck.

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Tree layer of Eucalyptus elata and E. ovata; a shrub layer of Acacia mearnsii and a herbaceous layer of Carex appressa, Cynodon dactylon, Gahnia clarkei, Pteridium esculentum, Viola hederacea and Dichondra repens. BT = Mines Road Big Tree - wet sclerorphyll forest, shrubby understory on south-facing slope. Tree layer of Eucalyptus muelleriana, E. obliqua and E. cypellocarpa; a shrub layer of Bedfordia arborescens, Lasiopetalum macrophyllum, Correa lawrenciana, Cyathea leichardtiana, Goodia lotifolia and Acmena smithii and a herbaceous layer of Eustrephus latifolius, Tetrarrhena juncea, Gahnia clarkei, Goodenia ovata, Senecio linearifolius and Viola hederacea FT = Mines Road Fire Trail - dry sclerophyll forest, heathy understory on stony ridge sloping upwards from Nadgee River. Tree layer of Eucalyptus sieberi and Angophora floribunda and with an understorey of Pultenaea daphnoides, Hibbertia empetrifolia, Allocasuarina littoralis, Acacia verticillata, Banksia cunninghamii, Hakea sericea, Lomatia ilicifolia and Correa reflexa and a herbaceous layer of Gonocarpus teucrioides, Tetrarrhena juncea, Tetratheca thymifolia, Schoenus melanostachys and Xanthorrhoea australis. SN = Sidling Swamp North - closed wet heath with dense Ti tree thickets grading into dry sclerophyll. Wet heath dominated by Melaleuca squarrosa and sloping uphill to the west into drier sclerophyll forest dominated by Eucalyptus consideniana. In addition to M. squarrosa, the shrub layer contained Xanthorrhoea australis, Banksia serrata and Leptospermum juniperinum and the herbaceous layer consists of Gonocarpus teucrioides, Pteridium esculentum, Gahnia clarkei, Lomandra glauca, Selaginella uliginosa, Burchardia umbellata and Gleichenia dicarpa. SS = Sidling Swamp South - closed graminoid heath. without dense Ti tree thickets grading into dry sclerophyll. Lowland heath with some overstorey layer of Eucalyptus consideniana, E. cypellocarpa and the unusual northern occurrence of E.conspicua. The shrub layer contained Melaleuca squarrosa, Pultenaea daphnoides, Leptospermum juniperinum, Callistemon citrinus and Kunzea spp. and a herbaceous layer of Empodisma minus, Cyathochaeta diandra, Pteridium esculentum, Xyris operculata, Lepidosperma flexuosum, Leptocarpus tenax, Lomandra spp., Schoenus apogon, Gahnia clarkei, Dampiera stricta, Drosera spatulata, Haloragis sp., Selaginella uliginosa and Trachymene incisa. WG = Watergums road - Open dry sclerophyll forest on steep north-facing slope extending down into a creekline. Dominant trees of Eucalyptus sieberi, E. globoidea and Angophora floribunda; a shrub layer of Melaleuca squarrosa, Pomaderris aspera and Pultenaea daphnoides and a herbaceous layer of Gonocarpus teucrioides, Entolasia spp, Goodenia ovata, Pteridium esculentum, Schoenus melanostachys, Brachycome sp. and Gahnia clarkei. th

Collection and examination of animals proceeded for 3.5 years up to the 18 of November 1980 when, in 42C temperatures, wildfire swept through 46,000 hectares of forest, stopping at the Pacific Ocean. Results Six mammal species were trapped in the 3.5 yrs pre-fire: two small dasyurids, the agile antechinus (Antechinus agilis) and the dusky antechinus (A. swainsonii), two peramelids, the long-nosed bandicoot (Perameles nasuta) and the southern brown bandicoot (Isoodon obesulus) and two native rodents, the bush rat (Rattus fuscipes) and the swamp rat (R. lutreolus) (Table 1).

44

Table 1. Numbers and species of mammals examined at six sites in Nadgee and Timbillica State Forests pre-wildfire, August 1977 - November 1980. species Totals Ludwigs Big Fire Sidling Sidling Watergums Tree Trail N S A. agilis 56 11 14 15 5 A. swainsonii 47 6 9 10 8 P. nasuta 8 6 0 0 0 I. obesulus 1 0 0 0 0 R. fuscipes 98 30 11 23 11 R. lutreolus 26 12 0 0 5 Totals 236 65 34 48 29 Overall prevalence of fleas on host species pre-wildfire was low (Table 2).

3 11 1 0 7 9 31

Table 2. Prevalence (%) of fleas on females and males of small mammal species and Timbillica State Forests pre-wildfire, August 1977 - November 1980. species Flea prev. Ludwigs Big Tree Fire Trail Sidling (%) N all all all all ♀♀ ♀♀ ♀♀ all ♀♀ ♂♂ ♂♂ ♂♂ ♂♂ ♀♀ ♂♂ A.agilis 0.0 13.3 42.9 14.3 0.0 0.0 0.0 0.0 14.3 n=56 4.6 20.6 0.0 18.2 71.4 0.0 A. 2.1 0.0 0.0 0.0 0.0 swainsonii 3.7 0.0 0.0 0.0 0.0 0.0 n=47 0.0 0.0 0.0 0.0 P .nasuta 12.5 16.7 n/a n/a n/a n=8 0.0 16.7 0.0 20.0 I. obesulus 100 n/a n/a n/a n/a n=1 n/a 100 R .fuscipes 0.0 13.0 0.0 9.5 13.3 0.0 18.2 0.0 n=98 12.3 6.5 21.4 0.0 8.3 0.0 6.3 R. lutreolus 4.2 0.0 n/a n/a 0.0 n=26 6.7 0.0 0.0 0.0 0.0 0.0 Total 8.9 7.7 17.6 10.4 0.0 N=236 7.8 10.0 10.7 6.3 9.5 0.0 5.4 29.4 11.1 0.0 n/a = no animals of that species/sex trapped at that site

8 3 1 1 16 0 29

at six sites in Nadgee Sidling S

Watergum s

all ♀♀ ♂♂ 0.0 0.0

all ♀♀

♂♂

0.0 0.0

0.0

0.0 0.0

0.0

0.0 n/a

0.0

9.1 16.7 0.0 0.0 n/a 0.0 n/a 14.3 0.0 25.0 12.5 25.0 0.0 10.0 15.4 5.6

0.0

100 n/a 6.3 10.0

100 0.0

n/a

6.9 6.3

7.7

At the sites, prevalence ranged from no fleas on mammals at Sidling North to 17.6% on mammals at Mines Road Big Tree. Prevalence generally was higher on females of both rat species. Fleas occurred exclusively on females of A. swainsonii but prevalence generally was much higher on males than females of A. agilis. Fleas occurred exclusively on male bandicoots. Overall diversity of flea species also was low; only five species were present and they were broadly host family specific rather than catholic, occurring on dasyurids, peramelids and murids (Table 3).

45

Table 3. Occurrence of flea species on small mammals at 6 sites pre-wildfire, July 1977 - November 1980. A. agilis A. P. I. R. R. lutreolus swainsonii nasuta obesulus fuscipes Acanthopsylla BT rothschildi FT FT* rothschildi Bibikovana nhr rainbowi SS* LU FT SS

Macropsylla hercules Stephanocircus dasyuri

WG* Stephanocircus pectinipes

WG

SS*

LU* SS*

nhr BT*

LU FT SS

LU=Ludwigs, BT=Big Tree, FT=Fire Trail, SS=Sidling South, WG=Watergums *=occurred only on a single host individual at this site nhr=new host record Stephanocircus dasyuri surprisingly was not observed on small dasyurids but on bandicoots and bush rats. Seven records of a flea species represent its occurrence on only a single host individual at a particular site (*). Findings pre-wildfire included two new host records (nhr) B. rainbowi on R. fuscipes and S. pectinipes on A. agilis . In contrast, the post-fire data over 11 years involved a greater diversity of both hosts and fleas. Nine host species were present including three small dasyurids with the addition of the white-footed dunnart, Sminthopsis leucopus, the two bandicoots, the pygmy possum, Cercartetus nanus, and three murid rodents with the addition of the house mouse, Mus domesticus.(Table 4). Table 4. Numbers and species of mammals examined at six sites in Nadgee and Timbillica State Forests post-wildfire, February 1981 - December 1991. Totals Ludwigs Big Tree Fire Trail Sidling N Sidling S Fire intensity* 3.0 1.5 1.5 3.0 3.0 A. agilis 452 43 87 145 59 67 A. swainsonii 141 3 26 87 6 16 S. leucopus 22 0 0 0 4 18 P. nasuta 8 1 1 6 0 0 I. obesulus 19 0 1 4 5 8 C. nanus 36 0 0 8 10 18 R. fuscipes 505 95 90 147 42 45 R. lutreolus 231 25 1 37 61 81 M. domesticus 164 34 7 9 25 43 Totals 1578 201 213 443 212 296 * Fire intensity: 1.5 = moderate, some refuge areas remain; 3.0 = severe, no refuge areas

Watergums 3.0 51 3 0 0 1 0 86 26 46 213

Mines Rd FT had the most substantial populations of four animal species (A. agilis, A. swainsonii, P. nasuta, R. fuscipes) and eight of nine species represented. The two Mines Rd sites had the smallest populations of M. domesticus possibly reflecting the less devastating effect of the wildfire at these sites i.e. proximity of unburnt refuge areas facilitating re-colonisation by native species. In contrast, Sidling South shares with the Watergums site (severe fire intensity) the largest and most similar sized

46

populations of M. domesticus. Eight of nine species also occurred at Sidling North and South and these were the only sites where S. leucopus was found, and then only in specific habitat. They contain the largest populations of R. lutreolus. Overall prevalence of fleas on host species was much higher than pre-fire; and there was a similar overall prevalence of fleas across the sites, except at Watergums (Table 5). Table 5. Prevalence (%) of fleas on males and females of small mammal species at and Timbillica State Forests post-wildfire, February 1981 - December 1991. species Flea Ludwigs Big Tree Fire Trail Sidling N prev.(%) all all all all all ♀♀ ♂♂ ♀♀ ♂♂ ♀♀ ♂♂ ♀♀ ♂♂ ♀♀ ♂♂ A. agilis 21.0 16.3 23.0 19.3 22.0 n=452 18.8 24.2 19.2 19.6 18.1 21.6 20.6 24.0 11.8 27.8 A. 24.1 33.3 26.9 23.0 16.7 swainsonii 23.4 25.0 n/a 23.1 22.0 24.3 0.0 25.0 n=141 33.3 30.8 S. leucopus 18.2 n/a n/a n/a 25.0 n=22 16.7 20.0 0.0 33.3 P. nasuta 37.5 0.0 100 33.3 n/a n=8 50.0 0.0 n/a 100 40.0 0.0 0.0 n/a I. obesulus 25.0 0.0 5.3 n/a 0.0 33.3 0.0 n/a 0.0 n=19 11.1 0.0 0.0 n/a C. nanus 13.9 n/a n/a 0.0 40.0 n=36 4.8 26.7 0.0 n/a 33.3 42.9 R. 27.7 28.4 30.0 29.9 35.7 fuscipes 24.6 34.1 28.9 28.1 23.8 n=505 30.7 23.5 31.6 31.1 47.6 R. 24.3 36.1 32.9 56.0 0.0 lutreolus 25.0 32.4 32.2 61.5 0.0 n/a 23.8 40.7 n=231 33.6 50.0 M. 0.0 0.0 0.0 0.0 0.0 domesticus 0.0 0.0 0.0 0.0 0.0 n=164 0.0 0.0 0.0 0.0 0.0 Total 22.7 25.1 25.8 23.5 25.2 N=1578 21.2 28.6 23.6 21.8 22.3 24.3 21.7 28.9 25.5 28.2 n/a = no animals of that species/sex trapped at that site

six sites in Nadgee Sidling S

Watergums

all ♀♀ ♂♂ 28.4 29.0 27.8

all ♀♀ ♂♂ 15.7 6.7 28.6

25.0 27.3 20.0

33.3 100

16.7 18.2 14.3 n/a

n/a n/a

0.0 0.0

0/0 n/a

5.6 0.0

0.0

12.5 20.0 15.0 24.0 32.1 30.8 33.3 0.0 0.0 0.0 21.5 21.3 21.6

0.0

0.0

n/a 20.9 12.0 33.3 19.2 22.2 12.5 0.0 0.0 0.0 15.6 11.0 21.8

There was a predominance of fleas on female hosts at Ludwigs and male hosts at Big Tree, Fire Trail, Sidling North and particularly Watergums. Highest prevalence of infection overall was on P. nasuta and R. lutreolus; in contrast, prevalence was low on I. obesulus. No fleas were found on M. domesticus. Fleas occurred exclusively on females in both bandicoot species, in marked contrast to the situation pre-fire, where they occurred exclusively on males. Fleas were predominant on males of C. nanus. Eleven species of flea were present post-wildfire and there were some surprising findings (Table 6).

47

Table 6. Occurrence of flea species on December 1991 Ante. Ante. Sminth. agilis swains leucop onii us A. rothschild i rothschild i

Acanthop sylla scintilla rectangul ata B. rainbowi

small mammals in 6 habitats post-wildfire, February 1981 Pera m nasut a

Isoodon obesulu s

Cercart etus nanus

Rattus fuscipe s

Rattus lutreol us

Mus domesti cus

nhr LU BT FT SN SS WG nhr FT SN SS

BT FT SS WG

SN*

SN SS

nhr

LU BT FT SN SS WG nhr

SN*

FT*

FT SN

SN SS nhr LU BT FT SN SS WG

FT*

Choristop sylla thomasi

nhr FT SN SS

nhr SN*

Coronap sylla jarvisi

BT FT SN SS WG

Leptopsyl la segnis SN* WG* M. hercules

nhr FT*

Pygiopsyl la hoplia

LU FT SN SS WG

LU BT FT SN SS

LU FT SN SS WG

nhr FT*

Pygiopsyl la zethi

LU BT FT SN SS WG

BT FT SS

FT

FT*

48

S. dasyuri

nhr FT

FT* SS* S. pectinipe s

nhr

FT*

LU BT FT SS

LU SN

nhr

LU LU FT BT SN FT SN* SS SN WG SS WG LU=Ludwigs, BT=Big Tree, FT=Fire Trail, SN=Sidling North, SS=Sidling South, WG=Watergums *=occurred only on a single host individual at this site nhr=new host record LU* BT FT

FT* SN

Acanthopsylla rothschildi rothschild, Pygiopsylla hoplia and Stephanocircus pectinipes were the most common and most catholic flea species, all three species occurring at all times of the year. P. hoplia is the most commonly collected of Australian fleas and known from more than 35 monotreme, marsupial and rodent species plus the rabbit and cattle. Acanthopsylla scintilla rectangulata here is predominantly a flea of the species of Antechinus although the type host of this flea is C. nanus. Bibikovana rainbowi and Macropsylla hercules here are predominantly fleas of the two species of Rattus and occurred throughout the year. B. rainbowi was known previously only from Rattus sordidus in the Atherton region of far North Queensland. M. hercules has been recorded previously almost exclusively from rodents with two records from A. swainsonii and a single record from A. flavipes. Both B. rainbowi and M. hercules occurred on a single Antechinus agilis at Mines Rd Fire Trail along with the only record of Pygiospylla hoplia from this host species, all three representing new host records. Choristopsylla thomasi was encountered only on the pygmy possum and is known also from sugar gliders (Petaurus breviceps) and feathertail gliders (Acrobates pygmaeus). Coronapsylla jarvisi occurred exclusively on A. agilis although A. swainsonii is the type host for the species. It was collected only during the autumn and winter collection periods (March to early July) and only on hosts from the Sidling Swamp and Mines Rd sites. It was not collected pre-fire, was first collected 22 June 1984 (3.5 years post-fire) and last collected 23 June 1989 (8.5 years post-fire) despite 86 A. agilis and 49 A. swainsonii being collected in 1990-1991 posing the question, what influences populations of this flea, or at least its presence on the host? Leptosylla segnis is primarily a flea of the introduced house mouse and introduced Rattus spp. It occurred on only two hosts within 18 months of wildfire when Mus domesticus populations were escalating and in the two habitats where the largest populations of M. domesticus occurred, yet fleas were never observed on 164 M. domesticus and neither of the introduced R. rattus nor R. norvegicus were trapped in this study. Pygiopsylla zethi occurred exclusively on P. nasuta and previously was well known from bandicoots and other marsupials. Stephanocircus dasyuri, despite its specific name, was rare on the small dasyurids and more common on bandicoots and rats. It is the second most commonly collected of Australian fleas occurring on more than 30 host species with a single record from A. swainsonii. There is a new host record here on A. agilis. Sixteen records of a flea species represent its occurrence on only a single host individual at a particular site, bringing the total in this study to a surprising twenty three such occurrences. Fourteen new host records occurred in the post-wildfire period bringing the total in this study to sixteen new host records.

49

While this is only a preliminary interrogation rather than rigorous analyses of the data, the results are indicative, often surprising and provide direction for future research. In most cases, only a very small proportion of an adult flea’s time is spent on the body of the host. When we capture an animal, we must presume that an unknown number of fleas remain in the “nest habitat” of that particular animal. But, we have no idea about that potential number nor the species composition, prompting the question, what intrinsic or extrinsic factors might influence the probability of adult fleas being on the body of their host? A potentially important aspect about this characteristic of infestation is that when a host animal disperses, it carries with it the potential colonisers for future flea generations. This prompts a further question, are there inherent characteristics of flea life history or behaviour that favour their dispersal with their hosts to new habitats? Conclusions These preliminary explorations were facilitated by the relational design of the database and by the features of the database management system. Parabase has saved this researcher considerable time, effort, pain, frustration and, worst of all, error. Future explorations and analyses of these data and those from other hosts and parasites will be quicker and easier than previously, but most importantly we can rely on the data and their aggregates to be as correct as is possible. The beauty of these kinds of exploratory analyses is that they highlight outliers which may point to errors in the data, and these can be corrected rapidly using other features of the database. Most importantly, you don’t have to correct it here and then go and find where it must be corrected somewhere else, the correction results in instant updating of the related numbers and aggregates throughout Parabase. We will never find all the “bugs”, perhaps, but use of these database management tools certainly assist us to examine them more effectively and use our research time to maximal advantage. References Dunnet, G. M. and Mardon, D.K. (1974). A monograph of Australian fleas (Siphonaptera). Australian Journal of Zoology, Supplementary Series No. 30, 1-273.

50

HEALTH MONITORING OF THE JENOLAN CAVES BRUSH-TAILED ROCK WALLABY (Petrogale penicillata) COLONY. Larry Vogelnest, Senior Veterinarian, Taronga Zoo Mikaylie Wilson, Veterinary Nurse, Taronga Zoo Brush-tailed Rock wallabies were once regarded as common in the Jenolan Caves area. In the 1950’s a rapid decline became apparent due to predation by foxes and feral cats. Competition from goats and rabbits, fire regimes, isolation and inbreeding also contributed toward the decline. In 1964 a large enclosure was built and a captive breeding program established to prevent localised extinction of the species. The wild population in this area has continued to decline. With the success of the captive breeding program animals were released ad hoc in 1988. Due to predation, the population of released animals declined dramatically. By 1992 the wild population was on the verge of local extinction. The remaining nine wild animals were captured and transferred to the enclosure. The Zoological Parks Board of NSW has assisted with the management of the colony since 1993. NPWS officers and Western Plains Zoo staff trapped, measured, sampled and micro-chipped individuals in order to build up a data base on the colony. This work ceased some years ago. Recently there has been renewed interest in the colony. Through the Brush-tailed Rock wallaby recovery plan there are plans to translocate and reintroduce animals from populations within NSW and even further a field. The Jenolan colony will be part of this. In order to do this successfully it is essential to establish the health and reproductive status, number of animals and the age and sex distribution within populations. Consequently trapping expeditions to the Jenolan colony have again commenced. Taronga Zoo veterinary staff has participated in these trapping expeditions to establish the health status of the colony. Animals are weighed, measured, checked for a microchip and one inserted if not present, examined and blood sampled. Other samples as indicated by the clinical examination are also collected.

51

CROSS-REACTIVE ANTIBODIES POTENTIALLY USEFUL IN CHARACTERISATION OF MARSUPIAL TUMOURS 1

2

2

S Hemsley , M Jones and J Cordell

1 Faculty of Veterinary Science B01, University of Sydney NSW 2006 [email protected] 2 LRF Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK Introduction The way by which tumours are generally diagnosed in veterinary species is by the examination of histochemically stained tissues1. While these staining procedures are useful in many cases, soft tissue sarcomas (including fibroma, fibrosarcoma, schwannoma and haemangiopericytoma), malignant histocytomas and round cell tumours are examples of tumours that can be difficult to diagnose by standard histological techniques. In addition, subclasses of lymphocytes involved in lymphosarcoma are impossible to differentiate histochemically. Classifying the phenotype and proliferative capacity of tumour cells is desirable because it can facilitate understanding of tumour behaviour and prognosis. Immunohistochemistry using specific 1 monoclonal or polyclonal antibodies can be utilised to help provide this information . In relation to tumour characterisation, tissue components that can be labelled include cytoskeletal components (e.g. actin, desmin, vimentin and cytokeratins), other nuclear and cytoplasmic components (e.g. tdt, S-100), enzymes (eg lysozyme) and surface markers (e.g. CD3 and CD79a/b). Quantitation of the Ki-67 epitope has been shown to be useful in determining the proliferative potential of neoplastic cells. This epitope is expressed in all tissue types, but exclusively in the nuclei of cycling cells2. Immunohistochemistry has been used for some time in the human field, but it has only fairly recently been used to any great extent in the investigation of neoplasia in 1 other animals . 3,4

Athough a range of tumours occur in Australian marsupials , use of immunohistochemistry has been confined 5,6 to determining the subtype of lymphocytes involved in koala lymphosarcoma , due to a lack of specific marsupial reagents. Spindle cell tumours, some round cell tumours, some epithelial tumours and nodular serosal proliferations in koalas are specific examples of marsupial tumours that may be difficult to classify3,4,7, P Canfield pers. comm. . This paper discusses preliminary results for immunohistochemical staining of marsupial tissue with a range of antibodies which have potential for tumour characterisation. Materials and Methods Sections from formalin-fixed paraffin embedded blocks of tissue were cut at 4 m and labelled immunohistochemically using an immunoperoxidase streptavidin-biotin horseradish peroxidase method8. Antibodies used are detailed in Table 1. Results Results are summarised in Table 2. Discussion This work, while only preliminary, shows that a number of antibodies potentially useful in tumour investigation can be successfully applied to immunohistochemistry of marsupial tissues. However, some antibodies which are potentially valuable did not stain marsupial cells, or appeared to only stain a subpopulation of the cells of interest (specifically; anti-von Willebrand Factor, anti-myeloid/histiocyte antigen, anti-LSP1 in possums and anti-Ki67). This does not mean, however, that extensions of this study might not result in successful labelling in these cases. For example, different antigen retrieval methods or different antibodies raised against the same antigen could result in positive labelling. In addition, there are a range of other potentially useful antibodies which have not been investigated yet, for example antibodies directed against other cytokeratins, glial fibrillary acidic protein, neurofilaments and neuronspecific enolase and antibodies against markers of metastasis such as E-cadherin1. The use of the

52

antibodies investigated in the current study, and potentially other antibodies, in the investigation of neoplasia in marsupials, could have implications both for the management of populations (the current problem with Tasmanian devils being a pertinent example) and individual animals. References 1. Rhind SM. Veterinary oncological pathology – current and future perspectives. The Veterinary Journal, 2002; 163: 7-18. 2. Brown DC and Gatter KC. Monoclonal antibody Ki-67: its use in histopathology. Histopathology, 1990; 17: 489-503 3. Canfield PJ, Hartley WJ and Reddacliff GL. Spontaneous proliferations in Australian Marsupials – a survey and review. 1. Macropods, koalas, wombats, possums and gliders. Journal of Comparative Pathology, 1990; 103: 135-146. 4. Canfield PJ, Hartley WJ and Reddacliff GL. Spontaneous proliferations in Australian Marsupials – a survey and review. 2. Dasyurids and bandicoots. Journal of Comparative Pathology, 1990; 103: 147-158. 5. Canfield PJ and Hemsley S. Thymic lymphosarcoma of T cell lineage in a koala (Phascolarctos cinereus). Australian Veterinary Journal, 1996; 74: 151-154. 6. Connolly JH, Canfield PJ, Hemsley S and Spencer AJ. Lymphoid neoplasia in the koala. Australian Veterinary Journal, 1998; 76: 819-825. 7. Canfield PJ, Hartley WJ, Gill PA, Miller R, Obendorf DL and Brown AS. Serosal proliferations in koalas. Australian Veterinary Journal, 1990; 67: 342-343. 8. Hemsley SW, Canfield PJ and Husband AJ. Immunohistological staining of lymphoid tissue in four Australian marsupial species using species cross-reactive antibodies. Immunology and Cell Biology, 1995; 73: 321-325. Table 1. Antibodies Used Antibody (clone) Anti-vimentin (V9)

Specificity/Use a

Cells of mesenchymal origin

Anti-swine desmin (DER11) Anti-human desmin (DE33)

a

Muscle cells

a

Muscle cells

Anti-human cytokeratin (LP34) a Anti-human cytokeratin (MNF116) Anti-bovine S-100 (polyclonal)

Cells of epithelial origin a

Cells of epithelial origin

b

Neuroectodermal tissue, melanocytes, Macrophages/dendritic cells Endothelial cells

Anti-human von Willebrand factor (polyclonal) b Anti- human myeloid/histiocyte antigen (MAC387) Anti-human Lymphocyte Specific Protein (LSP) 1 a (TPD153) b Anti-human CD3 (polyclonal) Anti-human CD79b (B29)

a

Anti-terminal deoxynucleotidyl transferase (tdt) (polyclonal) b c Anti-Ki-67 (MIB-1)

a

Granulocytes, monocytes, histiocytes Lymphocytes, blood monocytes, Granulocytes T lymphocytes B lymphocytes T and B cell precursors Proliferating cells (all tissue types)

a

LRF Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford DAKO c Immunotech b

53

Table 2: Results for immunohistochemical staining of tissues by species and site a

Ab Specificity

Species(No.)

Tissue (No.)

Result

Vimentin

Koala (2) Koala (1) Koala (5) BTP (4) Tas Devil*(2) Koala (3) BTP (3) Tas Devil (2) Koala (1) BTP (3) Tas Devil (1) Koala (1) BTP (3) Tas Devil (2) BTP (2) Tas Devil (2) Koala (1) EGK (1) EGK (1)

Spleen (1), lymph node (2) Lymphosarcoma (1) Nodular serosal proliferation (5) Spleen (3), tonsil (1) Tonsil (1), lymph node (1) Gut (1),lung (1),spleen (2),lymph node (1) Tonsil (3), lymph node (1), gut (1) Tonsil (1), spleen (1) Spleen (1), lymph node (1) Tonsil (2), spleen (1), gut (1) Tonsil (1) Pancreas (1) Tonsil (3), gut (1) Skin (1), pancreas (1) Tonsil (2), lymph node (1), gut (1) Skin (1), pancreas (1) Spleen (1), gut (1) Spleen (1), gut (1) Spleen (1), gut (1)

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative

Koala (3) BTP (5) Tas Devil (2) Koala (3) Koala (4) BTP (6) RTP (1) EGK (1) Tas Devil (1) Tas Devil (2)

Spleen (3), lymph node (3), gut (1) Spleen (4), lymph node (2), tonsil (1) Tonsil (1), lymph node (1) Spleen (3), lymph node (3) Lymphosarcoma Spleen (4), tonsil (2), lymph node (2) Thymus (1) Spleen (1), gut (1) Tonsil (1) Tonsil (1), lymph node (1)

(Positive) Negative (Positive) Positive Positive Negative (Positive) Positive Positive Positive

CD79bb

Tas Devil (2)

Tonsil (1), lymph node (1)

Positive

Tdt

RTP (1)

Thymus (1)

Positive

Ki-67

Koala (1) BTP (1) EGK (1)

Gut (1) Tonsil (1), lymph node (1) Spleen (1), gut (1)

Negative Negative Negative

Desmin (DER11)

Desmin (DE33)

Cytokeratin (LP34)

Cytokeratin (MNF116) S-100 Factor VIII Myeloid/histiocyte antigen

LSP1

CD3

b

a

Normal tissue unless stated otherwise Shown previously to cross-react in koalas, brushtail possums, ringtail possums and eastern grey 8 kangaroos BTP=brushtail possum; Tas Devil = Tasmanian Devil*; EGK = eastern grey kangaroo; RTP = ringtail possum; (positive) = scattered cells only positive b

*Tasmanian Devil tissues were kindly provided by Richmond Loh, Animal Health Laboratories Department of Primary Industries, Water & Environment, Kings Meadows, Tasmania

54

GAMMA INTERFERON ENZYME IMMUNOASSAYS AND THEIR USE IN THE INVESTIGATION OF TUBERCULOSIS IN A WESTERN LOWLAND GORILLA Helen E. McCracken, BSc(Vet), BVSc, MVS

1*

1

Senior Veterinarian, Melbourne Zoo, PO Box 74, Parkville, Victoria, 3052, Australia

Abstract Tuberculosis (TB) in non-human primates, caused by Mycobacterium tuberculosis or Mycobacterium bovis, is a disease of great concern in zoos due to its capacity for insidious spread to other collection animals, free-ranging animals in zoo grounds, and to human contacts. The majority of TB infections are controlled by the host’s immune defences and remain latent, however some such infections progress to active and contagious disease. It is very important therefore, to have a sensitive and specific diagnostic test to identify latent cases, so that further spread of disease may be prevented by either treatment or 4 culling of these individuals. Over the past decade, gamma interferon (IFN-γ) enzyme immunoassays (EIA) have been developed for rapid in vitro diagnosis of TB in several species, including humans and non-human primates. This paper reviews the relative merits of these assays and other currently available TB diagnostic techniques. Details are presented of the investigation of TB in a Western Lowland Gorilla at Melbourne Zoo, Australia, demonstrating the application of these assays. 1,5

The “gold standard” for TB diagnosis is isolation of the mycobacterial organism by culture. This process, however, can take up to 8 wk, and in 10-20% of human TB cases the organism is not successfully isolated.1 Furthermore, bacteria can only be cultured from cases of active disease, and the 1 test is therefore of no value in the diagnosis of latent TB. The screening test for both latent and active TB used for many years in both humans and non-human primates is the tuberculin skin test (TST). Intradermal injection of tuberculin purified protein derivative (PPD) will induce a cell-mediated immune (CMI) response in infected individuals, producing induration at 5 the injection site, measured at 72hr. Although this test has been used widely in humans for over a century, it is subject to considerable variation and other limitations. Inconsistencies and errors in the administration of the PPD and in the reading of results may produce either false negative or false positive results. False negatives may also occur due to anergy in immunocompromised individuals with a wide range of concurrent disease conditions. False positives may result from contact with environmental mycobacteria which share common antigens with M.tuberculosis and M.bovis, or from prior vaccination with Bacille Calmette Guérin (BCG), a strain of M.bovis which is also antigenically similar to the 1,3,4 pathogenic organisms. It is expected that these limitations also apply with use of the TST in nonhuman primates, including the effects of BCG vaccination which has historically been used occasionally in non-human primates (Andreas Knieriem, pers. comm.). Furthermore, the efficacy of this test has not been established for the vast majority of non-human primate species, and tuberculin testing practices vary widely between zoos, including differences in the PPD preparation(s) used, antigen strength and injection site.5 Comparative testing, involving the simultaneous administration of M.tuberculosis or M.bovis PPD and Mycobacterium avium PPD at two different sites, is used commonly in non-human primates to differentiate between TB and infection with environmental mycobacteria. However, interpretation of the results of such tests in humans has shown considerable variability1, and there have been no evaluations of the reliability of the procedure in non-human primates. The TST also has the disadvantage that it must be read after 72 hr. This introduces the issue of potential non-compliance in 4 human patients , and compromises the ability to accurately evaluate the result in non-human primates because frequently the injection site is only observed and not palpated, as it is preferable in most cases to avoid restraining animals again within such a short time period. Several alternate methods for indirect TB diagnosis have been developed. Lymphocyte transformation assays involve incubation of lymphocytes with mycobacterial antigens. In animals with mycobacterial infection, sensitised T-lymphocytes will undergo cell division in response to the antigens, and the expansion of these cell populations is measured using radiolabelling techniques. This test has been evaluated in deer and found to have 95% sensitivity and 92% specificity, however, its value in many

55

5

species remains to be demonstrated. The major disadvantages of this test are that strict protocols must be followed in the methods and timing of blood collection and laboratory submission, and it is a labour intensive and lengthy (approximately 7d) laboratory procedure (Jonathan Streeton, pers. comm.). It is therefore not very practical or cost effective as a routine screening test. Enzyme immunoassays (EIA) that detect circulating anti-mycobacterial antibodies have been developed for several animal species. These assays appear to be useful in detecting active TB in several species, 3, 5 but less effective in detecting latent infection. In cases of active TB, there is a heavy bacterial load and concomitant high levels of circulating antibody resulting from the inability of the immune system to control the infection. However, because mycobacteria are intracellular pathogens which replicate within host macrophages, host defences are believed to be largely dependent on T-lymphocytes, with antibodies 3 being of only minor importance. It is likely, therefore, that only low levels are present in cases of latent disease. The persistence of mycobacteria within macrophages is believed to be due to their ability to switch off the normal process of phagocytosis. The surrounding lymphocytes become cognisant of these persistent 6 bacteria and secrete cytokines, including IFN-γ, in an effort to stimulate the colonised macrophages. The CMI, measured by the TST, is dependent on the production of these cytokines by the sensitized 3, 6 lymphocytes at the site of the tuberculin injection, in recognition of the mycobacterial protein. Based on this phenomenon, Wood et al. developed an in vitro assay for measuring CMI responses. The assay involves overnight incubation of small aliquots of whole blood with tuberculin PPD antigens and mitogen (phytohaemagglutinin) to stimulate sensitised lymphocytes to produce IFN-γ. The blood is also cultured 2,3,7 with a nil antigen. The plasma supernatant is then assayed for IFN-γ using an EIA. The nil antigen control is used to detect IFN-γ in the circulation which, if present, may mask specific responses and make interpretation difficult. The mitogen antigen is used as a positive control to demonstrate that the blood contains immunologically competent T cells capable of producing IFN-γ. Inadequate response to this control may indicate immunosuppression or blood sample deterioration, and the test would be considered 3 invalid. This assay was first applied to TB diagnosis in cattle, using bovine and avian PPDs and an EIA specific for bovine IFN-γ (BOVIGAM™, CSL Animal Health, Parkville, Victoria 3052, Australia). This test has been extensively trialled and found to have 88-100% sensitivity for culture-confirmed bovine TB (compared with 72% for the skin test) and 94-100% specificity. It has been approved by the USDA, and is now used routinely in many countries for TB diagnosis in cattle, buffalo and goats. It is also known to 8 be effective in several exotic hoofstock species. The assay was subsequently applied to the diagnosis of TB in humans, using human and avian PPDs and an EIA specific for human IFN-γ (QuantiFERON®–TB, Cellestis Ltd., St. Kilda, Victoria 3182, Australia).3, 4 This test has been found to have 90% sensitivity and 6 98% specificity in the diagnosis of latent TB. It is now used as a diagnostic test in Australia and was approved by the US FDA in late 2001. Most recently, the assay has been modified for the diagnosis of TB in non-human primates, using bovine and avian PPDs and an EIA specific for primate IFN-γ 2 (PRIMAGAM™, CSL Animal Health, Parkville, Victoria 3052, Australia). It has been used successfully in gorillas, chimpanzees, orangutans, gibbons, macaques, baboons, mandrills, guenons, vervets, langurs, guerezas, squirrel monkeys, marmosets, tamarins, and lemurs (Stephen Jones, pers. comm.). It is currently used as a diagnostic test in Australia and, at the time of writing, has approval pending from the USDA. IFN-γ assays have several advantages over the TST and other indirect diagnostic techniques for TB. The tests require a single patient visit; they are not influenced by tester/observer error; they can be completed in less than 24 hr; the procedure is routine and can be handled by any standard serology laboratory performing EIA’s; they do not involve the introduction of foreign protein into an individual and therefore may be repeated as frequently as required; they differentiate between TB and exposure to environmental mycobacteria; and they include assessment of the patient’s current immune status, permitting 4, 6 assessment of the validity of the test response. The current QuantiFERON®-TB and PRIMAGAM™ tests, however, do not differentiate between IFN-γ responses generated by TB infection and BCG 1 vaccination, hence “false positives” may still occur in BCG vaccinees. Recently, specific antigens, ESAT-6 and CFP-10, have been identified which are present in the genome of M.tuberculosis and M.bovis (and three species of atypical mycobacteria rarely associated with disease), but not in BCG or

56

the other non-tuberculosis mycobacteria. Inclusion of ESAT-6 and/or CFP-10 in IFN-γ assays in the 1 future will permit differentiation of these responses. The following case investigation demonstrates the use of IFN-γ assays in TB diagnosis. A 20 yr old female Western Lowland Gorilla at Melbourne Zoo (“Julia”) was immobilised in Jan 2002 for investigation of chronic intermittent lameness. Osteoarthritis was diagnosed and routine TB tests performed, including a comparative TST and PRIMAGAM™ assay. These tests both detected a strong response to bovine PPD, indicating infection with M.bovis or M.tuberculosis (Table 1). This was a surprising result because there has been no known case of TB in any animals at the zoo in the past 30 years. Quarantine of all new arrivals to the zoo, including TB testing of all primates, pre-employment TB testing of all staff with animal contact, and regular surveillance of the TB status of individual primates have all been effective prophylactic measures against this disease entering the collection. TB is not reported in any native or feral animal species found in the zoo grounds, and bovine TB has officially been eradicated from Australia. Our gorilla group lives in a large open air display, and visitors view the animals either through glass or at a minimum distance of 10m. TB transmission from visitors, therefore, is not considered a possibility (Jonathan Streeton, pers. comm.). Julia was wild-caught as an infant in Gambia in 1982. She was subsequently housed with chimpanzees in a rehabilitation-release centre, then relocated to a European zoo in 1990. A single TST was performed on arrival there, with a negative result. She was relocated to Melbourne in Dec 1997; a comparative TST was performed while she was immobilised for placement in the shipment crate. Seventy-two hr later, there was slight induration at the bovine PPD injection site. She was immobilised 3wk later for TB investigations including repeat TST, PRIMAGAM™ assay, thoracic radiographs and specimen collection for acid fast smears and mycobacterial culture. On this occasion, the TST and PRIMAGAM™ were both negative and the other diagnostic procedures did not detect evidence of TB. She was therefore released from quarantine. Following the positive TST in Jan 2002, Julia was immobilised again for TB investigations, including all procedures undertaken in Jan 1998, with the addition of gastric and colonic endoscopy and biopsy, and PCR on specimens collected for culture. While the PRIMAGAM™ was positive, all other tests were negative for evidence of active disease. Furthermore, Julia has no clinical signs of active disease. A prophylactic course of isoniazid and rifampicin was implemented. IFN-γ assays proved very valuable in the investigation of this case: 1. There was a possibility that the TST result in Jan 1998 was a false negative, either due to errors in injection or reading techniques, or due to immunosuppression, possibly induced by the significant stress of relocation. These possibilities, however, were ruled out as the PRIMAGAM™ result was also negative, and the strong mitogen response indicated immunocompetence. It was concluded from this result that the earlier TST response was therefore likely to be a false positive, possible due to injection trauma or other trauma to the eyelid induced during the relocation process. 2. It was also possible that the TST result in Jan 2002 was a false positive, either due to injection technique, exposure to environmental mycobacteria, or previous BCG vaccination. The strong concurrence of the PRIMAGAM™ result with the TST ruled out the first two possibilities. The response to bovine PPD was significantly greater than that to avian PPD, indicating infection with M.bovis or M.tuberculosis. As it was plausible that Julia had been given BCG in Gambia, ESAT-6 and CFP-10 were included in the IFN-γ assay in Feb 2002. These are experimental antigens, provided by the Statens Serum Institute, Denmark, and Cellestis Ltd, Australia. Both produced strongly positive results which clearly ruled out the possibility of a BCG reaction because these antigens are not present in the BCG genome. Only the result for the ESAT-6 assay appears in Table 1 because there was insufficient blood to quantify the CFP-10 result. These results confirmed that Julia is infected with M.bovis or M.tuberculosis. There is no indirect diagnostic test that can differentiate between these two organisms. 3. The source of Julia’s infection has not been identified, however PRIMAGAM™ results from Jan 1998 are worth some attention. The OD Bovine PPD was > OD Avian PPD by 0.036, and OD Bovine PPD was > OD Nil antigen by 0.040. While both differences are less than the test cut-off point of 0.05, they are greater than those in all other adult gorillas tested (see Table 2), and these values may in fact be reflective of recent infection with TB. The protocol for screening humans following TB exposure is to

57

perform two TSTs 3 mo apart, because CMI does not develop immediately following infection, and a 6 case of early disease may be missed if the patient is only tested once. Australian quarantine protocols required Julia to be held in post arrival isolation for 60d, and a single TST performed in that time. The outcome of this case suggests that a longer post arrival quarantine period is appropriate for imported primates, including two TSTs 3 mo apart. 4. Subsequent to the diagnosis of TB in Julia, all other gorillas in the group and all personnel who had worked closely with her or her wastes were tested for TB using both TST and IFN-γ tests. Results of these tests are presented in Tables 2 and 3. All gorillas were negative for TB on both tests, but the IFN-γ results of Male #3 and Female #4 included a mild response to avian PPD which exceeded the bovine PPD response, indicating exposure to environmental mycobacteria. These are hand-reared infants which share an enclosure not accessed by other group members. The strong mitogen response reported for all gorillas ruled out the possibility of false negatives due to anergy. This is significant because all animals except Females #1 and #3 were infected with Varicella Zoster Virus either at, or close to, the time of testing. If TSTs alone had been performed, immunosupression by concurrent disease would have been considered a possibility. 5. Nineteen staff members were tested, all of whom had been mantoux tested 1-4 yr earlier. Three people had significant conversions in their mantoux responses. One other person (Veterinarian) did not present for mantoux test reading but had a positive QuantiFERON®-TB result. All four had histories of BCG vaccination, but as they had shown no or negligible response to their previous mantoux, these recent reactions caused some concern. The IFN-γ assays proved very useful in the interpretation of these responses (see Table 3). The QuantiFERON®-TB results of Gorilla Keeper #1 showed a dominant response to avian PPD, indicating that her TST conversion was due to exposure to environmental mycobacteria. The IFN-γ assays of the other three were all positive for M.tuberculosis complex organisms, however their ESAT-6 and CFP-10 assays were negative, clearly indicating that their responses were to BCG, and they are not infected with M.bovis or M.tuberculosis. If IFN-γ assays had not been available, Gorilla Keepers #1 and #2 would have been diagnosed as having latent TB on the basis of existing criteria (mantoux induration >15mm in BCG vaccinees) and prescribed a 9 mo course of isoniazid. All other tested staff members showed no recent mantoux conversions or significant IFN-γ results. The use of IFN-γ assays in this investigation has permitted us to conclude at this stage that there is no evidence of disease spread from the infected gorilla. All contact gorillas and staff will be retested 3 mo after their first test. Meanwhile strict quarantine procedures are in place within the zoo. ACKNOWLEDGEMENTS The author would like to thank Dr. Stephen Jones of CSL Animal Health for performing the PRIMAGAM™ ,ESAT-6 and CFP-10 assays, and for his invaluable advice in the investigation of this case. Sincere thanks are also due to Dr. Jonathan Streeton for his generous advice and support in the investigation and management of the gorilla and contact personnel. Thank you also to Dr. Peter Anderson, Statens Serum Institute (Copenhagen, Denmark) and Dr. Jim Rothel, Cellestis Ltd. (Melbourne, Australia) for providing the experimental ESAT-6 and CFP-10 antigens, and to the staff of Melbourne Zoo for their professionalism and cheerful co-operation throughout the investigation. LITERATURE CITED 1. Anderson, P., M.E. Munk, J.M. Pollock and T.M. Doherty. 2000. Specific immune-based diagnosis of tuberculosis. The Lancet 356:1099–1104. 2. C.S.L. Veterinary Division. 1998. Primagam™: Non-human primate gamma interferon test: an assay of cell mediated immunity and detection of tuberculosis infection in non-human primates. Manufacturers Instructions for Use. C.S.L. Limited, Melbourne, Australia. 3. Desem, N. and S.L. Jones. 1998. Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection. Clin. Diagn. Lab. Immunol. 5(4):531–536. 4. Mazurek, G.H., P.A. LoBue, C.L. Daley, J. Bernardo, A.A. Lardizabal. W.R. Bishai, M.F. Iademarco and J.S. Rothel. 2001. Comparison of a Whole Blood Interferon γ Assay with Tuberculin Skin Testing for Detecting Latent Mycobacterium tuberculosis Infection. J.A.M.A. 286(14):174 –1747.

58

5. Mikota, S.K. and J. Maslow. 1997. Theoretical and technical aspects of diagnostic techniques for mammalian tuberculosis. Proc. Amer. Assoc. Zoo Vet:162–165. 6. Streeton, J.A., N. Desem and S.L. Jones. 1998. Sensitivity and specificity of a gamma interferon blood test for tuberculosis infection. Int. J. Tuberc. Lung Dis. 2(6):443–450. 7. Wood, P.R., L.A. Corner and P. Plackett. 1990. Development of a simple, rapid in vitro cellular assay for bovine tuberculosis based on the production of γ interferon. Res. Vet. Sci. 49:46-49. 8. Wood, P.R. and S.L. Jones. 2001. Bovigam™: an in vitro diagnostic test for bovine tuberculosis. Tuberculosis 81 (1/2):147–155. Table 1: RESULTS OF TB SKIN IMMUNOASSAYS IN GORILLA “JULIA”

Date

TESTING

AND

GAMMA

INTERFERON

COMPARATIVE SKIN TESTING

PRIMAGAM™ ASSAY 1 2 (EIA ODs)

Bovine PPD

Avian PPD

Nil antigen

Bovine PPD

Avian PPD

Mitogen

Result

ENZYME

ESAT-63 Pg/ml

12.7.97

Weak +ve

-ve

-

-

-

-

-

-

1.6.98

-ve

-ve

0.026

0.066

0.030

1.473

-ve

-

1.31.02

Strong +ve

Weak +ve

0.043

0.591

0.103

3.233

+ve

-

2.22.02

-

-

0.051

0.938

0.182

4.300

+ve

83.5 (+ve)

Table 2:

RESULTS OF TB TESTING OF GORILLAS EXPOSED TO “JULIA” “POST-EXPOSURE” TB TESTING, FEBRUARY-APRIL 2002

ANIMAL (age)

Female #1 (44 y.o.) Female #2 (31 y.o.)

TB TEST HISTORY

Last tested April 94: ve. Last tested Nov 99: ve.

4

Comparative Skin Test Avia Bovin n e PPD PPD

PRIMAGAM™ Assay (EIA ODs)5 Avia Nil Bovin n Antigen e PPD PPD

-ve

-ve

0.048

0.045

-ve

-ve

0.032

0.032

Mitogen

Result

Thoracic radiographs

0.05 9

0.873

-ve

-

0.04 0

0.954

-ve

NSF

6

1

PRIMAGAM™ is a IFN-γ assay for the detection of TB in non-human primates (CSL, Australia). Results are interpreted as positive for exposure to M.tuberculosis complex organisms if ALL of the following criteria are met: OD Bovine PPD > OD Avian PPD by ≥ 0.05 OD Bovine PPD > OD Nil antigen by ≥ 0.05 OD Mitogen > 0.05 2 Enzyme Immunoassay Optical Densities 3 This is an assay for IFN-γ produced in response to the ESAT-6 antigen. Results are interpreted as positive when greater than 10 pg/ml

4

See Table 1, Footnote1 Enzyme Immunoassay Optical Densities 6 NSF: no significant findings; no evidence of TB lesions 5

59

“POST-EXPOSURE” TB TESTING, FEBRUARY-APRIL 2002 ANIMAL (age)

Female #3 (22 y.o.) Male #1 (19 y.o.) Male #2 (2.5 y.o.) Male #3 (2 y.o.) Female #4 (2 y.o.)

TB TEST HISTORY

Last tested April 00: ve. Last tested July 00: ve. No previous testing No previous testing No previous testing

Table 3:

4

Comparative Skin Test Avia Bovin n e PPD PPD

PRIMAGAM™ Assay 5 (EIA ODs) Avia Nil Bovin n Antigen e PPD PPD

-ve

-ve

0.029

0.037

-ve

-ve

0.051

-ve

-ve

-ve

-ve

Mitogen

Result

Thoracic radiographs

0.04 2

3.402

-ve

NSF

0.053

0.06 9

0.323

-ve

-

0.021

0.028

0.03 5

0.751

-ve

NSF

-ve

0.031

0.114

0.13 5

2.093

-ve

NSF

-ve

0.040

0.068

0.10 4

0.922

-ve

NSF

RESULTS OF TB TESTING OF PERSONNEL EXPOSED TO GORILLA “JULIA” “POST-EXPOSURE” TB TESTING, FEBRUARY-APRIL 2002 QuantiFERON®-TB Assay8

7

STAFF MEMBER

BCG VACCIN E STATU S

PREVIO US MANTO UX TEST

Manto ux test

Huma n PPD IU/ml

Avia n PPD IU/ml

Mitoge n IU/ml

% H/ M

% HA Resu lt H

ESAT6, CFP109 pg/ml

Thorac ic Radiographs

Gorilla Keeper #1

Yes

Oct 98 5mm

20mm

7.9

15.3

52.9

15

-94

-ve

5.6, 0 (-ve)

NSF

Gorilla Keeper #2

Yes

Dec 98 0mm

20mm

45.9

39.7

111.3

41

14

+ve

2.1, 0 (-ve)

Pendin g

Veterinarian

Yes

Feb 01 0mm

Not read

9.9

4.4

13.1

76

56

+ve

0.4, 0.7 (-ve)

Pendin g

10

7

Bacille Calmette Guérin QuantiFERON®-TB is an IFN-γ assay for the detection of TB in humans (Cellestis, Australia). Results are interpreted as positive for exposure to M. tuberculosis complex organisms if ALL of the following criteria are met: Human PPD > 1.5 IU/ml Mitogen > 1.5 IU/ml % H/M (% Human/Mitogen) > 15% % H – A / H (% Human – Avian / Human) > -10% 9 These are assays for IFN-γ produced in response to the ESAT-6 and CFP-10 antigens. Results for both assays are interpreted as positive when greater than 10 pg/ml 10 NSF: no significant findings; no evidence of TB lesions. 8

60

Waste Disposal Staff Member

Yes

Sept 01 0mm

13mm

5.8

3.4

12.1

48

41

+ve

1.2, 0 (-ve)

NSF

1

Bacille Calmette Guérin QuantiFERON®-TB is an IFN-γ assay for the detection of TB in humans (Cellestis, Australia). Results are interpreted as positive for exposure to M. tuberculosis complex organisms if ALL of the following criteria are met: Human PPD > 1.5 IU/ml Mitogen > 1.5 IU/ml % H/M (% Human/Mitogen) > 15% % H – A / H (% Human – Avian / Human) > -10% 1 These are assays for IFN-γ produced in response to the ESAT-6 and CFP-10 antigens. Results for both assays are interpreted as positive when greater than 10 pg/ml 1 NSF: no significant findings; no evidence of TB lesions. 1

61

SURVEILLANCE OF CHYTRID IN MOUNTAIN STREAMS IN SEQ – THE STORY OF GAP CREEK Pearl Symonds 62 Wondall Rd. Manly West, Qld 4179 There is a serial killer loose in the mountain streams of south east Queensland and as yet, despite knowing what the culprit is we still do not have enough evidence to arrest it’s progress! Gap creek is one such place draining the western side of Cunningham’s Gap, Main Range, the 300m transect for population counts is 700m above sea level and flows through world heritage rain forest. Chytrid has been recognized as a pathogen of the resident frog population of Mixophyes fleayi since 1996, when two M fleayi with two Litoria chloris where found terminally affected by the disease. This and the recognition that the species M fleayi was in decline prompted the 7 year population survey that has since been carried out on this section of creek. Mortalities involving small numbers of adults have occurred sporadically, despite this the adult population appears healthy with good recruitment of adult numbers, culminating with a big event witnessed Mar 2002, where 197 male adults were counted (plus another 170 downstream from transect). Only a limited number of metamorphs have been counted, suggesting that they may spend the early part of their lives some distance from the breeding sites. This M fleayi population has survived drought (Nov- Dec 2002) and this year a close bushfire. The breeding behavior and rainfall pattern is reasonably seasonally consistent, highest rainfall month in 2003 was January. Breeding follows the receding flood waters anytime between August and April. Water temperatures at this site range from 3.1 oC to 24.8 oC. Mixophyes fleayi are the only species that breed here with Lechriodus fletcheri and Litoria chloris observed breeding in isolated side pools. Using histological sections of M fleayi tadpoles as an indicator for chytrid presence and absence we have sampled one pool in this transect at regular intervals over a 12 month period, from August 2002 to September 2003. We have observed that: -Chytrid persists all year either cycling between tadpoles or between tadpoles and some other substrate in the pool environment. -There is a distinct annual pattern of infection. The younger (smaller sized) cohort becoming infected as the older (larger sized) tadpoles are at a stage of maximal chytrid burden and shedding of keratin mouthparts. -That there are strategies both physiological and behavioral that the infected tadpoles can employ to reduce chytrid burden at metamorphosis. This is in the form of an immune response both acute and chronic and complete shedding of keratin mouthparts. The ability to disperse may also be an important factor in reducing chytrid amplification and infective dose. -That the ability of chytrid to continue growing on affected tadpoles’ mouthparts despite sloughing of all keratinized tissue may be an expression of a virulence factor. Pathogen host relationships are complex and constantly in flux. The story of Gap creek shows that we need to constantly reassess this relationship. Regardless of when these two species first encountered one another, we now have a situation where chytrid is endemic, in a stable population. It has caused sporadic mortality events in the past, but what about the future? What we need to find out is the lifecycle of chytrid in the field and what factors contribute to mortality events. Special thanks to Harry Hines of Qld EPA, Terry Daley and Phil Bird from the oral biology dept UQ, and the technicians from UQ vet

62

ADVENTURES IN AVIAN ORTHOPAEDICS AND BEAKISTRY 1*

2*

Jennifer Youl and David Mclelland

1 University Avenue Veterinary Hosptial. 2 Litchfield Veterinary Hospital * current address: 13 Blue Gum Ave, Gymea, NSW 2227 Introduction: 1 The wedge-tailed eagle (Aquila audax) ranges across the Australian mainland and Tasmania . These birds have adapted well to human presence, and frequently take advantage of highways by scavenging on road-kill. Unfortunately this leads to birds being involved in motor-vehicle accidents (MVA) themselves. Two male wedge-tailed eagles were presented to University Avenue Veterinary Hospital (UAVH) in 2002 following MVA. The white-bellied sea eagle (Haliaeetus leucogaster) inhabits the coastal areas of mainland Australia and 1 Tasmania . It is not commonly a victim of MVA, however birds may be seen scavenging road kill in coastal areas. A male white-bellied sea eagle presented to Litchfield Veterinary Hospital following a MVA. Case 1“Heckle” ‘Heckle’, a 10 year old male wedge-tailed eagle, was presented to University Avenue Veterinary Hospital following a collision with a 4X4. A figure of 8 wing strap had been applied to the right wing by the wildlife rescuer. On initial examination the bird was bright and alert, respiratory noise and effort was slightly increased, there were multiple sites of bruising and grazing, and there was a transverse compound fracture of the mid-shaft of the right humerus. Meloxicam (0.2mg/kg) and enrofloxacin (10mg/kg) were administered daily by IM injection Two days following initial presentation, the bird was considered sufficiently stable to allow further examination. The bird was anaesthetised using isoflurane administered by mask. The fracture site was radiographed, then cleaned thoroughly and re-bandaged. On the third day, the eagle was again anaesthetised with isoflurane and intubated. Feathers were removed from the fracture site with primary flight feathers preserved. Via a medial approach to the fracture site a negatively threaded IM pin was placed retrograde through the distal humerus exiting cranial to the distal epicondyles. The fracture was reduced and the pin advanced normograde to seat the threaded portion of the pin in the proximal humeral metaphysis. A single cerclage wire was placed. Buprenorphine (0.2mg/kg) was administered peri-operatively with meloxicam and enrofloxacin. A figure of eight wing bandage was placed. ‘Heckle’ recovered well from the relatively long anaesthetic and accepted a hand-fed rat that evening. On day five it became evident that the IM pin would not provide sufficient rotational stability in the absence of a wing bandage. However, the use of wing bandages in the medium-to-long term is 2 disadvantageous as significant contraction of the patagium may result . The eagle was again anaesthetised with isoflurane via mask and intubated. A type I external fixator was applied with two pins in the proximal fragment and a single pin in the distal fragment. It was felt that the additional weight of the external fixator would not be a problem in such a large bird. A wing bandage was not reapplied. On day seven the fracture was stable and all minor injuries were healing well. Meloxicam (0.1mg/kg) was continued orally for a further five days and oral enrofloxacin (10mg/kg) for a further two weeks. ‘Heckle’ was sent to a raptor carer to be kept confined (4’x3’x4’) for two weeks and was intended to then be moved into a larger aviary (approximately 2mx4mx2m). However, the eagle remained in the smaller cage for a total of 6 weeks. It was unable to fully extend its wings in the cage resulting in some contraction of the right patagium and the loss or damage of a number of primary feathers. Healing of the fracture site may have been delayed as a result of the prolonged confinement.

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The surgery site was radiographed every two to three weeks to assess healing. The external fixator was removed six weeks post surgery and the IM pin removed after nine weeks. At six weeks post-surgery, ‘Heckle’ was placed in a flight aviary (14mx7.5m) with lowered perches. These were raised as strength improved. Eight months following the initial trauma, ‘Heckle’ was exercised on a long line until his flight fitness was considered sufficient for survival in the wild. He was subsequently released. Case 2 “Jeckle” A two year old male wedge-tailed eagle was presented to UAVH following a MVA in Western Queensland. When found by a member of the public he was reported to have been disorientated and easy to catch. He was subsequently driven several hundred kilometres to Darwin. On presentation he had head trauma resulting in depressed mentation. Anisocoria was observed, with the left pupil dilated and unresponsive to light with no appreciable menace reflex. Conversely the right eye exhibited a light responsive pupil and normal menace relfex. There was grazing and swelling of the tissues surrounding the left orbit, and a fluorescein positive ulcer approximately 1.5mm diameter was observed on the left cornea. The left carpus was grazed. No further injuries were observed at the time. The wounds were cleaned with chlorhexidine (0.5%) and meloxicam (0.2mg/kg) administered IM. The bird was sent to a raptor carer. Several days later the carer reported that the mentation of the bird had improved considerably, but he was unable to pre-hend food. Although he would eat it food was placed in his mouth, he would not take it with his beak. On re-examination it was observed that there was a split up either side of the hook of the beak. It was assumed that this was causing pain on prehension. The bird was anaesthetised with isoflurane via mask and endotracheal tube. Using a dental drill, holes were drilled rostral and caudal to the cracks on both side of the beak and cerclage wire placed to stabilise the fractures. Selley’s knead-it ™ was applied over the wire and the top of the beak. Four days following the surgery ‘Jeckle’ was taking food. By day seven the Selley’s knead-it had fallen off, and by day 14 the wire had grown out (or had been removed by the ‘Heckle’ who was being housed in the same aviary!) ‘Jeckle’ was subsequently able to eat unaided and was released with ‘Heckle’ in January 2004. Case 3 “Freckle” A four year old white-bellied sea eagle presented to Litchfield Veterinary Hospital in July 2003 following a MVA. Although very small for a sea eagle he was in good condition. A transverse fracture of the shaft of the right ulna was palpated and confirmed with radiography. Meloxicam (0.2mg/kg) was administered at presentation and a figure-of-eight wing bandage applied. Surgery was performed two days following presentation. The bird was anaesthetised with isofluorane administered via mask and endotracheal tube. An IM pin was placed retrograde through the fracture site and two cerclage wires placed for rotational stability. Meloxicam (0.2mg/kg) and enrofloxacin (10mg/kg) were administered IM. The eagle was kept in a confined area for two weeks then transferred to a larger aviary (4.5mX2.5m). The fracture site exhibited an excessive healing response, possibly due to some instability in the fracture site. The fracture was considered stable at four weeks and the pin was removed. The bird continued to intermittently exhibit a drooped right wing and did not adapt well to captivity. At the time of writing he is still in care and although the carer is keen to release him, there is a guarded poor prognosis for survival in the wild. DISCUSSION: It has been my experience that Large birds such as wedge-tailed eagles are much more likely to survive the initial impact with a car than smaller birds, making them better candidates for rehabilitation and

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release. When bringing a wild animal into a captive environment, it is important to consider the length of time it is going to remain captive and treat the injuries in such a way as to minimise this time. In case 1, a fixation technique that was going to provide good rotational stability as well as maintain alignment of the fracture site was paramount. To prevent contraction of the patagium, the wing had to be allowed and encouraged to extend normally. The initial intra-medullary pin technique with a single cerclage wire did not provide sufficient rotational stability for a fracture site with significant weight distal to it. The use of two cerclage wires may have provided this. The use of a type 1 external fixator enables the wing to extend and rotate normally without compromising the fracture site. Case 2 emphasised the importance of maintaining close contact with the rehabilitators that injured birds and animals are sent to. With retrospective examination of photographs taken at the initial presentation it is not possible to see the cracks in the beak. It is thought that attempted use of the beak created additional trauma exacerbating the injury. This case also highlights the importance of behavioural observations.

The conditions under which the birds will be kept during rehabilitation are also very important for reducing the amount of time in captivity. Prolonged confinement following surgery can result in problems that increase the bird’s captive time considerably. Suitable available aviary space and expertise for rehabilitation is essential for success. Acknowledgements The authors would like to thank University Avenue Veterinary Hospital for allowing a vet with a passion for wildlife free rein; Drs Jane Day and Jo Beckett for advice and surgical expertise; Robert Lawson and Kerry Turner for taking on the care of these high maintenance birds at their own cost; Healesville Sanctuary veterinarians for invaluable advice and support with all my wildlife cases; Stephany Philips for donating her time to nurse for the surgeries; Grant Bishop for recording everything on his trusty digital camera; Litchfield Veterinary Hospital; the NT News for publicity; and the general public for donations for wildlife treatment. References: 1. Pizzey, G. & Knight, F. 2000. The Field Guide to the Birds of Australia. Angus and Robertson. Pp. 132-135 2. Peter Holz, Personal communication.

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SHELL FRACTURE REPAIR USING GLASS IONOMER CEMENT IN THE LONG-NECK TORTOISE (Chelodina longicollis) Anne Fowler and Nick Magelakis Melrose Animal Hospital INTRODUCTION Long necked tortoises (Cheldonia longicollis) are a common inhabitant of the waterways of eastern Australia. Their range extends from Queensland to Victoria. Their habitat includes lakes, swamps, creeks and slow moving rivers. They only eat when submerged in water and their diet is based on invertebrates, small fish and tadpoles. Over winter, they aestivate at the bottom of drier waterholes, and emerge during warmer weather. Unfortunately, at this time, they often cross roads on their way to water and suffer injury by cars. The carapace of the shell is the most frequently traumatized area. However, damage to internal organs and bones is also common. In the past, there have been many materials used to repair shell fractures. These include fibreglass (H McCracken), methylmethacrylate (Ben Otten pers comm), bone cement and dental acrylics (M Cannon). These products are chosen to provide stability and are suitable to adhere to the shell. However, there are disadvantages to their use, especially fiberglass. If the resin gets in between the bone ends, it stops healing. The application is strongly exothermic, thus potentially damaging exposed tissue. It does not provide for growth. Due to these disadvantages, glass ionomer cement was considered in the repair on minor, non-displaced shell fractures. Glass ionomer cement (GIC) is a modern product used in dentistry for temporary fillings (Charlton). The generic name is aluminosilicate polyacrylate and it is composed of: 1. A silicate cement which releases Fluorine to provide a local antibacterial effect and increase precipitation of calcium to poorly mineralized tissue. 2. Polycarboxylate cement which binds by forming an ionic bond with the calcium in the hydroxyapatite component of the tooth. METHOD The initial phase is assessment and treatment of the tortoise. 1. A physical examination is performed. Condition and abnormalities such as reluctance to pull a leg back into the shell are noted. 2. The shell is dried. Moisture can permit the bacteria from the shell surface to colonise the fracture site. 3. The shell is cleaned with running water and a steel scrubbing brush. A 10% solution of iodine is applied to clean gently over the fractures. The full extent of fractures is only seen after cleaning. 4. Antibiotics are commenced upon the arrival of the tortoise using intramuscular enrofloxacin (Baytril, Bayer) at 5mg/kg every 48hours. Antibiotics are given for 7 – 10 days prior to shell repair and continued for a further 10 -14 days after shell repair. 5. A radiograph assesses damage to the girdles and limbs. 6. The tortoise is given a water bath for 1 hour a day in shallow water. 7. Once stabilization is performed with GIC, the tortoises are fed and housed by a wildlife rehabilitator. 8. Assessment of the fracture is also performed.The fractures are graded from Minor (1) – nondisplaced and involves either carapace or plastron. To Major (5) – displaced, missing pieces with exposure of guts, laceration of pleuroperiosteum. Grades 4-5 are euthanased.GIC is used in the treatment of minor to major non-displaced cracks in the shell.

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Application of GIC to the fractured shell. 1. Clean and dry shell. A 10% solution of Iodine is used. 2. Create a trough with a surgical burr either side of the fracture line. The burr removes the epidermis and devitalized tissue. Irrigation with saline is performed throughout to prevent heating of the bone. 3. Place either Dentine conditioner(10 - 40 % Polyacrylic Acid) or 10% Citric acid on surfaces to be bonded for 30 seconds. This will remove the smear layer (Smear layer is the layer of adherent debris produced when cutting the bone which varies in thickness and will consist primarily of ground shell substance). Rinse thoroughly. 4. Dry surfaces lightly. Exposed shell should be slightly glossy which indicates some moisture present. 5. Activate GIC capsule mix in Mixer (10 seconds) and then place into prepared trough. 6. Mould into trough by using finger lightly moistened in bonding resin. 7. Allow GIC to set and place a moisture sealant (bonding resin) over it. 8. The tortoise can be submerged in water after one hour. CONCLUSION GIC is showing promise as a new material in the repair of shell fractures in long-neck tortoises. It offers the advantages of promoting bone formation and preventing infection in the repair of fractured shells. Four case studies are presented with a discussion of the various degrees of fractures and associated problems associated with repair. REFERENCES Cannon M. Reptile Medicine. PGFVS. 2002, p136 Charlton D. Glass Ionomer Cement. From website: http://www.brooks.af.mil/dis/DMNOTES/gic.pdf Hulst F. Repair of Fractured Shells in Freshwater Turtles. In Wildlife and Ferrets Timeout PGFVS, 1999, p 115-119 James SB, Calle PP, Raphael BL, Papich M, Breheny J, Cook RA. Comparison of Injectable Versus Oral Enrofloxacin Pharmacokinetics in Red-Eared Slider Turtles, Trachemys scripta elegans J Herp Med Surg 13[1]:5-10 Spring 2003 McCracken H. Husbandry and Disease in Captive Reptiles. Pub Royal Melbourne Zoo,p 37 Marx KL, Roston MA. Exotic Animal Drug Compendium. Pub: Vet Learning Systems, 1996 Richards J. Metal Bridges – a new technique of turtle shell repair. J Herp Med Surg 11 (4) 31-34. 2001

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FURTHER INVESTIGATIONS INTO DISEASES OF THE KOALA (Phascolarctos cinereus) K. Stalder, G. Allen and M. Krockenberger 149 Peninsula Rd., Grays Point, NSW 2232 ABSTRACT A combined prospective and retrospective study was performed to further investigate diseases of the koala; their prevalence, diagnosis and epidemiology. The retrospective study consisted of post mortem records collected from 1061 koalas necropsied at the University of Sydney between 1980 and 2003. Summaries from these records containing the source of the cadaver, signalment of the koala and main gross and histopathological findings were collated and entered into an electronic database. From the summarised necropsy reports, 19 distinct disease types were identified and evaluated for prevalence within the necropsy population, and for epidemiological associations with factors such as age, sex, captive or free-living status and season of presentation. The population consisted of koalas from a variety of different sources, however the majority originated from free-living populations on the north coast of NSW. Both sexes were equally represented, and 65% of the koalas were older than four years, a figure unlikely to reflect the age distribution of koalas in the wild. Lesions attributable to urogenital disease (38.2%) and trauma (28.5%) were most commonly observed, a finding consistent with previous studies of a similar nature. Free-living, aged female koalas were over-represented within the urogenital disease group, possibly reflecting the prolonged deterioration of koalas with chronic urogenital disease or the reduced immune capacity of aging animals. Sub-adult male koalas were over-represented within the trauma group, presenting for necropsy primarily during spring. This correlates with the breeding season when there is increased roaming of these animals to find a mate and establish a home range. Neoplasia affected 12% of koalas, and lymphosarcoma was found to be the most common tumour type, accounting for 50.4% of these. The two most important infectious diseases were identified to be chlamydiosis and cryptococcosis, affecting approximately 35% and 2.5% of koalas respectively. Ultrasonography was proposed to be an effective method of detecting and grading the severity of chlamydial urogenital disease. Both normal koalas from Taronga Zoo and diseased cadavers were examined to establish the sonographic appearance of the normal and diseased urogenital tract. Koalas were placed in dorsal recumbency, and the organs of the urogenital tract examined systematically. The fur in the region to be examined was thoroughly wetted with alcohol and liberal quantities of acoustic coupling gel applied to ensure adequate contact of the transducer with the skin. Unique anatomical features of koalas including the glabrous pouch in females and lateral epipubic bones in both sexes respectively facilitated and hindered examination of the urinary bladder and lower genital tract. The kidneys and urinary bladder of normal koalas were sonographically comparable to those of small animals, and measurements of these organs were not correlated to age, sex, or weight. The ovaries, vaginal complex or urogenital sinus could not be detected in normal koalas, however the bilateral uteri were consistently visualised as small circular hypoechoic structures located dorsal to the bladder in the transverse plane. The prostate was difficult to visualise, often lying entirely within the pelvic cavity and was only definitively found in one normal koala. The sonographic appearance of testes and epididymis was comparable to that described in other species, whilst lateral bulbourethral glands were typically glandular in appearance with hypoechoic parenchyma of fine to medium texture. Structural changes in diseased cadavers that were readily detectable using ultrasonography included paraovarian cysts and thickening of the urinary bladder wall in animals with cystitis. Paraovarian cysts appeared as thin-walled simple or septate anechoic structures of varying shape and size that exhibited distal acoustic enhancement, whilst cystitis was recognisable by the measurable thickening of the bladder wall. Insufficient numbers of koalas available for necropsy examination, and poor quality of histopathological specimens precluded meaningful correlations between ultrasonographic and histopathological findings, however, should be undertaken in the future. Ultrasonography was found to be an inexpensive, noninvasive tool for evaluation of the urogenital tract of koalas, and should be developed further as a diagnostic test for assessment of structural urogenital tract disease.

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ABDOMINAL LUMPS IN THREE PYTHONS Teri Bellamy Austral Veterinary Clinic In the past 12 months I have seen 3 pythons present with almost identical clinical signs of a firm mid-body mass distending the diameter of the body by 1.5x and anorexia of several months duration. The pythons were all different species – 1 diamond python, 1 carpet python and 1 black-headed python. All were captive bred animals, 4-5 years old, 2 males and 1 female. Both the carpet python and diamond python were X-rayed and the black-headed python was ultrasounded to confirm the mass involved the gastrointestinal system. By measurements the masses were in the stomach or upper small intestine. The ultrasound indicated that there were multiple fluid filled spaces within the mass. In all three, samples were taken from the stomach/mass by stomach tube, percutaneous aspirate or laparotomy for examination for cryptosporidia and culture. The carpet python also had a biopsy sample taken for histopathology at the time of laparotomy. No cryptosporidia were found and a mixture of bacteria were cultured, mainly Gram negative organisms including Proteus mirabilis and mixed anaerobes. Sensitivities indicated the use of enrofloxacin and metronidazole as the drugs of choice. The biopsy in the carpet python indicated a granulomatous gastritis due to a bacterial infection but none of the animals responded to treatment. The diamond python died within a week. The carpet python, which had had surgical intervention and debridement of a large proportion of the mass, survived for several months on varying combinations of antibiotics but continued to be anorexic, lost weight and was eventually euthanased. The black-headed python had surgery after several weeks of antibiotic therapy to attempt total resection of the mass, removing the caudal part of the stomach and upper small intestine and re-anastomosis. All surgical interventions were performed under general anaesthesia using 2% lignocaine onto the tracheal opening and intubation, then 5% isoflurane and oxygen gas anaesthesia with intermittent positive pressure ventilation. The mass removed from the black-headed python was submitted for histopathology and the diagnosis was adenocarcinoma of the stomach. Samples from the post mortem of the carpet python were submitted to the Taronga Zoo Pathology Register and were identified as gastric granuloma and multifocal coelomic carcinoma. Samples of the first animal – the diamond python, have unfortunately been misplaced. All three animals were not old and no predisposing factors in the aetiology could be identified.

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FLUOROSIS AS A PROBABLE CAUSE OF CHRONIC LAMENESS IN EASTERN GREY KANGAROOS Emily Clarke 41 Allison Rd., Mont Albert Nth, Victoria 3129 Abstract: A population of eastern grey kangaroos (Macropus giganteus) inhabiting heathland and farmland surrounding an aluminium smelter at Portland, Victoria, Australia, exibited clinical signs of lameness. An investigation was undertaken to determine the cause of this lameness. Haematology, necropsy, histopathology, faecal egg counts, total worm counts, reproductive status and the population age range were examined and failed to reveal any additional underlying disease state. The specific problem of lameness was addressed using bone histopathology, radiography, quantitative ultrasonography, microradiography and multi-element analysis of bone ash samples. The significant lesions observed were: osteophytosis of the distal tibia and fibula, tarsal bones, metatarsus IV and proximal coccygeal vertebrae; osteopenia of the femur, tibia and metatarsus IV; incisor enamel hypoplasia, stained, uneven and abnormal teeth wear; abnormal bone matrix mineralisation and mottling; increased bone density; and elevated bone fluoride levels. Microradiography of affected kangaroos exhibited 'black osteons' which are a known manifestation of fluorosis. Collectively, these lesions were consistent with a diagnosis of fluorosis. Key words: Eastern grey kangaroo, Macropus giganteus, fluorosis, lameness. INTRODUCTION During a wildlife survey of the Portland aluminium smelter site at Portland, Victoria, Australia (Coulson et al. 2001), clinical signs of lameness, with visible signs of restricted movement, were observed in some individuals in the population of eastern grey kangaroos (Macropus giganteus). Relatively little information is available describing the clinical signs, pathological findings and the potential causes of lameness in macropods. The purpose of this study was to investigate and assess the general health of the eastern grey kangaroo population in Portland, Victoria, to describe any lesions discovered and to suggest a cause of lameness observed in some kangaroos. MATERIALS AND METHODS Eastern grey kangaroos were collected from two different locations: the Portland Aluminium smelter (38o23’S, 141o37'E), situated on the south-west coast of Victoria on a headland at the southern end of o o Portland Bay, and a cattle farm in Bridgewater (38 21'S, 141 23'E), situated 25 km west of Portland. The Bridgewater site provided a similar habitat to that in Portland and sufficient distance from the smelter to prevent contamination by airborne emissions containing fluoride. Professional kangaroo shooters were used to cull the eastern grey kangaroos with a single shot to the cranium. Carcasses were brought to a field laboratory for examination. Blood samples were collected immediately after death via cardiac puncture into EDTA tubes and were subsequently used for routine haematology.

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A detailed necropsy was performed and subsequently samples of adrenal, kidney, liver, lung, mesenteric lymph node, pancreas, spleen, thyroid and gonads were fixed in 10% neutral buffered formalin. After fixation and sectioning into cassettes, tissues were embedded in paraffin, and sections, cut at a thickness of 3µm, were stained with haematoxylin and eosin. A faecal sample was collected from the rectum of each kangaroo, and the gastrointestinal tract, from the stomach to the rectum, was removed from the cadaver and frozen. The skull, right hindlimb, left forelimb, a sample of the right ribs and a section of proximal coccygeal vertebrae were also collected and frozen. The incisors and molars were examined in situ for signs of enamel mottling (chalky white areas), staining (brown discoloration), hypoplasia (defective development) and abnormal wear. The premolar and molar series were also examined for uneven wear. The ages of mature kangaroos were calculated from de-fleshed skulls using their molar index (Kirkpatrick 1965) and the ages of pouch young were determined from head and foot lengths using sex-specific growth curves (Poole et al. 1982). The entire humerus, femur, tibia, tarsus, metatarsus IV, the fifth rib, and the proximal coccygeal vertebrae from four Portland and four Bridgewater kangaroos were placed in 10% neutral buffered formalin for 48 h. Using a bandsaw, 5mm sections were taken from the humerus (proximal epiphysis transversely and cross section of the diaphysis), femur (proximal and distal epiphysis transversely and cross section of the diaphysis), tibia (proximal and distal epiphysis transversely and cross section of the diaphysis), metatarsus IV (proximal epiphysis transversely and cross section of the diaphysis), proximal coccygeal vertebrae (cross section of the body) and the rib (costochondral junction transversely and cross section of the diaphysis) of each kangaroo. Sections of the tibio-tarsal joints from selected kangaroos were trimmed transversely. These samples were immersed in decalcifying solution (formic acid diluted with sodium formate and water) until the tissues could be cut with a scalpel blade. Sections were stained with haematoxylin and eosin. o

The feces collected were refrigerated at 4 C and examined two days later using a direct flotation method (Thienpont et al. 1979) to determine the number of nematode eggs. The remaining faecal samples were used to measure and identify the sporulated oocysts (Barker et al. 1989). The total number of nematodes found in the stomach and small intestine were estimated using a dilution technique (Beveridge and Arundel 1979). The stomach wall was examined for crateriform nodules associated with infection by larvae of Rugopharynx rosemariae infection (Presidente and Presidente 1978) and the small intestine was examined for nematodes and cestodes. If present, Labiostrongylus species were removed, fixed with 10% neutral buffered formalin and indentified (Smales 1995). Ultrasound speed measurements were made of the humerus, femur, tibia and metatarsus IV using a reflective ultrasound machine (Sunlight Omnisense, Sunlight Medical Ltd, Tel Aviv, Israel). The humerus was measured on the caudal aspect, the femur on the cranial, medial and lateral surface, the tibia on the cranial, caudal and medial aspects and the metatarsus IV on the dorsal surface. The microradiography technique was based on that of Feik et al. (2000). The 100µm bone sections were obtained using custom-made lapping tools (Manufacturing and Mechanical Engineering Workshop, University of Melbourne, Parkville, Victoria, Australia) in a Rolopol-21 lapping machine (Struers A/S, Copenhagen, Denmark) with Grit 1200 Waterproof Silicon Carbide paper, (Struers A/S, Copenhagen, Denmark). Microchrome high resolution glass plates (Microchrome Technology Inc., San Jose, California, USA) were used to capture the images. Sections were radiographed at 20 kV and 10 mA for 15 minutes. The plates were developed using D-5 negative developer (Microchrome Technology Inc., San Jose, California, USA), 2% acetate (Indicator Stop Bath, Kodak, Coburg, Victoria, Australia) and F-4 fixer (Microchrome Technology Inc., San Jose, California, USA). The plates were examined under a compound microscope to determine the bone structure. Female kangaroos from Portland and Bridgewater were selected for multi-element bone analysis. The femurs were cut distal to the head and proximal to the condyles to obtain bone samples weighing between 35 and 95 g. These samples were placed in a furnace (Combilator CL-V, Hanau, Germany) at

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800 C for three hours to obtain ashed samples. Multi-element analysis was performed on these samples and the concentrations of aluminium, antimony, arsenic, barium, boron, cadmium, calcium, cobalt, copper, chromium, fluoride, iron, lead, magnesium, manganese, mercury, molybdenum, selenium, sodium, phosphorus, potassium, thallium and zinc were determined. (The Animal Health Diagnostic Laboratory, Michigan State University, Michigan, USA). The mandibles from Portland and Bridgewater kangaroos were also analysed for fluoride levels. The ramus of the mandible was removed and the teeth extracted. The bone fluoride levels were determined using ion selective electrodes (Portland Aluminium Environmental Laboratory Test Methods). From three selected animals, a section of distal tibia was also analysed to allow comparison of the laboratories and their methods. Data analysis Descriptive statistics (mean, standard deviation and range) were determined for the haematological data using Microsoft Excel (Version 98, Microsoft Coorporation, USA). The mean values were compared statistically using a two-sample t-test with data published previously by Presidente (1978). The strongyle eggs per gram of faeces and the oocysts per gram of faeces data were plotted against the ages of the kangaroos. The Portland and Bridgewater kangaroos were plotted separately to determine if there was any difference between the two locations. A regression was fitted for log transformed data from Portland data for each graph and the regression analysis program was used to determine the effect of age. For the ultrasound measurements the normal male and normal female data were plotted separately on a graph against the ages of the kangaroos. Multiple regression was used to estimate the equation for normal male and female data: y = a + b.sex + c.Ln(age) + d.sex x Ln(age) where y = measured parameter, a, b, c, d = constants, sex = gender (1=male and 0=female), age = age in years A 95% prediction interval was calculated from the above equation (Minitab Version13.31, Minitab Inc, Pennsylvania, USA). The mean and standard deviation of the concentrations of elements detected in the femurs were calculated. The concentrations were also graphed against the age of the kangaroos. The Portland and Bridgewater data were plotted separately to determine if any differences existed between the two locations. A regression was fitted to the Portland data and regression analysis performed on the log transformed data. The fluoride concentrations detected in the femur and mandible were graphed against the age of the kangaroos. Regression analysis was performed using log transformed data to determine the equation for the femur fluoride concentration in the Portland females. Multiple regression was used to estimate the equation for the mandibular fluoride concentration in the entire Portland population. The 95% prediction interval was calculated for these equations to determine if there was a significant difference between the Portland and Bridgewater results. RESULTS Seventeen eastern grey kangaroos were collected from the Portland site: seven males aged between 1.3 and 7.8 years and ten females aged between 0.8 and 15.2 years. Three females aged between 10 and 15.2 years showed clinical signs of lameness. From the control site at Bridgewater, four kangaroos were collected: three males aged between 0.7 and 4.8 years and a female that was 2.8 years of age (Table 1). The haematological data was compared with previous studies on eastern grey kangaroos (Presidente 1978). A leucocytosis was observed in the Portland population. The three oldest females (P11, P12 and P13) had palpably enlarged tibio-tarsal joints. Gross examination of the region revealed severe osteophytosis on the distal tibia and distal fibula, on the periarticular aspects of the calcaneus and the distal calcaneal process. The metatarsus IV of kangaroo P12 was fused to the tarsal bones. The surface cartilage was yellow and was moderately fibrillated with

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focal areas of cartilage loss. Marked longitudinal grooves had developed on the joint surface, and several chondromas had developed at the transitional zone of the synoviae. A decrease in the viscosity of the synovial fluid was also observed. Kangaroo P12 also had extensive osteophytosis on the proximal coccygeal vertebrae, which had resulted in ankylosis of the vertebrae. (Table 2). The examination of the teeth revealed that the upper incisors of kangaroos P11 and P12 were roughened, dull and stained rather than smooth, shiny and white. They also displayed focal enamel hypoplasia, and kangaroo P11 had developed abnormal wear. The upper and lower molars of kangaroos P11, P12 and P13 were also worn unevenly. (Table 3). Histopathological examination revealed congestion in the liver, spleen and kidney of kangaroos P2, P5 and P13. Diffuse haemosiderosis was seen in the liver of kangaroos P11, P12 and P13, within the hepatocytes, portal triad macrophages and Kupffer cells. The cestode, Progamotaenia festiva, was observed within the bile duct of kangaroo B3; focal ductal ectasia had developed, but no inflammatory reaction was observed. Within the lungs of all kangaroos from both Portland and Bridgewater, the larger pulmonary veins had prominent muscular walls. The pulmonary veins in kangaroo P2 had thicker muscular walls and adventitia compared with the other kangaroos, both from Portland and Bridgewater. The regression analysis of faecal egg counts of Portland and Bridgewater kangaroos showed that there was no significant relationship between the number of strongyle eggs and oocysts per gram of faeces and age. A two-sample t-test revealed that there was no significant difference between the Portland and Bridgewater population for the number of strongyle eggs (p = 0.50) or oocysts (p = 0.73). Following sporulation, the oocysts were identified as Eimeria hestermani, E. macropodis, E. toganmainensis and E. yathongenesis. Sixteen species of helminths were encountered in the Portland and Bridgewater kangaroos. Regression analysis showed that there was no significant relationship between the number of worms, both in the stomach and large intestine, and age. Furthermore, a two-sample t-test revealed that there was no significant difference between the two populations in the total number of worms in the stomach (p = 0.48) and in the large intestine (p = 0.15). The nematodes found in the stomach were Rugopharynx macropodis, R. rosemariae, Pharyngostrangylus kappa, Alocostoma clelandi, Cloacina artemis, C. selene, C. pelops, C. hermes, C. obtusa, C. expansa, C. hera and Labiostrongylus kungi. The dominant nematode in the stomach was R. macropodis followed by P. kappa for both the Portland and the Bridgewater populations. The remaining nematodes constituted only a small proportion of the total worm burden. Labiostrongylus kungi was present in the stomachs of kangaroos P2, P3, P5, P6, P7, P9, P10, B2 and B3. Small nodules indicative of R. rosemariae infections were not found in the gastric mucosa of the kangaroos. The cestode, Progamotaenia ewersi, was found in the small intestine. The nematodes found in the large intestine were Macropoxyuris longigularis and M. brevigularis. M. longigularis was the dominant species present in the Portland kangaroos, but both species were present in equal numbers in the Bridgewater population. The radiographs of the femur, tibia and the metatarsal from kangaroos P11, P12 and P13 revealed severe osteopenia indicated by trabecular bone loss, thinning of the cortical bone and enlargement of the medullary cavity. Resorption was observed of the endosteal surface and the osteonal bone in the tibial microradiographs of kangaroos P11, P12 and P13. The osteonal bone had been replaced by abnormal bone matrix that was poorly mineralised and had a mottled appearance. The subperiosteal lamellar bone was also poorly mineralised and had a mottled appearance. Quantitative ultrasonography revealed that kangaroos P11, P12 and P13 had a decreased ultrasound speed of the lateral femur, but an increased ultrasound speed of the caudal tibia, kangaroo P13 also had an increased ultrasound speed of the cranial tibia (Table 4). The femora did not exhibit detectable levels of arsenic, cadmium, chromium, cobalt, lead, manganese or mercury. There was no change with age in the bone concentrations of barium, iron, calcium, magnesium, zinc, copper and sodium. However, the bone levels of phosphorus and potassium declined with age (p = 0.021 and p = 0.029 respectively).

74

The femur and mandible fluoride concentration for the Portland kangaroos were graphed against age. The curves of the Portland kangaroos were logarithmic and became flatter with increasing age. For the femur fluoride concentration, the Bridgewater female was within the 95% prediction intervals for the Portland females. However, the three Bridgewater kangaroos were outside the 95% prediction intervals for the mandible fluoride concentration. The femur, mandible and tibia fluoride levels of each kangaroo from Portland and Bridgewater are presented in Table 5. Compared with the known ranges of fluoride concentration in cattle metatarsal bones, the femora of kangaroos P11 and P13 had very high levels of fluoride, kangaroo P5 had high levels, kangaroos P3, P4, P6, P10, P12 and P15 had levels ranging from normal to high, whilst kangaroo B3 had normal fluoride levels. The tibiae of kangaroos P11, P12 and P13 were between the normal and high ranges. Furthermore, kangaroos P1, P5 and P11, P12, P13, P14 had high mandibular fluoride levels, kangaroos P3 and P6 and P7 had levels ranging from normal to high and kangaroos P2, P4, P8, P9, P10, P15, P16, P17, B1, B2 and B3 were within the normal fluoride range for cattle DISCUSSION General health The female kangaroos that were collected from Portland provided an adequate representation of the subpopulation, as their ages covered the entire range from 0.8 to 15.2 years of age, and the life span of eastern grey kangaroos is known to be up to 18 years (Quinn 1989). The Portland males and both the males and females from Bridgewater were generally younger, and may not have provided an adequate representation of their respective sub-populations. The number of kangaroos that could be collected from each site was restricted both by ethical and practical limitations. Unfortunately, these factors prevented examination of bones and joints for lesions that may occur in aged males in the Portland area. Age-control kangaroos to allow the comparison of lesions with the lame females were also not available. In spite of these limitations, the current investigation demonstrated significant osteological abnormalities in older kangaroos. The haematological results from the Portland population were not statistically different from those of Presidente (1978), with the exception of a slight leucocytosis. Overall, the haematological data failed to reveal any evidence of underlying disease. The histopathological findings revealed only incidental changes. Diffuse haemosiderosis of the liver is seen in haemolytic anaemias, aneamia of copper deficiency and cachexia, whereas localised haemosiderosis occurs following local haemorrhage. This chronic, low-grade haemosiderosis in the absence of other pathology appeared to have little clinical significance. The cestode, Progametaenia festiva, is considered non-pathogenic, (Presidente and Beveridge 1978). The muscular hypertrophy and thickened adventitia of pulmonary veins were observed in various degrees in all individuals but were more pronounced in the large males. This may be a physiological adaptation by kangaroos to maintain adequate blood supply throughout the body in response to their unique gait. However, there are no data available on the normal range of the pulmonary vasculature in eastern grey kangaroos. None of the observed histopathological lesions would have contributed to the lameness seen in the eastern grey kangaroos from Portland. The parasite infections in the eastern grey kangaroos from Portland and Bridgewater were not significantly different from those found in normal kangaroos from other localities in south-east Australia and were not considered detrimental to their general health (I. Beveridge, pers. obs.). Biomechanical measurements Quantitative ultrasonography has been used in humans and horses to study bone density and strength (Lepage et al. 2001). The decreased stiffness and density in the lateral femur, and an increase in the cranial and caudal tibia, could be due to changes in bone quality. In response to small increases in bone stress, normal primary lamellar bone is formed. However, if bone is distorted sufficiently, it changes shape rapidly and woven bone is produced, which is lower in quality and needs to have a high density to maintain its strength (Uhthoff 1982).

75

Significant lesions There are multiple potential causes of the observed bone and dental lesions, however, the analysis performed suggest fluorosis as the most likely cause. Following ingestion, fluorides are absorbed from the stomach and intestines and are distributed throughout the body. The concentration in most soft tissues is equivalent to the plasma concentration and there is no appreciable accumulation in any vital organ (Armstrong et al. 1970). Fluorides selectively accumulate in skeletal, dental and mineralising tissue (Shupe et al. 1979) and excess circulating fluoride is excreted in the urine. Constant, long-term fluoride ingestion results in the blood, bone and urine being in equilibrium (Blakemore et al. 1948). Fluoride ions are incorporated into the hydroxyapatite crystal of bone to form fluorapatite instead of carbapatite (Shupe et al. 1979), which causes an increase in crystal size and decreases the mineral stability (Zipkin et al. 1964). The level of fluoride storage in bone can increase over a period of time without evidence of changes to bone structure and function. However, structural bone changes will become evident if the fluoride levels being ingested are sufficiently high over an appreciable length of time (Johnson 1965; Shupe and Alther 1966; Shupe et al. 1963). Osteofluorotic bone changes occur in three recognisable grades. Firstly, bones may contain elevated levels of fluoride without detectable alterations to bone structure or function. The second grade is characterised by microscopic mottling, without alterations to bone function. This is due to damaged endosteal and periosteal osteoblasts, which result in the production of an abnormal bone matrix that fails to mineralise normally and is distinguished by brown discoloration (mottling) of osteonal and periosteal lamellae (Krook and Maylin 1979; Palmer 1993). This mottling is consistent with the microradiographic lesions observed in this study. The final grade is distinguished by sequential changes in bone structure that reflect altered mechanical properties of the preformed bone and production of new, abnormal bone. The changes result in an increased rate of remodelling and subsequent enlargement of the medullary cavity and osteoporosis (Johnson 1965; Palmer 1993; Shupe et al. 1979). The alterations in normal bone growth and development depend on the bone fluoride levels and occur in three stages: acceleration of cortical remodelling, dissociation of the normal sequences of osteogenesis and, finally, production of abnormal bone (Johnson 1965; Palmer 1993; Shupe et al. 1979). The osteophytosis observed in the lame kangaroos from Portland is consistent with previous studies that suggest that fluoride tends to accumulate in the periosteum and results in osteophyte development (Palmer 1993). Grossly, bones that are severely affected by fluoride become chalky white in appearance, with an increase in density and brittleness (Schmidt and Rand 1952; Shupe 1980; Shupe et al. 1979). Fluorosis can also induce the production of periosteal lamellar bone to cause osteosclerosis and mottled osteons. New periosteal bone and trabeculae can exhibit wide uncalcified osteoid seams that are indicative of osteomalacia (Johnson 1965; Shupe et al. 1979). The encroachment of osteophytes on tendons and ligaments with subsequent mineralisation and even ossification as a result of fluorosis can cause bilateral lameness and stiffness of gait (Palmer 1993; Shupe 1980). Fluorosis does not primarily affect intra-articular structures (Palmer 1993), but secondary peri-articular lesions may develop. Various degrees of osteoarthritis have been observed in animals severely affected by fluorosis. Whether a relationship exists between the two disease processes is unclear (Johnson 1965; Shupe et al. 1979; Shupe et al. 1984). The gross, radiographic and microradiographic lesions in the bones of the lame kangaroos are consistent with fluorosis as reported in other species. Developing teeth are extremely sensitive to excess levels of fluoride (Johnson 1965; Shupe et al. 1979). Incisor enamel hypoplasia, staining of molars and excessive abrasion are consistent with previous findings on osteofluorosis in humans, cattle and deer (Johnson 1965; Shupe et al. 1979; Vikøren and Stuve 1996). The severity and combination of dental fluorosis lesions depend upon the level of fluoride ingested during the development of the teeth (Shupe and Alther 1966). Ameloblasts and odontoblasts that are damaged by fluoride produce an abnormal matrix that fails to incorporate the required minerals. This leads to a reduction in both quantity and quality of mineralisation (Palmer 1993; Shupe and Alther 1966; Suttie 1980). Lesions of fluorosis develop later in premolars and molars than in incisors and are characterised by variation in abrasion (Shupe et al. 1979). The abrasion of teeth with age could also

76

account for the abnormal wear of the premolars and molars seen in the aged kangaroos. Natural tooth wear tends to be area specific, with greater rates of wear occurring in localities with more abrasive soils and plants (Kirkpatrick 1985). Alternately, after a certain degree of fluorosis has occurred in teeth, they are subjected to an increased rate of abrasion regardless of the type or quality of the feed ingested. However, more abrasive feeds will cause more of an increase in the rate of abrasion (Shupe et al. 1963). Therefore, the dental lesions are also consistent with fluoride toxicity. The fluoride concentrations in the femur show that many of the Portland kangaroos, three of which were showing the clinical signs of lameness, are within the high range for cattle. This suggests that fluorosis is a problem within this population of eastern grey kangaroos. The mandibular and femoral fluoride levels in the Bridgewater population are within the normal ranges for cattle (Animal Health Diagnostic Laboratory; Suttie et al. 1959). The mandibular fluoride concentration of Bridgewater kangaroos lies outside the 95% prediction interval for their age, but the femural fluoride level lies within the interval. This suggests that the Bridgewater control site has a high level of background fluoride or is close enough to the aluminium smelter to be affected by emissions containing fluorides. The Portland and Bridgewater kangaroos had significantly higher levels of fluoride in their femora than in their mandibles. This is not consistent with previous studies, which suggest the fluoride levels vary between different bones due to the degree of stress and strain imposed on them. Cancellous bones (pelvis, head, mandible, ribs and vertebrae) contain higher levels of fluoride than compact bones (metacarpus and metatarsus) (Johnson 1965; Palmer 1993; Shupe et al. 1979). The results showed that the fluoride was preferentially stored in the compact bone. The laboratories that performed the fluoride analyses are independent and may have different assay methods. Therefore, a difference in methodology may be an alternative explanation for the observed difference between these bone levels. The normal and high ranges of fluoride for eastern grey kangaroos have not been determined. Other wild herbivores, such as deer, are useful indicators of increased fluoride concentration in the environment (Suttie et al. 1987). However, deer have similar or higher levels of bone fluoride retention as cattle, but without signs of gross osteofluorosis (Vikøren and Stuve 1996). Therefore, they would be considered less sensitive to fluoride than cattle. The lame kangaroos showed clinical signs and lesions consistent with fluorosis at lower bone fluoride concentrations compared with those found in cattle. Therefore, kangaroos seem to be more sensitive than cattle to fluorosis and show signs of fluorosis at lower bone fluoride levels. This suggests that the local population of eastern grey kangaroos may be a more useful and more accurate indicator species for monitoring changes in fluoride contamination in the vicinity of the aluminium smelter rather than the herd of cattle currently maintained for this purpose. CONCLUSION The haematology, necropsy, histopathology and the parasitic burden results, the reproductive status and the high longevity of the Portland population suggest that no additional underlying disease states exist in these eastern grey kangaroos. Gross examination, histopathology, radiography, microradiography, biomechanical measurements, and multi-element and fluoride analysis of bones were procedures that were used to determine the possible cause of the observed lameness. The osteophytosis of the hindlimbs and proximal coccygeal vertebrae, osteoporotic hindlimb bones, incisor enamel hypoplasia, staining and abnormal wear, uneven molar wear, abnormal bone matrix mineralisation and mottling, increased bone density and the elevated bone fluoride levels are consistent with chronic fluoride poisoning. It is unclear whether the observed degenerative joint disease is an incidental finding related to the age of the lame kangaroos or a secondary effect of the fluorosis. In conclusion, the level of fluoride contamination from the Portland Aluminium smelter appears to result in clinical signs and lesions of fluorosis in the sedentary population of eastern grey kangaroos. These kangaroos seem to be more sensitive to the effects of chronic fluoride poisoning than cattle. Therefore, the local population of eastern grey kangaroos could be used to monitor the environmental effects of

77

fluoride contamination in the vicinity of the aluminium smelter rather than the herd of cattle currently maintained for this purpose. Acknowledgements: We would like to thank John Hill, Alcoa Australia for the opportunity to complete the project at Portland Aluminium smelter. Thanks to Ron Jeffries and Kevin Saunders for their assistance during the field work. Thanks also to Rodney Harris for allowing the use of his cattle farm. Prof. K. V. F. Jubb, Dr. Jenny Charles and Dr. Helen Davies for their assistance with the pathological and biomechanical sections. We wish to acknowledge Prof. J. G. Clement, Dr S. O'Connell, and D. Rowler of the OAMS Unit, School of Dentistry, The University of Melbourne and A. Obedoza for their interest in the project and their assistance with lapping, microradiography and the ashing of bones. J. Wilson and P. Benham for their assistance with histopathology. Thanks to G. Anderson for helping with the statistical analysis. Thanks also to C. Andersen, S. Meekings and B. Kehoe. We wish to acknowledge Sunlight Medical Ltd. for the use of the Sunlight Omnisense machine for the bone ultrasound speed measurements. LITERATURE CITED 1. Armstrong, W. D., I. Gedalia, L. Singer, J. A. Weatherell, and S. M. Weidmann. 1970. Distribution of Fluorides. In: Geneva: World Health Organisation (eds.). 1970. Fluoride and human health. Pp. 93-139. 2. Barker, I. K., M. G. O’Callaghan, and I. Beveridge. 1989. Host-parasite associations of Eimeria spp. (Apicomplexa: Eimeriidae) in kangaroos and wallabies of the genus Macropus (Marsupialia: Macropodidae). Int. J. Parasitol. 19(3): 241-263. 3. Beveridge, I., and J. H. Arundel. 1979. Helminth parasites of grey kangaroos, Macropus giganteus Shaw and M. fuliginosus (Desmarest), in Eastern Australia. Aust. Wildl. Res. 6: 69-77. 4. Blakemore, F., T. J. Bosworth, and H. H. Green.1948. Industrial fluorosis of farm animals in England, attributable to the manufacture of bricks, the calcining of ironstone, and to enamelling processes. J. Comp. Path. 58: 267-303. 5. Feik, S. A., C. D. Thomas, R. Bruns, and J. G. Clement. 2000. Regional variations in cortical modelling in the femoral midshaft: sex and age differences. Am J Phys Anthropol. 112(2):191205 6. Johnson. L. C. 1965. Histogenesis and mechanisms in the development of osteofluorosis. In: Simons, J.H (ed.). Fluorine Chemistry. Vol.IV, Academic Press, New York. Pp. 424-441. 7. Kirkpatrick, T. H. 1965. Studies of the Macropodidae in Queensland. 2. Age estimation in the grey kangaroo, the red kangaroo, the eastern wallaroo and the red-necked wallaby, with notes on dental abnormalities. Qd. J. Agr. An. Sci. 22:301-317. 8. Kirkpatrick, T. H. 1985. Biology for management. In: Lavery, H. J. (ed.) The kangaroo keepers. University of Queensland Press, Brisbane. Pp. 136-160.

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9. Krook, L., and G. A. Maylin. 1979. Industrial fluoride pollution. Chronic fluoride poisoning in Cornwell cattle. The Cornell Vet. 69 (Supplement 8):129-136. 10. Lepage, O. M., B. Carstanjen, and D. Uebelhart. 2001. Non-invasive assessment of equine bone: an update. Vet. J. 161:10-23. 11. Palmer, N. 1993. Bones and joints. In: Jubb, K. V. F., P. C. Kennedy, and N. Palmer (eds.). Pathology of Domestic Animals. 4th ed. Academic Press, New York. Vol. 1., Pp. 1-137. 12. Poole, W. E., S. M. Carpenter, and J. T. Wood. 1982. Growth of grey kangaroos and the reliability of age determination from body measurements I. The eastern grey kangaroo, Macropus giganteus. Aust. Wildl. Res. 9:9-20. 13. Presidente, P. J. A. 1978. Diseases seen in free-ranging marsupials and those held in captivity. In: Hungerford, T. G. (ed.). Proceedings no. 36 - Fauna. Postgraduate Committee in Veterinary Science, Sydney. Pp. 457-471. 14. Presidente, P. J. A., and I. Beveridge. 1978. Cholangitis associated with species of Progamotaenia (Cestoda: Anoplocephalidae) in the bile ducts of marsupials. J. Wildl. Dis. 14:371-377. 15. Quin, D. G. 1989. Age structures, reproduction and mortality of the eastern grey kangaroo (Macropus giganteus Shaw) from Yan Yean, Victoria. In: Grigg, G., P. Jarman, and I. Hume (eds.). Kangaroos, wallabies and rat-kangaroos. Surrey Beatty and Sons, New South Wales, Australia. Pp. 787-794 16. Schmidt, H. J., and W. E. Rand. 1952. A critical study of the literature on fluoride toxicology with respect to cattle damage. Am. J. Vet. Res. 13:38-49 17. Shupe, J. L. 1961. Arthritis in cattle. Can. Vet. J. 2(10):369-376. 18. Shupe, J. L. 1980. Clinicopathological features of fluoride toxicosis in cattle. 51(3):746-758.

J. Anim. Sci.

19. Shupe, J. L., and E. W. Alther. 1966. The effects of fluorides on livestock, with particular reference to cattle. In: Eichler, O., A. Farah, H. Herken, A. D. Welch, and F. A. Smith (eds.). Handbook of Environmental Pharmacology. Vol. 20. Springer-Verlag, New York. Part. 1, Pp. 307-354. 20. Shupe, J. L., M. L. Miner, D. A. Greenwood, L. E. Harris, and G. E. Stoddard. 1963. The effect of fluorine on dairy cattle II. Clinical and pathologic effects. Am. J. Vet. Res. 24(102):964-979. 21. Shupe, J. L., A. E. Olsen, and R. P. Sharma. 1979. Effects of fluorides in domestic and wild animals. In: Oehme, F. W. (ed.). Toxicity of heavy metals in the environment. Marcel Dekker, New York. Part 2. Pp. 517-540. 22. Shupe, J. L., A. E. Olsen, H. B. Peterson, and J. B. Low. 1984. Fluoride toxicosis in wild ungulates. J. Am. Vet. Med. Assoc. 185(11):1295-1300. 23. Smales, L. R. 1995. A revision of the subgenus Labiostrongylus (Labiosimplex) (Nematoda: Cloacinidae) from macropodid marsupials, with decriptions of twelve new species and a key to the species of the subgenus. Invertebr. Taxon. 9:181-242. 24. Suttie, J. W. 1980. Nutritional aspects of fluoride toxicosis. J. Anim. Sci. 51(3):759-766. 25. Suttie, J. and P. H. Phillips. 1959. Studies of the effects of dietary sodium fluoride on dairy cows. V. A Three-year study on mature animals. J. Dairy Sci. 42:1063-1069.

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26. Suttie, J. S., R. Dickie, A. B. Clay, P. Neilsen, W. E. Mahan, D. P. Baumann, and R. J. Hamilton. 1987. Effects of fluoride emissions from a modern primary aluminium smelter on a local population of white-tailed deer (Odocoileus virginianus). J. Wildl. Dis. 23(1):135-143. 27. Thienpont, D., F. Rochette, and O. F. J. Vanparijs. 1979. Diagnosing helminthiasis through coprological examination. Janssen Research Foundation, Beerse, Belgium. Pp. 187 28. Uhthoff, H. K. 1982. In: Martin, R. B. and D. B. Burr (eds.). 1989. Structure, Function, and Adaptation of Compact Bone. Raven Press, New York. P. 166. 29. Vikøren, T., and G. Stuve. 1996. Fluoride exposure in cervids inhabiting areas adjacent to aluminium smelters in Norway. II. Fluorosis. J. Wildl. Dis. 32(2):181-189. 30. Zipkin, I., E. D. Eanes, and J. L. Shupe. 1964. Effect of prolonged exposure to fluoride on the ash, fluoride, citrate and crystallinity of bovine bone. Am. J. Vet. Res. 25(109):1595-1597.

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Table 1. Sex, molar index, age and weight of eastern grey kangaroos, Macropus giganteus, collected at Portland and Bridgewater, Victoria. Kangaroo Number P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 B1 B2 B3 B4

Sex

MI

Male Male Female Female Female Female Male Male Male Female Female Female Female Male Female Male Female Male Male Female Male

0.7 3.4 1.9 0.6 2.7 2.0 2.5 2.1 3.2 2.6 4.4 4.0 3.9 2.3 2.4 1.5 2.7 1.5 1.9 -

Age (years) 1.3 7.8 2.8 1.2 4.8 3.0 4.2 3.2 6.8 4.5 15.2 11.6 10.9 3.7 3.9 2.1 0.82 4.8 2.1 2.8 0.72

Weight (kg) 16.5 78.0 25.0 13.0 32.0 31.0 65.0 43.5 71.0 28.0 28.0 29.0 28.0 50.0 30.5 23.0 3.0 69.0 34.0 30.0 1.8

Pouch Young Sex Age (years) Female 0.24 Female 0.65 Male 0.28 Female 0.31 Female (P17) Male (B4) -

0.82 0.72 -

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Table 2. Occurrence of lesions in the tibia and tarsal bones and joints in eastern grey kangaroos, Macropus giganteus, collected at Portland, Victoria. Kangaroo Number

Enlarged joint Yellowing of cartilage Fibrillation Foci of cartilage loss Physiological longitudinal grooves Chondroma Osteophytosis Decreased viscosity of synovial fluid * + ++ +++

P11

P12

P13

Distal Tibia * ++ +

Tarsal Bones ++ +

Distal Tibia +++ +

Tarsal Bones +++ +

Distal Tibia + +

Tarsal Bones + +

++ +

++ +

++ +

++ +

++ +

++ +

+

+

+

+

+

+

+++

++ +++

+++

+++

+++

++ +++

+

+

+

+

+

+

Mild Moderate Severe

Absent

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Table 3. Dental lesions in eastern grey kangaroos, Macropus giganteus, collected at Portland and Bridgewater, Victoria. INCISORS Upper -† +++ ++ -

†

+ ++ +++ * ** ***

Lower + + -

MOLARS Upper ** *** * -

Lower P1-10 P11 ** P12 *** P13 * P14-17 B1-4 Normal Not smooth, shiny and white Not smooth, shiny and white with enamel hypoplasia Not smooth, shiny and white with enamel hypoplasia and uneven wear Mild uneven wear Moderate uneven wear Severe uneven wear

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Table 4. Ultrasound speed of female eastern grey kangaroos, Macropus giganteus, collected at Portland, Victoria, showing clinical signs of lameness. Kangaroo Numbers

Ultrasound Speed

Humerus: Caudal Femur: Cranial Medial Lateral Tibia: Cranial Caudal Medial Metatarsus IV: Dorsal

P11 Value

95% Prediction Interval

Outlier

P12 Value

95% Prediction Interval

Outlier

P13 Value

4091.0

3100.9 - 5816.9

-

4523.0

3178.5 - 5614.7

-

4205.0 4386.0 2766.0

3161.9 - 4524.0 2896.6 - 5613.0 3460.1 - 5570.2

+

3918.0 4472.0 3126.0

3197.2 - 4419.0 3001.6 - 5438.3 3500.4 - 5393.1

4233.0 4259.0 4091.0

3607.1 - 4570.2 2789.6 - 3878.1 2882.0 - 5936.0

+ -

4426.0 4296.0 4523.0

3543.0

2901.5 - 4510.9

-

3497.0

95% Prediction Interval

Outlier

4060.0

3194.7 - 5569.8

-

+

3955.0 4036.0 2900.0

3204.6 - 4395.7 3024.2 - 5399.7 3508.4 - 5353.6

+

3625.7 - 4489.6 2930.4 - 3906.9 2958.0 - 5698.0

+ -

4660.0 4009.0 4060.0

3629.4 - 4471.6 2962.2 - 3914.2 2973.0 - 5645.0

+ + -

2942.7 - 4386.4

-

3596.0

2951.3 - 4358.7

-

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Table 5. Fluoride concentrations in the mandible, femur and tibia of eastern grey kangaroos, Macropus giganteus, collected at Portland and Bridgewater, Victoria, compared with cattle ranges. Kangaro Age Mandible Fluoride o (years Level 1 Numbers ) (ppm dry weight) 1.3 2535 (H) P1 7.8 1752 (N) P2 2.8 1967 (N) P3 1.2 680 (N) P4 4.8 2333 (H) P5 3.0 1989 (N) P6 4.2 1823 (N) P7 3.2 1333 (N) P8 6.8 1474 (N) P9 4.5 1794 (N) P10 15.2 3537 (H) P11 11.6 3656 (H) P12 10.9 3055 (H) P13 3.7 2110 (H) P14 3.9 1537 (N) P15 2.1 1478 (N) P16 0.8 137 (N) P17 4.8 208 (N) B1 2.1 161 (N) B2 2.8 212 (N) B3 1 Animal Health Diagnostic Laboratory 2 Suttie and Phillips (1959)

Femur Fluoride Level 2 (ppm dry weight) 3353 (N) 1859 (N) 4576 (H) 3025 (N) 3964 (N) 5833 (H) 3562 (N) 8672 (H) 3075 (N) 665 (N)

Tibia Fluoride Level 2 (ppm dry weight) 1515 (N) 2200 (N) 2832 (N) -

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ROSS RIVER VIRUS INFECTION IN CAPTIVE MACROPOD POPULATIONS IN URBAN NSW J. M. Old*, O. Chan and E. M. Deane Department of Biological Sciences, Macquarie University, NSW 2109, Australia Marsupials, especially macropods, have been suggested to be the major reservoirs for Ross river virus. Two large urban macropod populations were assessed for total antibodies to Ross river virus. One tammar wallaby population in north western Sydney at Macquarie University had 11.7% occurrence and another population from Newcastle N.S.W. had 33.3% prevalence. A small number of wallaroos, and parma wallabies from the Sydney area were also assessed for the prevalence of total antibodies to Ross river virus. All parma wallabies were negative with 44.4% of the wallaroos testing positive. The possibility of macropods as urban reservoirs for Ross river virus is discussed.

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IMMUNOPATHOLOGY OF LYMPHOID TISSUES FROM LONG-NOSED AND LONG-FOOTED POTOROOS L.J. Young1, P.H. Holz2, and E.M. Deane3 1

Central Queensland University Healesville Sanctuary 3 Macquarie University 2

Introduction Relatively little is known abou the immune systems of native, endangered species such as the long-footed potoroo (Potorous longipes) and the tammar wallaby (Macropus eugenii), although captive animals from the family Macropodidae are known to be susceptible to mycobacterial disease (Phelan, 1996; Buddle and Young, 2000). In order to manage captive marsupial populations more effectively, there is a need to clarify the factors determining their underlying immune status and hence, the causes for their aparent disease susceptibility. In this study, the examination of lymphoid tissues collected post-mortem from two species of potoroo, P. longipes and P. tridactylus (long-nosed potoroo) provided an opportunity to document the immunopathology resulting from infection with intracellular pathogens in these two closely related species. It also provided the first documented study of the lymphoid tissue architecture of these two species. Methods Tissue sections from animals suspected to be affected by mycobacterial disease were collected at autopsy and placed immediately into 10% neutral buffered formalin. Samples were transferred into ethanol, routinely processed and embedded in paraffin wax (Bancroft and Stevens, 1990). Serial sections of paraffin blocks were cut to a thickness of 4 or 6 μm, deparaffinised through xylene and rehydrated through a graded alcohol series before staining with haemotoxylin and eosin. Consecutive sections were also processed with Accustain Acid Fast Staining kit to detect the presence of acid-fast bacteria. Results In the majority of samples, acid-fast bacteria were sequestered in macrophages, although organisms were also found in short beaded segments throughout the lung and lymph node parenchyma and in the kidney of one wild-caught long-footed potoroo (LFP). The macrophages within lymph nodes of two long-nosed (LNP) potoroos contained large cytoplasmic bacterial loads, as did those of the facial lymph node and spleen of a third animal. Consistent with haematogenous spread of the mycobacterium, walls of most splenic blood vessels and venous sinuses appeared to be thickened and in some samples, extensive fibrosis was apparent. In others, lymphocytic infiltration into the splenic parenchyma was also evident. Whilst the underlying tissue architecture of potoroo lymph nodes was similar to other mammals, the presence of large numbers of secondary lymphoid follicles was indicative of an on-going humoral response. Small focal granulomas, composed primarily of epithelioid cells, were present in skin sections, whilst bronchus-associated lymphoid tissue (BALT) was identifeid in both LNP and LFP lung sections. Discussion and Conclusion After initial entry into the eutherian host, the Mycobacteriaceae are generally presented by macrophages to lymphoid cells in regions such as the BALT, the gut-associated lymphoid tissue and within lymph nodes that drain these sites (Thorel et al., 2001). If the bacteria are not contained within these regions, they will quickly spread via the lymphatic drainage systems to the bloodstream and eventually colonise the liver, kidneys and spleen. It was evident from the results of this study of potoroo immune tissues that these marsupials appear to respond to infection by intracellular pathogens in a manner that parallels eutherian mammals. These results suggest that factors related to immunological function and/or regulation rather than inherent defects in immunocapacity are most likely the cause of their apparent disease susceptibility.

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References: Bancroft, J.D., Stevens, A. (1990). Theory and Practice of Histological Techniques. (3rd ed.). Edinburgh: Churchill-Livingstone. Buddle, B.M., Young, L.J. (2000). Immunobiology of mycobacterial infections in Marsupials. Developmental and Comparative Immunology 24, 517-529. Phelan, J.R. (1996). Atypical Mycobacterial Infections in Captive Long-footed Potoroos (Potorous longipes). Proceedings of the American Association of Zoo Veterinarians, 443-445. Thorel, M.F., Huchzermeyer, H.F., Michel, A.L. (2001). Mycobacterium avium and Mycobacterium intracellulare infection in mammals. RevueScientifique et Technique (International Office of Epizootics) 20, 204-218. Acknowledgements: We thank Anne Peck and Caroline Wilson for assistance with tissue processing and sectioning.

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IDENTIFICATION OF IMMUNOREGULATORY MOLECULES IN SMALL WALLABY SPECIES L.J. Young1, G.A. Harrison2, P.H. Holz3, and E.M. Deane2 1

Central Queensland University Macquarie University 3 Healesville Sanctuary 2

Introduction The nature and type of immunoregulatory molecules (cytokines) synthesised in response to invading pathogens largely determined whether the host will effectively control an immune assault. In eutherian mammals that are infected with intracellular pathogens such as Mycobacterium avium, Coxiella burnetii and Toxoplasma gondii, cytokines play a key role in this response (Innes, 1997; Ghigo et al., 2001). Specific roles of these molecules include the activation of phagocytic cells, recruitment of effector cells, promotion of the specific memory response by lymphocytes, and production of other cytokines that influence both the differentiation, maturation and immunological responses of immune cells. Cytokines from the Tumour Necrosis Factor (TNF) and Interleukin-1 families are amongst the most important of these molecules in determining the primary and ongoing response to antigenic assault. IL-10 is also an important immunomodulator since it is known to inhibit the production of other cytokines. To date, a small number of cytokines have been identified in a select number of marsupial species. These molecules have been characterised using the reverse transcriptase polymerase chain reaction approach. Cytokines identified in metatherian species include: TNF-α from both the brushtail possum and the tammar wallaby, IL-10, IL-1β and Leukemia Inhibitory Factor from the brushtail possum, and IL-5, Lymphtoxin-α and β and Type I IFNs also from the tammar wallaby. The present study extends the list of species to include members of the potoroid family, specifically, the Long-footed potoroo, Potorous longipes and Long-nosed potoroo, P. tridactylus, both members of the family Macropodidae. The characterisation of immunoregulatory molecules in the potoroid species may shed some light on the reasons for their reported disease susceptibility. Method & Results: cDNA from various cell and tissue sources was prepared from isolated RNA using the Reverse Transcriptase System (Promega; USA) according to the manufacturer’s instructions. This cDNA was used as the DNA template for the polymerase chain reactions (PCR) performed in this study. PCR reaction cycling conditions for all amplifications included 5μL c DNA, 10 pmol primers, 200 μM dents, 25 mm MgCl2, 2.5 U Ta polymerase and nuclease-free water to 50 μL. Original PCR products were amplified at 45oC to facilitate screening. Once a product was obtained, the PCR was repeated on new cDNA at the higher temperature of 50oC to increase specificity. After gel electrophoresis, cloning and sequencing, partial cDNA sequence was identified for both TNF-α and IL-10 in tammar wallaby and potoroo tissues. In all cases, the sequence data was greater than 50% identical and greater than or equal to 80% similar to known eutherian cytokine sequences. Discussion & Future Work: In this study, the expression of the immunomodulatory cytokines TNF-α and IL-10 in the tissues of both healthy and disease-affected small wallaby species suggests that immunoregulation by cytokine molecules is a feature of immune responses in the family Macropodidae. Studies are currently underway to obtain the complete sequence for IL-1β and IL-10 from lymph node tissue of the tammar wallaby. This information should allow for a more complete analysis of the role of cytokines in the marsupial immune response and hence clarify some of the factors that determine their underlying immune status and hence their apparent susceptibility to disease. References: Innes, E.A. (1997). Toxoplasmosis: Comparative Species Susceptibility and Host immune Response. Comparative Immunology and Microbiology & Infectious Diseases 20, 131-138.

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Ghigo, E., Capo, C., Raoult, D., Mege, J. (2001). Interleukin-10 Stimulates Coxiella burnetii Replication in Human Monocytes through Tumor Necrosis Factor down-Modulation: Role in Microbicidal Defect of Q Fever. Infection and Immunity 69, 2345-2352.

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