KLOSSIELLA DULCIS N. SP. (APICOMPLEXA: KLOSSIELLIDAE) IN THE KIDNEYS OF PETAURUS BREVICEPS (MARSUPIALIA: PETAURIDAE).

Two cases of renal klossiellosis were diagnosed by histopathology in pet sugar gliders (Petaurus breviceps). In both cases, parasites were associated with tubular dilation and mild interstitial nephritis. Rare schizonts were seen in the proximal convoluted renal tubular epithelium, whereas all other life cycle stages were found within distal convoluted tubule cells or the urinary space of the structures distal to the loop of Henle. Conventional optical and transmission electron microscopies were used to assess the life stages of the parasite. The morphologic characteristics and measurements observed differ from those of previously described species of Klossiella infecting marsupial hosts, and the name Klossiella dulcis n. sp. is hereby proposed. This is the first report of a Klossiella sp. infection in Petaurus breviceps .

Eimeria tiliquae n. sp. (Apicomplexa: Eimeriidae) from the shingleback skink (Tiliqua rugosa rugosa).

A new species, Eimeria tiliquae n. sp. is described from a shingleback skink (Tiliqua rugosa rugosa). Sporulated oocysts (n=50) are spherical to subspherical, with colourless trilaminate oocyst wall, 0.7±0.1 (0.5-0.75) thick. Oocyst with 4 spheroidal to subspheroidal sporocysts. Oocyst length, 13.7±0.9 (12.0-16.3); oocyst width, 12.8±0.9 (11.5-15.0); oocyst length/width (L/W) ratio, 1.07±0.05 (1.0-1.2). Micropyle, oocyst residuum and polar granule absent. Sporocysts with globular sporocyst residuum and 2 sporozoites. Sporocyst length, 6.0±0.6 (5.0-7.5); sporocyst width, 5.4±0.6 (4.0-7.0); sporocyst L/W ratio, 1.11±0.11 (1.0-1.5). Stieda, parastieda and substieda bodies absent. Phylogenetic analysis of 18S rRNA sequences indicated that E. tiliquae n. sp. shared 96.4-96.5% genetic similarity to E. tropidura, its closest relative. Reptile-derived sequences were not available for the mitochondrial cytochrome oxidase gene (COI) and phylogenetic analysis at this locus placed E. tiliquae n. sp. in a clade by itself but grouping closest (92% similarity) with a novel isolate from a King's skink (Egernia kingii) from Western Australia. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that to date has only been found in shingleback skinks.

Novel Eimeria sp. isolated from a King's skink (Egernia kingii) in Western Australia.

A novel Eimeria sp. was identified in faeces collected from a King's skink (Egernia kingii) housed at the Kanyana Wildlife Rehabilitation Centre in Western Australia. Oocysts measure 17.0×15.0 µm with a length/width ratio (L/W) of 1.13. Phylogenetic analysis of 18S rRNA sequences indicated that the novel Eimeria sp. shared the highest genetic similarity to Eimeria antrozoi and Eimeria rioarribaensis from vespertilionid bats from North America (≥98.9%). At the COI locus, bat-derived sequences were not available and phylogenetic analysis placed the novel Eimeria sp. in a clade by itself and shared 98.8% similarity with the rodent-derived species E. falciformis and E. vermiformis. This suggests that the isolate from the King's skink's faeces was probably derived from a mammal, possibly a rodent or a bat.

Papillomavirus-associated multicentric squamous cell carcinoma in situ in a cat: an unusually extensive and progressive case with subsequent metastasis.

BACKGROUND: Multicentric squamous cell carcinoma in situ (MSCCIS) is an uncommon cutaneous disease of middle-aged to older cats, with some cases being linked to papillomavirus infection. The disease course is usually benign. Initial eruption of multifocal, pigmented, hyperkeratotic plaques is typical, with gradual progression to thickly crusted ulcerative lesions. ANIMAL: A 5-year-old male neutered Devon rex cat in apparent good health was initially presented with a 16 month history of over 40 nonpruritic dorsally distributed hyperpigmented patches. Lesions progressed gradually over 2 years to larger, more pigmented, crusted plaques and ulcerated nodules. At 7 years of age the cat developed neurological signs and systemic illness and was euthanized. METHODS AND RESULTS: Initial skin histopathology revealed discrete regions of epidermal and follicular epithelial hyperplasia, with moderate numbers of apoptotic keratinocytes, and mild focal epithelial dysplasia. A diagnosis of erythema multiforme was considered; feline herpesvirus-1 immunohistochemistry was negative. Repeat histopathology 22 months after initial presentation confirmed MSCCIS with foci of invasive squamous cell carcinoma (SCC). Postmortem examination 1 month later revealed SCC within the thoracic wall, lungs and vessels of the thoracic spinal cord and heart base, presumed to be metastases from skin lesions. Fluorescent in situ hybridization of initial and later histopathology samples was positive for Felis domesticus papillomavirus type 2. Immunoreactivity of p16 was prominent within early and late cutaneous lesions and internal SCCs. CONCLUSIONS: This case represents an unusual presentation of papillomavirus-associated MSCCIS with extensive lesions, atypical initial histopathology and progression to SCC with distant metastases.

Insights into Polyomaviridae microRNA function derived from study of the bandicoot papillomatosis carcinomatosis viruses.

Several different members of the Polyomaviridae, including some human pathogens, encode microRNAs (miRNAs) that lie antisense with respect to the early gene products, the tumor (T) antigens. These miRNAs negatively regulate T antigen expression by directing small interfering RNA (siRNA)-like cleavage of the early transcripts. miRNA mutant viruses of some members of the Polyomaviridae express increased levels of early proteins during lytic infection. However, the importance of miRNA-mediated negative regulation of the T antigens remains uncertain. Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) is associated with papillomas and carcinomas in the endangered marsupial the western barred bandicoot (Perameles bougainville). BPCV1 is the founding member of a new group of viruses that remarkably share distinct properties in common with both the polyomavirus and papillomavirus families. Here, we show that BPCV1 encodes, in the same orientation as the papillomavirus-like transcripts, a miRNA located within a long noncoding region (NCR) of the genome. Furthermore, this NCR serves the function of both promoter and template for the primary transcript that gives rise to the miRNA. Unlike the polyomavirus miRNAs, the BPCV1 miRNA is not encoded antisense to the T antigen transcripts but rather lies in a separate, proximal region of the genome. We have mapped the 3' untranslated region (UTR) of the BPCV1 large T antigen early transcript and identified a functional miRNA target site that is imperfectly complementary to the BPCV1 miRNA. Chimeric reporters containing the entire BPCV1 T antigen 3' UTR undergo negative regulation when coexpressed with the BPCV1 miRNA. Notably, the degree of negative regulation observed is equivalent to that of an identical reporter that is engineered to bind to the BPCV1 miRNA with perfect complementarity. We also show that this miRNA and this novel mode of early gene regulation are conserved with the related BPCV2. Finally, papillomatous lesions from a western barred bandicoot express readily detectable levels of this miRNA, stressing its likely importance in vivo. Combined, the alternative mechanisms of negative regulation of T antigen expression between the BPCVs and the polyomaviruses support the importance of miRNA-mediated autoregulation in the life cycles of some divergent polyomaviruses and polyomavirus-like viruses.

The first complete papillomavirus genome characterized from a marsupial host: a novel isolate from Bettongia penicillata.

The first fully sequenced papillomavirus (PV) of marsupials, tentatively named Bettongia penicillata papillomavirus type 1 (BpPV1), was detected in papillomas from a woylie (Bettongia penicillata ogilbyi). The circular, double-stranded DNA genome contains 7,737 bp and encodes 7 open reading frames (ORFs), E6, E7, E1, E2, E4, L2, and L1, in typical PV conformation. BpPV1 is a close-to-root PV with L1 and L2 ORFs most similar to European hedgehog PV and bandicoot papillomatosis carcinomatosis virus types 1 and 2 (BPCV1 and -2). It appears that the BPCVs arose by recombination between an ancient PV and an ancient polyomavirus more than 10 million years ago.

Use of fluorescence in situ hybridization to detect Felis catus papillomavirus type 2 in feline Bowenoid in situ carcinomas.

OBJECTIVES: Papillomaviruses (PVs) are ubiquitous host- and site-specific viruses. PV infections in cats are associated with oral papillomas, viral plaques, Bowenoid in situ carcinomas (BISCs), squamous cell carcinomas and sarcoids; this association is primarily based on PCR detection of PV DNA within said lesions. PV DNA is frequently detectable on normal feline skin; thus, it is possible that some of the implicated DNA is commensal rather than associated with lesion formation. Therefore, the aim of the present study was to use fluorescence in situ hybridization (FISH) to localize PV DNA within feline BISCs, to provide additional evidence that PV infection may influence the development of these neoplasms. METHODS: FISH probes targeting Felis catus papillomavirus type 2 (FcaPV2) DNA were used to localize FcaPV2 DNA within 42 BISCs from which FcaPV2 DNA had previously been amplified via PCR. RESULTS: Fifteen of 42 BISC lesions (35.7%) demonstrated intralesional FcaPV2 using FISH. Probe annealing was predominantly located within the nuclei of koilocytes found in the upper strata of the epidermis. Probes were typically scattered multifocally within the lesions; most commonly this was near the periphery of the BISCs. CONCLUSIONS AND RELEVANCE: These results confirm that a proportion of BISCs contain FcaPV2 DNA. These results further support a causative association between FcaPV2 and BISCs in cats.

A novel virus detected in papillomas and carcinomas of the endangered western barred bandicoot (Perameles bougainville) exhibits genomic features of both the Papillomaviridae and Polyomaviridae.

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size ( approximately 7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.

Hepatic intranuclear glycogen inclusions in western barred bandicoots (Perameles bougainville).

The western barred bandicoot, Perameles bougainville, is an endangered Australian marsupial species. Routine histology of liver samples collected at necropsy from 19 of 20 (95%) western barred bandicoots revealed the sporadic to common occurrence of abnormal hepatocyte nuclei characterized by margination of chromatin and concomitant central pallor. Some abnormal hepatocyte nuclei were mildly to markedly enlarged and irregularly shaped. Periodic acid-Schiff reagent stained 131 of 142 (92%) of these abnormal hepatocyte nuclei. Positive staining was completely eliminated by diastase pretreatment. Transmission electron microscopy revealed that abnormal hepatocyte nuclei with marginated chromatin did not contain viral particles. Rather, glycogen beta-particles and alpha-rosettes were identified within some abnormal hepatocyte nuclei. Glycogen intranuclear inclusions were an incidental finding in western barred bandicoot hepatocytes.

Clinical chemistry values and tissue enzyme activities in western barred bandicoots (Perameles bougainville).

BACKGROUND: The western barred bandicoot, Perameles bougainville, is an endangered Australian marsupial species whose survival is threatened by a papillomatosis and carcinomatosis syndrome. Investigations to characterize this syndrome would benefit from species-specific clinical chemistry data. OBJECTIVES: The purpose of this study was to determine plasma biochemical reference values and to determine enzyme activities in various tissues of P. bougainville. METHODS: Heparinized blood samples were collected by jugular venipuncture from 53 clinically healthy bandicoots of both sexes and at 3 geographic locations. Plasma was analyzed for routine clinical chemistry variables using an automated biochemistry analyzer. Tissues obtained following humane euthanasia of 3 bandicoots were analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase (CK), alpha-amylase (AML), and gamma-glutamyltransferase (GGT) activities. RESULTS: Significant differences in the results were found for animals based on geographic location and sex; hence, results were expressed as minimum and maximum values. A population reference interval was calculated for AST activity (20-283 U/L). ALT was found mainly in the liver, with lower levels in cardiac and skeletal muscle and kidneys. AST was detectable in many tissues, including the heart, liver, kidneys, and central nervous system; CK was found in skeletal and cardiac muscle and central nervous system; AML was found in the pancreas; and GGT was found mainly in kidneys with lower levels in the intestines and pancreas. CONCLUSIONS: These findings will facilitate the interpretation of clinical chemistry results from P. bougainville and thereby inform population management and clinical decision-making.

Hematologic characteristics of captive western barred bandicoots (Perameles bougainville) from Western Australia.

BACKGROUND: The western barred bandicoot (Perameles bougainville) is an Australian marsupial species now considered endangered as a consequence of habitat destruction and predation. A recently discovered papillomatosis syndrome is hindering efforts to repopulate this species. Hematology reference intervals have been lacking for P bougainville, preventing optimal interpretation of hematology results from wart-affected and clinically normal animals. OBJECTIVES: The purpose of this study was to establish hematology reference values and describe morphologic characteristics of blood cells of healthy western barred bandicoots. METHODS: Fifty-nine whole blood samples were collected by jugular venipuncture into EDTA from 47 clinically healthy captive western barred bandicoots at 3 locations on the Western Australian mainland. A CBC was performed using an ADVIA-120 analyzer. Data were compared on the basis of geographic location, sex, age, and lactation status, and reference intervals were calculated. Blood cell morphology was evaluated using light microscopy, and transmission and scanning electron microscopy. RESULTS: Significant differences were found based on sex (RBC indices, fibrinogen), age (% polychromatophilic RBCs), and geographic location (RBC, neutrophil, and lymphocyte counts, MCHC, % polychromatophilic RBCs, fibrinogen). Combined reference intervals were calculated for hemoglobin concentration (122-165 g/L), HCT (0.36-0.49 L/L), and total WBC (2.9-14.9 x 10(9)/L), monocyte (0-0.6 x 10(9)/L), eosinophil (0-0.9 x 10(9)/L), and total plasma protein (47-63 g/L) concentrations. Leukocyte, erythrocyte, and platelet morphology were similar to those of other marsupial peramelid species. Nuclei in neutrophils, monocytes, and eosinophils occasionally had an annular configuration. CONCLUSIONS: Reference intervals and blood cell morphology obtained in this study will be useful for the evaluation of laboratory data from ill animals and assist with population health monitoring of western barred bandicoots.

Development of loop-mediated isothermal amplification-based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia-associated P multocida serotype B:2.

OBJECTIVE To develop 2 rapid loop-mediated isothermal amplification (LAMP) assays for detection of Pasteurella multocida DNA (Pm-LAMP assay) and P multocida DNA from strains associated with hemorrhagic septicemia (HS) in cattle and buffalo (HS-LAMP assay). SAMPLE Solutions containing 16 P multocida strains and 9 other bacterial species at various concentrations. PROCEDURES Optimal conditions were determined for running the Pm-LAMP and HS-LAMP assays. The assays were then used to detect DNA of the test organisms. Results of LAMP assays were validated against conventional PCR assays designed for specific detection of P multocida and the B:2 serotype of HS-associated strains. RESULTS Following incubation of sample reaction mixtures for 27 minutes, specificity and sensitivity of the HS-LAMP assay at template DNA amounts as low as 5 pg were 93% and 97%, respectively. When duplicates of each sample were incubated for 28 minutes (a positive result defined as positive results for both reactions of a given sample), specificity and sensitivity of the HS-LAMP assay in the same conditions increased to 100%. The best specificity and sensitivity of Pm-LAMP single (93% and 91%) and duplicate (97% and 98%) reactions at template DNA amounts as low as 10 pg were achieved at 33 and 34 minutes, respectively. CONCLUSIONS AND CLINICAL RELEVANCE These preliminary findings suggested the developed HS-LAMP assay had high sensitivity and specificity for detection of HS-associated P multocida. Additional research is needed to determine the accuracy of the assay for use on clinical specimens obtained in HS-endemic countries such as Pakistan and Thailand.

Metastatic Squamous Cell Carcinoma in a Northern Brown Bandicoot (Isoodon macrourus).

Aside from a handful of notable exceptions, neoplasia is not reported as a major cause of mortality in wild animal populations and often goes undetected. For northern brown bandicoots specifically, there are few reported tumors in the literature and on file in the Australian Registry of Wildlife Health. This report describes a case of squamous cell carcinoma in a northern brown bandicoot (Isoodon macrourus), with metastases to the draining lymph nodes and lung. This neoplasm consisted predominantly of well-differentiated squamous cells and multifocal keratin pearls, with areas possibly consistent with epithelial to mesenchymal transition, as identified by positive immunohistochemical staining by both pancytokeratin (AE1/AE3) and vimentin. Additional investigations were negative for bandicoot papillomatosis carcinomatosis viruses.

Comparative Genomic Analysis of Asian Haemorrhagic Septicaemia-Associated Strains of Pasteurella multocida Identifies More than 90 Haemorrhagic Septicaemia-Specific Genes.

Pasteurella multocida is the primary causative agent of a range of economically important diseases in animals, including haemorrhagic septicaemia (HS), a rapidly fatal disease of ungulates. There is limited information available on the diversity of P. multocida strains that cause HS. Therefore, we determined draft genome sequences of ten disease-causing isolates and two vaccine strains and compared these genomes using a range of bioinformatic analyses. The draft genomes of the 12 HS strains were between 2,298,035 and 2,410,300 bp in length. Comparison of these genomes with the North American HS strain, M1404, and other available P. multocida genomes (Pm70, 3480, 36950 and HN06) identified a core set of 1,824 genes. A set of 96 genes was present in all HS isolates and vaccine strains examined in this study, but absent from Pm70, 3480, 36950 and HN06. Moreover, 59 genes were shared only by the Asian B:2 strains. In two Pakistani isolates, genes with high similarity to genes in the integrative and conjugative element, ICEPmu1 from strain 36950 were identified along with a range of other antimicrobial resistance genes. Phylogenetic analysis indicated that the HS strains formed clades based on their country of isolation. Future analysis of the 96 genes unique to the HS isolates will aid the identification of HS-specific virulence attributes and facilitate the development of disease-specific diagnostic tests.

Butcherbird polyomavirus isolated from a grey butcherbird (Cracticus torquatus) in Queensland, Australia.

A novel avian polyomavirus was detected in peri-ocular skin lesions collected from a grey butcherbird (Cracticus torquatus), using a combination of multiply primed rolling circle amplification, nested PCR and long range PCR. The sequence of Butcherbird polyomavirus was determined by combining next generation sequencing and primer walking techniques. The circular double-stranded DNA genome of Butcherbird polyomavirus consisted of 5084 bp, and encoded six open reading frames (ORF-X, VP2, VP3, VP1, small T-antigen and large T-antigen). Phylogenetic analysis placed it amongst other members of the genus Avipolyomavirus, most closely related to Crow polyomavirus. Next generation sequencing enabled the detection of DNA fragments similar to, but distinct from, Canarypox virus within the same lesion from which Butcherbird polyomavirus was amplified, thus confirming an avipolyomavirus-avipoxvirus co-infection in the peri-ocular skin lesions of this grey butcherbird.

Molecular typing of haemorrhagic septicaemia-associated Pasteurella multocida isolates from Pakistan and Thailand using multilocus sequence typing and pulsed-field gel electrophoresis.

A comparative genetic study of 23 field isolates and vaccine strains of Pasteurella multocida associated with haemorrhagic septicaemia cases from Pakistan and Thailand was done using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLST sequence type (ST) for all 20 of the 23 isolates tested was 122. The PFGE results showed one band difference between the Pakistani and the Thai isolates. Sequence type 122 is the dominant associated profile with haemorrhagic septicaemia (HS) cases in South Asia. The study supports the concept of using PFGE for short-term epidemiology and MLST for long-term epidemiology.

Diversity of Bartonella species detected in arthropod vectors from animals in Australia.

A variety of Bartonella species were detected in two species of ticks and three species of fleas collected from marsupial hosts; brush-tailed bettong or woylie (Bettongia penicillata) and western barred bandicoots (Perameles bougainville) and from a rodent host; Rattus fuscipes in Western Australia. Bartonella species were detected using nested-PCR of the gltA gene and the 16S-23S ribosomal internal transcribed spacer region (ITS), and species were characterized using DNA sequencing of the 16S rRNA, gltA, rpoB, ftsZ genes and the ITS region. Bartonella rattaustraliani and B. coopersplainsensis were detected in Ixodes spp. ticks and fleas (Stephanocircus pectinipes) respectively collected from rodents. Two novel Bartonella species were detected from marsupials; Candidatus Bartonella woyliei n. sp. was detected in both fleas (Pygiopsylla hilli) and ticks (Ixodes australiensis) collected from woylies and Candidatus Bartonella bandicootii n. sp. was detected in fleas (Pygiopsylla tunneyi) collected from western barred bandicoots. Concatenated phylogenetic analysis of all 5 loci clarified the marsupial cluster of Bartonella species in Australia and confirmed the species status of these two Bartonella species in ticks and fleas from woylies and western barred bandicoots, which are classified as threatened species and are vulnerable to extinction.

Genetic characterization of flea-derived Bartonella species from native animals in Australia suggests host-parasite co-evolution.

Fleas are important arthropod vectors for a variety of diseases in veterinary and human medicine, and bacteria belonging to the genus Bartonella are among the organisms most commonly transmitted by these ectoparasites. Recently, a number of novel Bartonella species and novel species candidates have been reported in marsupial fleas in Australia. In the present study the genetic diversity of marsupial fleas was investigated; 10 species of fleas were collected from seven different marsupial and placental mammal hosts in Western Australia including woylies (Bettongia penicillata), western barred bandicoots (Perameles bougainville), mardos (Antechinus flavipes), bush rats (Rattus fuscipes), red foxes (Vulpes vulpes), feral cats (Felis catus) and rabbits (Oryctolagus cuniculus). PCR and sequence analysis of the cytochrome oxidase subunit I (COI) and the 18S rRNA genes from these fleas was performed. Concatenated phylogenetic analysis of the COI and 18S rRNA genes revealed a close genetic relationship between marsupial fleas, with Pygiopsylla hilli from woylies, Pygiopsylla tunneyi from western barred bandicoots and Acanthopsylla jordani from mardos, forming a separate cluster from fleas collected from the placental mammals in the same geographical area. The clustering of Bartonella species with their marsupial flea hosts suggests co-evolution of marsupial hosts, marsupial fleas and Bartonella species in Australia.

Coxiella burnetii in western barred bandicoots (Perameles bougainville) from Bernier and Dorre Islands in Western Australia.

The aim of this work is to investigate the presence of Coxiella burnetii in Perameles bougainville and their ticks on two islands off Western Australia. Haemaphysalis humerosa, Haemaphysalis ratti, and Haemaphysalis lagostrophi were collected from P. bougainville on Bernier and Dorre Islands from 2005 to 2007; only Amblyomma limbatum was collected from humans over the same interval. One of 13 tick samples and 1 of 12 P. bougainville fecal samples were positive for C. burnetii DNA using quantitative polymerase chain reaction. DNA fragments had >99% similarity to published C. burnetii sequences. Three of 35 P. bougainville sera tested positive for anti-C. burnetii antibodies using enzyme-linked immunosorbent assay. C. burnetii was found in P. bougainville feces and a H. humerosa tick on Dorre Island and Bernier Island, respectively. This is the first reported use of enzyme-linked immunosorbent assay for screening of P. bougainville sera. The risk of zoonotic Q fever infection for human visitors to these islands is considered relatively low, however, appropriate precautions should be taken when handling western barred bandicoots, their feces and their ticks on Bernier and Dorre Islands.

In situ hybridization to detect bandicoot papillomatosis carcinomatosis virus type 1 in biopsies from endangered western barred bandicoots (Perameles bougainville).

The western barred bandicoot (Perameles bougainville) is an endangered Australian marsupial species in which a papillomatosis and carcinomatosis syndrome occurs. Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) is associated with the lesions of this progressively debilitating syndrome. Five digoxigenin-labelled DNA probes were generated for in situ hybridization (ISH) and the technique was optimized and performed on formalin-fixed paraffin-embedded (FFPE) biopsies. Staining of keratinocyte and sebocyte nuclei within lesions was achieved with all five probes. The sensitivity of ISH (76.9%) surpassed that of PCR (30.8%) for FFPE samples. The sensitivity of ISH varied from 81% (papillomas) and 70% (carcinoma in situ) to 29% (squamous cell carcinomas). The specificity of the test was confirmed using an irrelevant probe and papillomas from other species. These results strengthen the association between BPCV1 and the western barred bandicoot papillomatosis and carcinomatosis syndrome and give insight into the biology of the virus-host interaction.