Lucy Woolford

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

ABSTRACT 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 (∼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.

Genomic characterization of a novel virus found in papillomatous lesions from a southern brown bandicoot (Isoodon obesulus) in Western Australia

The genome of a novel virus, tentatively named bandicoot papillomatosis carcinomatosis virus type 2 (BPCV2), obtained from multicentric papillomatous lesions from an adult male southern brown bandicoot (Isoodon obesulus) was sequenced in its entirety. BPCV2 had a circular double-stranded DNA genome consisting of 7277 bp and open reading frames encoding putative L1 and L2 structural proteins and putative large T antigen and small t antigen transforming proteins. These genomic features, intermediate between Papillomaviridae and Polyomaviridae are most similar to BPCV1, recently described from papillomas and carcinomas in the endangered western barred bandicoot (Perameles bougainville). This study also employed in situ hybridization to definitively demonstrate BPCV2 DNA within lesion biopsies. The discovery of BPCV2 provides evidence of virus-host co-speciation between BPCVs and marsupial bandicoots and has important implications for the phylogeny and taxonomy of circular double-stranded DNA viruses infecting vertebrates.

Cutaneous Papillomatosis and Carcinomatosis in the Western Barred Bandicoot (Perameles bougainville)

A progressive wart-like syndrome in both captive and wild populations of the Western barred bandicoot (WBB) is hindering conservation efforts to prevent the extinction of this endangered marsupial. In this study, 42 WBBs exhibiting the papillomatosis and carcinomatosis syndrome were examined. The disease was characterized by multicentric proliferative lesions involving cutaneous and mucosal surfaces, which were seen clinically to increase in size with time. Grossly and histologically the smaller skin lesions resembled papillomas, whereas the larger lesions were most commonly observed to be squamous cell carcinomas. Large amphophilic intranuclear inclusion bodies were observed in hyperplastic conjunctival lesions of 8 WBBs under light microscopy. Conjunctival lesions from 2 WBBs examined using transmission electron microscopy contained a crystalline array of spherical electrondense particles of 45-nm diameter, within the nucleus of conjunctival epithelial cells, consistent with a papillomavirus or polyomavirus. Conjunctival samples from 3 bandicoots that contained intranuclear inclusion bodies also demonstrated a positive immunohistochemical reaction after indirect immunohis-tochemistry for papillomavirus structural antigens. Ultrastructural and/or immunohistochemical evidence of an etiologic agent was not identified in the nonconjunctival lesions examined. Here we describe the gross, histopathologic, ultrastructural, and immunohistochemical findings of a papillomatosis and carcinomatosis syndrome recently identified in the WBB.

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BackgroundChlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts.MethodsWhole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts.ResultsThree porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock.Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec.ConclusionsThis study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

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.

Pathological Features of Oxalate Nephrosis in a Population of Koalas (Phascolarctos cinereus) in South Australia

The wild and captive koala population of the Mt Lofty Ranges in South Australia has a high level of renal dysfunction in which crystals consistent with calcium oxalate have been observed in the kidneys. This study aimed to describe the pathological features of the renal disease in this population, confirm the composition of renal crystals as calcium oxalate, and determine whether any age or sex predispositions exist for this disease. A total of 51 koalas (28 wild rescues, 23 captive) were examined at necropsy, of which 28 (55%) were found to have gross and/or histological evidence of oxalate nephrosis. Histopathological features included intratubular and interstitial inflammation, tubule dilation, glomerular atrophy, tubule loss, and cortical fibrosis. Calcium oxalate crystals were demonstrated using a combination of polarization microscopy, alizarin red S staining, infrared spectroscopy, and energy-dispersive X-ray analysis with scanning electron microscopy. Uric acid and phosphate deposits were also shown to be present but were associated with minimal histopathological changes. No significant differences were found between the numbers of affected captive and wild rescued koalas; also, there were no sex or age predispositions identified, but it was found that oxalate nephrosis may affect koalas <2 years of age. The findings of this study suggest that oxalate nephrosis is a leading disease in this koala population. Possible causes of this disease are currently under investigation.

A NEW EIMERIA SPECIES PARASITIC IN WESTERN BARRED BANDICOOTS, PERAMELES BOUGAINVILLE (MARSUPIALIA: PERAMELIDAE), IN WESTERN AUSTRALIA

Feces from western barred bandicoots, Perameles bougainville, examined during routine monitoring of captive breeding colonies and wild populations were frequently found to contain oocysts. Fecal oocysts from 1 individual housed at Kanyana Wildlife Rehabilitation Centre were allowed to sporulate in 2% potassium dichromate (K2Cr2O7) at room temperature. Sporulated oocysts are subspheroidal 18.8 × 17.9 (16.9–21.0 × 16.0–19.9) μm, with length/width (L/W) ratio of 1.05 (1.00–1.15), lack a micropyle and oocyst residuum, but they usually have a polar granule within a smooth trilaminate oocyst wall 1.0 (0.7–1.3) μm thick. Sporocysts are ovoid, 9.1 × 7.0 (8.1–10.8 × 6.1–8.6) μm, with L/W ratio of 1.32 (1.04–1.51), have a Stieda body, sporocyst residuum, and 2 comma-shaped sporozoites, each containing 2 spheroidal refractile bodies. Sporulation takes 2–5 days at room temperature. This is the first formal description of an Eimeria species parasitic in the order Peramelemorphia.

Ventricular and Extraventricular Ependymal Tumors in 18 Cats

Ependymal tumors are reported rarely in domestic animals. The aims of this study were to examine the clinical and pathologic features of ventricular and extraventricular ependymomas and subependymomas in 18 domestic cats examined between 1978 and 2011. Parameters examined included age, sex, breed, clinical signs, and macroscopic and histopathologic features. The mean age of affected cats was 9 years, 4 months; median age, 8.5 years. There were 8 female and 4 male cats, and 6 cats for which sex was not recorded. Breeds included 10 domestic shorthaired, 2 domestic longhaired, 1 Persian, and 1 Siamese. Clinical signs included altered mentation or behavior, seizures, circling, propulsive gait, generalized discomfort, and loss of condition. The tumors often formed intraventricular masses and usually arose from the lining of the lateral or third ventricles, followed by the fourth ventricle, mesencephalic aqueduct, and spinal cord central canal. Three tumors were extraventricular, forming masses within the cerebrum and adjacent subarachnoid space. Histologically, 15 tumors were classified as variants of ependymomas (classic, papillary, tanycytic, or clear cell) and 3 as subependymomas. Tumors were generally well demarcated; however, 6 ependymomas focally or extensively infiltrated the adjacent neural parenchyma. Characteristic perivascular pseudorosettes were observed in all ependymomas; true rosettes were less common. Some tumors had areas of necrosis, mineralization, cholesterol clefts, and/or hemorrhage. This cohort study of feline ependymal tumors includes subependymoma and primary extraventricular ependymoma, variants not previously described in the veterinary literature but well recognized in humans.

Symmetry: the key to diagnosing propeller strike injuries in sea mammals

The fresh carcass of a neonatal Indo-Pacific bottlenose dolphin (Tursiops aduncus) was recovered from a suburban beach near Adelaide, South Australia, by officers of the Australian Marine Wildlife Research and Rescue Organisation (AMWRO). The carcass was incomplete with loss of the tail and three deep parallel incised/chop wounds to the torso and tail (Fig. 1). At necropsy the carcass was that of an otherwise healthy Indo-Pacific bottlenose dolphin neonate of around 1–2 weeks of age. The animal was normally formed and well nourished, with milk in its mouth and stomach indicating that recent breast feeding had occurred. Frothy white foam was present in the upper airway. The most significant findings consisted of a series of four curvilinear parallel incised/chop wounds predominately to the right side of the body (Fig. 2). The most rostral injury was a clean incised wound of the upper back and right side of the chest which shelved into paraspinal muscles (Fig. 3). No major vessels had been damaged and the chest cavity had not been opened. Within the chest cavity, however, the deepest part of the chop wound was associated with fracture-dislocation of the midthoracic spine (Fig. 4) with spinal cord transaction. There were also fractures of the posterior aspects of the first to sixth ribs on the right side with fresh paravertebal soft tissue hemorrhage bilaterally. Thoracic organs and vessels were intact. Fifty-nine centimeters caudal to this was a second incised/chop wound which passed from the right side through the anterior aspect of the dorsal fin (Figs. 1, 2). Although no body cavities or major vessels had been opened the wound shelved caudally with fracturing of the underlying spine and adjacent fresh interstitial hemorrhage. Caudal to this was a deep curved incised wound that also shelved caudally and extended into paravertebral muscles with a chopping injury to the lateral aspect of the vertebral bodies, with underlying fractures. The final injury consisted of amputation of the tail (Fig. 5). There were no congenital defects, underlying organic diseases or parasitic infestations identified that could have caused or contributed to death. Death was, therefore, due to multiple incised/chop wounds typical of boat propeller injuries with subsequent exsanguination. Amputation of the tail and transaction of the mid thoracic spinal cord would have also have prevented the animal from swimming.

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.