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http://vet.sagepub.com/content/45/1/95The online version of this article can be found at:

 DOI: 10.1354/vp.45-1-95

2008 45: 95Vet PatholWarren

L. Woolford, A. J. O'Hara, M. D. Bennett, M. Slaven, R. Swan, J. A. Friend, A. Ducki, C. Sims, S. Hill, P. K. Nicholls and K. S.)Perameles bougainvilleCutaneous Papillomatosis and Carcinomatosis in the Western Barred Bandicoot (

  

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Cutaneous Papillomatosis and Carcinomatosis in the Western BarredBandicoot (Perameles bougainville)

L. WOOLFORD, A. J. O’HARA, M. D. BENNETT, M. SLAVEN, R. SWAN, J. A. FRIEND, A. DUCKI, C. SIMS,S. HILL, P. K. NICHOLLS, AND K. S. WARREN

School of Veterinary and Biomedical Sciences, Murdoch University, Western Australia, Australia (LW,AJO, MDB, MS, RS, AD, PKN, KSW); and Department of Environment and Conservation, Western

Australia, Australia (JAF, CS, SH)

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

Key words: Carcinomatosis; papillomatosis; papillomavirus; polyomavirus; Western barredbandicoot.

Infectious diseases have the potential to drivepopulation declines and contribute to speciesextinction.14,15 The Western barred bandicoot(WBB), Perameles bougainville, is an endangeredAustralian marsupial that was once widespreadacross western and southern Australia. Nowextinct on the mainland, wild populations areknown only to exist on Bernier and Dorre Islands,in the World Heritage Area of Shark Bay WesternAustralia.37,41,42,49 Conservation efforts to preventthe extinction of this species are currently ham-pered by a progressively debilitating cutaneous andmuco-cutaneous papillomatosis and carcinomato-sis syndrome observed in both captive and wildWBBs. Affected animals develop multicentricpapillomatous lesions, which appear clinically toprogress to larger sizes. These lesions are extremelydebilitating. Involvement of the feet, eyes, andmouth leads to problems with ambulation, vision,and eating. Affected animals often die due tosecondary complications or are euthanatized onwelfare grounds. Awareness of the papillomatosissyndrome first arose in 1999, when lesions were

noticed developing on a captive adult femalehoused at Kanyana Wildlife Rehabilitation Center,Perth.48 Within a year, additional WBBs housed atthe facility were noticed to have developed lesions.Similar cutaneous lesions were also noticed oncaptive WBBs at another breeding colony in SharkBay and identified on wild WBBs on Bernier Islandby the Western Australian Department of Envi-ronment and Conservation (DEC) in 2001. Ahistoric trace back performed by one of the authors(SH) on 93 museum specimens from 5 museums inAustralia found similar lesions to be present onWestern Australian Museum WBB specimensdating back to 1982.22 The purpose of this reportis to describe the gross, histologic, ultrastructural,and immunohistochemical features of a multicentricpapillomatosis and carcinomatosis syndrome iden-tified in 42 WBBs.

Materials and Methods

Study population

Between 2000 and 2006, submissions from 42 WBBsdisplaying focal or multifocal cutaneous and/or muco-

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cutaneous thickenings or masses were received by thepathology section at the Murdoch University School ofVeterinary and Biomedical Sciences. Essential criteriafor inclusion of WBBs in this study were epitheliallesions with gross and histologic characteristics consis-tent with either papilloma formation and/or squamouscell carcinomas, as classified by Goldschmidt et al.1998.18 All animals in the study were sourced fromcaptive populations of WBBs held at either KanyanaWildlife Rehabilitation Center, Gooseberry Hill, West-ern Australia; Dryandra Field Breeding Center, nearNarrogin, Western Australia; the Peron Captive Breed-ing Center, Shark Bay, Western Australia; or theAdelaide and Monarto Zoological Gardens, SouthAustralia.

Submissions

Submissions consisted of either surgical biopsiescollected into 10% neutral buffered formalin or entiredeceased WBBs for postmortem examination. Entireanimals submitted for postmortem were subjected toa complete postmortem examination; tissue sampleswere collected from skin lesions and all the major organsystems into 10% neutral buffered formalin. Samplesfrom skin lesions were also trimmed to ,3 mm inthickness and immersed in 5% glutaraldehyde for thepurpose of transmission electron microscopy. Surgicalbiopsies and postmortem samples were formalin-fixedfor at least 24 hours before processing for routine lightmicroscopy.

Clinical records

Clinical records relating to skin disease onset andprogression in cohort individuals were sourced from theaforementioned captive facilities as well as VetPathDiagnostic Laboratory Western Australia, the WesternAustralian Department of Agriculture and Food, andthe Western Australian Department of Environmentand Conservation.

Sample processing and evaluation

Tissues fixed in 10%neutral buffered formalin, collectedas described above, were routinely processed for exami-nation by light microscopy.4 After fixation for a minimumof 24 hours, tissues were trimmed to 5-mm-thick sectionsand placed into labeled cassettes. Sections were dehy-drated through graded ethanol concentrations, clearedwith xylene, and embedded with paraffin wax in a LeicaEG 1150C automated processor (Leica Microsystems,Nussloch, Germany). Tissue blocks were sectioned at5 mmusing a Leica 2135microtome (LeicaMicrosystems).Slides were stained with Harris’s hematoxylin and 1%eosin (HE), dehydrated, cleared, and mounted witha cover-slip using dibutyl phthalate pix xylene (DPX).

For ultrastructural examination using transmissionelectron microscopy, tissue sections were immersed inglutaraldehyde solution (5% vol/vol) for at least24 hours prior to processing. For tissues fixed in 10%neutral buffered formalin, the formalin was removedand the tissue resuspended in Sorenson’s buffer for

a maximum of 30 minutes, following which the bufferwas removed and discarded. This process was repeated 3to 4 times. Tissues were then placed in glutaraldehydesolution (5% vol/vol). Tissues were postfixed in Dalton’schrome osmic acid (aqueous osmium tetroxide, potas-sium dichromate, aqueous sodium chloride), dehydratedin sequential ethanol concentrations, followed by treat-ment with propylene oxide and embedded in TAAB 812epoxy resin. Sections were cut to 1 mm in thickness usinga Reichart OM U3 ultramicrotome (Reichart, Vienna,Austria) and stained with 1% toluidine blue for exami-nation by light microscopy to select appropriate resin-embedded tissue blocks for transmission electron micros-copy. Ultrathin (90 nm) sections from selected blockswere cut onto grids, stained with lead citrate (0.1–0.4%wt/vol) and uranyl acetate (2% saturated aqueous wt/vol,pH 4.5), and examined using a transmission electronmicroscope (Philips CM100 BioTwin transmission elec-tron microscope, Eindhoven, The Netherlands). Tissuesections were then photographed (Kodak electronmicroscope film 4489, 6.5 3 9 cm, New Haven, CT).

Indirect immunohistochemistry using a polyclonalantibody recognizing conserved papillomavirus capsidantigens (rabbit anti-bovine papillomavirus [BPV-1],B0580, Dako Corporation, Carpinteria, CA)29 wasperformed on 5-mm paraffin sections according toa modified manufacturer’s protocol. Sections froma known papillomavirus-infected canine oral papillomawere used as a positive control. In brief, 5-mm paraffinsections were deparaffinized in xylene. Sections were thensubjected to antigen retrieval. The sections were placed ina pH 9 Tris-EDTA-sucrose (TES) buffer solution andmicrowaved twice on the reheat setting and twice on thelow setting: each cycle was for 4 minutes. The sectionswere then cooled by a gradual addition of de-ionizedwater. Following cooling, the sections were treated with3% hydrogen peroxide for 5 minutes then washed withdistilled water followed by pH 7.8 Tris/HCl-bufferedwashing solution. Sections were ringed with a wax penciland treated with serum-free protein block (Dako Corp.,X0909) for 10 minutes. Protein block was poured fromthe test slides without washing, and the primary antibodydiluted 1 : 600 with Dako or Chemicon (Temecula, CA)antibody diluent was applied for 10 minutes. Thenegative control slides remained in the protein block forthis 10-minute period. The slides were then washed inpH 7.8 Tris/HCl-buffered washing solution, and thentreated with anti-rabbit antibody (ENVISION+, K4003,Dako Corp.) for 30 minutes. Following this step, theywere treated with DAB+ substrate-chromogen system(K3468, Dako Corp.) solution for 3.5 minutes, changingthe solution twice. Slides were counterstained withHarris’s hematoxylin, dehydrated, cleared, and mountedwith a cover-slip using DPX.

Results

Disease onset, duration, and age affected

The study cohort comprised 15 affected malesand 25 affected females; the sex of 2 individuals was

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not recorded. The average age of onset of lesionswas 3 years and 2 months of age (median, 3 years;range, 1 year to 5 years and 9 months). The timeelapsed between the onset of skin lesions andeuthanasia or death due to other causes averaged1 year and 4.5 months (median, 1 year; range,2 weeks to 4 years and 6 months). The averageage at death of WBBs in the cohort was 4 years and6 months (median, 4 years and 9 months; range,2 years to 6 years and 6 months). The maximumlife expectancy of WBB in captivity is estimated at6 years of age, whereas in wild populations it isestimated at only 4 years of age (J. Butcher and N.Thomas, personal communication.) The age of 2individuals was unknown as these animals werecaptured in the wild as adults.

Gross morphologic appearance and lesion distribution

The cutaneous and/or muco-cutaneous lesions inaffected animals were variable in size and eithersolitary or multicentric. They ranged from regionsof alopecia and small (1–2 mm in diameter andheight) nodular papillomatous thickenings to largeexophytic masses (.53 53 5 mm) often with anexudative crust or ulceration. Sites at which lesionswere observed included skin of the digits (64% ofstudy cohort); haired skin of the neck, thorax, andabdomen (55%); the periaural region (52%); andmuco-cutaneous junctions of the lips (64%),conjunctiva (50%), and pouch/inguinal region(26%) (Figs. 1, 2). Within the cohort, 7 (16.7%)animals displayed a lesion at a single anatomiclocation only. The remaining 35 (83.3%) affectedWBBs displayed lesions with a multicentric distri-bution.

Histopathology of skin lesions

One hundred and seventeen skin and/or muco-cutaneous lesions were examined by light micros-copy. Of these, 58 were classified as epithelialhyperplasia (49.6%), 30 as carcinoma in situ(25.6%), and 28 as squamous cell carcinoma(SCC) (23.9%). Adenocarcinoma of the pouchwas recorded in one WBB.There were 35 WBBs in the study cohort with

multicentric lesions. Purely multicentric hyperplas-tic epithelial lesions were seen in 11 WBBs (31.4%),entirely malignant lesions were seen in 9 WBBs(25.7%), and both malignant and benign lesionswere seen concurrently in 15 WBBs (42.8%).Solitary lesions were seen in 7 animals andconsisted of either epithelial hyperplasia (4/7) orSCC (3/7); they were seen on the oral mucosa (3/7),the digital skin (3/7), or the haired skin of the body(1/7).

Thirty WBBs displayed 1 or more lesionsclassified as epithelial hyperplasia (n 5 58). Theselesions were characterized by mild, moderate, ormarked irregular hyperplasia of the stratifyingepithelium. The hyperplasia was attributable to anincrease in the number of suprabasilar epithelial cellsat mucosal sites and an increase in the number ofkeratinocytes within both the stratum spinosum andstratum granulosum of the epidermis. Hyperplasiaof the outer root sheath of hair follicles, and theglandular elements of sebaceous and sweat glands,was also seen. Koilocytes and epithelial cells withmarginated nuclear chromatin and large amphophi-lic inclusions (Fig. 3A) were evident in 8 cases withhyperplastic conjunctival lesions (Table 1).

Lesions categorized as carcinoma in situ (n5 30)were seen in 17 individuals. These lesions showedan expansion of the stratum spinosum and stratumgranulosum and mild to moderate dysplasticchanges in keratinocyte morphology and epidermalarchitecture, such as disturbed polarity of kerati-nocyte differentiation and suprabasilar mitoses.Individual cellular characteristics included hyper-chromasia of nuclei, prominent nucleoli, clear orvacuolated cytoplasm, and mild anisocytosis andanisokaryosis. In 1 conjunctival lesion classified ascarcinoma in situ, the nuclei of some keratinocytesin the stratum granulosum displayed marginationof nuclear chromatin and large amphophilic inclu-sions.

Lesions categorized as SCC (n 5 28) werelocated at cutaneous and muco-cutaneous sites in20 individuals (Fig. 4). Histologically, the carcino-mas consisted of invasive cords, islands, andtrabeculae of neoplastic epithelial cells showingvarying degrees of differentiation. In well-differen-tiated tumors, there was formation of keratin‘‘pearls’’ circumscribed by neoplastic keratinocytes.The neoplastic epithelium extended into the dermis,with or without association with the overlyingepidermis. The neoplastic cells themselves showedabundant eosinophilic cytoplasm with distinct cellborders, intercellular desmosomes, and varyingdegrees of anisocytosis and anisokaryosis. Nucleiwere often large, ovoid, and vesicular with prom-inent nucleoli. The squamous cell carcinomas werelocally aggressive, invading into the surroundingdermis, subcutis, and even bone (n 5 1). In-flammatory infiltrates were frequently observedwithin SCCs or in the surrounding subcutis,comprising neutrophilic, histiocytic, lymphoplas-macytic, or mixed cellular populations. In 3animals in this study with cutaneous and muco-cutaneous SCC, metastases were observed ina lymph node (n 5 1) and the lungs (n 5 2).

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Fig. 1. Skin of the eyelids, lips, forelimbs, and digits; Western barred bandicoot. Demonstrates nodular andirregular papillomatous thickening.

Fig. 2. Ventral abdomen; Western barred bandicoot. Irregular papillomatous thickening of the dorsal skin ofthe hind digits and muco-cutaneous junctions of the pouch and cloacal opening. A large exophytic papillomatousmass is seen arising from the muco-cutaneous junction of the pouch.

Fig. 3. Conjunctival epithelium; Western barred bandicoot. Fig. 3A. Irregular hyperplasia of the mucosalepithelium. Multiple keratinocytes demonstrate margination of nuclear chromatin and central glassy amphophilicinclusions. Koilocytes are also evident. HE. Bar5 100 mm. Fig. 3B. Serial section of Fig. 3A. Immunohistochemicaldemonstration of papillomavirus structural antigens by use of rabbit polyclonal antibody developed from bovine

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The distribution and histologic classification oflesions according to their anatomic site is detailedin Table 1. SCCs were seen most commonly overthe haired skin of the thorax and abdomen and inthe periaural region. Adenocarcinoma of the pouchmammary glands was observed in 1 female affectedby papillomatous and carcinomatous lesions atother anatomic sites. Conjunctival lesions wereleast frequently observed to undergo malignanttransformation, with only 2/21 lesions designatedcarcinoma in situ and no cases of SCC observed atthis site. However, these lesions exclusively dis-played hyperplastic epithelium (n 5 8) or carcino-ma in situ (n 5 1) in which keratinocytesdemonstrated nuclear chromatin margination andamphophilic intranuclear inclusions.

Ultrastructural examination of skin lesions

Ultrathin sections from a range of hyperplasticand malignant lesions from 11 WBBs wereexamined by transmission electron microscopy.Two hyperplastic conjunctival lesions from 2individuals that displayed keratinocyte intranuclearinclusions visible on the toluidine-blue-stainedslides displayed a crystalline array of sphericalelectron dense particles, approximately 45 nm indiameter (Fig. 5), within keratinocyte nuclei. Themorphology and size of these particles were

consistent with that of a papillomavirus or a poly-omavirus.

Immunohistochemistry

Indirect immunohistochemistry using a polyclon-al antibody against BPV-1 (Dako Corporation)was performed on skin lesions from 14 individuals.Positive staining was observed in conjunctivalepithelial lesions from 3 WBBs in which keratino-cytes displayed margination of nuclear chromatinand large glassy amphophilic inclusions (Fig. 3B).

Discussion

This report describes the pathologic features ofa recently recognized papillomatosis and carcino-matosis syndrome in 42 WBBs. The spectrum ofcutaneous and muco-cutaneous lesions recordedresembles the stepwise progression of epitheliafrom premalignant stages into invasive squamouscell carcinomas. A single case of adenocarcinomaof the pouch mucosa was observed; however, thesignificance of this tumor in relation to the other-wise exclusively squamous carcinomatosis syn-drome is still unclear. This disease occurs in adultindividuals, lesions increase in size with age, andmalignant transformation occurs in those lesions.53 53 5 mm. The discernable stages of lesiondevelopment and the progression in lesion size and

r

papillomavirus type 1 (BPV-1). Hematoxylin counterstain. Bar 5 100 mm.Fig. 4. Cutaneous squamous cell carcinoma; Western barred bandicoot. Islands of neoplastic epithelial cells

containing central cores of keratin invade the dermis. Cells are closely apposed with intercellular desmosomes visibleand have abundant eosinophilic cytoplasm and basophilic staining nuclei with prominent and often multiplenucleoli. There is moderate to marked anisokaryosis and anisocytosis and mitotic figures are observed frequently. Amild neutrophilic infiltrate is seen in the subcutis and neoplastic epithelium. HE. Bar 5 200 mm.

Table 1. Distribution and histopathological classification of cutaneous and mucosal lesions seen in the Westernbarred bandicoot (WBB).

Lesion SiteNo. Lesions

Examined at Site Hyperplasia

Hyperplasia +IntranuclearInclusions

CarcinomaIn Situ

Squamous CellCarcinoma

Adenocar-cinoma

ConjunctivalMuco-cutaneousJunction

21 (WBB 5 18) 11 (52.4%) 8 (38.1%) 2 (9.5%) 0 (0.0%) 0 (0.0%)

Oral Muco-cutaneousJunction

23 (WBB 5 21) 12 (52.2%) 0 (0.0%) 5 (21.7%) 6 (26.9%) 0 (0.0%)

Periaural Skin 17 (WBB 5 16) 5 (29.4%) 0 (0.0%) 4 (23.5%) 8 (47.1%) 0 (0.0%)Digital Skin 21 (WBB 5 18) 10 (47.7%) 0 (0.0%) 6 (28.6%) 5 (23.7%) 0 (0.0%)Haired Skin

(thorax/abdomen)25 (WBB 5 18) 7 (28.0%) 0 (0.0%) 10 (40.0%) 8 (32.0%) 0 (0.0%)

Pouch/Inguinal Region 10 (WBB 5 10) 5 (50.0%) 0 (0.0%) 3 (30.0%) 1 (10.0%) 1 (10.0%)Total 117 (WBB 5 42) 50 (36.0%) 8 (6.8%) 30 (25.6%) 28 (23.9%) 1 (0.9%)

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malignancy seen with increasing age is consistentwith a multistage model of carcinogenesis, reflect-ing the acquisition of genetic alterations by tumorcells driving progressive transformation into highlymalignant derivatives.21,53

The development of malignancy involves com-plex interactions between several factors, bothexogenous (environmental) and endogenous (ge-netic, hormonal, immunologic).53 Certain virusesare known to play key roles in carcinogenesis inhumans and various animal species.23 Gross andhistopathologic features of the skin and mucosallesions combined with the ultrastructural andimmunohistochemical features of conjunctival le-sions in affected WBBs in this study are supportiveof the involvement of either a papillomavirus, orless probably a polyomavirus, in the pathogenesisof this disease. In addition, the observation of skinlesion development in seemingly healthy, wildWBBs introduced into captive colonies of WBBsaffected with the skin lesions provides furthersupport that the syndrome may be caused by aninfectious agent. Fight wounds and incidental

trauma including loss of tails, ears, and eyes havebeen observed by the authors in both captive andwild WBBs. These incidents may facilitate the entryof an epitheliotropic agent such as a papillomavirusor polyomavirus, and thus it must be consideredthat a multicentric lesion distribution may berelated to sites of epithelial trauma. Alternatively,the multicentric distribution of lesions may havearisen secondary to autoinoculation or perhapsviremia as has been alluded to in other speciesinfected with papilloma- or polyomaviruses.2,3,6,17,40

In general, papillomaviruses are species specificand induce excessive irregular proliferation ofcutaneous and mucosal epithelia in both humansand a range of animal species.46,54 Over 100papillomavirus types have been completely charac-terized in humans alone.16 Cutaneous papilloma-tosis is not always associated with papillomavirusinfection, however, with a range of infectious aswell as noninfectious agents also recognized toinduce proliferative and papillomatous lesions inanimal species.46

Despite often appearing early in the course ofdisease (author observation, L. Woolford) and anincrease in lesion size seen clinically, conjunctivallesions were least frequently observed to undergomalignant transformation. In addition, all lesionsthat demonstrated ultrastructural (n 5 2) orimmunohistochemical evidence (n 5 3) of papillo-mavirus virions were derived from the conjunctivaonly. Nonconjunctival lesions examined in thisstudy may have lacked ultrastructural and immu-nohistochemical evidence for papillomavirus in-fection due to being intermittently infected, a con-dition described in papillomavirus-induced lesionsin other species.44 Alternatively, nonconjunctivallesions may have lacked evidence of virions ifsampled early in the course of development.Experimental infections in other species haveshown that lesions do not express the capsidantigen (L1) until 7 weeks or more after infection.Consequently, early proliferative lesions oftenprove negative by immunohistochemistry for L1capsid antigens and do not contain viral particleson ultrastructural examination.32 Furthermore,epithelia undergoing malignant transformation inassociation with papillomavirus infection may benegative for structural antigens by immunohisto-chemistry and be unremarkable on ultrastructuralexamination if viral DNA has become integratedinto the host genome, leading to a nonproductiveinfection. Integration of papillomavirus DNA intohost genomic DNA has been well documented inmalignant lesions associated with high risk humanpapillomavirus infections.12,24,26,27,36

Fig. 5. Conjunctival keratinocyte nuclei; Westernbarred bandicoot. Crystalline array of spherical electrondense virus-like particles ,45 nm in diameter seenwithin hyperplastic keratinocyte nuclei. Bar 5 500 nm.

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The development of malignancy involves a com-plex interaction between both exogenous andendogenous factors. Indeed the outcome of a pap-illomavirus infection and the role of the virus incarcinogenesis may be influenced by factors such asthe virulence of the particular papillomavirustype,8,43,45,54 compromise of the host immunesystem,7,33,34 host genetics,1,9,20,30,51 and the presenceof various chemical cofactors.10,25,38 Immunodefi-ciency and/or immunocompromise in the WBB areconsidered as potential risk factors for develop-ment of this disease. Assessment of immunocom-petence in the WBB and the factors by which itmay be influenced are being investigated concur-rently by the authors. An increased incidence ofpapillomavirus-related disease is being reported inother endangered animal populations6,28 withdecreases in genetic diversity a proposed cause ofincreased susceptibility to disease agents in someendangered species.47 In attempts to elucidate thepathogenesis of this disease in the WBB, it must beconsidered that a complex interaction between viraland other factors may be driving the developmentof malignant epithelial tumors.Immunohistochemical staining of WBB conjunc-

tival lesions in this study led us to consider theinvolvement of a papillomavirus in the pathogen-esis of this disease. However, we did not feel wecould rule out the involvement of a polyomaviruson the basis of immunohistochemical results alone.Significant antigen retrieval techniques were re-quired to demonstrate papillomavirus structuralantigens, which in the authors’ experience have notbeen required for papillomavirus-associated epi-thelial lesions from other species using an otherwiseidentical technique. In addition, gross, histologic,and ultrastructural features of lesions did notdiscount a polyomavirus as an alternative etiologicagent. The mammalian polyomaviruses displaya narrow host range and do not productively infectother species, typically causing apathogenic sub-clinical natural infections in immunocompetenthosts. These viruses may cause severe disease inthe immunocompromised host or can cause tumorformation when they are introduced into anunnatural host.11,31 The hyperplastic changes seenaffecting the root sheath epithelium in WBB lesionsfrom areas of haired skin was reminiscent of theappearance of polyomavirus-associated tricho-epitheliomas seen in hamsters.19 These tumors ofhamsters occur primarily on the skin of the head,chin, neck, back, and frequently around the eyesand external ears; histologically they demonstrateproliferation of the hair root epithelium, formingcyst-like masses filled with cornified material.39

Definitive evidence to support or refute thepresence of a specific etiologic agent in thissyndrome is also lacking at this stage of investiga-tions as nonconjunctival epithelial lesions did notdemonstrate intranuclear inclusion bodies norultrastructural or immunohistochemical evidenceof viral infection.

The papillomatosis and carcinomatosis syn-drome observed in the Western barred bandicootis a novel and emerging disease in this species and,to the authors’ knowledge, is unlike skin diseases ofAustralian marsupials previously documented. Nospecific skin diseases of Australian bandicootspecies have been documented to date; however,a conjunctivitis syndrome has been seen in captiveand wild WBBs associated with novel Chlamydialessp. isolated from the conjunctiva.5,52 Papillomatouslesions have been described in a common brushtailpossum (Trichosurus vulpeca) in association witha papillomavirus infection, this being the onlypapillomavirus-associated lesions documented inan Australian marsupial to the authors’ knowl-edge.35 Papillomatous lesions have also beenrecorded in a North American opossum (Didelphisvirginiana) associated with a papillomavirus.46

Captive breeding and reintroduction programsform the basis of many animal species conservationefforts, and vigilance must be maintained for theemergence of new diseases: these form a majorthreat to biodiversity and have been shown toundermine the success of breeding and reintroduc-tion programs.13–15,50 The papillomatosis and car-cinomatosis syndrome in captive and wild WBBs isa pertinent example of disease threatening thesurvival of a species. Future investigations into thepapillomatosis and carcinomatosis syndrome of theWestern barred bandicoot will involve pursuingdetection of an etiologic or contributing viral agentusing molecular techniques, studying the trans-missibility of the disease, and examining thepatterns of disease in wild and captive populationsto enable further elucidation of the pathogenesis ofthis novel and debilitating disease.

Acknowledgements

We thank Gerald Spoelstra of the Murdoch Univer-sity histopathology laboratory for his skillful productionof histology slides, and James Poynton for his technicalassistance in the Murdoch University necropsy suite. Weare grateful to June Butcher and the volunteers atKanyana Wildlife Rehabilitation Center for their careand monitoring of the Western barred bandicootshoused there, and the Denham and Narrogin De-partment of Environment and Conservation WesternAustralia (DEC) officers for the care and monitoring of

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Western barred bandicoots in the Peron and Dryandracaptive breeding facilities, respectively. This project isfunded by the Australian Research Council in partner-ship with Murdoch University and the Western Austra-lian Department of Environment and Conservation(DEC) under Linkage Project LP0455050. Lucy Wool-ford is funded by the Lorna Edith Murdoch VeterinaryTrust Scholarship.

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Request reprints from Dr. L. Woolford, Department of Veterinary Pathology, School of Veterinary and BiomedicalSciences, Murdoch University, Perth, WA 6150 (Australia). E-mail: l.woolford@murdoch.edu.au.

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