Journal of Wildlife Diseases, 47(4), 2011, pp. 1019–1025# Wildlife Disease Association 2011

Balanoposthitis, Dyspareunia, and Treponema in the Critically

Endangered Gilbert’s Potoroo (Potorous gilbertii)

Rebecca Vaughan-Higgins,1,2,5 Nicky Buller,3 J. Anthony Friend,4 Ian Robertson,2 Cree L. Monaghan,1,2

Stan Fenwick,2 and Kristin Warren2 1Veterinary Department, Perth Zoo, South Perth, 6151, Western Australia;2Conservation Medicine Program, School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch,6150, Western Australia; 3Animal Health Laboratories, Department of Agriculture and Food, Western Australia, SouthPerth, 6151, Western Australia; 4Department of Environment and Conservation, Albany, 6330, Western Australia;5Corresponding author (email: rebecca.vaughan-higgins@ioz.ac.uk)

ABSTRACT: The Gilbert’s potoroo (Potorousgilbertii) is one of Australia’s most criticallyendangered mammals with a current esti-mated population of 70 individuals. Both thewild and captive populations have a long his-tory of balanoposthitis with associated crusting,ulceration, and preputial discharge. We soughtto identify the microbial species found in thedischarge, determine their significance in caus-ing balanoposthitis, and correlate these findingswith reproductive success and survivorship.Bacteriologic examination revealed the dis-charge to be a polymicrobial infection involvingTreponema spp., Actinobacillus spp., and Pas-teurella spp. Preputial histopathology reporteda moderate, chronic, erosive inflammatoryresponse with diffuse, moderate to markedsecondary epithelial hyperplasia in conjunc-tion with moderate numbers of spirochetes,suggesting a causative relationship. Clinicalexamination, preputial biopsies, and serologicscreening found no evidence of associatedsystemic disease. The clinical investigation ofTreponema is significant with respect to theoverall recovery of Gilbert’s potoroo, giventhe clinical and histopathologic similaritiesto Treponema paraluis-cuniculi found in rab-bits, causing dyspareunia, and the severity ofthe associated balanoposthitis.

Key words: Balanoposthitis, conservationmedicine, dyspareunia, marsupials, Trepone-ma infection, urogenital disease.

A small colony of the presumed-extinctGilbert’s potoroo (Potorous gilbertii) wasdiscovered in 1994 at Two Peoples BayNature Reserve in the southwest of West-ern Australia (34u53.99S, 118u13.39E). Arecovery plan was immediately preparedto establish a captive breeding program forthe animals in order to establish newpopulations (Courtenay and Friend,2004). The Gilbert’s potoroo is classifiedas Critically Endangered (IUCN, 2011). Agenetic bottleneck, consistent with demo-graphic decline, highlights the concern

for the long-term survival of this species(Sinclair and Westermann, 1997).

A collaborative health and diseasestudy was undertaken from March 2005to March 2008 to determine the preva-lence of specific diseases in both the wildand captive populations and to correlatethe effects of identified diseases onpopulation dynamics. Results indicatedthe disease of greatest significance wasbalanoposthitis, associated with a crustygreen tenacious preputial discharge. Wesought to identify the microbial speciesfound in the discharge, determine theirsignificance in causing balanoposthitis,and correlate these findings with repro-ductive success and survivorship. In 2001,a Treponema-like organism associatedwith a mixed bacterial infection wasidentified in a captive Gilbert’s potoroowith balanoposthitis (A.-M. Horwitz andD. Forshaw, pers. com.). A clinicalcorrelation with venereal spirochetosiscaused by Treponema paraluis-cuniculiin rabbits was later noted. In rabbits,venereal spirochetosis presents with pre-putial lesions, beginning as areas ofhyperemia and edema, with the develop-ment of papules and vesicles followed byulceration, crustiness, and hyperkeratosis(Cunliffe-Beamer and Fox, 1981a). Thelesions are thought to be painful and toimpede breeding activity through dyspa-reunia, (Cunliffe-Beamer and Fox,1981a). Colony outbreaks have resultedin decreased rates of conception andincreased rates of metritis, placenta re-tention, infertility, and neonatal deaths(Saito et al., 2003). Defining the relation-ship between Treponema and balanopos-thitis in the Gilbert’s potoroo, if any, may

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be important for the health managementof this critically endangered species.

Wild and captive potoroos were rou-tinely trapped three times per year overthe study period (Vaughan et al., 2009). Acomplete health assessment was carriedout under isoflurane gaseous anesthesia.When a male potoroo was anaesthetized,the penis was exteriorized from its prepu-tial sheath and observed for exudates,swelling, and erythema. The level andnature of discharge was assigned a grade(0 [no discharge] to 3+ [severe discharge];Fig. 1A, B, C).

In females, it was difficult to qualify thenature of discharge due to their urogeni-tal anatomy, so females were assigned ashaving no discharge or discharge, thenature of which was described. A dryswab was taken from the prepuce of malesand from the urogenital sinus of femalesfor Treponema DNA amplification viaPCR. An Amie’s transport medium swab(TranswabH, Medical Wire and Equip-ment, Wiltshire, England) was taken formicrobiologic examination. A wet prepa-ration was made from this second swab forexamination for spirochetes by dark fieldmicroscopy. Both swabs were forwardedon ice (approximately 4 C) to the Depart-ment of Agriculture and Food WesternAustralia, South Perth, Australia for mi-crobiologic analysis and PCR. Samples wereanalyzed within 24 hr of collection. ATreponema sp. was detected under darkfield microscopy. Morphologically, theywere differentiated from Brachyspira bytheir eight tight spirals. Blood was collectedfor hematology, biochemistry, and bloodsmear examination as described by Vaughanet al. (2009).

In the final trapping session in March2008, four preputial biopsies and Trepo-nema serology from 15 individuals wereundertaken (Vaughan, 2008). We testedserum from known Treponema-infectedpotoroos and known Treponema-negativepotoroos. Infection status was based onPCR results and was undertaken to addfurther validity to the tests if similar

FIGURE 1. Grading scheme for balanoposthitis inGilbert’s potoroos (Potorous gilbertii) from WesternAustralia. (A) Mildly inflamed cloaca, mildly in-flamed prepuce, and minor nonodorous dischargewith an adherent crust were observed with 1+balanoposthitis. (B) moderately inflamed cloaca,moderately inflamed prepuce, with moderate associ-ated odor and moderate discharge, with a denseconsistency and adherent crust was observed with 2+balanoposthitis. (C) Severely inflamed cloaca, se-verely inflamed prepuce, with moderate associatedodor, and profuse fluorescent green or creamydischarge with an adherent crust, was observed with3+ balanoposthitis.

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results were seen. These results were alsocross-referenced with evidence of spir-ochetes detected on dark field microscopy.

Preputial biopsies and histopathologywere conducted under general anesthesiaon four captive potoroos, two with pre-putial discharge and two without. A smallpunch biopsy, 0.3 3 0.3 cm, of mucosaltissue was obtained at a tissue depth of0.2 cm. The edges of the biopsy site wereapposed with an absorbable suture. Thetissue biopsy was placed in formalin andsent to Murdoch University for histolo-gic examination and silver staining forspirochetes.

Prior sequencing attempts of a clinicalcase in a Gilbert’s potoroo in 2002 hadrevealed similarities to Treponema medi-um subsp. bovis, Treponema mediumgenomic RNA for 16S ribosomal RNA,and Treponema sp. oral clone JUO31using GenBank. Therefore, we choseprimers following a review of papersidentifying spirochetes associated withbovine digital dermatitis (Rijpkema et al.,1997; Table 1). Five primer combinationswere trialed (Table 2). Significant productwas only found using SPF5 and SPR4.When the resulting 190-base pair ampli-fied product sequence was subjected to anucleotide-nucleotide gene search withthe basic local alignment search tool, theclosest similarity to any bacterium was a92% match with an uncultured Trepone-ma clone. The lack of high sequencesimilarity would suggest the identificationof a new Treponema species from theGilbert’s potoroo. This primer pair wasused for all subsequent screening forTreponema infection in Gilbert’s potoroos.

In total, 44 potoroos (24 males, 20females) were sampled from 2005–2008.This included recruitment and those lost tothe population through death or dispersal.We collected 100 urogenital swabs forculture and PCR analysis, including sam-ples from three captive potoroos that wereswabbed six times each. The period prev-alence of Treponema infection was calcu-lated by adding new infected cases to

existing cases. However, because twoinitially infected females cleared infection(demonstrated through PCR), these indi-viduals were no longer classed as infectedand the true period prevalence was cor-rected to 30/44 (68%). No males clearedinfection over the study period.

In males, typical lesions included severepreputial and cloacal inflammation withulceration and associated tenacious greendischarge (Fig. 1B, C). In many cases, theseverity of the cloacal inflammation madeextrusion of the penis physically difficult.Preputial histopathology revealed a mod-erate chronic erosive inflammatory re-sponse with diffuse, moderate to markedsecondary epithelial hyperplasia in con-junction with moderate numbers of spir-ochetes, suggesting a causative relation-ship. The silver-stained spirochetes, 2–4 mm long, were diffusely scattered overthe mucosal surface and extended into thelamina propria in association with amoderate accumulation of inflammatorycells, predominantly macrophages, withlesser lymphocytes and plasma cells(Fig. 2). Culture yielded a heavy, mixedgrowth of the Actinobacillus-Pasteurellagroup and Escherichia coli. A secondbiopsy showed similar results in conjunc-tion with a low to moderate number ofneutrophils forming small micro-abscesseswithin the epithelium. No evidence ofspirochetes or histopathologic lesionswas found in the two potoroos withoutdischarge.

The presence of discharge was associ-ated with Treponema infection. Gilbert’spotoroos with Treponema infection were26.9 times more likely to have dischargethan those not infected with Treponema(x2587.98, df51, P50.00, odds ratio(OR)526.9, 95% CI58.6–83.5; Table 3).Actinobacillus and Pasteurella were culturedfrom all preputial biopsies and ORs fromurogenital swab cultures confirmed that theTreponema-infected population was morelikely to have an associated Actinobacillus(5.8 times) and Pasteurella spp. infection(3.3 times) than the noninfected Treponema

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population. Of these three microbes, Trep-onema was most positively associated withdischarge. However, those without Trepo-nema infection with Actinobacillus infection(OR525.7, 95% CI57.3–90.1) and Pasteu-rella infection (OR519.7, 95% CI56.2–63.1) still presented with discharge. Whenwe cross-referenced these PCR results withdark field microscopy results, spirocheteswere found, indicating these five individualslikely had false-negative PCR results. Thecourier service for these five samples wasdelayed, and this may have resulted inthawing and loss of genetic material.

A generalized trend of higher percentag-es of moderate (2+) and severe (3+) clinicalsigns and levels of discharge was observedin males with Treponema infection. Infemales, no cloacal inflammation was evi-dent and Treponema-infected females ei-ther showed no discharge or had discharge.Correspondingly, a significant relationshipbetween the level of discharge and genderwas found (x2562.53, df53, P50.00).Males with Treponema infection were 26.9times (95% CI59.2–78.3) more likely to

have discharge than did females withTreponema infection.

We were unable to culture the trepo-neme identified in the Gilbert’s potoroo,despite using selective enrichment media,and no antibody response was detected inany of the 15 samples submitted for therapid plasmid reagin total plasmid particleagglutination and the Treponema pallidumtotal antibody test (Saito et al., 2003), eventhough eight of these samples were PCR-positive for Treponema infection. Whencomparing the results of the hematologyand biochemistry of the Treponema-infect-ed to the non–Treponema-infected popu-lation, differences were minimal and werenot thought to be clinically significant(Vaughan et al., 2009). Clinically, therewere no statistically significant changes in

TABLE 1. Primers used in polymerase chain reaction for detection of a spirochetal 16S rRNA gene inGilbert’s potoroos (Potorous gilbertii) sampled in Western Australia, 2005–2008.

Primer name Location Specificity Sequence

EUBB 7–26 Bacteria 59-AGAGTTGATCMTGGCTCAGSPF1 49–68 Spirochetes 59-TAAGCATGCAAGTCGAGCGGGCSPF3 336–356 Spirochetes 59-TCCTACGGGAGGCAGCAGCTSPF4 833–854 Spirochetes 59-AGTGTACCGCCTGGGGACTATGCSPF5 168–183 Treponema 59-AATACCGAATACACTCAGTGCTTSPR1 859–835 Spirochetes 59-GCGGGCATACTCCCCAGGCGGYACASPR2 1202–1180 Spirochetes 59-TTGTAGCACGTGTGTAGCCCTGGASPR4 375–355 Treponema 59-CCATTGCGGAATATTCTTAGSPR5 987–965 Treponema 59-GTACGCTGCCATATCTCTAAGTC

TABLE 2. Primer combinations trialed for detectionof a spirochetal 16S rRNA gene in Gilbert’s potoroos(Potorous gilbertii) sampled in Western Australia,2005–2008.

Forward primer name Reverse primer name

EUBB SPR4EUBB SPR2SPF1 SPR4SPF1 SPR2SPF5 SPR2

FIGURE 2. Numerous, silver-stained spirochetespenetrating the penile epithelium of Gilbert’spotoroos (Potorous gilbertii) with balanoposthitisfrom Western Australia. Warthrin-Starry stain.1003. Bar5500 mm.

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body condition, rectal temperature, orpostanesthetic recovery in the Trepone-ma-infected compared to the non–Trepo-nema-infected population. These factscombined indicated that a localized inva-sion, rather than systemic disease, wasmore likely.

Three methods of diagnosis for Trepo-nema infection were compared (Table 3).A statistically significant relationship wasfound between the presence of dischargeand Treponema infection (x2545.27,df53, P50.00, OR526.9, 95% CI58.6–83.5); between PCR results and Trepone-ma infection (x2587.98, df51, P50.00,OR54.5, CI52.4–8.7); and between thepresence of spirochetes and Treponemainfection. All individuals with spirocheteshad Treponema infection. The threediagnostic techniques were analyzed forsensitivity and specificity; dark field mi-croscopy for spirochetes was the mostsensitive test and PCR was the mostspecific (Table 4).

While the disease does not cause severedebilitation, in males, the presence ofTreponema infection can be associatedwith severe balanoposthitis and severecloacal inflammation, often making extru-sion of the penis physically difficult. Theincreased prevalence of infection in malescould be explained by the fact that maleshave the ability to evert and retract theirgenitalia. This process could abrade mu-cosal barriers, creating an avenue formucosal colonization and commensalovergrowth which, in turn, promotes afavorable microenvironment for Trepone-ma growth.

Clinical signs were similar to those seenin rabbits where genital lesions contribut-ed to reduced reproductive capabilitiesthrough dyspareunia (Cunliffe-Beamerand Fox, 1981a, b). Furthermore, on histo-pathologic examination, the severe bala-noposthitis in two Treponema-infectedcaptive individuals was similar to thereported histopathology findings in rabbitswith Treponema paraluis-cuniculi. In bothinstances, the presence of spirochetes wasassociated with a chronic inflammatoryresponse, ulceration, and hyperkeratosis ofthe epithelium.

Overall, the presence of discharge inGilbert’s potoroos was associated with afacultative anaerobic polymicrobial environ-ment containing Treponema, Actinobacillus,and Pasteurella. This suggests that, likehuman periodontitis, enrichment for Trep-onema by preceding bacterial populationsand subsequent inflammatory events seemscrucial to the underlying pathology and alsosuggests that the presence of discharge,while often a precursor of infection, is not amethod of definitive diagnosis.

TABLE 3. Numbers of Treponema-infected and noninfected Gilbert’s potoroos (Potorous gilbertii) fromWestern Australia, assigned to each of four categories based on clinical signs.

Clinical signs and dischargea Total

Treponema status 0 1+ 2+ 3+Noninfected 31 0 5 0 36Infected 12 12 19 21 64

Total 43 12 24 21 100

a Level of discharge was scored from 0 (no discharge) to 3+ (severe discharge; see Fig. 1A–C for explanation of gradingscheme).

TABLE 4. Sensitivity and specificity of threetechniques used in this study for diagnosingTreponema infection in Gilbert’s potoroos (Potorousgilbertii) from Western Australia.

Diagnostic test Sensitivity (%) Specificity (%)

Clinical signs andpresence ofdischarge

91.2 27.9

Dark field microscopyfor spirochetes

100 0

Polymerase chainreaction

95.3 100

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Treponema in the Gilbert’s potoroo isbest screened for by PCR in conjunctionwith dark field microscopy for spirochetesto minimize the number of false negatives.Dark field microscopy is a relativelysimple technique to conduct. However,due to the fastidious nature of trepo-nemes, it is recommended that slides areanalyzed within 24 hr of collection by anoperator experienced in detecting thespiral-shaped organism. PCR is the mostcostly of all techniques and requiresexperience and a dedicated laboratory toprocess samples; however, this techniquecan provide rapid, specific results forprerelease screening prior to transloca-tion. The level of discharge should also betaken into account. Histopathology andsilver staining provide definitive diagnosis;however, these are not practical methodsto screen wild individuals prior to translo-cation because of the invasiveness and thetime lag for results.

It is difficult to comment on thereproductive and health effects of Trepo-nema infection in the Gilbert’s potoroo, asboth the wild and captive populations areinfected and there is no control group forcomparison. Adult females with evidenceof Treponema infection still appear to bereproducing. However, it is possible thedisease is contributing to reduced fertilityand fecundity, reducing the ability of theGilbert’s potoroo population to recoverquickly. This novel disease warrants fur-ther attention due to the significantproportion of the population infected(68%), the close clinical correlations tothe rabbit model, and the potential forTreponema to alter the immune response.Currently, there are three populations ofGilbert’s potoroo: the original populationat Two Peoples Bay and two translocatedpopulations, one on Bald Island and onewithin a 380-ha enclosure on the main-land. Balanoposthitis is evident in all threepopulations. Further research should bedirected to characterizing transmissionand the development of practical treat-ment regimes. Penicillin G benzathine-

penicillin G procaine at 84,000 IU/kgintramuscular at 7-day intervals for threeinjections, as used in rabbits (Cunliffe-Beamer, and Fox, 1981c), has beentrialed in analogous long-nosed potoroosfor the treatment of nonclinical Trepone-ma infection. Preliminary results indicatethis treatment may be effective; however,a controlled trial in Gilbert’s potoroos iswarranted. Treatment could practicallybe undertaken in quarantine prior totranslocation to create a disease-freepopulation to allow comparisons of thisdisease on population dynamics and, inparticular, would allow the reproductiveeffects on fertility and fecundity to beassessed. Such a trial would aid in theunderstanding of this novel disease andits importance to the ongoing recoveryof the critically endangered Gilbert’spotoroo.

Funding for this study was providedby the Department of Environment andConservation, Murdoch University, theDepartment of Agriculture and FoodWestern Australia (DAFWA), and thePerth Zoo. We also acknowledge the workand advice of Stephanie Hill, Tim Button,and the Gilbert’s Potoroo Recovery Team.Thanks to the staff of the Perth ZooVeterinary Department and the AnimalHealth section, DAFWA, especially SamTai and Shane Besier, for their histopath-ologic knowledge.

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Submitted for publication 8 September 2010.Accepted 30 June 2011.

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