health evaluation of free-ranging eastern bettongs ( bettongia gaimardi ) during translocation for...

HEALTH EVALUATION OF FREE-RANGING EASTERN BETTONGS

(BETTONGIA GAIMARDI) DURING TRANSLOCATION FOR

REINTRODUCTION IN AUSTRALIA

Timothy Portas,1,8 Don Fletcher,2 David Spratt,3 Andrea Reiss,1 Peter Holz,1

Kathryn Stalder,4,5 Joanne Devlin,5 David Taylor,6 David Dobroszczyk,1 andAdrian D. Manning7

1 Veterinary and Research Centre, Tidbinbilla Nature Reserve, RMB 141, Via Tharwa, Australian Capital Territory 2620,Australia2 Conservation Research, Environment and Sustainable Development Directorate, Australian Capital TerritoryGovernment, PO Box 158, Australian Capital Territory 2601, Australia3 CSIRO Ecosystem Sciences, Australian National Wildlife Collection, GPO Box 1700, Canberra, Australian CapitalTerritory 2601, Australia4 Australian Wildlife Health Centre, Healesville Sanctuary, Badger Creek Road, Healesville, Victoria 3777, Australia5 Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia6 Vetnostics, 60 Waterloo Road, North Ryde, New South Wales 2113, Australia7 Fenner School of Environment and Society, Australian National University College of Medicine, Biology andEnvironment, Canberra, Australian Capital Territory 0200, Australia8 Corresponding author (email: [email protected])

ABSTRACT: Sixty (19 male, 41 female) free-ranging adult eastern bettongs (Bettongia gaimardi)were captured in Tasmania and translocated to the Australian Capital Territory between July 2011and September 2012 for reintroduction into fenced, predator-proof reserves. The bettongs wereanesthetized for physical examination and screened for selected diseases during translocation.Reference ranges for hematologic and biochemical parameters were determined. Two bettongshad detectable antibodies to the alphaherpesviruses macropodid herpesvirus 1 and macropodidherpesvirus 2 by serum neutralization assay. A novel gammaherpesvirus was detected, via PCR,from pooled swabs collected from the nasal, conjunctival, and urogenital tract mucosa of four otherbettongs. Sera from 59 bettongs were negative for antibodies to Toxoplasma gondii as assessed byboth the modified agglutination test and the direct agglutination test (n553) or by the modifiedagglutination test only (n56). Rectal swabs from 14 bettongs were submitted for bacterial cultureand all were negative for Salmonella serovars. Ectoparasites identified on the bettongs includedfleas (Pygiopsylla zethi, Stephanocircus harrisoni), a louse (Paraheterodoxous sp.), mites (Guntheriacf. pertinax, Haemolaelaps hatteni, a suspected protonymph of Thadeua sp., Cytostethumtasmaniense, Cytostethum intermedium, Cytostethum thetis, Cytostethum wallabia), and ticks(Ixodes cornuatus, Ixodes trichosuri, Ixodes tasmani). An intraerythrocytic organism morpho-logically consistent with a Theileria species was identified in blood smears from four bettongs.These data provide baseline health and disease information for free-ranging eastern bettongsthat can be used for the conservation management of both the source and translocatedpopulations.

Key words: Bettongia gaimardi, eastern bettong, health evaluation, herpesvirus, parasite,toxoplasmosis, translocation.

INTRODUCTION

The eastern bettong (Bettongia gai-mardi) is a small, nocturnal, predomi-nantly mycophagous, potoroid marsupialthat inhabits dry sclerophyllous forestswith open understories (Rose, 1986).Formerly eastern bettongs were widelydistributed across coastal and adjacentareas of southeastern Australia but be-came extinct on the mainland by the1920s (Short, 1998). Three of the four

extant Bettongia species have undergonedramatic range and population declinessince the European colonization of Aus-tralia. Extinction of eastern bettongs onthe mainland was likely the result of acombination of factors including theintroduction of eutherian predators suchas the red fox (Vulpes vulpes); habitatmodification by introduced herbivores;habitat fragmentation; persecution asagricultural pests; and altered fire re-gimes (Short, 1998).

DOI: 10.7589/2013-08-202 Journal of Wildlife Diseases, 50(2), 2014, pp. 210–223# Wildlife Disease Association 2014

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Eastern bettongs are now restricted tothe island of Tasmania with an estimatedpopulation of 60,000 individuals (Claridgeet al., 2007). Threats to the remainingTasmanian population include the recentintroduction and establishment of the redfox in Tasmania (Sarre et al., 2012), poorhabitat preservation, and potentially, in-fectious disease. Two novel Trypanosomaspecies and the protozoan parasite Toxo-plasma gondii have been implicated aspossible contributing factors in the recentabrupt and precipitous decline of theclosely related brush-tailed bettong (Bet-tongia penicillata) in Western Australia(Smith, Clark et al., 2008; Thompson etal., 2013), although the precise cause ofthe decline remains unknown (Wayne etal., 2013). To date the health status offree-ranging eastern bettongs has not beenevaluated. An understanding of the base-line health and disease status of easternbettongs is a prerequisite for understand-ing the success or failure of reintroductionattempts (Mathews et al., 2006).

As part of an ecosystem restorationexperiment (Manning et al., 2011; Short-house et al., 2012) and to establish aninsurance population against the threat ofextinction, free-ranging eastern bettongsfrom Tasmania were translocated andreintroduced into two, predator-free,fenced reserves, Tidbinbilla Nature Re-serve (TNR) and Mulligan’s Flats Wood-land Sanctuary (MFWS) within theirformer range in the Australian CapitalTerritory (ACT). Health assessment ofeastern bettongs was undertaken duringtranslocation to establish baseline healthand disease data for the species. Weprovide hematologic and serum biochem-istry reference ranges for free-rangingeastern bettongs obtained during translo-cation. We evaluate the health status ofeastern bettongs, detail the parasitespresent, and investigate the prevalence ofinfectious disease agents that may impactthe health of translocated bettongs orsympatric macropodoids at the reintro-duction sites.

MATERIALS AND METHODS

Trapping, sedation, and transportation

We live-trapped eastern bettongs July–October 2011 and April–September 2012 ateight locations in Tasmania (Fig. 1). Thesesites included a mix of remnant native forest,forestry plantations, and agricultural land.Bettongs were captured at night in paddedcage traps baited with a mixture of bread,peanut butter, and truffle oil. Traps were set atdusk and checked approximately 4 hr later.Bettongs were removed from traps, placed incloth bags, and assessed for suitability fortranslocation. Bettongs selected for transloca-tion were weighed and administered diazepam(Pamlin Injection, Parnell Australia Pty. Ltd.,Alexandria, Australia) 1 mg/kg intramuscularly(IM). For transportation, cloth bags containingindividual bettongs were suspended from theceiling and anchored to the sides and the baseof purpose-built wooden crates. Bettongs weretransported by road to one of two Tasmanianairports and freighted by air to the ACT (totaltransport times ranged 7–18 hr). A repeat doseof diazepam was administered prior to loadingfor air transportation (3–8 hr after the firstinjection). Sixty adult bettongs (19 male, 41female) were translocated. All bettongs un-derwent health evaluation at TNR immediate-ly following translocation and prior to releaseat either TNR or MFWS. Bettongs werereleased at TNR (n528) in 2011 and 2012following a 30-day quarantine. Bettongs werereleased at MFWS (n532) in 2012 only; thisincluded animals that were translocated in2011 and held at TNR (n516) until releaseand animals released directly at MFWSfollowing health evaluation (n516).

Anesthesia, sample and data collection,and analyses

On arrival at TNR, bettongs were anesthe-tized with isoflurane (I.S.O. Inhalation Anaes-thetic, Veterinary Companies of Australia Pty.Ltd., Kings Park, Australia) in oxygen deliv-ered via mask. All bettongs received acomplete physical examination under anesthe-sia; weight was determined to the nearestgram using an electronic scale; body conditionwas assessed on a scale of 1 to 4 (1 beingemaciated and 4 being ideal); tooth wear wasassessed as none, mild, moderate, or heavy;the presence of mites (via microscopic exam-ination of fur pluck and skin scrape samples),ticks, fleas, and lice was noted, and theectoparasite burden assessed as none, low,moderate, or heavy. Any physical abnormali-ties were noted and investigated as required.

PORTAS ET AL.—HEALTH EVALUATION OF BETTONGS 211

Up to 10 mL of blood were collected fromthe lateral coccygeal vein and placed intoethylenediaminetetraacetic acid (EDTA) andserum separator tubes (BD Vacutainer, Bec-ton, Dickinson and Company, Plymouth, UK).Serum tubes were allowed to clot for 1–4 hrand centrifuged. Sera for biochemical analysisand T. gondii serology were stored at 4 C untilprocessed. Sera for herpesvirus serology werestored at 220 C until processed. Blood smearswere made using fresh blood, stained with amodified Wright-Giemsa stain (Diff Quik, LabAids Pty. Ltd., Narrabeen, Australia), andexamined (200 nonconsecutive fields per slide)for hemoparasites at 4003 magnification usingoil immersion. Samples for detection ofherpesvirus DNA using PCR were collectedusing aluminum-shafted swabs (Sterile SwabApplicators, Copan Italia, Bressica, Italy) fromthe conjunctival, nasal, and urogenital tractmucosa and stored at 220 C until processed.Rectal swabs (n514) were collected usingsterile bacteriologic swabs (Sterile TransportSwabs, Interpath Service Pty. Ltd., Mel-bourne, Australia) and submitted for Salmo-nella culture. Representative samples of ecto-parasites were collected and stored in 70%ethanol until identification using publishedliterature (Roberts, 1970; von Keler, 1971;

Dunnet and Mardon, 1974; Fain and Dom-row, 1974a, b). Fecal samples were collectedfrom the trap or transport bag and assessedusing the sodium nitrate flotation techniquefor endoparasite ova. Bettongs translocated in2011 (n523) received ivermectin (IvomecAntiparasitic Injection for Cattle, MerialAustralia Pty. Ltd., Parramatta, Australia) at200 mg/kg subcutaneously.

In four bettongs with dermatitis, represen-tative sections of affected skin were collectedusing a 4-mm biopsy punch (DisposableBiopsy Punch, Kai Industries Co. Ltd., SekiCity, Japan). Biopsy sites were closed with anabsorbable suture (4-0 Coated Vicryl, EthiconInc., Somerville, New Jersey, USA), and asingle IM dose of a long-acting penicillinpreparation (Norocillin L. A. Injection, Nor-brook Laboratories Ltd., Corby, UK) contain-ing procaine penicillin G (15 mg/kg) andbenzathine penicillin G (11.25 mg/kg) wasadministered. Tissue was placed into 10%neutral buffered formalin, routinely processed,and embedded in paraffin. Sections were cutat 4–6 mm and stained with hematoxylin andeosin prior to histologic examination.

Hematologic and biochemical analyses andSalmonella culture were performed within24 hr of collection by Vetnostics, North Ryde,

FIGURE 1. Location of trapping sites (numbered circles) for free-ranging eastern bettongs (Bettongiagaimardi) selected for translocation in Tasmania, Australia, July 2011–September 2012. 1 5 Long Hill, 2 5

Pipers River, 3 5 Mt. Morrison, 4 5 Bangor, 5 5 The Lea, 6 5 Grove, 7 5 Castle Forbes Bay, 8 5

Geeveston. Five collecting regions were identified to ensure adequate genetic variation of the sourcepopulation; NW 5 North West, NE 5 North East, SE 5 South East, C 5 Central Southern, SW 5 SouthWest. Within the Central Southern region no bettong population outside conservation reserves wassufficiently abundant to meet collecting requirements.

212 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014

Australia. Hematologic analysis was performedon EDTA whole blood with initial processingon a Sysmex XT-2000iTM automated hematol-ogy analyzer (Sysmex America Inc., Lincoln-shire, Illinois, USA) followed by manualdifferential. Hemoglobin, red cell count, meancorpuscular volume, mean corpuscular hemo-globin, mean corpuscular hemoglobin content,platelet count, neutrophils, lymphocytes,monocytes, eosinophils, and basophils weremeasured. Serum biochemistry was performedon the Roche Modular EVO Analyzer (RocheProducts Pty. Ltd., Castle Hill, Australia) withthe exception of total protein and albumin,which were performed on the Advia Chemis-try 2400 System (Siemens AG, Erlangen,Germany). Sodium, potassium, chloride, bi-carbonate, anion gap, urea, creatinine, glu-cose, bilirubin, aspartate amino transferase(AST), alanine transaminase (ALT), gammaglutamyl transferase (GGT), alkaline phospha-tase (ALP), globulin, albumin/globulin ratio,calcium, phosphate, creatine kinase, choles-terol, and triglyceride were also measured. ForSalmonella culture swabs were inoculated ontoxylose lysine deoxycholate (XLD) agar mediaand Mannitol Selenite broth and incubated at35 C for 48 hr in an aerobic atmosphere. TheMannitol Selenite broth was then subculturedonto an additional XLD agar plate at 24 hrpostinoculation. Both XLD plates were read at48 hr postinoculation.

Descriptive statistics were performed forhematologic and biochemical values. One-wayanalysis of variance was used to determine ifthere were significant differences betweenvalues for males and females. Where nosignificant difference existed the data werepooled. Statistical analyses were performedusing GenStat 15th Edition (VSN Internation-al Ltd., Hemel Hempstead, UK).

Toxoplasma gondii serology was performedat the Department of Primary Industries, Waterand Environment, Mount Pleasant Laborato-ries, Launceston, Australia using the direct(DAT) and modified agglutination tests (MAT)for antibodies to T. gondii (Johnson et al., 1989).Both tests were performed on sera from 53bettongs, after which time the laboratory ceasedto offer the DAT, and sera from six bettongswere assayed using the MAT only.

Herpesvirus serology and detection ofherpesvirus DNA by PCR were performed atthe Faculty of Veterinary Science, Universityof Melbourne, Parkville, Australia. Serum-virus neutralization assays were performedagainst the alphaherpesviruses macropodidherpesvirus 1 (MaHV-1) and macropodidherpesvirus 2 (MaHV-2) as described (Warneret al., 2001; Vaz et al., 2013) using wallaby

JU56 fibroblast cells (Uren et al., 1966). Serawere tested at a dilution of 1:2 and wells wereexamined for herpesvirus-induced cytopathiceffect after 4 days of incubation. For thedetection of herpesviruses in swab samples,DNA was extracted from swab samples pooledfrom each animal as previously described (Vazet al., 2011). Extracted DNA was used as thetemplate in a generic pan-herpes PCR usingprimers targeting a conserved region of theherpesvirus DNA polymerase gene, approxi-mately 210–230 base pairs long (Chmielewicz etal., 2003). The PCR products were purified andtheir DNA sequence was determined. Nucleo-tide sequencing and phylogenetic analysis wereperformed using ClustalW2 (Larkin et al., 2007)and GENEious software (Biomatters Ltd.,Auckland, New Zealand). The predicted aminoacid sequence was compared with publiclyavailable sequences in GenBank (NationalCenter for Biotechnology Information [NCBI],Bethesda, Maryland) using the blastx onlinealgorithm and were subsequently aligned withrepresentative members from the three Her-pesviridae subfamilies from a range of hostspecies. An unrooted maximum-likelihood phy-logenetic tree was generated from this sequencealignment using the Jones-Taylor-Thorntonmodel of amino acid replacement. Followingtranslocation, bettongs were monitored bycamera (n544) or telemetry in combinationwith live trapping (n516) for 90 days.

RESULTS

Physical examination

On the basis of physical examination, 32of 60 bettongs (53%) were subjectivelyassessed as being in less than ideal bodycondition. Ocular abnormalities (cornealulceration, corneal scarring, and cataractformation) were noted in four bettongs(7%); dental disease (fractured teeth andmild gingivitis) was noted in four bettongs(7%); and one male had an enlargedepididymis and a swollen left hock, withradiographs of the hock joint revealingmarked degenerative joint disease.Mean6SD body weights for male andfemale bettongs were 1.7160.23 kg and1.6560.17 kg, respectively.

Hematologic and serum biochemistry variables

Clotting of two hematologic samplesand insufficient sample volume meant that


not all tests were performed for allbettongs. Hematologic and biochemistryvariables were comparable to publishedvalues for other potoroids (Tables 1, 2)with the exception of an inverted neutro-phil/lymphocyte ratio, a marked elevationin creatine kinase, and a moderate eleva-tion in ALP (Moore and Gillespie, 1968;Vaughan et al. 2009).

Salmonella serovars culture

All 14 rectal swab samples were culturenegative for Salmonella serovars.

Serology and PCR

Sera were negative for antibodies to T.gondii as assessed by both the MAT andDAT (n553) or by the MAT only (n56).Two of 50 bettongs (4%) had antibodies toMaHV-1 or MaHV-2 as assessed byserum-virus neutralization assay. Herpes-virus serology results for five animals couldnot be determined due to cytotoxicity or

contamination, and insufficient serum sam-ples precluded serology for five animals.Four bettongs were PCR positive forherpesvirus DNA as determined by se-quence analysis of PCR products. Align-ment of the nucleotide sequence (approx-imately 150 nucleotides) to those of othermarsupial herpesviruses showed that thedetected virus was distinct from all otherknown marsupial herpesviruses. The first100 matches with the deduced amino acidsequence generated from this nucleotidesequence in blastx were to the DNApolymerase gene of herpesviruses withinthe subfamily Gammaherpesvirinae. Thedetected virus (tentatively designated po-toroid herpesvirus 1, PotHV-1) groupedwith other gammaherpesviruses (Fig. 2).

Parasites

Fleas, lice, mites, and ticks recoveredfrom bettongs were deposited in theAustralian National Wildlife Collection

TABLE 1. Hematologic variables from free-ranging adult eastern bettongs (Bettongia gaimardi) sampledduring translocation from Tasmania to the Australian Capital Territory, Australia.

Parameter n Mean (SD) Reference interval (5–95%)

Hematocrit (%) 58 0.40 (4.47) 0.3440–0.4720Hemoglobin (g/L)a

Males 17 141.4 (8.32) 124.8–153.6Females 41 129.7 (17.83) 107.7–152.9

Red cell count (31012/L)a

Males 17 9.959 (0.62) 8.705–10.80Females 41 9.076 (1.51) 6.855–11.14

Mean corpuscular volume (fL)a

Males 17 40.29 (5.67) 26.85–47.60Females 41 43.98 (4.71) 37.00–53.90

Mean corpuscular hemoglobin (pg) 58 14.44 (1.01) 13.00–16.00Mean corpuscular hemoglobin content (g/L)a

Males 17 343.3 (15.11) 322.8–372.3Females 41 330.3 (17.78) 303.5–357.9

Platelets (3109/L) 51 377 (118.36) 135.7–618.7White blood cell count (3109/L) 58 3.78 (0.93) 1.98–6.46Neutrophils (3109/L) 58 2.17 (0.93) 0.90–3.93Lymphocytes (3109/L) 58 1.41 (0.73) 0.5–2.97Monocytes (3109/L) 58 0.15 (0.08) 0–0.3Eosinophils (3109/L) 58 0.05 (0.07) 0–0.2Basophils (3109/L) 58 0.01 (0.03) 0–0.1

a Significant difference (P,0.05) between male and female values. Data are presented separately for males and females.


(CSIRO, Canberra; Table 3). Forty-nine(82%) bettongs had a low ectoparasiteburden, nine (15%) had a moderateectoparasite burden, one (2%) had a heavyectoparasite burden, and one (2%) had novisible ectoparasites. Ticks were found on55 (92%) bettongs, the most commonbeing Ixodes tasmani. Lice and fleas werepresent on 24 (40%) and 25 (42%)bettongs, respectively. Fur mites of thegenus Cytostethum were present on 37 of38 (97%) bettongs from which fur plucksamples were collected. Fecal samplesfrom 43 bettongs were examined forendoparasites. Eimeria gaimardi werepresent in six (14%) samples. Capillariid-like eggs were present in two (5%) fecalsamples. Strongylid eggs were present in29 (67%) samples and, although severalmorphotypes were present, identificationto the genus level was not possible.Strongylid larvae were present in six(14%) fecal samples. Examination of blood

films revealed round to ovoid intraeryth-rocytic organisms morphologically consis-tent with a Theileria species in four (7%)bettongs.

Mild to moderate dermatitis affectingthe medial aspects of both hind limbs fromthe hock to the inguinal area was presentin seven (12%) bettongs. Gross lesionsincluded alopecia, diffuse erythema, andmultifocal scaling, crusting, pustules, andulceration. Histologic examination of theaffected areas of skin in four bettongsrevealed moderately irregular acanthosis,hyperkeratosis superficial pustules, andparakeratotic crusting. A diffuse perivas-cular to interstitial infiltrate comprising avariable number and mixture of neutro-phils, mast cells, lymphocytes, plasmacells, eosinophils, and macrophages waspresent in the dermis. Eosinophil andneutrophil infiltration was predomi-nant within the vicinity of intraepithelialmites (Fig. 3). The mites had a chitinous

TABLE 2. Biochemical variables obtained from free-ranging adult eastern bettongs (Bettongia gaimardi)sampled during translocation from Tasmania to the Australian Capital Territory, Australia.

Parameter n Mean (6SD) Reference interval (5–95%)

Sodium (mmol/L)a

Male 18 145.67 (4.42) 135.4–151.2Female 40 147.57 (1.98) 144.0–151.0

Potassium (mmol/L) 57 3.62 (1.62) 2.500–4.260Chloride (mmol/L) 58 106.44 (3.28) 101.0–112.2Bicarbonate (mmol/L) 58 28.96 (2.39) 25.00–33.20Anion gap (mmol/L) 58 15.30 (3.01) 10.00–20.00Urea (mmol/L) 58 6.17 (1.76) 4.260–10.120Creatinine (mmol/L) 58 41.81 (7.18) 32.00–55.00Glucose (U/L) 58 11.05 (3.24) 8.08–15.96Bilirubin (U/L) 58 1.05 (0.39) 0.40–2.00AST (U/L) 58 181.3 (64.63) 106–316.20ALT (U/L) 58 82.07 (32.91) 42.35–136.25GGT (U/L) 58 16.19 (4.32) 10.40–24.20ALP (U/L) 58 1564 (1638.07) 315–5,481Protein (g/L) 58 56.02 (5.04) 49.0–64.10Albumin (g/L) 58 40.37 (5.32) 33–49.55Globulin (g/L) 58 15.66 (4.07) 6.35–22.55Albumin/globulin ratio 58 3.17 (2.43) 1.7–7.81Calcium (mmol/L) 58 2.31 (0.13) 2.13–2.52Phosphate (mmol/L) 58 2.46 (0.77) 1.39–3.90Creatine kinase (U/L) 58 23,391.0 (13,101.77) 8,276–50,260Cholesterol (mmol/L) 57 3.46 (0.90) 2.44–5.14Triglyceride (mmol/L) 58 1.06 (1.51) 0.44–1.86

a Significant difference (P,0.05) between male and female values. Data are presented separately for males and females.


FIGURE 2. The relationship between potoroid herpesvirus 1 and herpesviruses from the Alphaherpesvir-inae (a), Betaherpesvirinae (b), and Gammaherpesvirinae (c) subfamilies. (A) Alignment of predicted aminoacid sequences of the available DNA polymerase gene sequence data from PotHV-1 and the correspondingregions of DNA polymerase gene sequences from other herpesviruses. Asterisks indicate amino acid identity.Hyphens (-) indicate alignment gaps. (B) Unrooted maximum-likelihood phylogenetic tree generated usingthe above amino acid alignment. Divergences between pairs of aligned sequences were calculated anddistance trees derived using GENEious software and the Jones-Taylor-Thornton model of amino acidreplacement. The reliability of each tree branch was tested using 100 replicates in a bootstrapping analysis.Bootstrapping values (n5100) are shown for each branch. Abbreviations and GenBank accession details:


exoskeleton, body cavity, striated muscle,intestinal and reproductive structures, andfocal accumulations of deeply basophilicnuclei. Mite tunnels extended from thesuperficial epidermis to basal regions withulceration.

Posttranslocation survival

Four bettongs (7%) died in the 90 daysfollowing translocation. Two deaths werethe result of trauma; a fractured cervicalspine following collision with a fence andasphyxiation following telemetry collarentanglement. A male bettong died 5 daysposttranslocation; microscopic findings in-cluded a bacterial pneumonia and thepresence of nematodes in the airways. Afemale bettong was found dead 37 daysposttranslocation, but advanced decompo-sition of the carcass precluded postmor-tem examination.

DISCUSSION

The eastern bettongs in this study weregenerally considered to be clinicallyhealthy, despite approximately half of thetranslocated animals being assessed to bein less than ideal body condition duringinitial physical examination. Trapping ofbettongs occurred in areas of disturbedand fragmented habitat or on agriculturalland. While no attempt was made to assessthe suitability of the habitat at these sites,it is possible that the habitat may havebeen less than ideal, resulting in nutri-tional stress for some of the sourcepopulations. A small number of individu-als had minor injuries likely associatedwith conspecific or environmental trauma.No clinical signs of infectious disease wereevident in any of the bettongs examined.

The significant, sex-related differencesbetween hematologic and biochemical

r

bovine herpesvirus 4 (BoHV-4: NP_076501.1), macacine herpesvirus 5 (McHV-5: NC_003401.1), macropodidherpesvirus 3 (MaHV-3: EF467663.1), phascolarctid herpesvirus 1 (PhaHV-1: JN585829), phascolarctidherpesvirus 2 (PhaHV-2: JQ996387), porcine cytomegalovirus (PCMV: AF268042.1), human herpesvirus 6(HHV-6: NP_042931.1), human herpesvirus 1 (HHV-1: HQ123098), and human herpesvirus 2 (HHV-2: M16321.1).

TABLE 3. Ectoparasites from free-ranging eastern bettongs (Bettongia gaimardi) sampled duringtranslocation from Tasmania to the Australian Capital Territory, Australia.

Parasite order, family Species Accession numbera

Acari

Listrophoridae Paraheterodoxous ? n. sp. (see Green and Munday, 1971) AR 1572, 1574Trombiculidae Guntheria cf. pertinaxb AR 1579Laelapidae Haemolaelaps hatteni AR 1575

? Thadeua sp. protonymph AR1585Atopomelidae Cytostethum (Metacytostethum) tasmaniense AR 1576

Cytostethum (Metacytostethum) intermedium AR 1576Cytostethum (Metacytostethum) thetisb AR 1576Cytostethum (Metacytostethum) wallabiab AR 1576

Ixodidae Ixodes cornuatus AR 1586Ixodes trichosuri AR 1571Ixodes tasmani AR 1587

Phthiraptera

Pygiopsyllidae Pygiopsylla zethi AR 1573Stephanocercidae Stephanocircus harrisonib AR 1588

a Wildlife Parasite Collection, Australian National Wildlife Collection, CSIRO Ecosystem Sciences, Canberra, Australia.b New host record.


parameters were limited to some red cellindices and sodium. Sex-related differenc-es in hematologic parameters have beenpreviously reported in marsupials (Barneset al., 2008) although obvious sex-relatedpatterns are not apparent (Clark, 2004).The most notable biochemical finding wasthe elevation in creatine kinase. Meancreatine kinase values were elevated over20-fold compared with mean values forGilbert’s potoroo (Potorous gilbertii)(Vaughan et al., 2009). The creatine kinasevalues most likely reflect exertional myop-athy associated with capture and trans-port. Despite the severity of the changes,clinical signs of exertional myopathy werenot evident. The use of benzodiazepineshas been advocated for the prevention ofexertional myopathy (Vogelnest and Por-tas, 2008), and diazepam administrationmay have ameliorated the development ofclinical signs despite the physiologicchanges present.

The absence of antibodies to T. gondiiin sera from the bettongs is an unexpectedfinding because domestic cats (Felis catus)are widespread across Tasmania. Whileserosurveys for T. gondii in easternbettongs have not been conducted previ-ously, toxoplasmosis is a well-recognizeddisease of both free-ranging and captivemacropodoids. Previous serologic surveysof other free-ranging Tasmanian macro-podoids have demonstrated variable prev-alence between and within species from1.2–42.9% (Munday, 1972; Johnson et al.,1988; Johnson et al., 1989). Of potoroidsexamined elsewhere in Australia, 5.8% ofbrush-tailed bettongs from one mainlandsite were antibody positive while brush-tailed bettongs and burrowing bettongs(Bettongia lesueur) from cat-free offshoreislands had prevalences of 1.4% and 0%,respectively (Parameswaran, 2010).

Eastern bettongs may be highly suscep-tible to T. gondii with infection resulting

FIGURE 3. Histologic section of skin from a free-ranging eastern bettong (Bettongia gaimardi) withdermatitis examined during translocation from Tasmania to the Australian Capital Territory, Australia. Anintraepithelial mite is present associated with infiltration by eosinophils and neutrophils. 2003. Bar 5 100 mm.


in death rather than latent infection.However, the presence of antibodies inother potoroids suggests this is unlikely. Itis plausible that disease recrudescencemay have occurred in antibody-positiveand, therefore, latently infected bettongsunder nutritional stress, resulting in clin-ical toxoplasmosis and subsequent deathof affected animals. Recrudescence oflatent toxoplasmosis during periods ofstress or immunosuppression has beenpreviously reported in macropodoids, andthis may offer an explanation for the lackof antibody-positive animals (Bermudezet al., 2009). Alternatively, if latent diseaseoccurs at a low prevalence in this popu-lation, the sample size may have beeninsufficient to detect infected animals.

The identification of a previously unde-scribed gammaherpesvirus is not unex-pected given that many mammal specieshave at least one host-adapted herpesvi-rus. Macropodid herpesviruses 1 and 2(subfamily Alphaherpesvirinae) have beenassociated with morbidity and mortality inparma wallabies (Macropus parma,MaHV-1), quokkas (Setonix brachyurus,MaHV-2), and brown dorcopsis wallabies(Dorcopsis muelleri, MaHV-2; Aclandet al., 1981; Callinan and Kefford, 1981;Wilks et al., 1981). A related but geneticallydistinct alphaherpesvirus was recently re-ported in a free-ranging eastern greykangaroo (Macropus giganteus) with clinicaldisease (Vaz et al., 2013). Macropodidherpesvirus 3 (Gammaherpesvirinae) isassociated with morbidity and mortality incaptive and free-ranging eastern grey kan-garoos (Smith, Wellehan et al., 2008; Wilcoxet al., 2011). An outbreak of herpesvirus-associated disease characterized by a per-acute course and high mortality was report-ed to affect a captive colony of brush-tailedand rufous (Aepyprymnus rufescens) bet-tongs, but the herpesvirus responsible wasnot reported (Dickson et al., 1980).

Reactivation of latent macropodid her-pesvirus infections has been demonstratedin eastern grey kangaroos following corti-costeroid-induced immunosuppression

(Guliani et al., 1999). Field capture,transportation, and translocation wereconsidered potential stressors that couldeither reactivate latent herpesvirus infec-tions in translocated bettongs or renderthem more susceptible if exposed toinfected and shedding macropodoids atthe translocation sites. No clinical evi-dence of disease consistent with herpesvi-rus infection was detected in bettongs thatwere directly examined or remotely mon-itored by camera in the months followingtranslocation, although there was onedeath for which a diagnosis was notobtained. The lack of clinical disease inthe bettongs from which the herpesviruswas detected, despite the stress of captureand translocation, suggests this herpesvirusis of low pathogenicity in its host. Theserum-virus neutralization assay utilizedMaHV-1 and MaHV-2, both alphaherpes-viruses, while the herpesvirus recoveredfrom ocular, nasal, and urogenital swabswas a gammaherpesvirus. It is unsurprising,therefore, that despite being PCR positive,all four bettongs were negative using thisserologic assay. The identification of twobettongs with antibodies against MaHV-1or MaHV-2 suggests prior exposure to oneof these herpesviruses or to a closelyrelated alphaherpesvirus. However, wedid not detect an alphaherpesvirus beingshed at the time of sampling. Serial testingof these individuals and other conspecificsmay further elucidate the significance ofthese results.

Free-ranging macropodoids have beenidentified as asymptomatic carriers of awide range of Salmonella serovars (Speareet al., 1989; Potter et al., 2011). Theabsence of Salmonella serovars in any ofthe bettongs assessed in this study couldbe explained by small sample size (n514)or limitations associated with rectal sam-pling or culture. A seasonal influence isalso possible because the translocationswere planned specifically to avoid the hotsummer. A marked seasonal incidence inthe prevalence of Salmonella infections infree-ranging quokkas on Rottnest Island


has been demonstrated with a highprevalence of infection in summer (Hartet al., 1985). Alternatively, the results mayreflect a true low incidence of infectionwith Salmonella serovars in this species.

Clinical manifestations of disease asso-ciated with the diverse ectoparasite faunaon the majority of bettongs was limited todermatitis associated with mites in sevenindividuals and anemia (hematocrit 20%,red cell count 831012/L and hemoglobin57 g/L) in one female with a heavy burdenof I. tasmani and I. trichosuri. Inguinalectoparasitic dermatitis has been reportedin free-ranging macropodoids with lesionsdescribed in Proserpine rock wallabies(Petrogale persephone), black-striped wal-labies (Macropus dorsalis; Skerratt et al.,2007), swamp wallabies (Wallabia bicolor;Ladds, 2009), and brush-tailed rock wal-labies (Petrogale penicillata; Barnes et al.,2010) associated with mites of the genusThadeua; in yellow-footed rock wallabies(Petrogale xanthopus) associated withOdontacarus adelaideae (O’Callaghanet al., 1994); and in swamp wallabiesassociated with Macropodicoptes mironovi(Portas et al., 2009; Bochkov, 2012).However, this is the first report ofectoparasitic dermatitis in a potoroidmarsupial, although it was not possible toidentify the mites on the basis of histologicappearance, and the species associatedwith the dermatitis is unknown. FourCytostethum species were identified inthe fur of bettongs. Multiple speciation offur mites from the genus Cytostethum hasbeen reported previously in another po-toroid, the long-nosed potoroo (Potoroustridactylus), from which 21 species ofCytostethum have been recorded (Fainand Domrow, 1974b).

Verminous bronchitis and bronchiolitisassociated with the capillariid parasiteEucoleus potoroi have been reported incaptive rufous and brush-tailed bettongsand long-nosed potoroos but has not beenreported from free-ranging potoroids(Love and Redacliff, 1992; Spratt, 2007).The presence of capillariid-like eggs in the

feces of two bettongs and nematodes inhistologic sections of the airways of onebettong that died suggest that E. potoroior a related species is present in free-ranging eastern bettongs.

The morphology of the intraerythrocyticpiroplasm observed in blood smears fromfour bettongs most closely resembled thatof a Theileria species. Theileria species/genotypes have been reported from fourother potoroids; Theileria penicillata fromthe brush-tailed bettong (Clark and Spen-cer, 2007), Theileria gilberti in Gilbert’spotoroo (Potorous gilbertii; Lee et al.,2009), and two novel Theileria genotypesfrom the long-nosed potoroo and theburrowing bettong (Paparini et al., 2012).Given the presence of Theileria in four ofeight extant potoroids, the presence of thispiroplasm in the eastern bettong is notunusual; however, molecular analyseswere not done, so the identity of thisorganism remains speculative. There wereno clinical signs associated with thepresence of these organisms, and all fourbettongs had hematocrits and red cellparameters within the calculated refer-ence range.

Our data provide baseline health anddisease information for free-ranging east-ern bettongs that can be used in theconservation management of both thesource and translocated populations. Thisinformation is a prerequisite to under-standing the potential role of disease inthe long-term success or failure of thistranslocation. Ongoing monitoring of thetranslocated populations and sympatricmacropodoids at the release sites will alsobe required to understand more fully thepotential impact of disease on transloca-tion success in potoroids.

ACKNOWLEDGMENTS

The translocation was carried out underlicense from the Tasmanian Department ofPrimary Industries, Parks, Water and Envi-ronment and all procedures were approved byits associated Animal Ethics Committee (AECProject 18/2010-2011). Postrelease researchwas approved by the Australian National


University Animal Experimentation EthicsCommittee (ethics protocol A2011/017). Fund-ing was provided by the ACT Government andan Australian Research Council Linkage Grant(LP110100126). A.D.M. was supported by anAustralian Research Council Future Fellow-ship (FT100100358). We acknowledge NickMooney for significant advice and logisticsupport in trapping bettongs; Matt Pauza andAnnie Phillips for assistance in Tasmania;Claire Wimpenny, Elyce Fraser, Ani Kunz,Brian Phillips, Scott Ryan, Kym Birgan, PeterMills, Grant Woodbridge, John Lawler, StuartJeffress, Jenny Newport, Will Batson, HelenCrisp, and Arianne Lowe for logistic support;and Ross Cunningham and Christine Donnellyfor assistance with statistical analysis. We ac-knowledge Mulligans Flat–Goorooyarroo Wood-land Experiment and Mulligans Flat WoodlandSanctuary for supporting the translocation. Wethank Forestry Tasmania, Gunns Ltd., The ScoutAssociation, and Oak Tasmania’s Tahune FieldNursery for site access to trap bettongs.

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Submitted for publication 5 August 2013.Accepted 29 October 2013.


health evaluation of free-ranging eastern bettongs ( bettongia gaimardi ) during translocation for...

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