HEMATOLOGIC AND SERUM BIOCHEMICAL REFERENCE RANGES AND AN ASSESSMENT OF EXPOSURE TO INFECTIOUS DISEASES PRIOR TO TRANSLOCATION OF THE THREATENED WESTERN RINGTAIL POSSUM (PSEUDOCHEIRUS OCCIDENTALIS)

Minimization of risk for disease transmission must be considered during reintroduction programs (Viggers et al., 1993; Woodford and Rossiter, 1994), and examination of hematologic and serum biochemical parameters complements clinical assessments during health surveys of wild animal populations (Kock et al., 2007). Translocation has featured in management of the western ringtail possum, Pseudocheirus occidentalis, which is listed as threatened under the Commonwealth of Australia's Environment Protection and Biodiversity Conservation Act, 1999 (Department of the Environment, Water, Heritage and the Arts, 2009) and by the International Union for the Conservation of Nature (IUCN, 2009). Land clearing for building development threatens the last major coastal population stronghold around Busselton in southwestern Australia (de Tores, 2008).

Although there is no current evidence that disease limits persistence of marsupial populations in southwestern Australia, there are anecdotal historical reports of widespread disease-induced mortality of native species, including possums, soon after European colonization (Abbott, 2006). The unexplained decline of a translocated P. occidentalis population between 1998 and 2002 (de Tores et al., 2004) prompted this study in which we investigated pathogens most likely to affect possum survival and fecundity: Salmonella spp., Toxoplasma gondii, Leptospira spp., Cryptococcus spp., and Chlamydophila spp. These organisms are capable of causing disease in stressed hosts, especially those immunosuppressed following capture.

Knowledge of species-specific reference ranges from healthy wild populations facilitates identification of diseased individuals. Data on hematologic and serum biochemical parameters are scarce for P. occidentalis (Clark, 2004); therefore, establishment of baseline reference ranges was important. Our aims were to 1) determine levels of exposure of P. occidentalis and coexisting Trichosurus vulpecula hypoleucus to the infectious diseases most likely to adversely affect their survival or fecundity, 2) assess fecal shedding of Salmonella spp. by these possums, and 3) establish hematologic and biochemical reference ranges for coastal populations of P. occidentalis.

We sampled 36, 22, and 15 clinically healthy wild P. occidentalis from Busselton (33°39′S, 115°21′E), Tuart Forest National Park (TFNP, 33°38′S, 115°25′E) and Gelorup (33°25′S, 115°37′E), respectively, between November 2005 and March 2008. Possums were captured by darting using 1∶1 tiletamine and zolazepam (Zoletil 100®, Virbac, Milperra, New South Wales, Australia) delivered at 0.1 mg/kg. We also sampled 24 orphaned or displaced captive P. occidentalis held by local wildlife rehabilitators. Samples were collected from 19 Busselton-born P. occidentalis ≥5 mo after translocation 80–150 km north to Leschenault Peninsula Conservation Park (33°14′S, 115°41′E) and Yalgorup National Park (32°50′S, 115°40′E). Samples were collected from 68 T. v. hypoleucus at the translocation sites. Thirty of the P. occidentalis and 17 of the T. v. hypoleucus were sampled on 2–4 occasions, at least 3 mo apart.

Possums were anesthetized with isoflurane delivered via facemask using an anesthetic machine employing an open, non-rebreathing circuit system (Stinger®, Advanced Anaesthesia Specialists, Sydney, Australia). Blood samples, to a maximum of 4 mL, were collected from the tail vein into ethylenediaminetetraacetic acid–coated tubes (Becton, Dickenson and Co., North Ryde, New South Wales, Australia) for hematology and into untreated tubes for serum biochemistry and antibody-antigen testing. Cloacal swabs for microbiologic culture were taken into MW 170 transport medium (Transwab®, Medical Wire and Equipment Co. Ltd., Corsham, Wiltshire, England). Epithelial cell samples for Chlamydophila testing were collected onto sterile dry rayon swabs (COPAN® Italia, Brescia, Italy) from the conjunctiva of eyes, pharynx, and cloaca. Wild and captive possums from Busselton and the translocation sites were sampled for all tests; some more than once. Samples from P. occidentalis at TFNP and Gelorup were collected for hematologic and serum biochemical analyses only.

Hematologic and serum biochemical analyses were carried out using an automated Advia 120 Hematology System® (Siemens Healthcare Diagnostics, Deerfield, Illinois, USA) and an RX Daytona™ (Randox Laboratories, Crumlin, Co. Antrim, UK) autoanalyzer, respectively, to determine parameters listed in Tables 1 and 2. Differential white cell counts were performed on blood smears stained with Wright's Giemsa stain, spun packed cell volume was measured manually, and total solids protein was read by refractometry. Fibrinogen was measured by heat precipitation (Schalm, 1980).

Cloacal swabs were plated onto sheep blood agar and MacConkey agar. Bacteria were identified by Gram reaction and standard biochemical tests and diagnostic kits (MB12A Microbact®, Oxoid Ltd., Cambridge, UK, and API-20E®, bioMérieux Clinical Diagnostics, worldwide). Each sample was tested for Salmonella spp. using Salmonella-Shigella agar and brilliant green agar. Any Salmonella spp. found were checked by Poly O (A–S) and Poly H antiserum agglutination testing and then identified to species. Antibody testing for T. gondii used direct agglutination (DAT) and modified agglutination (MAT) tests (Antigen Toxo AD kit, BioMerieux, Charbonnieres les Bains, France). A titer of ≥1/64 was considered positive. Sera were screened, using standard microscopic agglutination tests (Levett, 2001), against a reference panel of 23 Leptospira interrogans serovars (pomona, hardjo, tarassovi, grippotyphosa, celledoni, copenhageni, australis, zanoni, robinsoni, canicola, kremastos, szwajizak, medanensis, bulgarica, cynopteri, ballum, bataviae, djasiman, javanica, panama, shermani, topaz, and balcanica). Sera were tested for antigens of Cryptococcus neoformans and Cryptococcus gattii using slide latex cryptococcal antigen tests (Crypto-LA Test, Wampole Laboratories, Cranbury, New Jersey, USA). We detected Chlamydophila spp. in mucosal swabs using PCR (Markey et al., 2007).

We used only samples from clinically healthy possums >600 g to calculate reference ranges. Wild-caught and captive P. occidentalis were considered separately and juveniles (<600g) were not included. Outlying data points were identified if either (x_(n)−x_(n-1))/(x_(n)−x₍₁₎) or (x₍₂₎−x₍₁₎)/(x_(n)−x₍₁₎) was >1/3 (Lumsden and Mullen, 1978) and then removed. Box plots, normal probability plots, and Shapiro-Wilk normality test statistics were assessed to check for parametric distribution of continuous variables. Nonnormal variables were logarithm or square-root transformed. For wild P. occidentalis means, SD, and Gaussian tolerance intervals (Lumsden and Mullen, 1978) were calculated for variables conforming to parametric distributions before or after transformation. Medians and central 95% intervals were calculated for all variables. Sample sizes of at least 120 are recommended for reliable inference from empirical quantiles (Reed et al., 1971); sample sizes in this study ranged from 107–122. Only means, medians, and ranges were calculated for variables from captive P. occidentalis because samples sizes were small (19–23).

We assessed effects of age (subadult [developing pouch or testicles] vs. adult), sex, lactation, and location on hematologic and serum biochemical parameters from wild P. occidentalis using multilevel mixed effects linear regression with restricted maximum likelihood model fitting (Bolker et al., 2009); animal ID was modeled as the random effect. Wald test statistics of fixed effects (age, sex, lactation, and location) were examined (α = 0.05) to determine groupings for comparing variables. Effects of wild vs. captive status on hematologic and serum biochemical parameters were similarly assessed.

We analyzed 138 blood and 145 serum samples from clinically healthy P. occidentalis for hematology and biochemistry, respectively. Three outlying data points were removed (a low creatinine value, a low albumin value, and a high number of disintegrated cells in the differential white blood cell count). Hematologic and serum biochemical reference ranges from wild-caught and captive P. occidentalis are provided in Tables 1 and 2. Values for neutrophil count, total solids protein (TSP), fibrinogen, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine kinase (CK), bilirubin, cholesterol, protein, urea, and globulin for captive P. occidentalis were all lower than for wild P. occidentalis while alkaline phosphatase (ALP) and glucose values were higher (Wald tests, P<0.05).

We found effects of sex, age, and lactation status for some parameters (Table 3). Red blood cell (RBC) counts were lower for nonlactating females than for lactating females (Wald statistic = 7.25, P = 0.007, n = 69) or males (Wald statistic = 9.21, P = 0.002, n = 71). Albumin levels were higher in lactating females than in males (Wald statistic = 13.48, P<0.001, n = 93) or nonlactating females (Wald statistic = 19.54, P<0.001, n = 75). Creatinine kinase levels were higher in nonlactating females than in males (Wald statistic = 7.76, P = 0.005, n = 75) or lactating females (Wald statistic = 10.88, P = 0.001, n = 74). Urea levels differed between lactating and nonlactating females (Wald statistic = 12.41, P<0.001, n = 75). Levels of ALP and phosphorus were higher in subadults than in adults (Wald statistic = 20.23, P<0.001, n = 122; Wald statistic = 21.53, P<0.001, n = 122, respectively), while bilirubin levels were higher in adults than in younger animals (Wald statistic = 7.45, P = 0.006, n = 119). Levels of AST were higher in males than in females (Wald statistic = 7.61, P = 0.006, n = 122).

We identified site differences for RBC count (Wald statistic = 20.85, P<0.001, n = 115), hemoglobin (Wald statistic = 14.30, P<0.001, n = 115), albumin (Wald statistic = 9.75, P = 0.002, n = 121), urea (Wald statistic = 6.61, P = 0.01, n = 122), and globulin (Wald statistic = 6.62, P = 0.01, n = 121; Table 4). Red blood cell counts and levels of hemoglobin and albumin followed similar patterns among the four sites with values lowest at TFNP and highest at Busselton development sites. Urea and globulin levels were lower at Busselton than at TFNP (Table 4).

We isolated bacteria from 79 of 99 swabs from P. occidentalis and from 96 of 97 cloacal swabs from T. v. hypoleucus. Bacterial growth was heavier for T. v. hypoleucus than for P. occidentalis and mixed bacterial growths were common. Coliform bacteria were most frequently isolated from T. v. hypoleucus samples while Streptococcus spp. and Coryneform bacilli were more common in P. occidentalis. No Salmonella spp. were cultured from either possum species.

We tested 99 and 95 serum samples from P. occidentalis and T. v. hypoleucus, respectively, for antibodies to T. gondii. Twenty-one individual P. occidentalis and 17 T. v. hypoleucus were tested more than once, at least 3 mo apart. All T. v. hypoleucus samples were negative on DAT and MAT tests. All P. occidentalis samples were negative on MAT; however, three samples (from two different P. occidentalis) gave mild positive reactions to DAT (titers of 1:64 and 1:256) without evidence of seroconversion. Neither of the two animals became ill and the MAT status of one retested after 3 mo remained negative.

We tested 89 serum samples from P. occidentalis and 89 from T. v. hypoleucus for antibodies to 23 serovars of Leptospira interrogans. No possums of either species were positive. Eighty-four and 92 serum samples from P. occidentalis and T. v. hypoleucus, respectively, were tested for antigen of serovars of C. neoformans and C. gattii. All, except one T. v. hypoleucus, were negative. The positive T. v. hypoleucus that had a low titer of 1:8 was negative when resampled after 10 and 14 mo. No chlamydial DNA was amplified by PCR from swabs from 95 P. occidentalis and 97 T. v. hypoleucus.

We found age, sex, and lactation effects in several parameters. Age differences in ALP and phosphorus occur as a function of increased levels of the bone isoenzyme of ALP in growing animals (Willard and Tvedten, 2004). Age differences in bilirubin levels could be due to different degrees of fasting or differing levels of endoparasite-induced hemolysis, although evidence for either is lacking. Sex differences in red cell counts are common in animals, with males usually having higher values (Clark, 2004). Nonlactating female P. occidentalis had lower red cell counts and albumin than did males and lactating females; this may have been partly an age effect, although we found no age differences in males. Lactating females had the highest albumin levels, which may have been partly a function of hydration status. Levels of urea in nonlactating females were higher than in males or lactating females. Urea values generally reflect protein intake for healthy animals in neutral or positive energy balance (Stirrat, 2003). Those in negative energy balance may catabolize their muscle protein, leading to high blood urea levels even with low protein intake (Reiss et al., 2008). Given the lack of sex and lactation differences in glucose levels, it is likely that the difference in urea levels between the three groups resulted from differing protein intake and nitrogen balance.

We found statistical differences between wild and captive populations and variation between sites for wild P. occidentalis. Nutritional factors may explain most of the former. Captive animals cannot select foliage quality and are likely to have diets lower in protein and available nitrogen, resulting in lower blood values for protein- and nitrogen-breakdown products. Their diets are often supplemented with fruit and flowers which increase their blood glucose. Enzymes elevated during stress include AST and CK (Jain, 1993); wild P. occidentalis are likely to produce higher levels of these enzymes following capture than do captive individuals that are used to people. Similar differences in AST, CK, bilirubin, urea, and globulin were found between captive and wild populations of Gilbert's potoroos (Potorous gilbertii) (Vaughan et al., 2007).

Differences in hematologic and serum biochemical parameters may reflect variations in habitat quality and foliage nutritional status, as observed for other marsupials (Stirrat, 2003; Clark and Spencer, 2006). The Busselton development sites are in prime habitat that supports high population densities of P. occidentalis (Jones et al., 1994a, b) while densities at Gelorup, TFNP, and the translocation sites are lower than elsewhere (de Tores and Elscot, 2010; Clarke, 2011). Values for RBC, hemoglobin, and albumin were higher at the Busselton development sites than at TFNP and the translocation sites, and levels of urea and globulin were lower at Busselton than at TFNP and Gelorup. The variation in urea levels could represent lower protein intake at Busselton. Alternatively, P. occidentalis at TFNP and Gelorup could be in negative energy balance, having higher urea values due to muscle protein catabolism. Glucose levels at the four sites, although not statistically different, followed the reverse pattern to urea, supporting the latter explanation.

The variety of gastroenteric bacteria cultured from cloacal swabs was normal for small marsupials (Heard et al., 1997) and none appeared associated with disease. Although several serovars of Salmonella enterica, including Typhimurium have been isolated from free-ranging possums elsewhere (Presidente, 1984), Salmonella spp. were absent in cultures from cloacal swabs of our animals. An asymptomatic carrier status for Salmonella spp. can exist in free-ranging possums (Johnson and Hemsley, 2008); this could develop into clinical disease under stress. It is therefore encouraging that we found no fecal evidence of such carrier status. These findings also suggest a low zoonotic risk of Salmonella transmission.

We found no evidence in either possum species of infection with T. gondii, Leptospira spp., or Chlamydophila spp., indicating that the health and survival of possums in this region are unaffected by these pathogens and that transmission between translocated and resident individuals is not a risk for either species, despite the prevalence of toxoplasmosis in cats in southern Western Australia (Jakob-Hoff and Dunsmore, 1983). Reaction to nonspecific immunoglobulin (Desmonts and Remington, 1980) is the most likely reason for the low positive DAT titers observed in two P. occidentalis, as MAT titers were negative. Leptospira serovars occur in domestic stock in Western Australia but few wildlife surveys have occurred prior to this study. Recent surveys have increased the known wildlife host range for Chlamydophila spp.; however, in possums chlamydial DNA has only been detected in the mountain brushtail possum (Trichosurus caninus; Bodetti et al., 2003). Clinical disease has been recorded only in the koala (Phascolarctos cinereus), greater glider (Petauroides volans), and western barred bandicoot (Perameles bougainville; Warren et al., 2005).

Although one T. v. hypoleucus was positive for Cryptococcus antigen on the first of three sampling occasions, the titer was low and subsequent samples were negative, consistent with subclinical infection (Malik et al., 1999) or a false-positive result. If present, the causal organism was most likely C. gattii, which has been found in association with tuart trees (Ellis and Pfeiffer, 1996), common at the field sites. Subclinical infection with C. gattii may reduce reproductive output in T. v. hypoleucus; the animal with the low titer in this study was in poor condition and nonbreeding. Alternatively, mild cryptococcal infection could follow immunosuppression due to parasitism and poor body condition. Our generally negative findings are encouraging for wildlife conservation and translocation outcomes, as there appears little likelihood of activation of latent disease by the stress associated with relocation of possums. The low translocation success of P. occidentalis in this region is related to inadequate control of introduced predators, high numbers of T. v. hypoleucus, and limited high-quality habitat (Clarke, 2011) rather than to infectious disease. However, the lack of evidence of exposure to T. gondii, Leptospira, Cryptococcus, and Chlamydophila indicates that possums in this area may be particularly susceptible to introductions of these diseases. This emphasizes the need for vigilance and continued surveillance of wildlife health when managing threatened species.

Murdoch University, the Western Australian Department of Environment and Conservation (DEC), and the Australian Research Council funded the work which was conducted under Murdoch University Animal Ethics Permits W1062/04 and W2028/07 and DEC Animal Ethics Permit 2006/55. We thank the Veterinary Clinical Pathology Laboratory at Murdoch University and the State reference laboratory at PathWest Laboratory Medicine, Western Australia, for hematologic, serum biochemical, and microbiologic analyses; Pat Statham at the Department of Primary Industries, Parks, Water and Environment, Mt. Pleasant Laboratories, Launceston, Tasmania, Australia for toxoplasmosis testing; Lee Smythe at the World Health Organization/Food and Agriculture Organization/World Organisation for Animal Health Collaborating Centre for Reference and Research on Leptospirosis at Queensland Health Scientific Services, Coopers Plains, Queensland, Australia for leptospirosis testing; Mark Krockenberger at the Koala Infectious Diseases Research Group in the Faculty of Veterinary Science at the University of Sydney, New South Wales, Australia for cryptococcosis testing; and Peter Timms at the Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia for chlamydophila testing. We are also grateful to Helen Grimm for collecting some P. occidentalis samples and to our many field volunteers.