Abstract
The Pallas’ cat, a small-sized felid species, is threatened with extinction in its natural habitat in Asia. Causes of population decline include habitat loss, vermin control programs, and hunting for the fur trade.9 To establish a genetically representative founder population in captivity, a total of 24 wild-born Pallas’ cats were imported from Russia and Mongolia into North American zoos between 1995–2000. Captive breeding of these Pallas’ cats has been very productive during the past 6 yr, with the birth of 65 kittens in 17 litters. Unfortunately, there has been extraordinarily high (∼60%) mortality of newborn kittens within 4 months of birth. In the majority of deceased kittens evaluated at necropsy, infection with Toxoplasma gondii has been identified as the cause of death.11
Almost all adult Pallas’ cats (>80%) in North American zoos are chronically seropositive for anti-T. gondii antibodies, but, like most adult felids, only rarely display clinical signs of infection.3,10 However, during pregnancy, maternal immune responses apparently are not protective in Pallas’ cats, unlike in pregnant domestic cats and other cat species with previous T. gondii exposure.11 High neonatal mortality in Pallas’ cats is jeopardizing the maintenance of a viable captive population, but the basis for this extreme susceptibility to fatal toxoplasmosis is unknown. Comparative information from wild Pallas’ cats would be invaluable for determining whether toxoplasmosis is endemic to wild populations or if it represents an emerging disease primarily associated with captivity.
In this study, our specific objectives were to:
1. Evaluate the general health status of wild Pallas’ cats in Mongolia, including normative blood values, viral serology, and fecal parasite loads
2. Determine prevalence of T. gondii exposure in Mongolian Pallas’ cats, domestic cats and prey species
3. Assess fecal cortisol metabolite levels as an indirect indicator of basal stress
4. Compare findings to those from concurrent studies of wild-born and captive-born Pallas’ cats maintained in North American zoos
Biologic samples were obtained from wild Pallas’ cats (n=15) captured in the central province of Mongolia during the summer months of 2000 and 2001. For capture, active Pallas’ cat den sites were identified and excavated. Pallas’ cats were manually restrained and anesthetized (ketamine hydrochloride; 15 mg/kg body mass; IM) by hand syringe. Blood (8–12 ml) was collected via jugular venipuncture, placed into EDTA tubes, and centrifuged. Plasma, red and white blood cells, and whole blood samples were frozen in liquid nitrogen (-196°C). A separate whole blood sample (1 ml; in EDTA) was chilled (4°C) for CBC analysis. Fecal samples were obtained via a lubricated rectal loop and stored frozen (-20°C). When fully recovered from anesthesia (after ∼2 hours), cats were released close to their point of capture. Comparative data were obtained from nine captive Pallas’ cats maintained in three North American zoos (Birmingham Zoo, Cincinnati Zoo and Botanical Garden, Denver Zoological Gardens). Blood and fecal samples were collected during 1999–2000 and subjected to similar laboratory analyses (as described below).
Blood samples also were collected from domestic cats (Felis catus; n=15) captured at the study sites in Mongolia, nearby villages, and in Ulaanbaatar during the summer of 2001. Cats were anesthetized (ketamine hydrochloride; 15 mg/kg body mass; IM) and blood (2–4 ml) collected via venipuncture, placed into EDTA tubes and centrifuged. Plasma and whole blood from each cat were frozen and stored in a liquid nitrogen tank. Prey species (pikas and rodents; n=45) were manually captured by local Mongolian herdsmen. For anesthesia, each animal was placed into a nylon bag containing a cotton ball soaked with halothane. Blood samples (2 ml) were obtained via cardiac puncture, placed into EDTA tubes, centrifuged and stored in liquid nitrogen. Immediately following blood sample collection, prey animals were euthanatized by decapitation. Whole brains were isolated, sectioned, and stored in buffered formalin for later PCR analysis.
Sample analysis included assessments of blood cell and biochemical parameters, viral serology (FIV, FeLV, FIP), T. gondii exposure, and determination of fecal cortisol metabolite concentrations. Complete blood counts were performed at a human diagnostic lab in Ulaanbaatar on fresh (<24h) chilled samples. Biochemical analysis and enzyme-linked immunoassays (EIA) for viral antibodies and/or antigens (FIV, FeLV, FIP) in frozen serum samples were conducted at the Cincinnati Veterinary Diagnostic Lab. Fecal flotation for presence of T. gondii oocysts was conducted at The Ohio State University College of Veterinary Medicine. PCR testing of whole blood/brain tissue for T. gondii DNA and EIA/latex agglutination testing of plasma samples for T. gondii antibodies were conducted at Colorado State University.7,8 Fecal extractions and corticosterone radioimmunoassays were performed at the National Zoo’s Conservation and Research Center.4
Comparing wild to captive Pallas’ cats, there were no differences (p>0.05) in any hematologic values nor in most biochemical parameters, with the following exceptions. Wild Pallas’ cats had higher (p<0.05; mean ± SEM) serum glutamic-oxaloacetic transaminase (SGOT; 119.4±20.0 IU/L) and total bilirubin (0.70±0.12 mg/dl) and lower (p<0.05) glucose (47.0±10.9 mg/dl) levels than that found in captive cats (35.8±14.1 IU/L, 0.19±0.06 mg/dl, 121.1±11.0 mg/dl, respectively). Viral screening via domestic cat EIA revealed no evidence of FIV, FeLV, or FIP in the wild population.
Of the fifteen wild Pallas’ cats, thirteen (87%) were negative for anti-T. gondii antibodies via EIA. The two seropositive cats had moderately elevated IgG titers (1:512–1:1024). In contrast, all (9/9, 100%) of the comparative captive population were seropositive and all had high IgG titers (1:2048–1:8192). Assessment of blood samples from Mongolian domestic cats and prey items indicated a complete absence of T. gondii exposure based on antibody titers. Furthermore, PCR analysis of whole blood samples from all wild Pallas’ cats, domestic cats, and prey items, and of brain tissue from prey species, found no evidence of T. gondii DNA. Comparison of fecal cortisol metabolite concentrations revealed no difference (p>0.05) between wild (48.6±9.2 ng/g wet feces) and captive (56.8±7.9 ng/g wet feces) Pallas’ cats. No T. gondii oocysts were found in fecal samples from any wild Pallas’ cat.
Our findings suggest that the general health status of wild and captive Pallas’ cats are comparable, based on physical exams, complete blood counts, and blood biochemistry analyses. Furthermore, fecal cortisol metabolite levels did not differ between populations, indicating that Pallas’ cats in captivity and the wild exhibit similar adrenocortical activity levels. These observations provide evidence that general health status, stress levels and other captive husbandry issues are not the primary factors causing the extreme susceptibility to toxoplasmosis.
One pronounced difference between wild and captive populations was the seroprevalence to T. gondii. Whereas all captive Pallas’ cats in this study exhibited pronounced antibody titers to this organism, only two of 15 (13%) wild Pallas’ cats were seropositive. An additional four wild-caught Pallas’ cats imported directly from Mongolia in 2000 also were seronegative (John Aynes, personal communication). These findings suggest that wild Pallas’ cats have minimal opportunity for exposure to T. gondii in their natural habitat and likely do not become infected with this parasite until being brought into captivity. Our results from concurrent evaluations of Mongolian domestic cats and prey species support this conclusion. Analysis of both blood products and brain tissue found no evidence of T. gondii exposure in any of the domestic cats, pikas, or rodents sampled in Mongolia. In contrast, studies of domestic cats and prey species in other geographic regions have reported T. gondii seroprevalence ranging from 33.7%–80% and 2.1%–24%, respectively.1,2,5,6
In conclusion, evidence suggests that the extreme susceptibility of Pallas’ cats to toxoplasmosis is a consequence of evolving in a biologically unique environment. In their culture, Mongolians do not typically maintain domestic cats as companion animals, limiting the number of feral cats available to serve as definitive hosts for T. gondii. In addition, the severity of the Mongolian winters likely reduces the viability of any T. gondii oocysts that may be shed into the environment by domestic cats. The combination of limited numbers of definitive hosts and low oocyst survival during the winter months could impair the ability of T. gondii to complete its natural life cycle and persist in the wild. Accordingly, Pallas’ cats may not have co-evolved with this specific parasite, creating a degree of susceptibility to toxoplasmosis that is most similar to that observed in several island species, such as macropods and lemurs.
The implications of this finding are profound for the captive population. If zoos are to maintain Pallas’ cats in captivity, it is imperative that research be directed at developing management strategies to prevent exposure of naive Pallas’ cats to T. gondii without compromising reproduction.13 For Pallas’ cats previously infected with T. gondii, pharmaceutical options need to be explored that will limit disease transmission to offspring in utero or postpartum or provide effective therapy for clinically ill neonates.12 Until this disease issue is adequately resolved, importation of additional Pallas’ cats from the wild is unlikely to benefit the conservation of this threatened species.
Acknowledgments
The authors would like to thank Drs. Mel Shaw, Mark Campbell, and David Kenny, and the animal keeper staffs at the Birmingham Zoo, the Cincinnati Zoo & Botanical Garden, and Denver Zoological Gardens for providing blood and fecal samples from captive Pallas’ cats. We also thank Dr. Amanda Fine, Dominic O’Neill, and staff at the McGayls Medical Center, and International Snow Leopard Trust Mongolia for assistance in Mongolia. Finally, thank you to Drs. Nongnuch Inpanbutr and Cliff Monahan from The Ohio State University. Funding for this study was provided, in part, by The Ohio State University SOAR program and Alumni Society, Columbus Zoo and Aquarium, Wild about Cats, Woodland Park Zoological Gardens, and Disney’s Animal Kingdom.
Literature Cited
1. Chhabra MB, Gupta SL, Gautam OP. Toxoplasma seroprevalence in animals in northern India. Int J Zoonoses. 1985;12:136–142.
2. Dubey JP, Weigel RM, Siegel AM, Thulliez P, Kitron UD, Mitchell MA, et al. Sources and reservoirs of Toxoplasma gondii infection on 47 swine farms in Illinois. J Parasitol. 1995;81:723–729.
3. Dubey JP, Gendron-Fitzpatrick AP, Lenhard AL, Bowman D. Fatal toxoplasmosis and enteroepithelial stages of Toxoplasma gondii in a Pallas’ cat (Felis manul). J Protozool. 1988;35:528–530.
4. Graham LH, Brown JL. Cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids. Zoo Biol. 1996;15:71–82.
5. Hejlicek KVL, Nezval J. Toxoplasmosis in wild mammals from the Czech Republic. J Wildl Dis. 1997;33:480–485.
6. Hill RE, Zimmerman JJ, Wills RW, Patton S, Clark WR. Seroprevalence of antibodies against Toxoplasma gondii in free-ranging mammals in Iowa. J Wildl Dis. 1998;34:811–815.
7. Howan WL, Vercammen M, de Braekeleer J, Verschueren H. Identification of a 200 to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol. 2000;30:69–75.
8. Lappin MR, Jacobson ER, Kollias GV, Powell CC, Stover J. Comparison of serologic assays for the diagnosis of toxoplasmosis in nondomestic felids. J Zoo Wildl Med. 1991;22:169–174.
9. Nowell K, Jackson P. Wild cats: Status survey and conservation action plan. IUCN/SSC Cat Specialist Group: Gland, Switzerland; 1996:97–99.
10. Riemann HP, Fowler ME, Schulz T, Lock A, Thilsted J, Pulley LT, et al. Toxoplasmosis in Pallas’ cats. J Wildl Dis. 1974;10:471–477.
11. Swanson WF. Toxoplasmosis and neonatal mortality in Pallas’ cats: a survey of North American zoological institutions. In: Proceedings of the American Association of Zoo Veterinarians. 1999:347–350.
12. Swanson WF, Bond J, Bush M. Assessment of diclazuril toxicity in neonatal domestic cats (Felis catus) and initial application for prevention and treatment of toxoplasmosis in neonatal Pallas’ cats (Otocolobus manul). In: Proceedings of the American Association of Zoo Veterinarians. 2001:390–391.
13. Swanson WF, Kennedy-Stoskopf S. Reproductive seasonality of male Pallas’ cats (Otocolobus manul) maintained under artificial lighting with simulated natural photoperiods. In: Proceedings of the Society for the Study of Reproduction. (in press).