Australian marsupials are thought to be particularly vulnerable to pathologic impacts of Toxoplasma gondii, and they may be similarly affected by Neospora caninum. Pathology due to either organism could be expressed as reduced female reproductive performance. We studied adult female western grey kangaroos (Macropus fuliginosus ocydromus) from suburban Perth, Western Australia, between May 2006 and October 2008. We used indirect fluorescent antibody tests to look for evidence of exposure to T. gondii and N. caninum in M. fuliginosus ocydromus and tested the association between their reproductive performance and a positive test result. Although 20% of plasma samples collected from 102 female kangaroos were positive for T. gondii and 18% were positive for N. caninum, we found no association between positive results and reproductive performance. Further study will be required to clarify if, and under what circumstances, T. gondii and N. caninum are pathogenic to macropod marsupials.
Emerging infectious diseases are a major threat to the health of humans, domestic animals, and wildlife populations globally (Daszak et al. 2000). In Australia, the introduction of domestic and feral animals has led to some spillover of their pathogens to native animal populations. Two notable examples are the protozoan parasites Toxoplasma gondii and Neospora caninum, which probably arrived with their respective definitive hosts, cats (Felis catus; Fayer 1981) and dogs (Canis familiaris; McAllister et al. 1998), within the last 300 and 5,000 yr (Tenter et al. 2000; Savolainen et al. 2004), respectively. Infection of adult cats and dogs is usually asymptomatic, but infection of juveniles and other vertebrates can be either asymptomatic or lead variously to encephalitis, systemic disease, abortion, or death. Further, nondefinitive hosts that survive infection become chronically infected with parasitic cysts in their tissues, providing possible sources for spill-back to domestic animals and humans via food-chain effects (Fayer 1981; Dubey and Lindsay 1996). Toxoplasma gondii and N. caninum are implicated in having negative impacts on Australian marsupials (Basso et al. 2007; Parameswaran et al. 2008), but there is a dearth of studies that explore such effects. We investigated the relationship between reproductive performance of the western subspecies of the western grey kangaroo, Macropus fuliginosus ocydromus, and serologic evidence of exposure to T. gondii and N. caninum at four sites within the greater metropolitan area of Perth, Western Australia.
Macropus fuliginosus ocydromus breeds from October to May, with most conceptions occurring from November to March (Mayberry et al. 2010). Births occur 1 mo later, and the young permanently vacate the pouch after 10 mo (Poole 1975). Since adult females typically average around 90% fertility (Arnold et al. 1991), most are expected to have pouch-young from May to September.
We collected blood samples (n = 102) to test for T. gondii and N. caninum exposure and information on the reproductive status of adult female M. fuliginosus ocydromus from two reserves and two golf courses in the Perth metropolitan area. From Thomsons Lake Nature Reserve, on the Swan coastal plain 22 km south of the center of Perth, we sampled 47 individuals between May and July 2006. Serial samples from 24 individuals were collected from the neighboring Harry Waring Marsupial Reserve in February, May, and October 2007, although not every kangaroo was sampled on every occasion. A further 15 individuals were sampled on Melville Glades Golf Club, 14 km south of Perth city center, and 16 from Marangaroo Golf Course, 14 km north of Perth city center, in September 2008. Dogs and foxes (Vulpes vulpes) were removed from the two reserves through a 1080 (sodium monofluoracetate) baiting program at the time they were surrounded by dog- and fox-proof fences in the early 1990s, but some cats remain. Cats and dogs are regularly seen on both golf courses.
Blood samples were separated by centrifugation at 1,880 × G for 15 min within 3 hr of collection and the plasma was stored at −20 C until assayed for serologic evidence of infection using the routine methods of the Animal Health Laboratories, Department of Agriculture and Food, Western Australia (National Association of Testing Authorities, Australia, accreditation no. 13724). These involve indirect fluorescent-antibody tests as previously published for T. gondii (Buxton and Maley 2004) and N. caninum (Conrad et al. 1993) with a standard modification used when fluorescein-conjugated antibodies are not available. Tachyzoites of each parasite maintained in Vero cell cultures were layered into E0R-202B teflon-coated 12-well multi-test slides (Thermo Fisher Scientific Australia, Pty. Ltd., Scoresby, Victoria, Australia), air-dried, fixed in methanol, air-dried again, packaged in sealed plastic bags, and stored at −20 C. Diluted test sera (20 µL) were added to the wells, followed by rabbit anti-kangaroo immunoglobulin G (IgG) (Kangaroo IgG - heavy and light chain antibody, Bethyl Laboratories Inc., Montgomery, Texas, USA) (20 µL), then fluorescein isothiocyanate–conjugated polyclonal anti-rabbit IgG (Dako Australia Pty. Ltd., Kingsgrove, New South Wales, Australia) (20 µL). The slides were incubated for 45–60 min at room temperature, washed with Bacto™ FA buffer (a 0.85% saline solution phosphate-buffered to pH 7.2, BD Australia, North Ryde, New South Wales, Australia), and air-dried. One drop of para-phenylenediamine and a cover slip were added, and the slides were examined under a fluorescence microscope. A positive result shows as a complete halo of green fluorescence around 80% of the tachyzoites. Cross-reactions between T. gondii, N. caninum, and other similar organisms show as a zone of apical fluorescence. We used a cutoff dilution of 1∶50 for discrimination between positive and negative, at which point specificity and sensitivity are both very high (Conrad et al. 1993; Packham et al. 1998; Buxton and Maley 2004). A kangaroo sample found positive at a dilution of 1∶256,000 was used as a positive control. For kangaroos on Harry Waring Marsupial Reserve, a positive test result in February, May, or both was counted as a positive result. We used Fisher's exact test to compare the numbers of kangaroos with pouch-young among the golf courses and the reserves, and to check for any association between presence of pouch-young and serologic evidence of infection by either T. gondii or N. caninum.
There were marked differences between the four sites in female reproductive performance and antibody prevalence to both parasites. Overall, 20% of the kangaroos were positive for T. gondii and 18% were positive for N. caninum (Table 1). No kangaroos were positive for both T. gondii and N. caninum. Female kangaroos on Harry Waring Marsupial Reserve with positive tests for either parasite generally fluctuated between positive and negative. Six seroconverted from negative to positive for N. caninum, one seroconverted from positive to negative for N. caninum, and one that was positive in February and October 2007 was negative in May 2007. One female kangaroo seroconverted from negative to positive for T. gondii, while another seroconverted from positive to negative. One female kangaroo was positive for N. caninum on all three occasions. The fertility of 15 of the 24 female kangaroos on Harry Waring Marsupial Reserve was confounded by another experiment so their data have been excluded from analyses of pathogen exposure on reproductive performance.
The percentage of kangaroos with pouch-young was significantly higher on the golf courses (96%) than on the reserves (49%) (P<0.001). This may be due to nutritional stress in the reserves, but this has been discussed and discounted elsewhere (Mayberry et al. 2010). The only female kangaroo at Melville Glades found positive for N. caninum lost her pouch-young in August, 2 wk before being blood-sampled, and was counted as infertile. There was no other evidence of an association between absence of pouch-young and serologic evidence of infection by either T. gondii or N. caninum (Table 2). A kangaroo that was antibody positive for either T. gondii or N. caninum was just as likely to have a pouch young as a kangaroo that was negative for both parasites.
It is likely that circulating antibodies to T. gondii and N. caninum in western grey kangaroos normally fluctuate between detectable and undetectable levels, as seen with antibodies to T. gondii in Bennett's wallaby (M. rufogriseus; Dubey and Crutchley 2008), making the use of rising titers as evidence of recent infection suspect. Vertical transmission of N. caninum has been reported in cattle (Conrad et al. 1993), and vertical transmission of T. gondii is now known to occur in some Australian marsupials (Parameswaran et al. 2009), but no evidence of horizontal transmission of either parasite between herbivores has been reported. Because both of the metropolitan reserves have been virtually free of dogs for >16 yr, it is likely that any challenge with N. caninum would be by vertical transmission.
The 20% prevalence of antibody to T. gondii in western grey kangaroos in our study is similar to that previously reported for kangaroos in the Perth metropolitan area (Parameswaran et al. 2009). Because infected adult western grey kangaroos appear to be asymptomatic (Parameswaran et al. 2009), and the carcasses of infected kangaroos culled from reserves can end up in the pet food trade (Massam et al. 2006), the potential for the spill-back of T. gondii and N. caninum from western grey kangaroos to domestic pets and humans warrants further investigation.
In conclusion, exposure to T. gondii and N. caninum does not appear to impact the reproductive performance of western grey kangaroos. This study thereby fills a gap in understanding how these novel pathogens might affect macropod marsupial populations. Future research could focus on the extent to which kangaroos might serve as a reservoir for these parasites to spill-back to domestic animals and humans, or other evidence of pathogenicity, such as analyses of survivorship associated with exposure.
This research was supported by the Australian Research Council's Linkage Projects Funding scheme (project number LP0560344, entitled Fertility management of koalas, kangaroos and wallabies) and assisted with funding from The University of Western Australia, through a University Post Graduate Award, and made possible by the cooperation of staff from the Animal Health Laboratory of the Department of Agriculture and Food, and from the Regional Parks Office of the Department of Environment and Conservation. Tachyzoites of T. gondii and N. caninum were provided by Nevi Parameswaran and the Murdoch University Veterinary Hospital.