NEOSPORA CANINUM AND TOXOPLASMA GONDII ANTIBODY PREVALENCE IN ALASKA WILDLIFE

Journal of Wildlife Diseases, 46(2), 2010, pp. 348 355 # Wildlife Disease Association 2010 NEOSPORA CANINUM AND TOXOPLASMA GONDII ANTIBODY PREVALENCE IN ALASKA WILDLIFE Erica Stieve, 1 Kimberlee Beckmen, 2 Stephen A. Kania, 1 Amanda Widner, 1 and Sharon Patton 1,3 1 Department of Comparative Medicine, University of Tennessee College of Veterinary Medicine, 2407 River Drive, Room A205, Knoxville, Tennessee 37996-4543, USA 2 Division of Wildlife Conservation, Alaska Department of Fish and Game, 1300 College Road, Fairbanks, Alaska 99701-1599, USA 3 Corresponding author (email: spatton@utk.edu) ABSTRACT: Free-ranging caribou and moose populations in some regions of Alaska undergo periodic declines in numbers. Caribou and moose are managed by the state as valuable resources for not only sustenance and subsistence, but also for cultural heritage. Incidence and prevalence of diseases that may impact herd health and recruitment from year to year are relevant to management decisions aimed to protect the long-term viability of these herds. Neospora caninum and Toxoplasma gondii are two apicomplexan parasites that can cause neurologic disease and abortions in their intermediate hosts and less frequently cause disease in their definitive hosts. The definitive hosts of N. caninum and T. gondii are canids and felids, respectively, and prevalence in the environment is in part dependent on maintenance of the life cycle through the definitive hosts. Serum samples from caribou (Rangifer tarandus, n5453), wolf (Canis lupus, n5324), moose (Alces alces, n5201), black-tailed deer (Odocoileus hemionus, n555), coyote (Canis latrans, n512), and fox (Vulpes vulpes, n59) collected in Alaska were assayed for N. caninum and T. gondii reactive antibodies with an immunofluorescent antibody test (IFAT) and a modified agglutination test (MAT), respectively. Seroprevalence of N. caninum was greater in caribou (11.5%) than in wolves (9.0%), moose (0.5%), or black-tailed deer (0%). Seroprevalence of T. gondii was greater in wolves (17.8%) than in caribou (0.4%), moose (0%), or black-tailed deer (0%). Seroprevalence of N. caninum and T. gondii were 16.7% and 0.0% in coyotes and 0.0% and 12.5% in fox, but small sample sizes prevented further analysis. Antibodies to N. caninum in young caribou compared to adult caribou suggest that vertical transmission may be an important component of new infections in Alaskan caribou. The spatial distribution of antibody-positive individuals across Alaska may reflect differences in frequency of definitive hosts and alteration of predation patterns among regions. Key words: Alaska wildlife, antibody prevalence, apicomplexa, Neospora caninum, Toxoplasma gondii. INTRODUCTION Neospora caninum and Toxoplasma gondii are important apicomplexan parasites that may cause abortion or neurologic disease in their intermediate hosts. The similarities in clinical presentation and life cycle caused N. caninum to be misdiagnosed as T. gondii until 1988 (Dubey et al., 1988). Dogs and coyotes are definitive hosts for N. caninum (McAllister et al., 1998; Gondim et al., 2004b), whereas felid species are definitive hosts for T. gondii. Both parasites infect a wide range of intermediate hosts (Chomel et al., 1995; Zarnke et al., 2000; Kutz et al., 2001; Dubey et al., 2003a, b; Yai et al., 2003; Gondim et al., 2004a; Moore, 2005) and are present in both domestic and wildlife populations. The abortogenic potential of these parasites can impact production of livestock and wildlife and is important to the management of both populations. Both parasites can be transmitted between domestic and wildlife hosts where their ranges overlap. Prevalence of the parasite in a host population will influence the rate of transmission to other species. Determination of parasite prevalence in populations can provide insight into population fitness and likelihood of disease transmission. Our objectives in this study were to 1) estimate the antibody prevalence of N. caninum and T. gondii in six species of Alaskan wildlife, and 2) describe the spatial distribution of antibody-positive individuals. 348

STIEVE ET AL. NEOSPORA CANINUM AND TOXOPLASMA GONDII IN ALASKA 349 MATERIALS AND METHODS Serum samples were collected by the Alaska Department of Fish and Game and the Yukon Department of the Environment. Serum samples from wolf (Canis lupus, n5324; collected 1996 2008), caribou (Rangifer tarandus, n5453; 1994 2006), moose (Alces alces, n5201; 2001 2005), black-tailed deer (Odocoileus hemionus, n555; 1980 2000), fox (Vulpes vulpes, n59; 1985 2006), and coyote (Canis latrans, n512; 2005) were assayed for N. caninum and T. gondii reactive antibodies. Some caribou samples (n564) were available for the N. caninum assay. The efficacy of using rabbit antideer IgG to bind moose and caribou antibodies was evaluated with the use of an enzyme-linked immunosorbent assay (ELISA). The reagents were optimized with a series of titration experiments. ELISA plate (Immulon, Thermo Scientific, Milford, Massachusetts, USA) wells were coated with rabbit antideer (2 mg/ml in phosphate-buffered saline [PBS], Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA) and allowed to bind overnight at 4 C. Plates were washed three times with PBS containing 0.5% polyoxyethylene sorbitan monolaurate (PBStween, Sigma Life Sciences, St. Louis, Missouri, USA). A pool of several deer sera was used to prevent bias from any one serum. Similarly, multiple samples of moose serum and caribou serum were pooled by species. The pooled samples of deer serum, caribou serum, and moose serum were serially diluted with PBStween (1:10,000 to 1:1,280,000). Each dilution was dispensed into separate wells in triplicate, 100 ml per well. Each plate was washed again with PBStween, and 100 ml of biotin-conjugated, rabbit antideer IgG (Kirkegaard and Perry; 1:400 in PBStween) was added per well. The plate was incubated at 37 C for 1 hr, the wash was repeated, and 100 ml of avidin-horseradish peroxidase (diluted 1/2,000, Sigma) was added to each well. The samples were incubated again at 37 C for 1 hr. The substrate 3,39,5,59- tetramethyl benzidine (TMB, Thermo Scientific, Rockford, Illinois, USA) was added and the plate was incubated for 15 30 min at room temperature. The color reaction was halted with 100 ml of stop solution (0.18 M H 2 SO 4 ) and the ELISA plate was read on an EL x 800 Universal Microplate Reader (Bio-Tek Instruments, Inc, Winooski, Vermont, USA). Titers for N. caninum antibodies were determined using an indirect fluorescent antibody test (IFAT) on slides coated with fixed N. caninum tachyzoites (VMRD, Pullman, Washington, USA). The positive and negative controls for the canid species were commercial positive and negative dog sera (VMRD). Initial controls for the cervid species were known N. caninum-positive and negative deer sera provided by Dr. Milton McAllister, University of Illinois, Department of Pathobiology, Urbana, Illinois. Positive caribou samples from the first year of the study were used during the second year when control deer sera were no longer available. Cervid antibodies bound to N. caninum tachyzoites were visualized with fluorescein isothiocyanate (FITC) - rabbit anti-deer IgG. Canid antibodies were detected with FITC-goat anti-dog IgG. The samples were screened at a 1:25 dilution for specific fluorescence to N. caninum (Pare et al., 1995), and samples with specific staining were diluted to endpoint reactivity for determination of titer. Apical fluorescence of the tachyzooite was classified as nonspecific staining, while peripheral staining of the tachyzooite was classified as specific (Pare et al., 1995). An individual was considered positive for N. caninum antibody if the serum had specific fluorescence at $1:100 dilution. This dilution was chosen to minimize nonspecific staining interfering with interpretation and to minimize false positives. Serum samples were assayed for T. gondii antibodies with the use of the modified agglutination technique (MAT) with formalin-fixed tachyzoites (BioMerieux Laboratory Reagents, Marcy-l Etoile, France) as antigen (Desmonts and Remmington, 1980; Patton et al., 1990, 1991). Samples were considered positive if they demonstrated agglutination at $1:32 dilution. Information regarding age, sex, and location of capture, when available, was used to describe the distribution of seropositive individuals. Antibody prevalence among species was compared with chi-square tests or Fisher s exact test (alpha,0.05) for statistical significance. RESULTS The ELISA comparison of the binding of rabbit anti-deer to moose, caribou, and deer sera demonstrated that the anti-deer bound with the deer, caribou, and moose antibodies (Fig. 1). Serologic reactivity data for all species examined are presented in Tables 1 and 2. Neospora. caninum antibody prevalence (Table 1) was greater in caribou (13.6%) than wolves (9.0%), black-tailed deer (0.0%), or moose (0.5%). Toxoplasma gondii seroprevalence was

350 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 2, APRIL 2010 FIGURE 1. Binding of biotin-labeled rabbit antideer with antibodies in deer, caribou, and moose sera. Sera were serially diluted from 1:10,000 to 1:1,200,000 and the optical density of the reaction measured. greater in wolves (17.8%) than in caribou (0.4%), and no antibody was detected in moose or black-tailed deer (Table 2). The small number of coyote (n512, 2 positive) and fox (n59, 0 positive) samples precluded statistical evaluation. Age data were unavailable for many of the samples, but could be compared in 405 caribou (Table 3). Seroprevalence of N. caninum was the same in caribou,1 yr old (12.0%, 26/216) compared to caribou $1 yr or old (10.1%, 19/189). The percent of adult caribou positive for T. gondii (1.1%, 2/189) was higher than that found in caribou,1 yr old (0.0%, 0/216). The spatial distribution of antibody prevalence for T. gondii and N. caninum is mapped in Figure 2. The maps show antibody prevalence within an estimated herd range for caribou and indicate prevalence for moose, black-tailed deer, and wolf within specific game management units (GMU). The figure emphasizes the low antibody prevalence of T. gondii in moose, caribou, and black-tailed deer, the presence of N. caninum antibody in caribou, and the presence of antibodies to both parasites in wolves. DISCUSSION The presence or absence of definitive hosts of T. gondii and N. caninum is a factor in the prevalence of infected Alaskan wildlife. Felids, the definitive host of T. gondii, are represented in Alaska only by lynx and domestic cats. The spatial distribution (Fig. 1) of T. gondii antibody prevalence in wolves reflects the distribution of lynx in Alaska as described by Zarnke et al. (2001) insofar as lynx are found throughout most of the mainland and do not occur on the Kodiak islands or the islands of the southeast region. In addition, population density of lynx is particularly low on the southeastern mainland, on the Alaska Peninsula, and north of the Brooks Range. Population densities of lynx are low throughout Alaska and TABLE 1. Prevalence of antibody (Ab) to Neospora caninum in select wildlife of Alaska, USA and Yukon, Canada. Samples were considered positive if they demonstrated specific immunofluorescent antibody staining at a titer of $100. Other studies have used threshold titers as low as 50 to identify positive samples. Fisher s exact test was used instead of chi-square when expected frequencies in the 232 table were,5. Species Antibody prevalence (%) Number positive n Titer 50 100 200 400 $800 Caribou a (Rangifer tarandus) 11.5 45 390 3 12 15 10 8 Moose b (Alces alces) 0.5 1 202 0 0 0 0 1 Black-tailed deer (Odocoileus hemionus) 0.0 0 54 0 0 0 0 0 Wolf (Canis lupus) 9.0 29 324 9 10 10 6 3 Coyote (Canis latrans) 16.7 2 12 0 1 1 0 0 Fox (Vulpes vulpes) 0.0 0 9 0 0 0 0 0 a Neospora caninum antibody prevalence greater in caribou than wolves (P50.01, chi-square test). b Neospora caninum antibody prevalence greater in caribou than moose or black-tailed deer (P,0.007, Fisher s exact test).

STIEVE ET AL. NEOSPORA CANINUM AND TOXOPLASMA GONDII IN ALASKA 351 TABLE 2. Prevalence of antibody to Toxoplasma gondii in wildlife in Alaska, USA, and Yukon, Canada. Samples were considered positive if they demonstrated agglutination with modified agglutination test at a titer $32. Antibody prevalence (%) Number positive n Titer 32 64 128 256 $512 Caribou (Rangifer tarandus) 0.4 2 452 0 2 0 0 0 Moose (Alces alces) 0.0 0 202 0 0 0 0 0 Black-tailed deer (Odocoileus hemionus) 0.0 0 55 0 0 0 0 0 Wolf a (Canis lupus) 17.8 57 320 13 10 7 13 14 Coyote (Canis latrans) 0.0 0 12 0 0 0 0 0 Fox (Vulpes vulpes) 12.5 1 8 0 1 0 0 0 a Toxoplasma gondii antibody prevalence greater in wolves than caribou, moose, or black-tailed deer (P,0.0001, Fisher s exact test). Canada, with a range of 2.0 44.9 per 100 km 2 (Slough and Mowat, 1996) and antibody prevalence of T. gondii in lynx populations of Interior Alaska is estimated at 15% (Zarnke et al., 2001). Feral domestic cats do not survive outside of human establishments (Zarnke et al., 2000, 2001). Fewer definitive hosts in the environment decrease the potential number of oocysts shed in the environment and potentially limit the rate of infection of intermediate hosts through the consumption of contaminated forage and other infected vertebrate hosts. In contrast, N. caninum has many potential definitive hosts in Alaska, represented by coyotes (Gondim et al., 2004b), dogs (McAllister et al., 1998), and possibly wolves (Gondim, 2004a). In our study, antibody prevalence for N. caninum in caribou (11.5%) was greater than for T. gondii (0.4%). This is possibly the result of the greater density of hosts shedding N. caninum oocysts compared to the relative paucity of hosts shedding T. gondii oocysts. The lack of black-tailed deer positive for T. gondi is consistent with the low density of lynx, the definitive host. Differing foraging patterns of herbivores may alter risk of infection from the ingestion of oocysts of both parasites. Only one moose serum sample was positive for TABLE 3. Prevalence of antibody to Neospora caninum and Toxoplasma gondii in age classes of caribou (n5number of caribou identified in each class). Age class n No. positive for Neospora caninum % No. positive for Toxoplasma gondii % Adult 124 17 13.7 2 1.6 4 6 yr 5 1 20.0 0 0.0 3 yr 13 0 0.0 0 0.0 2 yr 21 1 4.8 0 0.0 1 yr 26 0 0.0 0 0.0.12 mo 189 19 10.1 a 2 1.1 Calf 20 1 5.0 0 0.0 11 mo 32 3 9.4 0 0.0 10 mo 14 0 0.0 0 0.0 9 mo 49 12 24.5 0 0.0 6 mo 3 0 0.0 0 0.0 5 mo 59 7 11.9 0 0.0 4 mo 39 3 7.7 0 0.0,1 yr 216 26 12.0 a 0 0.0 a Prevalence of antibody to N. caninum not significantly different between adults and calves (P50.63, chi-square test).

352 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 2, APRIL 2010 FIGURE 2. Maps of Neospora caninum and Toxoplasma gondii antibody prevalence in moose, caribou, black-tailed deer, and wolf populations in Alaska, USA and the Yukon Territory, Canada. Relative antibody prevalence within each game management unit or within each herd range is indicated by shading. N. caninum antibodies (antibody prevalence50.5%), whereas 11.5% of the caribou had antibody to N. caninum. Moose eat aquatic vegetation and browse on leaves from taller shrubs, and caribou graze low-growing vegetation, lichens in particular, that may be easily contaminated by feces and oocysts. We speculate that by browsing from vegetation less likely to be contaminated by fecal material, moose may have limited exposure to oocysts and risk of infection. A similar finding might be expected with antibody prevalence of T. gondii in these samples, but the previously mentioned paucity of definitive hosts we speculate has led to minimal infection in both cervids. Antibody prevalence of N. caninum and T. gondii in Minnesota moose was much lower than in white-tailed deer, whose foraging habits are more similar to that of caribou (Gondim et al., 2004a, Table 4). A difference in foraging patterns is an inadequate explanation for the finding that blacktailed deer sera in this project were negative for both N. caninum antibodies and T. gondii. Carnivores, in addition to being infected by ingesting oocysts, can potentially become infected and develop antibodies to T. gondii and N. caninum by consuming meat containing bradyzooites (Dubey, 1982, 1983; Lindsay et al., 1996; Gondim et al., 2005). This increased risk compared to noncarnivores alters the expected antibody prevalence of N. caninum and T. gondii in carnivores compared to the herbivores that they consume. In this study carnivores had the greatest prevalence of antibody to T. gondii but not N. caninum. If wolves preyed primarily upon caribou, we would expect antibody prevalence for both N. caninum and T. gondii in wolves to be greater than in caribou. Our data suggest that wolves in Alaska prey significantly on species other than caribou. Particular prey species vary in availability by region. This variation in prey choice may be reflected in the spatial distribution of antibody positive wolf

STIEVE ET AL. NEOSPORA CANINUM AND TOXOPLASMA GONDII IN ALASKA 353 TABLE 4. Comparison of data from current study to data from the literature. Tests used were indirect immunofluorescent test (IFAT), modified agglutination test (MAT), or Neospora agglutination test (NAT). Subscript indicates threshold titer for a positive sample. Antibody prevalence (%) by species is given. Agent Assay Caribou Deer Moose Wolf Coyote Fox Neospora caninum Current study, Alaska IFA 100 11.5 0.5 5.6 16.7 0.0 Gondim et al. (2004), Minnesota IFA 100 20.0 13.1 Current study, Alaska IFA 50 12.3 0.0 8.5 16.7 0.0 Gondim et al.(2004), Minnesota and Colorado IFA 50-39.0 a 17.9 b Dubey et al. (2005), Alaska NAT 40 3.1 0.0 3.2 Toxoplasma gondii Current study, Alaska MAT 32 0.4 0.0 0.0 13.6 0.0 12.5 Gondim et al. (2004), Minnesota IFA 50 4.6 1.6 28.7 Zarnke et al. (2000), Alaska MAT 25 6.0 1.0 0.1 a Data from the state of Minnesota, USA. b Data from the state of Colorado, USA. populations (Fig. 2). Prevalence of antibody to T. gondii in wolves in some regions of the interior and on the Northern Alaska Peninsula is particularly high (GMU25C 2/5 [40%], and GMU25B 3/7 [42%], and Northern Alaska Peninsula 18/ 39 [46%]). This most likely reflects their consumption of infected prey of different species. On the Northern Alaska Peninsula, for instance, wolves have been observed to scavenge on beached marine mammal carcasses (Dominique Watts, USFWS, pers. comm.). Marine mammals from Alaska and Canada have been antibody positive for T. gondii and N. caninum (Dubey et al., 2003b; Measures et al., 2004), but there were no T. gondii antibody-positive caribou (n562) from the Northern Alaskan Peninsula. Prevalence of antibody to T. gondii within human populations increases with age (Roughmann et al., 1999; Jones et al., 2001; Asthana et al., 2006), possibly as the summation of constant risk of exposure over a lifetime. This trend is an epidemiologic feature of a parasite infection primarily dependent upon horizontal transmission with lasting seroconversion. Two adult caribou in this study were antibody positive for T. gondii and no calves were positive (Table 3), but the low numbers of positive animals precludes statistical analysis. There was no difference in prevalence of antibody to N. caninum between adult and immature caribou (Table 3). This is relevant because, in bovine herds, N. caninum can be maintained through vertical transmission (French et al., 1999; Gay, 2006). Vertical transmission increases the prevalence of infection in young animals. We suggest that the lack of increase in prevalence of antibody to N. caninum with age in caribou reflects the importance of vertical transmission of N. caninum within these herds. It is likely that N. caninum may have reproductive consequences in these caribou herds as it does in bovine herds. There are published reports of antibody prevalence of N. caninum and T. gondii in similar populations to those sampled in this study. In Minnesota and Colorado, both herbivores and carnivores had greater antibody prevalence than similar species in this study (Gondim et al., 2004a; Table 4). This may reflect decreasing survival of oocysts in colder winter temperatures or lower densities of intermediate and definitive hosts decreasing the rate of infection. Prevalence of antibody to T. gondii in herbivores in our study was less than that found by Zarnke et al. (2000) in Alaska (Table 4). In caribou the difference is unlikely to be explained solely by a

354 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 2, APRIL 2010 difference in the threshold of positive titers (positive at 1:25 dilution in Zarnke et al., 2000, and positive at 1:32 in the current study). At higher concentration of serum to antigen, the sensitivity of the method increases but the specificity decreases. For this reason more samples are likely to be positive at a 1:25 dilution compared to a 1:32 dilution. Perhaps individuals of the current study are younger than those tested in Zarnke et al. (2000). Ages are not available for the animals from the Zarnke et al. (2000) study for comparison. A third explanation is that T. gondii infection is decreasing in these populations. The samples from Zarnke et al. (2000) were collected between 1976 and 1996, and samples from this study were taken between 1994 and 2006. Dubey and Thulleiz (2005) assayed antibody prevalence of N. caninum in the same population as the Zarnke et al. (2000) study (Table 4) with the use of a Neospora agglutination test. Their results were similar to those in this study. In conclusion, prevalences of antibody to N. caninum and T. gondii were low in the populations of Alaskan caribou, moose, wolves, coyotes, and foxes that we surveyed. The black-tailed deer samples were negative for serum antibodies to N. caninum and T. gondii. Neospora caninum may impact the reproductive health of infected caribou herds through vertical transmission. The spatial pattern of N. caninum and T. gondii antibody prevalence reflects a distribution associated with the presence of the definitive hosts and, in carnivores, the consumption of infected prey species. ACKNOWLEDGMENTS We thank the University of Tennessee College of Veterinary Medicine Center of Excellence program for funding during the summers of 2005 and 2006. Rodney Boertje, John Burch, Lem Butler, Mark McNay, Toby Boudreau, John Crouse, Kevin White, Mark Keech, Jim Woolington, Bruce Dale, Jim Dau, and Kimberlee Beckmen (Alaska Department of Fish and Game, Fairbanks, AK) collected a significant proportion of the samples used in this research. Rick Farnell and Robert Hayes of the Yukon Department of the Environment provide the samples for caribou, moose, and wolves in the Yukon Territory, Canada. The University of Tennessee Virology Laboratory shared the FITC labeled rabbit anti-deer and offered advice when it was needed. Milton McAllister (University of Illinois, Department of Pathobiology, Urbana) furnished positive and negative deer controls. The staff of the University of Tennessee Parasitology Laboratory offered generous help and support throughout the years of this project. LITERATURE CITED ASTHANA, S. P., C. N. L. MACPHERSON,S.H.WEISS,R. STEPHENS, T. N. DENNY, R. N. SHARMA, AND J. P. DUBEY. 2006. Seroprevalence of Toxoplasma gondii in pregnant women and cats in Grenada, West Indies. Journal of Parasitology 92: 644 645. CHOMEL, B. B., R. L. ZARNKE, R. W. KASTEN, P. H. KASS, AND E. MENDES. 1995. Serologic survey of Toxoplasma gondii in grizzly bears (Ursus arctos) and black bears (Ursus americanus) from Alaska, 1988 to 1991. Journal of Wildlife Diseases 31: 472 479. DESMONTS, G., AND J. S. REMMINGTON. 1980. Direct agglutination test for diagnosis of Toxoplasma infection; method for increasing sensitivity and specificity. Journal of Clinical Microbiology 11: 562 568. DUBEY, J. P. 1982. Induced Toxoplasma gondii, Toxocara canis and Isospora canis infections in coyotes. Journal of the American Veterinary Medical Association 181: 1268 1269.. 1983. Experimental infections of Sarcocystis cruzi, Sarcocystis tenella, Sarcocystis capracanis and Toxoplasma gondii in red foxes (Vulpes vulpes). Journal of Wildlife Diseases 19: 200 203., AND P. THULLIEZ. 2005. Prevalence to antibodies to Neospora caninum in wild animals. Journal of Parasitology 91: 1217 1218., J. L. CARPENTER, C. A. SPEER, M. J. TOPPER, AND A. UGGLA. 1988. Newly recognized fatal protozoan disease of dogs. Journal of the American Veterinary Medical Association 192: 1269 1285., S. M. MITCHELL, J. K. MORROW, J. C. RHYAN, L. M. STEWART, D.E.GRANSTROM, S.ROMAND, P. THULLIEZ, W. J. SAVILLE, AND D. S. LINDSAY. 2003a. Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming. Journal of Parasitology 89: 716 720., R. ZARNKE, N. J. THOMAS, S. K. WONG, W. VAN BONN,M.BRIGGS, J.W.DAVIS,R.EWING,M.

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