PREVALENCE OF ANTIBODIES TO TOXOPLASMA GONDII IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) IN NEW YORK STATE, USA

Toxoplasmosis, caused by infection with Toxoplasma gondii, is one of the most common protozoal infections in the world and the third leading cause of human death from foodborne disease in the United States (Mead et al., 1999; Lindsay et al., 2003). Human infection is common, but geographically variable (Dubey, 2010). Exposure can be due to vertical or horizontal transmission, with the latter arising from consumption of tissue from an infected intermediate host or ingestion of oocysts produced by the definitive hosts, domestic and wild felids (Dubey, 2008). Significant morbidity from Toxoplasma is uncommon, with the exception of fetal infection, due to exposure of the mother, and infection in immunocompromised individuals. Similarly, animal infection is common across a broad range of domestic and wild, warm-blooded species, with little pathology typically noted. A particular concern is the infection of food animals, both because of potential reproductive effects in the animals and the risk to consumers of tissue cysts in meat or other tissues (Dubey and Jones, 2008). Toxoplasmosis is of known economic concern among goat and sheep producers and an emerging concern among camelid producers (More et al., 2008).

Barring supplementation with, or inadvertent ingestion of, animal products, grazing animals are primarily exposed by vertical transmission from infected females or by consuming oocysts. Their population density and tendency to frequent livestock pastures make deer (Cervidae) a suitable proxy to characterize risk factors for infection in common with other grazing animals (Kirchgessner et al., 2012), and the use of their meat for human and animal consumption makes their infection with Toxoplasma a point of interest for human and domestic animal health. The importance of deer as a potential source of infection has been suggested by research noting a higher degree of Toxoplasma exposure among consumers of venison than among the general population (Sacks et al., 1983; Ross et al., 2001).

White-tailed deer (Odocoileus virginianus) are often subject to intensive management related to their propensity to cause damage to property and their contribution to the spread and maintenance of zoonotic and economically significant diseases (Forrester, 1992; DeNicola et al., 2000). Control efforts aimed at reducing Ixodes scapularis (black-legged or deer tick) populations in the northeastern US by targeting deer have met with some success, but also have emphasized the difficulties of control of such an abundant species. Deer live in close association with human and domestic animal populations, making some lethal control efforts difficult and often unpalatable to local residents (Porter et al., 1991). These factors make deer a useful sentinel species studying the prevalence of pathogenic agents that pose a risk to humans and other animals, and the ready availability of tissue samples during hunting season provides a unique opportunity to test large numbers. Researchers have reported Toxoplasma antibody prevalences in white-tailed deer ranging from 28.7% to 64.2% in US surveys (Lindsay et al., 1991; Brillhart et al., 1994; Vanek et al., 1996; Dubey, 2010; Dubey et al., 2004, 2009, 2013). An antibody prevalence of 60% was detected among deer killed in Pennsylvania, the nearest previous study to the present population (Humphreys et al., 1995). There are no reports of clinical toxoplasmosis in white-tailed deer, suggesting that infection is not a predisposing factor in being harvested by hunters, although more research on this point is of interest given behavior anomalies observed in other intermediate hosts (Afonso et al., 2012; Gatkowska et al., 2012).

The examination of Toxoplasma antibody prevalence in the population of hunter-harvested New York State deer can provide insight into the dynamics of infection among wildlife and domestic animal populations. In 2010, the year of collection of these samples, 230,100 deer were harvested in the state from an estimated population of 923,466 (NYS DEC, 2011). Inferences can be made regarding the level of exposure based on sex, age, and the human population density of the town in which the deer were killed. Differences in male vs. female levels of infection may suggest sex-based risk factors promoting exposure. Age-based differences in infection level can help to determine how the population of deer has been exposed. For example, a constant level of infection across age groups would suggest a larger role for vertical transmission, whereas an increasing level of infection with age would suggest horizontal transmission through environmental exposure as a more important route. Finally, the interaction between the local human population density and infection levels in the associated deer may provide insight into the importance of domestic cats (Felis catus) in serving as a source of exposure. We used the human population as a proxy for cat population because accurate estimates of the latter are lacking, whereas census-based data for the former are available. However, discrepant results between cat population density and antibody prevalence in deer may be due to management factors; rural owners may be more likely to allow cats to roam freely, leading to more potential oocyst contamination than would be seen with urban, primarily indoor cats. Additionally, a high level of Toxoplasma infection was reported in Pennsylvania bobcats (Lynx rufus rufus), which shed oocysts into the environment (Mucker et al., 2006). This contribution is expected to be minimal compared with that of domestic cats because the bobcat population in New York State is estimated at only 5,000 (NYS DEC, 2012).

We describe analysis of 299 white-tailed deer serum samples from animals harvested during November 2010 across New York State for antibodies to Toxoplasma as an indication of their exposure history. Serum samples were assayed using an enzyme-linked immunosorbent assay (ELISA) effective for the detection of immunoglobulin (Ig) G antibody in a broad range of animal species (Schaefer et al., 2012). Demographic information collected for the deer was contrasted between antibody-positive and antibody-negative deer to identify significant risk factors. Results suggest broad characterizations that can distinguish antibody-positive from antibody-negative deer and lend themselves to risk analysis for meat producers and consumers of venison.

Blood samples were collected from the thoracic cavity of hunter-harvested white-tailed deer at private deer processing facilities and New York State Department of Environmental Conservation deer-check stations across New York State. Collections were performed 20–21 November 2010, the first weekend of the 2010–2011 regular firearm season in most of the state, during which the daily temperature range was −4 to 9 C in the collection area (National Weather Service, 2013). Blood was transferred to 10-mL glass serum tubes and stored at 4 C before centrifugation at 1,300 × G for 10 min. Supernatant (serum) was collected and frozen at −80 C pending analysis (Kirchgessner et al., 2012).

Serum samples were tested for anti-Toxoplasma IgG using the technique described by Schaefer et al. (2012). The Wampole Laboratories' Toxoplasma gondii IgG ELISA II kit (Princeton, New Jersey, USA) provided antigen-coated wells that were filled with 100 µL of the serum of interest diluted 1∶21 with kit-supplied diluent, were allowed to incubate, and were washed with kit-supplied wash buffer. Protein A/G conjugate diluted to 1∶40,000 with phosphate-buffered saline was used for the detection of IgG antibody instead of the kit-supplied anti-human IgG conjugate. Following incubation and a second wash step, tetramethylbenzidine was added as an indicator of conjugate and color intensity was measured on a microplate reader at 450 µm. Optical density ratios were calculated using kit-supplied standards and were used to determine antibody status.

Samples were categorized as “positive,” “negative,” or “equivocal,” based on the results of the ELISA. Positive samples were considered cases, and negative samples were considered controls for purposes of risk factor interpretation. Logistic regression was performed using JMP9 statistical software (SAS Institute, Carey, North Carolina, USA) with variables of age (<1 yr old vs. >1 yr old, based on body morphology), sex, and human population density in the town in which each deer was killed, with output of antibody status. Statistically significant (P<0.05) factors contributing to antibody status were identified and further analyzed by determining odds ratios with 95% confidence intervals (CI). ArcGIS10 (esri, Redlands, California, USA) was used to produce a New York State map indicating towns from which antibody-positive deer were identified and those in which none of the deer sampled were antibody-positive.

Of the 299 serum samples evaluated, 31 (10.4%) produced optical density ratio values in the equivocal range and were excluded from analysis, leaving 113 samples (42.2%) that were positive and 155 (57.8%) that were negative for Toxoplasma antibody, for an apparent prevalence of 42%. The ELISA is known to have a sensitivity and specificity of 92% and 89%, respectively, using agglutination testing as a gold standard (Schaefer et al., 2012). Using calculations for true prevalence based on test performance, our testing indicates a prevalence of 38.5% (95% CI, 31.2–45.8%) for this population of deer (Rogan and Gladen, 1978). The 268 serum samples that were used for analysis were from 90 does and 178 bucks; 34 samples were from deer <1 yr old and 218 from adult deer (age data for 16 samples was not available).

In the logistic regression, age of >1 yr was a significant factor in increasing risk of being antibody-positive (P = 0.0013); the effects of sex and local human population density were not significant (P = 0.4831 and 0.1285, respectively). Antibody prevalences were 21% (95% CI = 10.4–36.8%) for the younger group vs. 46.8% (95% CI = 40.3–53.4%) for the older group. The odds ratio for antibody-positive deer that were >1 yr old to those that <1 yr old was 3.39 (95% CI = 1.42–8.12). When distinguished by sex, 39% (95% CI = 29.5–49.2%) of females were ELISA-positive vs. 43.8% (95% CI = 36.7–51.2%) of males. Human population density was analyzed as a continuous variable, with no significant trend in relation to antibody prevalence detected. Animals were found to be seropositive across the range of areas sampled, including areas of low and high human population density (Fig. 1).

Widespread exposure of deer to Toxoplasma in New York State is indicated by an antibody prevalence of 38.5% our sample of deer. This is consistent with prevalences described from other states, but lower than the 60% reported from Pennsylvania in 1995 (Humphreys et al., 1995; Dubey, 2010). Deer >1 yr old had a significantly higher risk of having antibody, whereas no significant difference was detected between male and female prevalences. This is consistent with horizontal transmission, with the risk of having ingested oocysts increasing with age. The local human population density did not appear to be a risk factor for infection. Our sampling was opportunistic, and the number of samples from each site was not standardized. No conclusions regarding these specific locations and the presence or absence of Toxoplasma oocysts in the environment can be derived from these results beyond noting the widespread nature of antibody-positive sites.

The relation to human population analysis was performed based on the town in which the deer was killed. Average deer ranges are 2.8–4.9 km² in yearling male white-tailed deer, whereas female deer have a more-limited range (McCoy et al., 2005). A New York study conducted in the Adirondacks showed an average range for white-tailed deer of 2.25 km² in the summer and 1.35 km² in the winter (Tierson et al., 1985). Landscape fragmentation was a negative predictor of the size of white-tailed deer home ranges (Quinn et al., 2013). The towns from which these deer were collected are generally >100 km² in area, making it likely that most Toxoplasma exposures occurred in the town of collection, but overlap with neighboring towns is certain to be seen, and the description of male dispersal distances of greater than 70 km also complicates that prediction (Nixon et al., 1994). Human population density was not a significant predictor of antibody prevalence. This group of samples was collected from rural towns with a median human population density of 25 residents/km². In one measure, the US Census considers urban areas as those with at least 386 residents/km². Future sample collections including more populated Hudson River Valley suburban townships that allow hunting will allow comparison of Toxoplasma prevalence in deer from towns with a wider range of population densities.

A potential relationship between human population density and Toxoplasma antibody prevalence in deer may be due to a greater domestic feline population density and therefore greater environmental oocyst contamination. Because this is the expected route of exposure for deer, it should be investigated further. Deer feeding behavior in areas of greater human population density may be expected to include grazing closer to human habitations, areas in which owned cats are more likely to defecate. This scenario is based on domestic cats being the singular source of Toxoplasma oocysts. Bobcats, the only other expected feline source of oocysts in the region, comprise a fraction of the domestic cat population. Conversely, rural areas might be expected to harbor more cats that spend time outdoors and participate in rodent consumption, thereby being more likely to be infected with Toxoplasma. Interestingly, a study in Ohio noted greater exposure in deer from areas of lower human population density (Crist et al., 1999), potentially supporting the latter scenario. Further investigation including deer from more-diverse areas in the state will help to clarify this question, and thereby provide a more accurate estimate of factors that contribute to Toxoplasma infection in deer and their role in human exposure.

Thanks to the New York State Department of Environmental Conservation and undergraduate students at the State University of New York College of Environmental Science and Forestry for assistance with sample collection.