Seroepidemiology of Coxiella burnetii in Wild White-Tailed Deer ( Odocoileus virginianus ) in New York,United States

Abstract

Coxiella burnetii is an environmentally resistant bacterium that has been reported in wildlife populations. Frequent contact on pasture between white-tailed deer (Odocoileus virginianus) and cattle has been reported by farmers in the Northeast U.S., and transmission of C. burnetii is thought to occur between wild deer and domestic livestock such as cows, sheep, and goats. Blood samples were collected from white-tailed deer throughout New York State in 2009 and 2010 and examined for anti-C. burnetii phase II antibodies via indirect microimmunofluorescence assays. Exploratory spatial cluster analysis revealed a lack of significant clustering of C. burnetii-seropositive deer. Logistic regression analysis revealed a significant association between the C. burnetii serostatus of deer and sex, percent agriculture, shrub, and forest cover, and townships with more than 10 bovine herds. A lack of significant association was revealed between the serostatus of deer and the year of sampling, soil type, percent wetland and open water cover, total annual precipitation, and townships with more than two sheep or goat herds. Because four different land cover types were associated with a higher probability of C. burnetii seropositivity, it is likely that land cover is not a discriminating factor in C. burnetii exposure. This is probably because C. burnetii environmental contamination is widespread and not localized to certain cover types. The social behavior of male deer may contribute to the lack of spatial clustering. Bucks typically travel over greater distances, which leads to a greater variety of encountered environments and a greater chance for exposure to C. burnetii. Because increasing agricultural land cover and townships with greater than 10 bovine herds are associated with an increased probability of diagnosing a seropositive deer, it appears likely that transmission of C. burnetii between domestic livestock and white-tailed deer may occur.

Introduction

M any pathogens capable of infecting wildlife such as white-tailed deer (Odocoileus virginianus) have the potential to negatively affect the health of humans, domestic livestock, and other wildlife species. Bohm and associates (2007) report that 77% of livestock pathogens are generalists that infect multiple species, and although an estimated 59% of human diseases are zoonotic (Daszak et al. 2001; Bengis et al. 2004), 73% of human emerging or re-emerging infectious diseases of the last decade affect a wildlife host (Bengis et al. 2004). Coxiella burnetii, an environmentally resistant obligate intracellular gram-negative bacterium found throughout the world (McCaul and Williams 1981), is known to infect domestic livestock, pets, and humans. It has also been reported in wildlife populations (Kersh et al. 2010). Frequent contact on pasture between deer and cattle has been reported by dairy farmers in the Northeast U.S. (United States Department of Agriculture [USDA], 2007), and interspecies transmission of C. burnetii is hypothesized to occur between wild deer and domestic livestock (Enright et al. 1969; Ruiz-Fons et al. 2008). Antibodies to C. burnetii have been documented in various wild ruminant species throughout the world, including Hokkaido deer (Cervus nippon yesoensis; 69%), Japanese deer (Cervus Nippon centralis; 56%), Columbia black-tailed deer (Odocoileus hemionus columbianus; 22%), European roe deer (Capreolus capreolus; 15.4%), Iberian red deer (Cervus elaphus; 5.6%), and white-tailed deer (1.5%; Ejercito et al. 1993; Marrie et al. 1993; Ruiz-Fons et al. 2008). The threat that C. burnetii-infected wildlife pose to humans is uncertain.

Transmission of infectious agents between wildlife and cattle, or vice versa, occurs mainly on pasture (Van Campen and Rhyan 2010), but interspecies transmission may also occur when wildlife gains access to haystacks, open silage pits, feed bunks, mineral blocks, and water sources. Approximately 63% of dairy operators located in the Northeast report definitive contact between deer and cattle (USDA 2007), and C. burnetii seroprevalence is reported to be fairly high in bovine, goat, and sheep herds in the United States (McQuiston and Childs 2002; Kim et al. 2005). Kim and colleagues (2005) report a prevalence of greater than 94% in bulk milk tank samples from U.S. dairy herds, and some studies suggest that dairy cows are more frequently chronically infected than sheep, and therefore represent the greatest source for human infection (Maurin and Raoult 1999). A meta-analysis by McQuiston and Childs (2002) reported individual sheep and goat seroprevalences of 16.5% and 41.6%, respectively. Additionally, considering the frequently reported contact between wild deer and domestic livestock in the Northeast, and the high levels of C. burnetii seroprevalence in U.S. domestic livestock herds, serological evidence of infection seroconversion of white-tailed deer is not a surprise.

In addition to the high seroprevalences reported in domestic livestock herds, C. burnetii environmental contamination is widespread in the United States (Kersh et al. 2010). More than 23% of environmental samples tested for the presence of C. burnetii DNA were positive, and a statistically significant difference was reported between the percentage of positive samples collected from locations with livestock and those without livestock (Kersh et al. 2010). The small-cell variant of the bacterium is reported to be able to withstand a variety of environmental conditions (Rodolakis 2009), and is able to survive for extended periods of time, so prolonged environmental persistence and widespread environmental contamination is not surprising. Due to the potential for the small-cell variant of C. burnetii to persist in the environment, different climatic factors may influence the persistence of the bacterium. Soil type, total annual precipitation, and type of land cover, may influence the ability of the bacterium to survive in the environment, thereby affecting the potential for wild white-tailed deer to become seropositive secondary to environmental contamination.

The first step in assessing the risk of transmission of C. burnetii between wild white-tailed deer and domestic livestock is to determine the prevalence of anti-C. burnetii antibodies in wild white-tailed deer. The aims of this study were to investigate via indirect microimmunofluorescence assays (MIA) the seroprevalence of anti-C. burnetii antibodies in hunter-harvested white-tailed deer; to investigate the relationship between the probability of diagnosing a C. burnetii-seropositive deer and the soil type, total annual precipitation, and type of cover within a particular township; and to assess the risk of interspecies C. burnetii transmission by analyzing the spatial epidemiologic relationship between seropositive deer and domestic livestock herds.

Materials and Methods

Study site

New York State encompasses 18.6 million acres of forest, with 83% of the forested land composed of timberland (USDA Forest Service 2005). Land cover in New York is classified as approximately 53% forest, 22% pasture or hay and cultivated crops, 9% developed, 8% wetlands, 4% shrub/scrub or herbaceous, and 3% open water (Fry et al. 2011). The 2007 USDA Agricultural Census reported an estimated 103,102 sheep and goats distributed over 2273 sheep and/or goat herds in New York, along with 1.44 million head of cattle (including calves) distributed over 13,520 herds (USDA National Agricultural Statistics Service 2009).

Sample processing

Wild white-tailed deer were surveyed for antibodies to C. burnetii in central New York State in 2009 and throughout the entire state in 2010 (Fig. 1). Hunter-harvested deer were sampled at private deer processing facilities and New York State Department of Environmental Conservation (NYSDEC) deer check stations on the first weekend of the New York State regular firearm season in 2009 and 2010. In 2009, 333 white-tailed deer (219 males and 114 females) were sampled. In 2010, 726 deer (350 males and 376 females) were tested. Blood samples were obtained either from the thoracic cavity or as it drained from the nasal and oral cavities (Passler et al. 2008). The blood was transferred to 10-mL glass serum tubes and stored at 4°C prior to centrifugation at 1300 g for 10 min. After separation, the serum was collected and frozen at −80°C (Martinez et al. 1999; Duncan et al. 2008).

FIG. 1.

Map of New York State showing the locations of white-tailed deer sample collections. Filled circles indicate the sites of Coxiella burnetii-seropositive deer, and open circles represent the locations of C. burnetii-seronegative deer.

Laboratory analysis

Laboratory procedures were performed as described in Kirchgessner and associates (in press). In brief, C. burnetii phase II whole-cell antigen-coated slides were used to screen sera via MIA. Phase II C. burnetii strain RSA439 (from James Samuel, Texas A&M University, College Station, TX), stored at −80°C until just prior to inoculation, was used to infect 1-day-old cells of the Vero line in Eagle's minimum essential medium (EMEM) plus 10% fetal bovine serum (FBS). No antibiotics were added to the medium. The flasks were incubated at 37°C for 72 h in 2009 and 96 h in 2010. The protocol was amended when it was determined that an additional 24 h of incubation led to the development of larger intracellular vacuoles with greater numbers of C. burnetii microorganisms. Trypsin was added and the cells were suspended in 16 mL of EMEM plus 10% FBS. A glass pipette was used to dispense approximately 0.4 mL of the suspension to each well of a Teflon-coated slide (Teflon-coated immunological slides with multiple wells; Cel-Line Associates, Newfield, NJ). In 2009, the slides were incubated at 37°C for 3 h, then fixed for 5 min in 10% formalin, rinsed in 0.01 M phosphate-buffered saline (PBS), and then immersed for an additional 5 min in 100% methanol. The fixative procedure in 2010 remained the same, with the exception that 3.75% formalin was used. The protocol change in formalin concentration was enacted in order to augment the degree of fluorescence and facilitate reading of the MIA. The slides were stored at −20°C until staining.

MIA tests were carried out as follows. Approximately 0.4 mL of sample sera diluted 1:20 in blocking buffer (PBS, 1% bovine serum albumin, and 0.1% Tween 20) was added to each slide well and incubated at 37°C for 30 min in a humidified chamber. The wells were rinsed with PBS and washed in a PBS bath for 10 min. After air drying, fluorescein isothiocyanate-conjugated rabbit anti-deer Ig G (anti-deer IgG [H + L] antibody, FITC labeled; Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) diluted 1:80 in blocking buffer was applied to each well and the slide was incubated at 37°C for 30 min in a humidified chamber. The slides were rinsed and washed again with PBS using the same method, and then dipped in Evans blue stain for 5 min. The slides were rinsed in distilled water, allowed to air dry, and then mounted in 50% glycerol-PBS mounting medium. The slides were examined using fluorescence microscopy at 400× magnification on a Leitz Wetzlar fluorescence microscope.

Statistical analysis

SaTScan version 9.0 (Kulldorff 1997) was used to determine the locations of clusters of C. burnetii-seropositive white-tailed deer at the township level. This software used the centroid of each township as a focal point, then employed circles of varying diameter to check for the presence of clusters of seropositive deer. The Bernoulli method was utilized to compare the locations of seropositive deer to the locations of seronegative deer; seropositive deer were identified as “cases,” while seronegative deer were recognized as “controls.” The Bernoulli method compared the number of seropositive and seronegative deer within each arbitrarily sized circle to the number of seropositive and seronegative deer remaining outside the circle (Olea-Popelka et al. 2003). A likelihood ratio was calculated for each possible circle, and a cluster of seropositive deer was determined to be significant if fewer than 5% of the 1000 Monte Carlo trials produced the observed or more extreme result (Olea-Popelka et al. 2003). A maximum population size window of 25% (approximately twice the seroprevalence determined in 2009 and 2010) was selected.

Logistic regression analysis was completed in R version 2.13.0 (R Development Core Team 2011) using the serostatus of each white-tailed deer as the dependent variable. In order to find the most parsimonious model, the stepAIC function from the MASS package (Venables and Ripley 2002) was utilized. The stepAIC function uses a stepwise model selection procedure that removes and adds explanatory variables from the full model until the model with the lowest Akaike information criterion (AIC) is found (Venables and Ripley 2002). The 2007 Census of Agriculture data were used to determine the number of bovine and sheep/goat herds in each township (USDA National Agricultural Statistics Service 2009). Spatial predictor variables (precipitation, land cover, and soil type) were acquired as Geographical Information Systems datasets and imported into ArcMap version 10.0 (Environmental Systems Research Institute (ESRI), Redlands, CA). The datasets were overlaid on the township boundaries to compute descriptive summaries for each township. Total annual precipitation data were compiled for 1961–1990 (Daly et al. 2001), and were summarized as mean values (cm) for each township. Land cover (Fry et al. 2011) and soil type (United Nations Educational Scientific and Cultural Organization 2005) data were summarized as the percentage of each township occupied by the various land cover classes (agricultural, developed, forest, open water, shrub, and wetlands), and soil types (alfisols, inceptisols, and spodosols). The explanatory variables were coded as follows: (1) sex (binary, reference level: “female”); (2) year (binary, reference level: “2009”); (3) presence of bovine herds per township (binary, reference level: “10 or fewer bovine herds”); (4) presence of sheep/goat herds per township (binary, reference level: “2 or fewer sheep/goat herds”); (5) all other explanatory variables (continuous). The Le Cessie and Houwelingen test (α=0.05) was used to assess the goodness-of-fit of the most parsimonious model (Hosmer et al. 1997).

Results

Overall, 14.64% (155/1059; 95% confidence interval [CI] 12.64%,16.90%) of the tested white-tailed deer were C. burnetii phase II-seropositive. The male seroprevalence rate was 17.57% (100/569; 195% CI 14.66%,20.91%), and the female seroprevalence rate was 11.22% (55/490; 95% CI 8.72%,14.32%). The exploratory spatial cluster analysis revealed a lack of significant clustering of C. burnetii-seropositive deer.

Six explanatory variables were significantly related to C. burnetii serostatus in white-tailed deer. Males were more likely to be seropositive (odds ratio [OR]=1.643, 95% CI 1.144–2.384), and the probability of seropositivity significantly increased with increasing percentage of agriculture (OR=1.071, 95% CI 1.038,1.107), developed (OR=1.076, 95% CI 1.041,1.115), shrub (OR=1.062, 95% CI 1.006,1.122), and forest cover (OR=1.056, 95% CI 1.027,1.090). Townships with greater than 10 bovine herds had a higher exposure rate than townships with fewer than 10 herds (OR=1.457, 95% CI 1.015–2.104; Table 1). The Le Cessie and Houwelingen test for the model was 0.980, indicating an adequate fit (Table 2). The soil type, percentage of wetlands and open water cover, year of sampling, more than two sheep or goat herds per township, and total annual precipitation, were not significant predictors of the C. burnetii serostatus of deer.

Table 1.

Logistic Regression Analysis Results for the Most Parsimonious Model as Determined via Stepwise Regression

Parameter	Standard error	Wald statistic	p Value	Odds ratio	Confidence interval of odds ratio
Constant	1.470	−5.428	<0.0001	−	<0.001,0.005
Greater than 10 bovine herds	0.186	2.027	0.043	1.457	1.015,2.104
Sex status	0.187	2.656	0.008	0.1.643	1.144,2.384
Agriculture cover	0.016	4.141	<0.0001	1.071	1.038,1.107
Developed cover	0.018	4.168	<0.001	1.076	1.041,1.115
Forest cover	0.015	3.606	0.0003	1.056	1.027,1.090
Shrub cover	0.028	2.175	0.030	1.062	1.006,1.122

Table 2.

Model Fit Tests for the Most Parsimonious Model as Determined via Stepwise Regression Analysis

Test	Chi-square value	Z value	p Value	Degrees of freedom
Likelihood ratio test	38.916	–	<0.0001	6
Le Cessie and Houwelingen test	–	−0.025	0.980	1058

Discussion

Deer sampled in townships with greater than 10 bovine herds were significantly more likely to be C. burnetii seropositive than deer sampled from townships with fewer than 10 herds. This is not surprising, considering the high prevalence of C. burnetii-contaminated bulk milk tanks diagnosed via polymerase chain reaction in New York; between 90 and 95% of tested bovine farms were C. burnetii-positive over a consecutive 3-year period (Kim et al. 2005). Additionally, chronically-infected cows are considered by some to be the most important source of human infection because they excrete the bacterium in milk and birth products for many years (Maurin and Raoult 1999; Woldehiwet 2004), which leads to contamination of both manure and the fields upon which it is spread (Rodolakis 2009). The fact that increasing agricultural land cover is associated with an increased probability of diagnosing a C. burnetii seropositive deer is also not surprising, considering that there is an increased probability of diagnosing a seropositive deer in townships with greater than 10 bovine herds. The increased probability of diagnosing a seropositive deer in townships with increasing forest, shrub, and developed land cover are less clear. Because four different land cover types are associated with a higher probability of C. burnetii seropositivite deer, it is likely that land cover is not a discriminating factor in C. burnetii exposure. The ORs for the significant land cover types range from 1.056 to 1.076, further supporting the conclusion that, although significant, the type of land cover in a township does not dramatically influence the serostatus of deer.

In support of the conclusion that land cover type does not considerably influence the serostatus of deer, the spatial data analysis revealed a lack of spatial clustering of C. burnetii-seropositive deer. This is likely attributable to the fact that environmental contamination is widespread and not localized to certain cover types. Due to the presumed pathogenesis of C. burnetii in white-tailed deer and reported social behavior in deer, it was hypothesized a priori that deer most likely became infected with C. burnetii as a result of environmental exposure, as opposed to contact with infected conspecifics. The pathogenesis of C. burnetii infection in white-tailed deer has not been reported; however, it has been extensively studied in domestic livestock. It has been reported that infected female cows, sheep, and goats excrete an enormous amount of C. burnetii in birth products (Luoto and Huebner 1950; Norlander 2000; Rousset et al. 2009). Smaller numbers of the bacterium are shed in urine, feces, and milk, and shedding may continue for several months, particularly in feces, milk, and vaginal secretions (Arricau Bouvery et al. 2003; Kim et al. 2005; Rousset et al. 2009), even in seronegative animals (Berri et al. 2001). Because sites contaminated by infectious birth products may serve as sources of infection for weeks or months (Yanase et al. 1998), the birth of an infected doe could contribute to environmental contamination. But, since female ungulates seek total seclusion for parturition (Fraser 1968), it would likely not result in a rapid increase in the number of C. burnetii-infected deer within a herd as a result of direct contact with an infectious fawn or birth products. As a result of low rates of dispersal by does (Dusek et al. 1989), and the establishment of overlapping home ranges close to those of the mothers (Porter et al. 1991), the social structure of female white-tailed deer may facilitate perpetuation of C. burnetii infections among certain social groups. However, these potential small clusters of C. burnetii exposure would likely not be reflected in the spatial analysis due to the coarse nature of the spatial investigation. Because environmental background levels of C. burnetii are quite high in the United States (Kersh et al. 2010), minimal clustering of exposed deer is not unexpected.

The social behavior of male deer, which are 64% more likely to be C. burnetii-seropositive than females, may also contribute to the lack of spatial clustering. In contrast to males, female white-tailed deer offspring typically exhibit less than 5% rates of dispersal (Dusek et al. 1989), and tend to establish overlapping home ranges in close proximity to those of the mothers (Porter et al. 1991). In other words, does do not tend to roam and expose themselves to a variety of locales or landscapes. Bucks on the other hand typically disperse between 5 and 10 km at approximately 12–18 months of age, and dispersal distances up to 40 km have been reported (Long et al. 2005; McCoy et al. 2005). Average male dispersal behavior leads to a greater variety of encountered environments and landscapes compared to females, and likely leads to a greater chance for exposure to C. burnetii.

In contrast to bovine herds, deer sampled in townships with more than two sheep or goat herds did not have a higher probability of seropositivity. This lack of significance may be a result of the fact that sheep and goat herds are far less common in New York than bovine herds; the average number of bovine herds per township is 17.42, as compared to 3.42 sheep and/or goat herds per township.

It is recognized that some aspects of the study design may have impacted the logistic regression model results. A more precise determination of the locations of the sampled white-tailed deer and the livestock herds, rather than using the centroid of a township, would be desirable for estimating more precisely the association between domestic livestock herds and the C. burnetii serostatus of wild deer. Also, due to both the lack of effort to determine the C. burnetii status of most domestic livestock herds, and the low level of diagnostic testing for the presence of C. burnetii in individual clinical cases, particularly in bovines, the locations of infected premises are largely unknown (E.J. Dubovi, unpublished data). Consequently, direct associations between infected livestock herds and exposed deer could not be assessed. In addition, the associations between the serostatus of deer with both deer density per township and the age of the sampled deer would ideally also have been examined. However, deer density estimates for New York are not currently available, and the manner in which the blood samples were collected precluded age determination. Because all of the blood samples were collected from harvested deer, hemolysis was observed in many of the samples post-centrifugation. Boadella and Gortazar (2011) report no difference in seroprevalence in hemolyzed versus high-quality sera in samples collected from harvested wild boars (Sus scrofa).

There are few reports of C. burnetii infection or seroprevalence surveys in wildlife, and it is not known if infected wildlife pose a threat to either humans or domestic livestock (Ejercito et al. 1993). This aims of this study were to investigate the seroprevalence of anti-C. burnetii antibodies in wild white-tailed deer, and to assess the risk of interspecies C. burnetii transmission. Male deer were determined to be approximately 63% more likely than females to be C. burnetii-seropositive, which is indicative of a higher exposure rate for male deer. Deer sampled from townships with greater than 10 bovine herds were estimated to be 46% more likely to be seropositive than deer sampled from townships with fewer than 10 bovine herds. Because several land cover types were significantly associated with the C. burnetti serostatus of deer, we concluded that widespread C. burnetii environmental contamination and the presence of greater than 10 bovine herds per township likely had more of an effect on the serostatus of deer than particular land cover types. Our study confirmed that white-tailed deer are exposed to C. burnetii in New York, and may serve as potential sources of C. burnetii for both domestic livestock and humans. Additionally, infected domestic bovines are potential sources of infection for wild white-tailed deer.

Footnotes

Acknowledgments

Funding for this project was provided in part by the Edna Bailey Sussman Foundation and Sigma Xi. Many thanks to Elizabeth Bunting, NYSDEC, and undergraduate students at the State University of New York College of Environmental Science and Forestry, for assisting with sample collection. Special thanks to Nancy Zylich and the staff at the Animal Health Diagnostic Laboratory at Cornell University for assistance with the development of the C. burnetii MIA. Many thanks also to Kendell Ryan and Ed Laube for assistance with the GIS mapping layers.

Author Disclosure Statement

No conflicting financial interests exist.

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