• View in gallery

    Map of counties in southeastern Ohio showing the study area. Five concentric rings (gray region), centered around the Père David deer enclosure within The Wilds (a large wild animal conservation center represented by the central area outlined in black), were drawn from coyote home-range estimates to establish the study area. Each buffer had a radius of 5 km (total area, approx 1,964 km2). Although this is obscured by the concentric rings, The Wilds is situated among 4 counties.

  • View in gallery

    Photographs of examples of coyote feces for which freshness was scored as 1 (fresh; A), 2 (intermediate; B), or 3 (old; C). Fresh was defined as black, wet feces with no visible undigested material. Old was defined as white, dry feces with only undigested material visible. All other feces were classified as having intermediate freshness.

  • View in gallery

    Map showing the relative density of wild canids in southeastern Ohio as estimated on the basis of morphological classification of collected fecal samples (n = 285). The index of wild canid relative density ranged from 0 to 1.5 fecal samples/km/d (demonstrated by the size of the circle). Coyote relative density ranged from 0 to 1.2 fecal samples/km/d. Fox relative density ranged from 0 to 0.21 fecal samples/km/d.

  • View in gallery

    Map of the distributions of observed proportions (prevalence) of collected wild canid fecal samples (n = 285) in which coccidian DNA was identified. The size of each circle reflects the number of samples tested (n = 1 to 23) at each evaluated transect. The number of positive samples per transect ranged from 0 to 7, and the number of negative samples ranged from 0 to 17.

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Risk of environmental exposure to small coccidia from wild canid feces in rural Ohio

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  • 1 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210.
  • | 2 College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 3 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210.
  • | 4 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210.
  • | 5 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210.
  • | 6 Department of Anthropology, College of Arts and Sciences, The Ohio State University, Columbus, OH 43210.
  • | 7 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210.

Abstract

OBJECTIVE To determine the extent of environmental exposure to heteroxenous coccidia from wild canid feces in southeastern Ohio.

SAMPLE 285 presumed wild canid fecal samples collected across an ecological system in southeastern Ohio.

PROCEDURES Morphological classification and molecular analysis were used to determine the canid genus for collected fecal samples. Microscopic and molecular analysis were used to detect coccidian oocysts and DNA. Several variables were analyzed for associations with coccidian DNA detection or prevalence.

RESULTS Coccidian DNA was detected in 51 of 285 (17.9%) fecal samples. Of those positive samples, 1% (95% confidence interval, 0.4% to 3%) had positive results for Hammondia heydorni and none had positive results for Neospora caninum, for an estimated environmental N caninum prevalence of 0% (95% confidence interval, 0% to 7%)/1-km2 hexagonal area evaluated. Morphological classification revealed that 78.9% (225/285) of fecal samples were from coyotes and 17.2% (49/285) were from foxes. No difference in proportions of coccidian DNA-positive fecal samples was identified among canid species. Environmental temperature and fecal freshness were associated with coccidian DNA detection. Land use type, relative canid density, and cattle density were not associated with the prevalence of coccidian DNA-positive samples.

CONCLUSIONS AND CLINICAL RELEVANCE The low prevalence of coccidia shed in wild canid feces in this study, including the estimated 0% environmental prevalence of N caninum, suggested that the role of the oocyst environmental phase in coccidia transmission to ruminants is likely minor in rural southeastern Ohio.

Abstract

OBJECTIVE To determine the extent of environmental exposure to heteroxenous coccidia from wild canid feces in southeastern Ohio.

SAMPLE 285 presumed wild canid fecal samples collected across an ecological system in southeastern Ohio.

PROCEDURES Morphological classification and molecular analysis were used to determine the canid genus for collected fecal samples. Microscopic and molecular analysis were used to detect coccidian oocysts and DNA. Several variables were analyzed for associations with coccidian DNA detection or prevalence.

RESULTS Coccidian DNA was detected in 51 of 285 (17.9%) fecal samples. Of those positive samples, 1% (95% confidence interval, 0.4% to 3%) had positive results for Hammondia heydorni and none had positive results for Neospora caninum, for an estimated environmental N caninum prevalence of 0% (95% confidence interval, 0% to 7%)/1-km2 hexagonal area evaluated. Morphological classification revealed that 78.9% (225/285) of fecal samples were from coyotes and 17.2% (49/285) were from foxes. No difference in proportions of coccidian DNA-positive fecal samples was identified among canid species. Environmental temperature and fecal freshness were associated with coccidian DNA detection. Land use type, relative canid density, and cattle density were not associated with the prevalence of coccidian DNA-positive samples.

CONCLUSIONS AND CLINICAL RELEVANCE The low prevalence of coccidia shed in wild canid feces in this study, including the estimated 0% environmental prevalence of N caninum, suggested that the role of the oocyst environmental phase in coccidia transmission to ruminants is likely minor in rural southeastern Ohio.

The 3 most common wildlife-livestock interfaces for the spread of infectious diseases are between birds and poultry, artiodactyls and cattle, and carnivores and cattle.1 In North America, the presence of coyotes (Canis latrans) and foxes (Vulpes spp) has been considered a risk factor for heteroxenous coccidian infections in cattle.23 Coyotes are suggested to be definitive hosts of Neospora caninum4; however, the role of free-ranging carnivores such as coyotes in the epidemiology of ungulate coccidiosis and, specifically, the dynamics of N caninum transmission has yet to be established in natural settings.

Historically, coyotes have been restricted mostly to the prairies of central North America. A recent dramatic increase of coyote ranges and populations has enabled them to expand throughout the contiguous United States and Mexico, a vast area of Canada, and Central America.5 Therefore, an increase is expected in the probability of contact between coyotes and livestock.4,6 An indication of this probability of contact is the rate of cattle losses by predation, which in the United States increased from 2.4% in 1991 to 5.5% in 2010.7,8 Approximately 90% of this predation was due to coyotes.7,8 In Ohio specifically, the coyote population has increased 3-fold since 1990, and the rate of cattle losses by predation has increased from 0.9% in 1995 to 5.6% in 2010.8–10

Although predation is an obvious result of the expanding coyote population, coyotes also influence livestock production through their roles as disease hosts and vectors. On the other hand, coyotes serve an essential ecological function of regulating populations of species, such as rodents and foxes, that can also serve as hosts and vectors of disease. Furthermore, coyotes serve as a biocontrol for overabundant species, such as Canada geese (Branta canadensis) and white-tailed deer (Odocoileus virginianus).11 For example, the role of wild-prey biomass in decreasing sheep predation by coyotes has been described,12 highlighting the importance of healthy food web dynamics in the overall sustainability of an ecosystem.

In previous and ongoing investigations in southeastern Ohio, positive results of serologic testing for N caninum have been obtained for captive ungulates such as Père David deer (Elaphurus davidianus), bison (Bison bison), domestic cattle (Bos taurus), and white-tailed deer.13,14,a,b Although a preliminary studyc revealed small (< 20 μm) coccidia in a small number (n = 29) of coyote fecal samples by microscopic examination, molecular methods were used only to rule out N caninum and not to identify the responsible coccidian species. The purpose of the study reported here was to determine the role of coyote feces in the transmission dynamics of heteroxenous coccidia that may infect ruminant species in southeastern Ohio by estimating relative density of wild canids in that region, assessing the prevalence of environmental contamination with DNA of coccidian oocysts shed by wild canids, and measuring associations between detection of coccidian oocysts and putative risk factors for transmission to livestock (ie, cattle density, wild canid relative density, and land use).

Materials and Methods

Study area and population

The study was conducted within the Ohio Appalachian bioregion, which intersects 4 counties (Muskingum, Morgan, Noble, and Guernsey) and includes the largest conservation center in North America, the International Center for the Preservation of Wild Animals (also known as The Wilds). The area surrounding The Wilds is rural; livestock farms include cattle, sheep, and goats. Domestic dogs (Canis familiaris) are also kept on farms mainly as companions or livestock guards. Wild carnivores such as coyotes and foxes in the area are part of the free-ranging wildlife that are considered pests, tourist attractions, and a recreation source for the local community. This particular region was selected because it allowed exploration of the disease ecology of multihost pathogens such as heteroxenous coccidia and evaluation of the role of species such as coyotes at the wildlife-livestock interface. Within this region, the area selected for the study was based on the home range of coyotes (3 to 42 km2; mean, 17.5 km2)15 and consisted of a circle with a 5-km radius surrounded by 4 concentric annuli, each with a 5-km width (adding to a total circular study area of 25-km radius) and centered around the Père David deer enclosure at The Wilds (Figure 1).

Figure 1—
Figure 1—

Map of counties in southeastern Ohio showing the study area. Five concentric rings (gray region), centered around the Père David deer enclosure within The Wilds (a large wild animal conservation center represented by the central area outlined in black), were drawn from coyote home-range estimates to establish the study area. Each buffer had a radius of 5 km (total area, approx 1,964 km2). Although this is obscured by the concentric rings, The Wilds is situated among 4 counties.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1179

Study design and sample collection

A spatial sample collection strategy was used to determine the relative density of coyotes and the environmental prevalence and distribution of coccidian parasites through noninvasive fecal sample collection. Sample collection took place from May through August 2014. At this time of year in the US Midwest (Ohio), coyotes should have fixed home ranges as they rear pups in dens.5 Therefore, an assumption of independence for the analysis was made for samples collected between locations that were at least 5 km distant from each other. Coyotes are known to frequent linear features such as roads, trails, firebreaks, and field edges, so these features were surveyed within the study area.16 Transects (linear features) were selected arbitrarily from the available linear features on properties in the study area where owners agreed to participate in the study. Fifty-six transects corresponding to a total of 89.4 km were surveyed for coyote feces and included The Wilds and 29 farm or recreational properties. Transect sizes ranged from 0.45 to 4.5 km. Each transect was surveyed, and feces were collected twice by at least 2 researchers. On the first day, transects were walked to collect feces and clear any feces from the area. Fourteen days later, fecal samples for the study were collected again along the transects and feces were counted for estimating relative densities of wild canids.

When wild canid feces were identified, global positioning system coordinates were recorded and the feces were collected and stored at 4°C in individual sealed plastic bags17 until processing (2 to 3 days later). Fecal freshness was recorded by use of a numeric scale on the basis of fecal color, wetness, and grossly apparent content (eg, digested prey tissue versus undigested material such as hair, bones, and feathers). A score of 1 (fresh) was assigned to black, wet feces with no visible undigested material; a score of 3 (old) was assigned to white, dry feces with only undigested material visible; and a score of 2 (intermediate) was assigned to all other feces (Figure 2). Additionally, morphological characteristics as described elsewhere18 were used to identify the host species and the observer's certainty level (70%, 90%, or 100%) for that judgment (ie, how well the feces conformed to the ideal for that species). To confirm accuracy in host species identification, molecular analysis (considered the more specific method) was performed on a subset of samples. Both of these methods (morphological and molecular) included differentiation of coyote feces from domestic dog feces. No domestic dog feces were included in this study.

Figure 2—
Figure 2—

Photographs of examples of coyote feces for which freshness was scored as 1 (fresh; A), 2 (intermediate; B), or 3 (old; C). Fresh was defined as black, wet feces with no visible undigested material. Old was defined as white, dry feces with only undigested material visible. All other feces were classified as having intermediate freshness.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1179

The mean environmental temperature, humidity, and precipitation for the 15 days prior to fecal sample collection were obtained from the National Oceanic and Atmospheric Association databased (data for the Zanesville municipal airport in Ohio) to determine the environmental conditions that the feces were exposed to prior to collection. Land-use data for the areas of sample collection were recorded by the investigators on the basis of visual identification as crop (cultivated agricultural use), pasture (livestock grazing or unoccupied grassland), or woods (forested). Finally, property owners were surveyed to determine the number of cattle per farm and number of acres per farm at the evaluated transects.

Restriction enzyme analysis for host genetic identification

To confirm results of morphological identification of wild canid fecal samples, a section of mitochondrial DNA was evaluated from a subset of 50 coccidia-positive and 53 coccidia-negative fecal samples. For this test, DNA was extracted from 1 g of each fecal sample by use of a fecal DNA kit.e The DNA was purified and concentrated with an ethanol precipitation process.19 For positive control samples, DNA was extracted from banked blood samples (100 μL each) from a coyote, red fox (Vulpes vulpes), and gray fox (Urocyon cinereoargenteus) that had been provided by the USDA Wildlife Services, National Wildlife Disease Program by use of a blood and tissue DNA kit.f

A modified version of a previously reported digestion enzyme analysis approach20 was used. Briefly, identification primers developed previously20 were used to amplify 200 bp of the cytochrome b region of mitochondrial DNA in 20-μL PCR reactions consisting of a final concentration mix containing 200μM dNTP, 0.5μM forward primer, 0.5μM reverse primer, 1X reaction buffer,g 0.2 μL of DNA polymeraseh (1.0 U/50-μL PCR reaction), and 2.3 μL of template DNA (DNA concentration ranged from 0.5 to 8 ng/μL). The PCR conditions were set on a thermal cycleri as follows: initial denaturation at 98°C for 30 seconds; 35 cycles of 98°C for 10 seconds, 50°C for 45 seconds, and 72°C for 15 seconds; and final extension at 72°C for 5 minutes. A positive control sample and negative control sample (ultrapure DNase and RNase-free distilled water) were included in each batch.

Amplified products were run on 2% standard agarosej gels and stained with ethidium bromide solution. This method allowed visual differentiation of feces of coyotes or foxes from feces of domestic dogs and other more distant species. Then, to differentiate between feces from red foxes and feces from coyotes or gray foxes, 6.7 μL of PCR product was digested with 0.8 μL of 1X bufferk and 0.5 μL (1.0 U/50 μL) of TaqαI restriction enzymel in a 65°C-dwell cycle for 2 hours (final volume, 8 μL) and inactivated at 80°C for 20 minutes. Digested products were run on 3% standard agarosej gels and stained with ethidium bromide solution. Finally, to differentiate between feces from coyotes and gray foxes, 6.8 μL of PCR product was digested with 0.8 μL of 1X bufferk and 0.4 μL (1.0 U/50 μL) of Hinf I restriction enzymel at 37°C overnight (final volume, 8 μL), then inactivated at 80°C for 20 minutes. Digested products were run on 3% standard agarosej gels and stained with ethidium bromide solution. Sizes of the separated fragments were compared with a 50-bp DNA ladder.m

Fecal examination and molecular identification of parasites

Sheather sugar solution and light microscopy21 were used to identify oocysts matching the morphological and morphometric characteristics of small coccidia.17,21 Other parasites and ova were recorded but not enumerated. For differentiation of N caninum oocysts from other morphologically similar small coccidia such as Hammondia spp and Toxoplasma gondii,22 oocysts were isolated from 5 g of each fecal sample by means of a standard sucrose fecal flotation technique and placed in 6 mL of 2.5% potassium dichromate to inhibit fungal and bacterial growth. They were then stored at 4°C until processed for DNA extraction (7 to 13 months later).

Previously described DNA extraction and parasite PCR methods23 were used to identify small coccidia (10 to 15 μm) in fecal samples. Briefly, detection of coccidian DNA by PCR with melting curve analysis was performed by use of a universal coccidian primer cocktail24 to amplify an approximately 315-bp region of 18S rDNA. Detection of coccidian DNA via this assay may have included detection of coccidian oocysts with animal and human health implications as well as other coccidian species passing through the carnivore host following ingestion of the intermediate host. Coccidian DNA-positive samples underwent further identification testing with the species-specific primers Np6+/Np21+ (to target the Nc5 region of N caninum25) and JS4/JS5 (to target the ITS1 region of Hammondia heydorni).26 The detection limit of this universal coccidian PCR assay was the equivalent of DNA derived from 20 N caninum tachyzoites and 2 H heydorni oocysts.23 A subset (n = 3) of coccidian DNA-positive amplicons that had negative results for both N caninum and H heydorni DNA was further analyzed. These templates and applicable primers were submitted to The Ohio State University Plant-Microbe Genomics Facility for sequencing. The resulting sequences were aligned with sequence analysis software.n The DNA sequences were compared with published sequences by use of a bioinformatics search tool.o

Statistical analysis

To estimate the relative density of wild canids between transects (rather than the absolute count or density of wild canids), the rate of fecal deposition per kilometer per day was used. This rate (R) was calculated as R = S/(L × D), where S is the number of fecal samples found on the second walk in the transect, L is the length of transect, and D is the number of days elapsed between the 2 observations (date of initial fecal removal and date that feces were counted [ie, 14 days]).27 Cattle density was estimated as the number of cattle per farm acre.

The potential maximum prevalence of N caninum DNA when all samples had negative results was calculated as follows28:

article image

where α is the calculated upper or lower 95% confidence limit, N is the estimated population size, n is the sample size, and β is 1 - n. In this equation, the population size was calculated as the total number of 1-km2 hexagonal areas within the entire study area (N = 2,223),p and the sample size was the number of hexagonal areas occupied by the evaluated transects (n = 40). The size of the hexagon (1 km2) for this analysis was chosen on the basis of field collection logistics, such as how much a field assistant could walk in a given day. For the coccidian DNA and H heydorni DNA prevalence values, we estimated upper and lower 95% confidence limits by use of the so-called score method29,30 and statistical software.q,r

To measure agreement beyond chance between microscopic detection of Neospora-like oocysts and detection of coccidian DNA, the Cohen κ coefficient was calculated.31,s To determine how well observer certainty in their morphological classification of host species for fecal samples matched results from the molecular analysis, the exact multinomial test was used.32,t To evaluate whether a difference existed among wild canid species (as identified through morphological classification) in proportions of coccidian DNA-positive fecal samples, the Pearson χ2 test with Yates continuity correction was used.

To determine whether detection of coccidian DNA could be affected by the weather conditions to which the wild canid fecal samples had been exposed prior to collection and by the freshness of the fecal samples at the time of collection, logistic regression was performed, followed by the Hosmer-Lemeshow goodness-of-fit test.u Results are reported as ORs and 95% CIs.

To confirm the assumption of spatial independence, an explanatory analysis was performed to determine the spatial correlation of the observed proportions of coccidian DNA-positive samples, and a variogram was plotted to assess the spatial autocorrelation by distance between global positioning system-recorded sample collection sites.v,w To model the variation in the prevalence of coccidian DNA and identify risk factors for coccidia transmission between wild canids and livestock, a binomial logistic model was constructed by use of the Monte Carlo maximum likelihood method.w Putative risk factors included in the model were cattle density, relative density of wild canids (coyotes and foxes), and land use.

Results

Samples and environment

A total of 285 fecal samples from presumed wild canids were collected. Cattle densities in the study area ranged from 0 to 1.4 cattle/acre. The environmental conditions to which samples were exposed included a temperature range of 16.4° to 24.1°C, humidity range of 67% to 77%, and precipitation range of 1.03 to 13.6 mm.

Wild canid relative density and host identification

Wild canid relative density in the evaluated areas as estimated from collected fecal samples ranged from 0 to 1.5 fecal samples/km/d (Figure 3). Morphological classification of the 285 fecal samples on the basis of visual inspection indicated that 78.9% (n = 225) were from coyotes, 17.2% (49) were from foxes, and 3.9% (11) were from other species that could not be identified. Amplification of mitochondrial DNA extracted from a subset of samples (70/103 that were genetically analyzed) revealed that 70% (49/70) of fecal samples were from coyotes, 30% (21/70) were from red foxes, and none (0/70) were from domestic dogs or other species. No significant (P = 0.61) difference was identified between observed (morphological identification) and expected (based on molecular classification) proportions of coyotes and noncoyotes for each observer certainty level (100%, 90%, or 70%; Supplementary Table S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.11.1179).

Figure 3—
Figure 3—

Map showing the relative density of wild canids in southeastern Ohio as estimated on the basis of morphological classification of collected fecal samples (n = 285). The index of wild canid relative density ranged from 0 to 1.5 fecal samples/km/d (demonstrated by the size of the circle). Coyote relative density ranged from 0 to 1.2 fecal samples/km/d. Fox relative density ranged from 0 to 0.21 fecal samples/km/d.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1179

Coccidian prevalence

Results of the PCR assay to detect coccidian DNA were positive for 51 of 285 (17.9%) fecal samples (95% CI, 13.8% to 22.7%). Forty-five of the 225 (20%) morphologically identified coyote fecal samples and 6 of the 49 (12%) morphologically identified fox fecal samples were positive for coccidian DNA (P = 0.32).

Microscopic examination revealed small coccidian oocysts in 125 of 285 (43.9%) fecal samples; of these, 32 (25.6%) had positive results for coccidian DNA. For 19 of 285 (6.7%) samples, results of coccidian DNA testing were positive but results of microscopic examination were negative. On the other hand, 93 (32.6%) samples had microscopic oocysts but no coccidian DNA identified, yielding only slight agreement in results between the 2 tests (κ = 0.15; 95% CI, 0.049 to 0.24).

Polymerase chain reaction assays for detection of N caninum and H heydorni specifically were performed on the 51 coccidian DNA-positive samples, yielding few positive results. All samples were negative for N caninum DNA, and 3 coyote samples (2 genetically and 1 morphologically identified; 1%; 95% CI, 0.4% to 3.0%) were positive for H heydorni DNA. The N caninum-specific assay could detect DNA from as few as the equivalent of 2 tachyzoites and did not amplify negative control samples.23 The H heydorni-specific assay was able to detect DNA from 2 H heydorni oocysts and did not amplify negative control samples.23 The upper 95% confidence limit calculated for the environmental prevalence of N caninum DNA was 7.2%, indicating that a potential maximum of 160 1-km2 hexagonal areas might contain N caninum DNA in fecal samples among the 2,223 total hexagonal areas within the study area. Sequencing of a subset of the coccidian DNA-positive sample amplicons that were negative for N caninum and H heydorni DNA revealed Sarcocystis, Eimeria, and Cystoisospora DNA.

Coccidian DNA was identified via PCR assay in 13 of 73 (18%) fecal samples collected in areas classified as used for crops, 18 of 101 (18%) samples collected in areas classified as pasture, and 20 of 111 (18%) samples classified as collected in wooded areas. Overall, 18.0% (46/256) of fecal samples collected from transects with a cattle density < 0.5 cattle/acre had positive results of coccidian DNA testing, compared with 17.2% (5/29) of fecal samples collected from transects with a cattle density ≥ 0.5 cattle/acre.

Risk factors

Multivariate logistic regression revealed that for every 1°C increase in environmental temperature, the odds of detecting coccidian DNA-positive fecal samples increased by 2% (OR, 1.02; 95% CI, 1.01 to 1.04). Additionally, fecal sample freshness was associated with coccidian DNA detection in that fecal samples scored as 1 (most fresh) had an OR of 2.43 (95% CI, 1.14 to 5.48) and those scored as 2 had an OR of 1.04 (95% CI, 0.40 to 2.53), compared with fecal samples scored as 3. The model including these 2 variables fit the data well, as indicated by the Hosmer-Lemeshow goodness-of-fit test (P = 0.90). No significant association with this outcome was identified for environmental precipitation or humidity.

The observed proportions of coccidian DNA-positive samples were graphically mapped (Figure 4). A variogram revealed low spatial correlation in the prevalence of coccidian DNA, confirming the assumption of spatial independence. In logistic regression models to identify factors associated with the prevalence of coccidian DNA-positive fecal samples, none of the evaluated variables (cattle density, wild canid relative density, or land use) were significant.

Figure 4—
Figure 4—

Map of the distributions of observed proportions (prevalence) of collected wild canid fecal samples (n = 285) in which coccidian DNA was identified. The size of each circle reflects the number of samples tested (n = 1 to 23) at each evaluated transect. The number of positive samples per transect ranged from 0 to 7, and the number of negative samples ranged from 0 to 17.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1179

Discussion

The purpose of the present study was to explore the role of wild-canid feces in the transmission of heteroxenous coccidia to ruminant species in southeastern Ohio. Considered an important terrestrial predator in North America, coyotes are believed to play a role in the transmission of several infectious diseases at the wildlife-livestock interface. For example, coyotes are being used as sentinels to monitor bovine tuberculosis in deer.33 Coyotes are also a potential definitive host of N caninum4; that is, they host the sexually mature organism, allowing reproduction and possible environmental dispersion of the oocysts.34 In contrast, red foxes may serve as an intermediate rather than definitive host for N caninum.35–38

Unlike for coyotes, the relative abundance of red foxes in Ohio decreased 3-fold from 1990 to 2011.10,39 The estimated proportion of coyote fecal samples (70%) and fox fecal samples (30%) as determined by genetic analysis in the present study reflected these differences in relative abundance. Therefore, we believe that the differences between these 2 species in the transmission cycle of N caninum and their relative densities in the present study suggested that continued changes in wild canid diversity might affect the observed epidemiology of this parasite in southeastern Ohio. Although no difference was identified between coyotes and red foxes in proportions of coccidian DNA-positive fecal samples, more thorough testing for specific species of coccidia (eg, low-prevalence pathogens such as N caninum) might reveal temporal differences that are relevant across the wildlife-domestic animal interface.

An unspeciated Sarcocystis organism was identified through sequencing of coccidian-positive but N caninum- and H heydorni-negative samples. Because Sarcocystis cruzi has a life cycle that includes both cattle and coyotes,40 this parasite might be interesting to consider in future studies of similar wildlife-livestock interfaces.

Although results of experimental research suggest that coyotes are a definitive host of N caninum,4 these findings have not been confirmed in natural settings. Detection of N caninum oocysts in feces of wild coyotes may be plausible,17 but it has been difficult to demonstrate whether oocyst excretion was the result of infection rather than ingestion of infected prey. In the present study, the estimated maximum environmental prevalence of N caninum was 7.2% in southeastern Ohio, but no evidence of N caninum was identified in fecal samples from the study area during the collection period. In other studies17,41–45 based on canid fecal analyses performed in other parts of the word, the prevalence of N caninum was also low, suggesting that infection is uncommon among wild canids. The low prevalence estimate for the environmental phase of N caninum shed by coyotes and foxes in the present study may have been influenced by the shedding pattern and time of collection, type of prey or food source ingested, reliability of the laboratory tests used, environmental conditions to which the feces were exposed, or a truly low infection rate of canids with N caninum at endemic equilibrium.

In experimentally infected dogs, shedding of N caninum oocysts has been suggested to be affected by prior exposure of dogs to the parasite, and various shedding patterns and oocyst loads have been observed.46,47 The diet of coyotes and red foxes varies with seasonal availability. Although rodents, birds, rabbits, and fruit are common diet components, these wild canids also feed on white-tailed deer and domestic ruminants.5,48 Dogs fed N caninum-infected calf tissue reportedly shed more oocysts than those fed infected mouse tissue.21 Moreover in Spain, differences between red foxes and wolves in the seroprevalence of N caninum might be attributable to the diet of wolves being primarily based on ruminants, compared with the omnivorous diet of red foxes.49 Therefore, fecal shedding of or infection with N caninum in carnivores might vary with the intermediate host ingested.37,50,51 The sample collection period in the present study was after the deer hunting season in Ohio and corresponded to birthing of white-tailed deer fawns.52 Thus, deer offal left in the field (prior exposure) and deer placentas (current exposure) might provide a source of an environmental variation in oocyst prevalence for both N caninum and other coccidia, but this would need to be investigated further.

Similar to methods used in previous studies,44,45 the methods used to identify and differentiate coccidian oocysts in the present study were based on microscopy followed by molecular analysis. As reported by others,53 low agreement was achieved between results of microscopic and molecular testing to identify small coccidian oocysts, suggesting that investigations that rely solely on microscopic examination to detect small coccidian oocysts may underestimate or overestimate prevalence.

In a previous study,54 exposure of environmental N caninum to a temperature of 100°C for 1 minute and 10% sodium hypochlorite for 1 hour permanently inactivated the organisms. Moreover, a study55 of T gondii oocysts in soil showed that damp conditions result in greater viability of the organism than dry conditions. Expanding the discussion on environmental contamination with coccidian oocysts and detection of coccidian DNA, the present study showed that increasing environmental temperature (within the evaluated range of 16.4° to 24.1°C) increased the probability that a given fecal sample would contain coccidian DNA. Therefore, a temperature threshold for survival or detectability might exist. Additionally, we found that the odds of fecal samples having positive results of coccidian DNA testing decreased as sample freshness decreased, which supported other reported findings for coccidian oocysts.56,57 However, neither precipitation nor humidity was significantly associated with coccidian DNA detection. An initial suspension of fecal samples in a detergent-based solution might have resulted in a better extraction of parasite ova from dry or older feces than what was observed.56 These environmental effects on recovery of oocysts or coccidian DNA might be useful in designing sample collection strategies and analyses to adjust observed prevalences in future studies.

The prevalence of coccidian DNA was associated with none of the potential risk factors evaluated in the present study (ie, cattle density, wild canid relative density, and land use). The many hosts and coccidian species involved and the complex life cycles of those species could explain the observed spatial distribution of the parasites. Additional research involving specific coccidian genera and larger sample sizes per area evaluated might be needed to elucidate the mechanisms of the persistence of coccidia in the environment and in the populations of southeastern Ohio. Because the study area houses various ruminant species and different degrees of coccidia exposure could be expected depending on herd management protocols, a combined empirical and theoretical approach to understanding the role of different ruminant species (ie, host-related immunity) would help clarify the dynamics of heteroxenous coccidia at the community level.

We believe that the low prevalence of small coccidia shed in wild canid fecal samples, including the 0% environmental N caninum prevalence, suggested that the role of the environmental phase of coccidian oocysts in transmission to ruminants is likely minor in rural southeastern Ohio and possibly other areas with low endemicity. Understanding environmental contamination with N caninum attributable to carnivores may lead to improvements in ungulate reproduction by directing appropriate interventions. Although some ungulates (ie, cattle and white-tailed deer) in southeastern Ohio have had N caninum detected by ELISA,14 no existing scientific evidence, including that reported here, suggests that control of coyotes or foxes will control heteroxenous coccidia in ungulates in that region. This conflicting information between N caninum identification in ungulates but not in wild canid fecal samples in southeastern Ohio has been explored further by investigating the role of intermediate host species heterogeneity.58 However, additional epidemiological studies in natural settings with higher endemicity or outbreaks in ruminants might help to characterize the complex interactions between environmental conditions and the diversity of hosts that drive the transmission of infectious diseases at the wildlife-livestock interface.

Acknowledgments

This manuscript represents a modified portion of a thesis submitted by Dr. Moreno-Torres to The Ohio State University Department of Veterinary Preventive Medicine as partial fulfillment of the requirements for a Doctor in Philosophy degree.

Funded by a gift from Duncan Alexander, grants from OSU CARES and Columbus Zoo & Aquarium, and a National Institutes of Health summer research fellowship (No. T35 OD010977).

Funding sources had no involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

The authors thank Craig Hicks for providing advice on wildlife collection permits and providing control samples and Jean Dubach for molecular support on genetic analysis support for host identification.

ABBREVIATIONS

CI

Confidence interval

Footnotes

a.

Hinrichs KC. Seroprevalence of antibodies to Neospora caninum in Ohio cattle. MS thesis. Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, Ohio, 2003.

b.

Shoemaker ME. The effect of stress on the ecology of Neospora caninum in bison (Bison bison). MS thesis. Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, Ohio, 2014.

c.

Gupta S, Wolfe B, Rajala-Schultz P. Identification of Neospora caninum oocysts from feces of Ohio coyotes (Canis latrans) using quantitative PCR (poster and abstract). The Ohio State University College of Veterinary Medicine Research Symposium, April 2011.

d.

National Oceanic and Atmospheric Association climate data online. Available at: www.ncdc.noaa.gov/cdo-web. Accessed Nov 4, 2014.

e.

QIAamp DNA stool mini kit, Qiagen, Hilden, Germany.

f.

Qiagen DNeasy blood and tissue kit, Qiagen, Hilden, Germany.

g.

1X Phusion HF buffer, New England BioLabs Inc, Ipswich, Mass.

h.

Phusion DNA polymerase, New England BioLabs Inc, Ipswich, Mass.

i.

Dyad PTC-220 thermal cycler, MJ Research Inc, Waltham, Mass.

j.

IBI Scientific, Kapp Court Peosta, Iowa.

k.

1X CutSmart buffer, New England Biolabs Inc, Ipswich, Mass.

l.

New England Biolabs Inc, Ipswich, Mass.

m.

Invitrogen, Carlsbad, Calif.

n.

Vector NTI Advance software, version 11.5.4, Thermo Fisher Scientific, Waltham, Mass.

o.

BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Jul 27, 2015.

p.

Repeating shapes file, ArcGIS, version 9.3, Environmental Systems Research Institute Inc, Redlands, Calif.

q.

Devleesschauwer B, Torgerson P, Charlier J, et al. Prevalence: tools for prevalence assessment studies, R package version 0.4.0, 2014.

r.

R, version 3.4.0, R Foundation for Statistical Computing, Vienna, Austria.

s.

Revelle W. Psych: Procedures for personality and psychological research. R package version 1.5.8, 2015.

t.

Engels WR. XNomial: exact goodness-of-fit test for multinomial data with fixed probabilities. R package version 1.0.1, 2014.

u.

R Core Team. Stats package, 2015. R Foundation for Statistical Computing, Vienna, Austria.

v.

Ribeiro PJ Jr, Diggle PJ. geoR: Analysis of geostatistical data. R package version 1.7–5.1, 2015.

w.

Giorgi RE, Diggle Peter J. PrevMap: geostatistical modelling of spatially referenced prevalence data. R package version 1.2.3, 2015.

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Supplementary Materials

Contributor Notes

Dr. Moreno-Torres’ present address is Center for Epidemiology and Animal Health, Monitoring and Modeling, APHIS, USDA, 2150 Centre Ave, Fort Collins, CO 80526.

Address correspondence to Dr. Garabed (garabed.l@osu.edu).