Parasitology, Virology, and Serology of Free-Ranging Coyotes (Canis latrans) from Central Georgia, USA Author(s): Michelle Gates, Richard W. Gerhold, Rebecca P. Wilkes, William D. Gulsby, Lauren Maestas, Alexa Rosypal, Karl V. Miller, and Debra L. Miller Source: Journal of Wildlife Diseases, 50(4):896-901. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/2013-10-283 URL: http://www.bioone.org/doi/full/10.7589/2013-10-283

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DOI: 10.7589/2013-10-283

Journal of Wildlife Diseases, 50(4), 2014, pp. 896–901 # Wildlife Disease Association 2014

Parasitology, Virology, and Serology of Free-Ranging Coyotes (Canis latrans) from Central Georgia, USA Michelle Gates,1,2 Richard W. Gerhold,1,2 Rebecca P. Wilkes,2 William D. Gulsby,3 Lauren Maestas,1 Alexa Rosypal,4 Karl V. Miller,3 and Debra L. Miller1,2,3,5 1Center for Wildlife Health, Department of Forestry, Wildlife, and Fisheries, The University of Tennessee, 274 Ellington PSB, Knoxville, Tennessee 37996, USA; 2 Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, The University of Tennessee, 2407 River Drive, Knoxville, Tennessee 37996, USA; 3Warnell School of Forestry and Natural Resources, The University of Georgia, 180 E Green Street, Athens, Georgia 30602, USA; 4Department of Natural Sciences and Mathematics, Johnson C. Smith University, 100 Beatties Ford Road, Charlotte, North Carolina 28216, USA; 5Corresponding author (email: [email protected])

Management Area (BFGWMA; 33u409N, 83u499W) and Cedar Creek Wildlife Management Area (CCWMA; 33u259N, 83u509W) in Georgia’s Piedmont region. Blood collected at time of death was processed, as described by Miller et al. (2009). Carcasses were frozen until necropsy at the University of Tennessee Center for Wildlife Health (Knoxville, Tennessee, USA). Necropsies were performed, and representative tissue sections were collected into 10% buffered formalin and processed for histologic evaluation. Ticks were placed in 70% ethanol and identified using a dichotomous key (Keirans and Litwak 1989). All heartworms and a subset of intestinal helminthes were collected into 70% ethanol and identified. The sex of each heartworm was morphologically determined according to Orihel (1961). Indirect immunofluorescent assay (VMRD Inc., Pullman, Washington, USA) was used to detect serum antibodies to CPV, CDV, and canine adenovirus (CAV). Sera were tested for Leishmania spp. and Trypanosoma cruzi at Johnson C. Smith University, Charlotte, North Carolina, USA, as described by Rosypal et al. (2007). Toxoplasma gondii serum screening was performed using the modified agglutination test (MAT; Dubey et al. 1995). To screen for parvoviruses, DNA was extracted from a 200-mL mixture of fecal material and an equal volume of phosphate-buffered saline using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, California, USA), according to manufacturer’s instructions,

ABSTRACT: We examined 31 free-ranging coyotes (Canis latrans) from central Georgia, USA, for select parasites and viral agents. Sixteen coyotes had adult heartworms (Dirofilaria immitis). Serum samples from 27 animals revealed antibodies against canine parvovirus (100%), canine distemper virus (48%), canine adenovirus (37%), and Trypanosoma cruzi (7%); none were detected against Leishmania spp. Twenty-two of 24 (92%) coyotes were positive for Toxoplasma gondii. Real-time PCR of feces revealed 32% of coyotes were shedding canine parvovirus, and sequencing revealed type 2b and 2c. Because coyotes could be a spillover host of domestic dog (Canis lupus familiaris) pathogens, studies of the transmission of pathogens between coyotes and domestic dogs are warranted. Key words: Canine parvovirus, coyote (Canis latrans), heartworm (Dirofilaria immitis), parasite, Toxoplasma gondii, Trypanosoma cruzi.

Coyote (Canis latrans) populations have increased in the southeastern US in recent decades. Dietary and behavioral plasticity allows coyotes to live in close association with humans and domestic animals (Gehrt et al. 2010). Because there is concern that some pathogens, such as canine distemper virus (CDV), can be transmitted between domestic and wild canines, we performed a population health survey of coyotes in central Georgia, USA, to determine the prevalence of selected pathogens. We focused on heartworms (Dirofilaria immitis) and canine parvovirus (CPV), due to previous disease reports in coyotes and domestic dogs (Canis lupus familiaris). The Georgia Wildlife Resources Division experimentally reduced coyote populations from March to June 2011 and again in March 2012 on B.F. Grant Wildlife 896

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TABLE 1. Capture area, date, sex, weight, and age of coyotes (Canis latrans) collected, March–June 2011 and March 2012 in central Georgia, USA. Coyote identification

Sitea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

BF BF CC CC BF BF CC CC BF BF BF CC CC BF BF BF BF BF BF CC CC BF CC BF BF BF BF BF BF BF CC

a

Capture date

4 5 2 6 3 3 6 8 9 10 12 13 13 15 16 16 17 21 14 30 17 22 3 1 17 17 20 20 29 30 22

March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 March 2011 April 2011 April 2011 May 2011 May 2011 March 2011 June 2011 March 2012 March 2012 March 2012 March 2012 March 2012 March 2012 March 2012

Sexb

Weight (kg)

Age (years)c

F M F F F F M F M F M M M M F M M F M M M F M M M F M F M F F

12.08 15.21 14.06 11.99 13.96 11.39 14.95 10.06 11.45 16.15 12.7 16.11 10.3 10.56 11.35 13.44 12.75 10.5 16.73 14.42 16.09 10.8 12.82 14 14.53 10.84 16.49 15.73 11.47 9.87 12.14

2 4 ND 1 2 ,1 6 ,1 ,1 3 ND 6 ,1 ,1 ,1 ,1 ,1 ,1 5 ,1 5 1 1 1 ,1 ,1 ,1 ,1 1 ,1 1

BF 5 B.F. Grant Wildlife Management Area; CC 5 Cedar Creek Wildlife Management Area.

b

F 5 female; M 5 male.

c

ND 5 no data.

and processed by real-time PCR, following diagnostic protocol at the University of Tennessee College of Veterinary Medicine. Parvovirus typing was performed on PCRpositive samples, following Buonavoglia et al. (2001). We examined 31 coyotes (17 females and 14 males; Table 1). Blood smear slides were available for 26 coyotes and serum for 27. Sixteen (52%) had adult heartworms, with a mean intensity of 8.8 and abundance of 4.5 (Table 2). Five coyotes (16%) had lung and heart lesions consistent with heartworm disease (Fig. 1A) and microfilariae present in blood vessels

(Fig. 1B). Eleven coyotes (68%) infected with heartworms had both male and female heartworms, and the remaining five coyotes had only female heartworms. Microfilariae were only present in coyotes with both male and female heartworms (Table 2). All coyotes were examined for external parasites, but feces for intestinal parasites were only collected from 24. All coyotes had ticks, with most (66%) having .1 species; 93% (n528) had Amblyomma americanum, 60% (n518) Ixodes scapularis, 30% (n59) Amblyomma maculatum, 16% (n55) Ixodes cookei, and 6% (n52)

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TABLE 2. Heartworm (Dirofilaria immitis) infection at necropsy and microfilariae in blood smears for 31 coyotes (Canis latrans) collected March–June 2011 and March 2012 in central Georgia, USA. Animals not listed were negative. Coyote identification (age in years)

Microfilariaea

No. of female adult heartworms

No. of male adult heartworms

Total heartworm intensity

2 (4) 3 (unknown) 4 (1) 5 (2) 6 (,1) 7 (6) 10 (3) 12 (6) 13 (,1) 19 (5) 23 (1) 24 (1) 26 (,1) 28 (,1) 29 (1) 30 (,1)

Negative ND Positive Negative Negative ND Positive Positive Positive Positive Positive Negative ND Positive Positive ND

1 7 3 2 1 2 3 23 2 1 9 2 4 3 12 2

0 3 5 0 0 3 3 30 5 1 7 0 2 1 5 0

1 10 8 2 1 5 6 53 7 2 16 2 6 4 17 2

a

ND 5 no data (blood slide not collected).

Dermacentor variabilis. Ten of 24 coyotes (42%) were negative for intestinal parasites, but nine (37%) were infected with Ancylostoma caninum, eight (33%) with Sarcocystis spp., five (20%) with Trichuris vulpis, and three (12%) with Capillaria spp. Results of serum from 27 coyotes tested for Leishmania spp., T. cruzi, T. gondii,

CPV, CDV, and CAV are summarized in Table 3. Additionally, 10 of the 31 coyotes (32%) had parvovirus DNA in their feces with threshold cycle values of 29.52–38.80. Strain typing of parvovirus in four coyotes using fecal and fresh-tissue DNA disclosed three CPV 2c from BFGWMA and one CPV 2b from CCWMA.

FIGURE 1. Histopathologic lesions consistent with heartworm (Dirofilaria immitis) disease in the organs of coyotes (Canis latrans) from central Georgia, USA, showing (A) area of inflammatory tissue with increased eosinophils in lung tissue (arrows; bar520 mm) and (B) microfilariae within blood vessels of lung tissue (arrows; bar520 mm).

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TABLE 3. Select serology tests and numbers of antibody-positive coyotes (Canis latrans) collected March– June 2011 and March 2012 from central Georgia, USA. Test

Leishmania Trypanosoma cruzi Toxoplasma Canine parvovirus Canine distemper virus Canine adenovirus a

No. positive (n)

0 2 22 27 13 10

(27) (27) (24) (27) (27) (27)

Titer rangea

ND ND 128–.8,192 20–.60 20–.60 20–.60

Test

Indirect immunofluorescent Indirect immunofluorescent Modified agglutination test Indirect immunofluorescent Indirect immunofluorescent Indirect immunofluorescent

assay assay assay assay assay

ND 5 no data.

The heartworm prevalence we found (52%) is similar to prevalence reported in other coyote populations in the southeastern US (Holzman et al. 1992; Miller et al. 2009). However, previous authors only examined blood smears for microfilariae. Our results disclosed some coyotes had only adult female heartworms and, thus, no circulating microfilariae, suggesting that prevalences previously reported could be low. The evidence of heartworm disease found in the coyotes in our and other studies suggests that heartworms are a significant pathogen of coyotes in the southeastern US. Indeed, one report of a coyote death with a ruptured aortic aneurysm was suspected from heartworm disease (Miller et al. 2007). Furthermore, increasing urban coyote populations bring them into proximity with domestic dogs, increasing the potential for mosquito transmission of heartworms among the two species. The ticks and intestinal parasites we recovered were similar to those previously reported for the southeastern US. Previous authors investigated the prevalence of T. gondii, Leishmania spp., and T. cruzi in coyotes. Toxoplasma gondii is a globally distributed, zoonotic protozoan, which can infect all warm-blooded vertebrates (Dubey 2008), resulting in encephalitis, pneumonia, and other lesions. We found 92% (n522) of coyotes positive for T. gondii by MAT; however, there were no histologic lesions. Thus, the health impact of T. gondii infection in coyotes remains unknown. Leishmania spp. and T. cruzi

are endemic parasites in the US and Canada (Duprey et al. 2006) and can cause fatal infection in humans and dogs. Dogs with canine visceral leishmaniasis are a major reservoir host for human infections (Rosypal et al. 2003), and T. cruzi has been described in a variety of wild animals in the southeastern US (Brown et al. 2010). Our results (two coyotes positive for T. cruzi antibodies and none for Leishmania) were similar to those of other investigators in the southeastern US (Rosypal et al. 2007; Brown et al. 2010). However, prevalence may vary elsewhere. For example, Gro¨gl et al. (1984) reported 14% of 19 coyotes positive for T. cruzi antibodies in Texas. Expansion of sampling in other geographic regions may shed light on distribution patterns for these parasites. In our study, 100% of coyotes had detectable parvovirus antibody, and 32% were positive by real-time PCR testing on feces, suggesting they were shedding parvovirus. Similarly, Holzman et al. (1992), Gese et al. (1997), and Grinder and Krausman (2001) found 64, 100, and 100%, respectively, of coyotes antibodypositive for CPV but did not investigate shedding. Miller et al. (2009) did not test for antibodies but found one in 36 coyotes from South Carolina to be shedding parvovirus particles on electron microscopic examination of feces. In 1978, CPV type 2 (CPV-2) was found in domestic dogs and in wild canines 1 yr later (Hoelzer and Parrish 2010). By 2000, there were three variants of CPV-2 (2a,

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2b, and 2c), and all three are now found in multiple hosts worldwide (Hoelzer et al. 2008). Our PCR and sequencing results show three coyotes were infected with CPV-2c and one with CPV-2b, and both subtypes have been found in domestic dogs in Georgia (Hong et al. 2007; Kapil et al. 2007). Thus, there is potential for coyotes to be a reservoir or spillover host for this pathogen. Similar concern is given to CDV and CAV because of their impact on domestic dogs and other wildlife species. Although a recent study of coyotes from the southeastern US showed similar results to ours (Miller et al. 2009), Holzman et al. (1992) did not find antibodies for CDV and did not report on CAV. It is possible that this may represent a recent spillover of CDV into coyotes in the southeastern US. Domestic dogs are considered to be the primary reservoir of CDV, but the virus disseminates among free-ranging, unvaccinated, or incompletely vaccinated dogs and urban or rural wildlife (Kapil and Yeary 2011). The virus is highly labile outside of the host and could be maintained in wildlife populations between outbreaks (Trebbien et al. 2014). Surveillance of CDV genotypes in domestic dogs and wildlife within a discrete territory over an extended period would increase our understanding of how the virus spreads and evolves among species (Kapil and Yeary 2011). Coyote populations are increasing in urban areas, resulting in closer contact with domestic species and humans. Our research, as well as others’, reveals that coyotes are infected with common pathogens that can be transmitted to or from domestic dogs. It is unknown how coyotes are involved in pathogen transmission, and further research investigating the likelihood of transmission of pathogens among coyotes and domestic dogs is warranted. Funding for this project was provided by the Georgia Wildlife Resources Division through the Wildlife Restoration Program. M.G. was funded through the Summer Student Research Program by

the University of Tennessee Center of Excellence in Livestock Diseases and Human Health. We acknowledge Rusty Johnson, the Georgia Trapper’s Association, and the following students who assisted on the project: Rebecca Hardman, Conner England, Diana Gibbs, Lauren Henderson, Caroline Brown, and Jill Wilson. We also thank Aly Chapman and Heidi Wyrosdick for assistance with parasite identification. LITERATURE CITED Brown EL, Roellig DM, Gompper ME, Monello JR, Wenning KM, Gabriel MW, Yabsley MJ. 2010. Seroprevalence of Trypanosoma cruzi among eleven potential reservoir species from six states across the southern United States. Vector Borne Zoonotic Dis 10:757–763. Buonavoglia C, Martella V, Pratelli A, Tempesta M, Cavalli A, Buonavoglia D, Bozzo G, Elia G, Decaro N, Carmichael L. 2001. Evidence for evolution of canine parvovirus type 2 in Italy. J Gen Virol 82:3021–3025. Dubey JP. 2008. The history of Toxoplasma gondii— The first 100 years. J Eukaryot Microbiol 55:467–475. Dubey JP, Thulliez P, Weigel RM, Andrews CD, Lind P, Powell EC. 1995. Sensitivity and specificity of various serologic tests for detection of Toxoplasma gondii infection in naturally infected sows. Am J Vet Res 56:1030–1036. Duprey ZH, Steurer FJ, Rooney JA, Kirchhoff LV, Jackson JE, Rowton ED, Schantz PM. 2006. Canine visceral leishmaniasis, United States and Canada, 2000–2003. Emerg Infect Dis 12:440– 446. Gehrt SD, Riley SPD. 2010. Coyotes (Canis latrans). In: Urban carnivores: Ecology, conflict, and conservation, Gehrt SD, Riley SPD, Cypher BL, editors. The Johns Hopkins University Press, Baltimore, Maryland, pp. 78–95. Gese EM, Schultz RD, Johnson MR, Williams ES, Grabtree RL, Ruff RL. 1997. Serological survey for diseases in free ranging coyotes (Canis latrans) in Yellowstone National Park, Wyoming. J Wildl Dis 33:47–56. Grinder M, Krausman PR. 2001. Morbility: Mortality factors and survival of an urban coyote population in Arizona. J Wildl Dis 37:312–317. Gro¨gl M, Kuhn RE, Davis DS, Green GE. 1984. Antibodies to Trypanosoma cruzi in coyotes in Texas. J Parasitol 70:189–191. Hoelzer K, Parrish CR. 2010. The emergence of parvoviruses of carnivores. Vet Res 41:39. Hoelzer K, Shackelton LA, Parrish CR, Holmes EC. 2008. Phylogenetic analysis reveals the emergence,

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