Echinococcus Species Infections among Wild Canids in Pennsylvania, USA

Echinococcus species are zoonotic tapeworms of veterinary and public health concern. Two species in North America are Echinococcus multilocularis and Echinococcus canadensis (part of the Echinococcus granulosus sensu lato). Currently, E. granulosus sensu lato is composed of 10 genotypes (G1–10) with G8 and G10 recognized as E. canadensis (Cerda et al. 2018; Dell et al. 2020). In the US, red foxes (Vulpes vulpes), gray foxes (Urocyon cinereoargenteus), coyotes (Canis latrans), and wolves (Canis lupus) are the primary wild definitive hosts for Echinococcus spp. Domestic dogs (Canis familiaris) can also serve as definitive hosts (Schurer et al. 2013; Cerda et al. 2018). The E. multilocularis life cycle includes small mammals (i.e., rodents and insectivores) as intermediate hosts, and E. canadensis uses both wild and domestic artiodactlyid species such as white-tailed deer (Odocoileus virginianus), elk (Cervus elaphus), and sheep (Ovis sp.; Schurer et al. 2013; Cerda et al. 2018).

Echinococcus spp. infections in canids are generally restricted to the intestinal tract and not associated with overt disease. Recently in North America, however, alveolar echinococcosis (parasitic lesions in organs that are filled with developing protoscolices) has been reported in domestic dogs (Peregrine et al. 2012; Zajac et al. 2020). In intermediate hosts, larval tapeworm cysts form in a variety of organs (e.g., liver or lungs) resulting in morbidity and mortality. Echinococcus spp. are potential threats to wildlife, livestock, and human health. Further, these parasites threaten livestock production systems, especially in European and Asian countries, where production losses can total $1.5–2 billion USD annually (Cerda et al. 2018).

In North America, Echinococcus spp. are primarily reported from northern regions of the midwest and western US, Alaska, and Canada (Massolo et al. 2014); however, E. multilocularis was recently reported in eastern US states: a dog in Virginia, and a human in Vermont, and E. canadensis was reported in moose and coyotes in Maine and translocated elk in Tennessee (Catalano et al. 2012; Schurer et al. 2018; Dell et al. 2020; Zajac et al. 2020). These recent detections suggest that the distribution of Echinococcus spp. are more widespread than originally thought, emphasizing the need for increased surveillance in the eastern US. Our study tested wild canids from Pennsylvania for Echinococcus spp. to determine geographical presence and the definitive host species involved in transmission.

Between January 2019 to October 2020, 308 gastrointestinal tracts (GITs; stomach to rectum) were collected from coyotes, red foxes, and gray foxes from 48 counties in Pennsylvania, USA (Fig. 1, Supplementary Material Table S1). Samples were collected at check stations from organized recreational predator hunts, and fresh road-killed animals. No animals were euthanized specifically for this study. All samples were frozen at –20 C and shipped to the Southeastern Cooperative Wildlife Disease Study, University of Georgia (UGA; Athens, Georgia, USA) where sample processing occurred following biosafety protocols as described (Gesy et al. 2013). All methods were reviewed and approved by UGA's International Animal Care and Use (A2020 11-010-Y2-A3) and biosafety committees. The GITs were screened for adult Echinococcus spp. by scraping and washing small and large intestinal contents into a sieve and visually inspecting the contents for Echinococcus spp. as described (Gesy et al. 2013), with one modification: the mucosa of examined intestines were scraped to dislodge parasites as opposed to shaken in jars. This method was chosen because the scraping and counting technique (SCT) is considered the gold standard; however, Gesy et al. (2013) found that including a filtration step does not reduce the sensitivity and reduces the time to screen samples. Feces were collected from the large intestine and colon during this process. Any Echinococcus spp. suspects were preserved in 70% ethanol and identified to genus by morphology (i.e., 2–6 proglottids and scolex) using a dissecting microscope. Morphology was used to identify suspect worms only to genus level, as freeze–thaw led to some degradation of cestodes. To identify species, DNA was extracted from a subset of Echinococcus spp. (15 individual worms from a suspect positive sample) using DNeasy Blood and Tissue extraction kits (Qiagen, Germantown, Maryland, USA) to identify species and confirm positive Echinococcus sp. infection. Genus-wide and species-specific PCRs were used (Table 1). Amplicons were gel purified (Qiagen) and submitted to GENEWIZ (Azenta Life Sciences, South Plainfield, New Jersey, USA) for bidirectional sequencing. Consensus sequences were generated using Geneious Prime (Dotmatics, San Diego, California, USA).

In addition to intestinal sieving, a random subset of fecal samples (n=139) were PCR tested to compare the two methods. Fecal samples from the two animals detected as infected with Echinococcus spp. by morphology and PCR were included. For these two positive canids, three subsets of the feces were tested via molecular analysis to confirm species of Echinococcus. If only one sample was collected from a county, it was included. Before DNA extraction, 1 g of feces was frozen for 24 h at –80 C then heated at 105 C for 10 min to fracture eggshells. The DNA was extracted using a miniStool kit (Qiagen). The same PCR protocols were used to test feces as to test individual worms (Table 1). Negative water controls were included for the DNA extraction and PCR to ensure no contamination occurred during molecular analyses. Positive controls were E. multilocularis from a previous study for the conventional PCR analysis and E. granulosus and E. multilocularis commercially available gene fragments (gBlocks, Integrated Data Technologies Inc., Coralville, Iowa, USA) for RT-PCR. The gBlock for E. canadensis does not amplify using the E. multilocularis RT-PCR and vice versa.

Two of 155 coyotes (1.29%; 95% confidence interval [CI], –0.5%, 3.09%), both from 2020, each had adult Echinococcus spp. Using the intestinal sieving method, one adult male coyote from Bradford County was positive for both E. multilocularis (100% nucleotide match; GenBank accession nos. OP068158 and OP081143) and E. canadensis. G8 (99% match; nos. OP068161 and OP068166); one adult female coyote from Wyoming county was infected with E. canadensis G8 (99% match; nos. OP068160 and OP068164; Fig. 1, Supplementary Material Table S2). Echinococcus spp. were not enumerated in either coyote because of high infection intensities. None of the 137 red foxes or 16 gray foxes were positive via GIT sieving or fecal PCR. Two of 139 fecal samples, both from the positive coyotes mentioned, were Echinococcus spp. positive by PCR test and yielded co-infections of Echinococcus spp.: E. multilocularis (100% match; nos. OP068157, OP081141, and OP081142) and E. canadensis G8 (99% match; nos OP068159, OP068162, OP068163, and OP068165); see Supplementary Material Table S2.

The presence of Echinococcus spp. in Pennsylvania represents a historically unrecognized disease risk to humans, livestock, domestic animals, and wildlife. Echinococcus spp. eggs are relatively persistent, which may lead to human contact and infection in urbanized areas where indirect interactions with wildlife probably increases risk of exposure to humans (Veit et al. 1995). Intestinal Echinococcus spp. infections in domestic dogs also may elevate this risk, as humans might be exposed through interactions with their pets (Carmena and Cardona, 2013).

Domestic dogs classically serve as definitive hosts for Echinococcus spp. and do not typically develop clinical disease with intestinal infections. However, they do pose a peridomestic source for environmental contamination. Interestingly, there have been recent reports of domestic dogs developing alveolar echinococcosis, suggesting that these hosts can also act as aberrant intermediate hosts (Peregrine et al. 2012; Skelding et al. 2014; Pinard et al. 2019; Zajac et al. 2020). It is not fully understood why some domestic dogs develop alveolar echinococcosis; it has been hypothesized that either ingestion of eggs from infected wild canid feces and subsequent intermediate host-like infection occurs, or they have an existing intestinal infection from ingestion of infected cysts that results in autoinfection (Weiss et al. 2010; Pinard et al. 2019). It is currently unknown if wild canids can also serve as aberrant intermediate hosts.

Echinococcus canadensis poses a threat to wildlife and domestic animals that can serve as intermediate hosts. Although E. canadensis is traditionally found in sylvatic cycles with wild cervids acting as the intermediate hosts, infections have been reported in domestic muskox (Ovibos moschatus) from Quebec, Canada, and sheep from China (Schurer et al. 2013; Hua et al. 2019). When considering wild ruminants and wildlife management, E. canadensis infections in cervids could affect declining moose populations and restoration efforts for eastern elk populations (Musante et al. 2010; Schurer et al. 2013). Similarly, North American E. multilocularis infections in humans and dogs pose not only a veterinary and human health threat, but also indicate another risk for wildlife populations such as the Allegheny woodrat (Neotoma magister; Skelding et al. 2014; Zajac et al. 2020; Polish et al. 2021).

Although only two coyotes were detected to be infected with Echinococcus spp., this might be because of the relatively small sample sizes surveyed in the different regions. There is a need for continued surveillance of wild definitive and intermediate hosts to better define geographic prevalence and distribution, and to inform risk communication and preventative measures. Furthermore, the findings of both Echinococcus spp. in each infected coyote confirms co-infections in this region. Although such co-infections may not be common, they have also been noted in coyotes and red foxes in Alberta, Canada where they found both singly and co-infected individuals (Santa et al. 2018). Coyotes predate on both cervids and rodent hosts, and therefore may be exposed to both species of Echinococcus. It is important to remember that E. granulosus may be relatively small in coyotes and mistaken for E. multilocularis if no molecular confirmation is conducted (Santa et al. 2018). In the face of changing climate and landscape alteration, surveillance, research strategies, and informed management approaches for this important group of zoonotic cestodes are greatly needed.

We thank Alec Smith (Keystone College) and Nate Beard (USDA-APHIS-WS) for sample collection assistance and the Pennsylvania hunters who provided samples from organized hunts, the Mosquito Creek Sportsmen's Association, Cresson Sportsmen's Association, and the District 9 PA Trappers Association. We also thank Southeastern Cooperative Wildlife Disease Study member agencies and states for their financial support.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-22-00042.

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