Coyotes as Reservoirs for Onchocerca lupi, United States, 2015–2018

The Onchocerca lupi nematode infects dogs, cats, and humans, but whether it can be spread by coyotes has been unknown. We conducted surveillance for O. lupi nematode infection in coyotes in the southwestern United States. We identified multiple coyote populations in Arizona and New Mexico as probable reservoirs for this species.

co, Texas, and Utah (2,4,5,6). This parasitic nematode is now endemic in domesticated canines in the southwestern United States (7). Reports of O. lupi infection in Canada (Alberta and Prince Edward Island) (7) associated with dog importation from the southwestern United States and travel of US companion animals suggest an anthropogenic spread of the O. lupi nematode. Whether wild canids, including coyotes (C. latrans), might be reservoirs for the O. lupi nematode is unknown.
Because of the growing number of O. lupi infections in canines and humans, public health officials must understand the prevalence and distribution of this parasite in wildlife. Toward that goal, we investigated coyote populations in Arizona, New Mexico, and Nevada as potential primary hosts and natural reservoirs for the O. lupi nematode.

The Study
From December 2015 through July 2018, we collected skin tissue samples from coyotes harvested for predation management conducted by the Arizona Game and Fish Department, and from hunters in Arizona, New Mexico, and Nevada. We did not euthanize any coyotes for the specific purpose of this study. Skin tissue from the interocular frontal area of the animal's head was removed and stored in 80% ethanol until we extracted the DNA. We screened 707 DNA sequences for an O. lupi cytochrome c oxidase (COI) gene (8) using SYBR Green-real-time PCR on a QuantStudio 7 Flex Real Time PCR System (Thermo Fisher Scientific, https://www.thermofisher.com). We used DNA from an adult worm from an infected dog in northern Arizona as a positive control (GenBank accession no. MT878136). We included a no-template control in every real-time PCR reaction plate. We compared the product's melting curve to the positive control using a dissociation curve. We prepared every sample that had a melting curve resembling that of the positive     highest number (5) of coyotes that tested positive for the O. lupi nematode but a lower positivity rate (2.7%) than other counties. For example, in Hidalgo County we sampled only 2 coyotes, 1 of which tested positive.

Coyotes as Reservoirs for
We produced a phylogenetic tree of our 43 COI sequences (including 4 that were isolated from infected dogs and 2 from humans [GenBank accession nos. ) in addition to 30 O. lupi COI genes on GenBank spanning 432 total bases containing 12 single-nucleotide polymorphisms (SNPs) (Figure 2) using IQTREE version 1.6.9 (9) software with 1,000 bootstrap replicates. Examining only this region of the COI gene, we determined the US samples (from dogs, cats, coyotes, and humans) clustered within a single clade with dog samples from Germany, Romania, and Greece. Within this clade, we detected no SNP differences. This clade was separated from a sample from Hungary by 1 SNP and from a single clade containing a human isolate from Turkey and a dog sample from Greece by 1 SNP.

Conclusions
O. lupi infection has been reported mainly in domestic dogs and cats in the southwestern United States (4,2). However, international transportation (purchasing, adopting, and exporting) of dogs from that area has introduced this parasite into environments to which it is not endemic (7,10). We hypothesize coyotes are reservoirs for the O. lupi nematode and could spread this parasite throughout the southwestern United States.
We consider the probable importance of coyotes as natural reservoirs and dispersal agents. In the United States, the average home territory covered by a resident coyote population (either a pack or lone coyote) is 5-41 km 2 , whereas solitary transient coyote territories are up to 155 km 2 (11,12). The large geographic range and widespread occurrence of not only coyotes, but also the putative black fly vector (Diptera: Simuliidae) (13), might facilitate the spread and establishment of the O. lupi nematode in the southwestern United States. Furthermore, many North American wild canids, such as wolves and foxes, have never been assessed for the O. lupi nematode but also should be considered as potential reservoirs. Although the O. lupi nematode is only endemic to the southwestern United States, without appropriate surveillance and mitigation strategies it might spread across the United States and into Canada and Mexico. We are not aware of any reports of O. lupi nematodes in Mexico; however, we identified a coyote that tested positive for O. lupi infection in Hildago County, which borders Mexico. Increasing surveil-lance in nearby counties upon identification of O. lupi nematode-positive coyotes would be prudent. Furthermore, the overlap of rural human residences with coyote and black fly populations probably increases the risk for human exposure.
In summary, canine onchocerciasis is an ongoing emerging infectious threat to wildlife, companion animals, and humans. The expanding range to which the O. lupi nematode is endemic, coupled with increased incidence of onchocerciasis in humans and canines in the southwestern United States, reinforces the need to understand, respond to, and potentially mitigate this threat. This understanding will enable the development of surveillance and mitigation strategies, determine the risk of spread to nonendemic regions, and identify human populations at high risk of infection.