SENTINEL COYOTE PATHOGEN SURVEY TO ASSESS DECLINING BLACK-FOOTED FERRET (MUSTELA NIGRIPES) POPULATION IN SOUTH DAKOTA, USA

The black-footed ferret (Mustela nigripes), once considered extinct in the wild, is one of the most endangered mammals in North America. Historically, the black-footed ferret ranged from Canada to Mexico, coinciding with the habitat of prairie dogs (Cynomys spp.; Belant et al. 2015). Reliant on prairie dogs for burrows and prey, black-footed ferrets were driven to near extinction by the loss of prairie dogs and their habitat due to agricultural development throughout the 1900s coupled with disease outbreaks (Biggins and Godbey 2003). The last known population of wild black-footed ferrets was discovered in Meeteetse, Wyoming in 1981. Due to declines in this population caused by canine distemper virus (CDV) and sylvatic plague (Yersinia pestis), the last 18 ferrets were captured and brought into captivity by 1987 (Lockhart et al. 2006), forming the basis of a captive breeding and reintroduction program. As of 2018, more than 4,300 black-footed ferrets have been released at 30 different sites in the US (Santymire and Graves 2019).

Release of black-footed ferrets onto the Cheyenne River Sioux Reservation in South Dakota first occurred in 2000. The reintroduction site lies along the Moreau River and contains 5,739 ha of black-tailed prairie dog (Cynomys ludovicianus) colonies. The site was chosen due to the distribution and density of prairie dogs and the lack of sylvatic plague and CDV in the area (Lockhart et al. 2000). By 2008, this reintroduction site was classified as successful, with an estimated 150 animals and 75 breeding adults. Despite this initial success, numbers of black-footed ferrets declined, with only 25 black-footed ferrets seen in a partial survey in 2011 (USFWS 2013). In an effort to understand and mitigate the decline in the population, we investigated potential pathogens that could be contributing to the decline in numbers of black-footed ferrets at this site. The pathogens selected for testing included Yersinia pestis (sylvatic plague), CDV, West Nile virus (WNV), Francisella tularensis (tularemia), and Dirofilaria immitis (canine heartworm disease).

Due to the difficulty of sampling black-footed ferrets, the coyote (Canis latrans) was selected to determine disease prevalence in the areas occupied by the black-footed ferret population. Previous studies have used coyotes as a sentinel species due to their widespread distribution and exposure to multiple rodents, such as black-tailed prairie dogs, in their diet (Willeberg et al. 1979; Bischof and Rogers 2005). Coyotes may also be directly exposed to diseases present in the black-footed ferret population, as they accounted for up to 67% of predation on black-footed ferrets in one study (Biggins et al. 1999). The resistance of coyotes to mortality caused by tularemia, sylvatic plague, and canine distemper (Lundgren et al. 1957; Williams et al. 1988; Baeten et al. 2013), and their production of antibodies, makes them an efficient means of estimating the prevalence of these diseases in an area of study.

In December 2012, blood samples were recovered from coyote carcasses removed by aerial gunning in the southwest quadrant of the Cheyenne River Sioux Reservation in South Dakota (Fig. 1). Gunning and sample collection were performed by Cheyenne River Sioux Tribe Game, Fish and Parks Department personnel as part of management activities to control coyote populations. Free blood was collected from the chest cavity and aliquoted into whole and ethylenediaminetetracetic (EDTA) blood tubes. Samples were shipped chilled overnight to the Animal Health Diagnostic Center at the Cornell University College of Veterinary Medicine (Ithaca, New York, USA) for processing and distribution. Serum and plasma were removed following centrifugation of whole blood and stored frozen at –80 C until testing. Whole blood treated with EDTA was refrigerated until testing. Serum samples were tested for antibodies to WNV and CDV using serum virus neutralization assays at the Animal Health Diagnostic Center (Sheldon et al. 2017). For WNV, the challenge virus had the specificity of WNV-NY99 and the serum-virus mixture contained 5% guinea pig complement. Serum samples were tested for antibodies to Yersinia pestis F1 and V antigens using lateral flow cassettes and scored on a scale of 0–4+ according to the darkness of the test line, 0 being negative and 4+ being strongly positive (Abbott et al. 2014), at the US Geological Survey National Wildlife Health Center (Madison, Wisconsin, USA). We extracted DNA from whole EDTA-treated blood and tested for F. tularensis by real-time PCR at the New York State Department of Health—Wadsworth Center (Albany, New York, USA). Multiple assays were used at the Cornell University College of Veterinary Medicine to detect D. immitis infection to minimize false-positive and false-negative results associated with certain types of infections (American Heartworm Society 2018). Enzyme-linked immunosorbent assays (ELISA; DiroCHEK, Zoetis, Parsippany, New Jersey, USA and SNAP 4Dx, Idexx, West-brook, Maine, USA) were used to detect D. immitis antigen in plasma and serum. Polymerase chain reaction testing was used to detect D. immitis DNA that was extracted from EDTA-treated whole blood. The presence of D. immitis microfilariae in EDTA-treated whole blood was assessed using a Modified Knott's Heartworm test and examining Giemsa-stained blood smears. Results of pathogen testing of males and females were compared using a Fisher's exact two-tailed test (α=0.025).

Blood samples were collected from 15 adult male and 15 adult female coyotes. Assays and positive criteria used in the study are summarized in Table 1. Antibodies to one or more pathogens (WNV, CDV, or Y. pestis) were found in 77% (23/30) of the coyotes. West Nile virus had the highest seroprevalence (Table 2) with 71% (20/28) of coyotes testing antibody-positive; two animals were excluded from the WNV results due to nonspecific cytotoxicity with the WNV serum neutralization assay. Four (14%) coyotes were considered suspect-positive for WNV. Eight coyotes (27%) tested antibody-positive for CDV. Thirteen (43%) coyotes were considered suspect-positive for CDV. Four (13%) coyotes had positive results on the F1 lateral flow cassette and were considered positive for antibodies to Y. pestis; one of these coyotes also had a positive result on the V cassette. Three samples (10%) were considered suspect-positive for Y. pestis antibodies. There were no significant differences by sex for any of the pathogens (P>0.025). All samples were negative for F. tularensis DNA and D. immitis antigen, DNA, and microfilariae.

The use of coyotes as disease sentinels revealed a novel disease threat to black-footed ferret populations. First detected in the US in New York in 1999, WNV quickly spread west to reach South Dakota by 2002. Our 71% WNV seroprevalence in coyotes (n=28) was considerably higher than reported previously in other studies of wild canids, suggesting that exposure of mesopredators to WNV on the Cheyenne River Sioux Reservation may have been common in 2012. Studies in gray wolves (Canis lupus) in Minnesota (2007–13) and coyotes in Nebraska (2002–03) reported a WNV seroprevalence of 37% (n=257 adults) and 48% (n=67), respectively (Bischof and Rogers 2005; Carstensen et al. 2017). These studies used a plaque reduction neutralization test with a positive cut-off titer of ≥1:10, which would give results comparable to those we obtained using a serum virus neutralization assay (Di Gennaro et al. 2014). There was also a higher than average number of human cases of WNV-neuroinvasive disease reported in South Dakota in 2012 and 2013, with 203 and 149 cases, respectively (Kightlinger 2017). Increased average maximum temperature for the Cheyenne River Sioux Reservation in 2012 (National Oceanic and Atmospheric Administration 2019) may have been a factor, as replication and transmission of WNV by mosquitoes has been shown to increase with temperature (Kilpatrick et al. 2008). Local conditions, combined with the proximity of the site to the Moreau River that may have provided breeding sites for mosquitoes, could explain why we found a high prevalence in coyotes. In addition, a laboratory study of WNV infection in Vesper Sparrows (Pooecetes gramineus), a species that breeds throughout South Dakota, showed that this species may contribute to the amplification of WNV in this area (Hofmeister et al. 2016). Although studies of WNV have not been conducted in black-footed ferrets, it may be a concern for the success of black-footed ferrets due to the potential for infection to cause fatal neurologic disease in some mammalian species (Root and Bosco-Lauth 2019).

While the main route of exposure to WNV for most mammals is believed to be from a mosquito vector, recent studies have shown that oral transmission of the virus from an infected prey item is possible in domestic cats (Felis catus), golden hamsters (Mesocricetus auratus), and American alligators (Alligator mississippiensis; Austgen et al. 2004; Klenk et al. 2004; Sbrana et al. 2005). Wild birds are considered the main reservoir and amplifying host for WNV. However, recent studies have shown that eastern chipmunks (Tamias striatus) and eastern cottontail rabbits (Sylvilagus floridanus) produce viremias up to 10^7.8 plaque forming units (pfu)/mL and 10^5.8 pfu/ mL, respectively (Tiawsirisup et al. 2005; Platt et al. 2007). These viremias surpass the common threshold of greater than 10^5.0 pfu/ mL for considering a species to be an amplifying host for WNV (Root 2013). Fox squirrels (Sciurius niger) have been shown to produce a viremia of 10^4.98 pfu/mL when infected with WNV (Root et al. 2006). Both eastern chipmunks and fox squirrels belong to the family Sciuridae, which also includes prairie dogs. No studies have been undertaken to determine the susceptibility of either black-footed ferrets or black-tailed prairie dogs to WNV; however, transmission of the virus to black-footed ferrets from both mosquitoes and ingestion of infected prairie dogs warrants consideration. Future research could investigate the epizootiology of WNV in the prairie habitat of South Dakota to determine the potential threat of WNV to the endangered black-footed ferret as well as the role that black-tailed prairie dogs may play in amplifying and transmitting the virus.

Sylvatic plague, caused by Y. pestis, is considered one of the major threats to the recovery of the black-footed ferret and is of concern regarding the recent declines in this population (USFWS 2013). The disease can be fatal in black-footed ferrets themselves, but also causes high mortality among prairie dogs. Plague was first detected in wild animals in South Dakota in 2004 (South Dakota Department of Health 2019). Our seroprevalence of plague of 13% in coyotes corresponds to the overall seroprevalence (14%; n=17,403) seen in coyotes from western states, although the seroprevalence in specific states varied widely (0–98%) and occurred over a long span of time (1970s–2000s; Salkeld and Stapp 2006). The individual studies included in this review used hemagglutination inhibition and ELISA assays with positive cut-offs of ≥1:8 to >1:32. The lateral flow cassette used in this study showed results of antibodies to F1 antigen that strongly correlated to hemagglutination inhibition results with a positive cut-off of ≥1:16 (Abbott et al. 2014), making our results generally comparable to past results. No positive coyotes were found in a previous serosurvey of 166 coyotes from South Dakota (Salkeld and Stapp 2006); details about this survey were not given, but it may have been performed prior to the introduction of plague into the area.

Although most serosurveys of coyotes test for anti-F1 antibodies, it may be more useful to include other antigens, such as V, as we did, to avoid false negative results, especially for cases involving Y. pestis strains that do not express F1 antigen (Friedlander et al. 1995). Vernati et al. (2011) showed, using western blot testing, that 90% of coyotes, previously confirmed positive for exposure to plague by ELISA, had antibodies to antigens other than F1. Anti-F1 antibodies were found in only 35% of the coyotes compared to 55% that had anti-V antibodies (Vernati et al. 2011). This difference may be explained by anti-F1 antibodies being more short-lived in coyotes than are antibodies to other antigens (Gese et al. 1997). If we combine our positive (anti-F1) and suspect-positive results (anti-V), seven (23%) of the coyotes tested in this study showed evidence of exposure to Y. pestis, which may indicate a higher risk of plague in the area.

Coyotes are useful sentinel animals for plague surveillance because they ingest large numbers of rodents and may be exposed to plague even if the prevalence in rodents is low (Gage et al. 1994). Plague may exist at an enzootic level in some areas and affect black-footed ferret survival without noticeable prairie dog die-offs (Matchett et al. 2010). Seropositive coyotes in our study may represent local or introduced risk of plague to black-footed ferrets. Estimated antibody titers in our study were low, ≤1:32, which may indicate waning titers or low-exposure doses (Baeten et al. 2013). Because coyotes may travel long distances (Kolbe and Squires 2004), the location of exposure to Y. pestis may be far from the location of sampling. However, coyotes may also transport infected fleas, introducing the risk of plague exposure to new areas. The disease can be transmitted from prairie dogs to black-footed ferrets via a flea vector, but can also be transmitted via ingestion, causing mortality within 48 h (Godbey et al. 2006). Sylvatic plague has caused an estimated mortality of 75% among black-footed ferrets at Conata Basin, South Dakota, about 240 km from our study site, and has been implicated as one factor leading to a 40% reduction in breeding adults between 2008 and 2015 (Belant et al. 2015). Thus, although evidence of plague exposure at this site is indirect, efforts to protect black-footed ferrets from sylvatic plague, such as dusting prairie dog burrows with insecticides and use of vaccines for black-footed ferrets (Rocke et al. 2008) and prairie dogs (Rocke et al. 2014, 2017), may be warranted.

Canine distemper virus, a morbillivirus that infects a wide range of carnivores, is transmitted mainly via direct contact with infectious saliva and nasal exudates. The virus causes fatal neurologic disease in black-footed ferrets (Williams et al. 1988). We found antibodies to CDV in 27% of the coyotes sampled, a moderate seroprevalence rate compared to those found in Utah and Wyoming, 16% (n=43 adults) and 88% (n=33 adults), respectively (Gese et al. 1997; Arjo et al. 2003). A study of wolves in Minnesota found 19% (n=255 adults) positive for CDV antibodies (Carstensen et al. 2017). These studies also used serum virus neutralization tests with similar cut-offs (≥1:10 to ≥1:25). A study of the persistence of CDV in wolves in Yellowstone Park suggested that multiple hosts, including coyotes, may be needed for long-term persistence of CDV in a region (Almberg et al. 2010). Coyotes, swift foxes (Vulpes velox), and badgers (Taxidea taxus) are carnivores that all frequent prairie dog colonies and have been shown to be susceptible to CDV (Vanmoll et al. 1995; Miller et al. 2000). Thus, coyotes that are seropositive to CDV may indicate that CDV is circulating in habitat used by black-footed ferrets. The impact that CDV may have on black-footed ferrets can be highlighted by the decline of the Meteetsee population before the initiation of the captive breeding and reintroduction program (Carr 1986). Due to the potential impact of CDV on reintroduced ferrets, an effective vaccine for CDV was developed for black-footed ferrets and is now routinely administered to the captive population (Wimsatt et al. 2006). However, vaccinating all wild-born kits against CDV is not feasible, and thus wild populations are still susceptible to the disease.

Although none of the coyotes we sampled were positive for F. tularensis DNA by real-time PCR, other studies have shown serologic evidence of this pathogen in coyotes in neighboring states. In Wyoming and Nebraska, 21% (n=110) and 32% (n=67), respectively, of coyotes sampled had antibodies to F. tularensis (Gese et al. 1997; Bischof and Rogers 2005). Only 2% (n=43) of adult coyotes sampled in Utah were seropositive for F. tularensis (Arjo et al. 2003). Francisella tularensis can be transmitted by multiple routes including via arthropod vectors, such as ticks, which could be transported by coyotes coming from areas with active tularemia.

In addition, none of the coyotes we tested showed evidence of heartworm infection. However, exposure to D. immitis was detected in 6.5% (n=77) of coyotes sampled in Oklahoma and Texas and 7% (n=387) of coyotes in Minnesota (Paras et al. 2012; Carstensen et al. 2017). In the US, 21–42% of coyotes are infected with heartworm (McCall et al. 2008) and have been shown to carry heavy worm burdens with microfilariae in their peripheral blood, enabling them to be reservoirs of the parasite (Weinmann and Garcia 1980). Like domestic ferrets, black-footed ferrets are susceptible to heartworm disease (McCall 1998; USFWS 2017). Because heartworm is present in neighboring states, coyotes and black-footed ferrets in South Dakota may also be at risk of exposure and infection. The lack of tularemia and canine heartworm disease in our study could be due to small sample size and low prevalence in the population of study instead of to absence of the disease. Collection of samples in December should not have limited our ability to detect F. tularensis, which is associated with winter hunting of rabbits in humans, or D. immitis, which should persist once infection occurs in warmer months when mosquitoes are active. Our negative results may be useful as a baseline level for future pathogen surveys.

Although our study detected WNV, sylvatic plague, and CDV antibodies in coyotes on the Cheyenne River Sioux Reservation, the small sample size and potential for antibodies to be indicative of past infection instead of current disease dynamics is of note. As this study did not sample black-footed ferrets themselves or their prey base directly, the definitive cause for the decline of this population cannot be determined. However, the detection of antibodies to both sylvatic plague and CDV among coyotes in the area indicates the potential for black-footed ferrets in the region to be exposed to both. Either or both of these diseases could be causing the decline in ferret numbers observed in this population, as could other diseases or factors not measured, such as inadequate habitat or concomitant declines in prairie dogs. The high prevalence of WNV among coyotes and the potential for this virus to be impacting black-footed ferrets also warrant consideration. The high seroprevalence of WNV in the area indicates a wide exposure of coyotes to the virus; therefore, exposure of black-footed ferrets to the virus may be likely. Due to the ability of WNV to cause fatal disease in other species, studies would be useful to elucidate the impact that WNV could have on the success of reintroduced black-footed ferrets as well as factors influencing the spread and incidence of the disease in a prairie ecosystem.

We thank the dedicated staff at Cornell University's Animal Health Diagnostic Center, Cornell Wildlife Health Lab (including Nicholas Hollingshead who created the map), and the Veterinary Parasitology Laboratory; US Geological Survey National Wildlife Health Center; and New York State Department of Health—Wads-worth Laboratory. The editorial comments of Ariel E. Leon, Janet M. Carter, and two anonymous reviewers were greatly appreciated. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the US Government.