Members of the genus Orthopoxvirus (OPXV) infect a variety of mammalian taxa as both enzootic and zoonotic pathogens, with some species associated with severe febrile rash illness in humans (monkeypox virus). Other OPXVs are known to cause significant economic losses from infected livestock (vaccinia virus), or are recognized to infect pets and zoo animals (cowpox virus; Damon 2007). The distribution of OPXV is worldwide, with this naturally endemic virus genus occurring on all continents except Australia and Antarctica (Emerson et al. 2009). Three species of this genus have been recognized to be endemic to North America: skunkpox virus (Emerson et al. 2009), raccoonpox virus (Alexander et al. 1972), and volepox virus (Regnery 1987). In 2017 a human poxvirus infection from a novel OPXV was discovered in Alaska; however, it is possible that this may have been the result of an introduction from the Old World (Springer et al. 2017). This is the latest in an increasing number of reports of novel emerging OPXVs (Osadebe et al. 2014; Hoffman et al. 2015; Vora et al. 2015) that add to the increase in detected cases of human monkeypox disease in Africa (Rimoin et al. 2010; Durski et al. 2018). Administration of routine smallpox vaccinations ceased shortly after the eradication of smallpox in the 1980s. The smallpox vaccination afforded human populations with cross-protection to other related OPXV infections such as cowpox, vaccinia, and monkeypox viruses (Fulginiti et al. 2003). There is an increased proportion of unvaccinated people as the global population grows, further increasing the risks of human infection from new and emerging OPXVs.

Although the natural reservoir hosts of North American OPXVs remain unknown, anti-OPXV antibodies have been detected in many mammal species across the continent, and rodents are thought to be the primary reservoir hosts. North American OPXVs have been identified and isolated from many animals including rodents such as pinyon mouse (Peromyscus truei), California vole (Microtus californicus), and northern pygmy mouse (Baiomys taylori; Regnery 1987; Martínez-Duque et al. 2014; Gallardo-Romero et al. 2016; Hodo et al. 2018). Isolates of OPXVs have been obtained from different carnivores such as raccoons (Procyon lotor) and skunks (Mephitis spp.); seropositive samples have also been collected from coatis (Nasua spp.), and ring-tailed cats (Bassariscus spp.).

There are many predator–infected-prey interaction studies that have concluded that predators (carnivores) take a disproportionate number of prey that are diseased and infected with parasites (Hethcote et al. 2004). Infected prey are typically sick and more vulnerable to predation, and in some cases infected prey will alter their behavior, making them more easily preyed upon (Lafferty 1992). An increase in vulnerability of the infected prey, coupled with the tendency for predators to forage for easily available prey, can lead to the spread of disease to and among the susceptible predator population (Krebs 1978). Carnivores adapt well to urban and periurban environments (Gehrt et al. 2010) and can facilitate cross-species disease transmission with domestic dogs (Canis lupus familiaris) and cats (Felis catus) and their owners (Deem et al. 2001; Millan et al. 2016). There could be a potential risk to the human population in a given region where a carnivore population exhibits a high OPXV seroprevalence.

Alternatively, the presence of antirabies and anti-OPXV antibodies in carnivores of northwestern Mexico could be indicative of an exposure to oral rabies vaccine (ORV, a vaccinia-vectored rabies glycoprotein vaccine) because of the proximity to the US Department of Agriculture’s (USDA) bait drop zones along the US–Mexico border, and not to an exposure to a natural endemic OPXV. In this study, we tested sera collected from wild carnivore species in northwestern Mexico to further assess the distribution and prevalence of OPXV in North America, and to investigate differential species-specific exposures in this region.

Trapping efforts yielded a total of 51 carnivores representing seven species (Table 1) including: coyote (Canis latrans), gray fox (Urocyon cinereoargenteus), kit fox (Vulpes macrotis), bobcat (Lynx rufus), raccoon, American badger (Taxidea taxus), and striped skunk (Mephitis mephitis). Of the samples tested by WB, 59% (30/51) showed bands consistent with bands commonly found in OPXV infections (Demkowics et al. 1992; Hutson et al. 2007; Gallardo-Romero et al. 2012, 2016; Salzer et al. 2013). The 62-kDa band was present in 43% (22/51) of animals, the 39-kDa bands in 25% (13/51), and 49-kDa band in 35% (18/51). The bands at 75 kDa and 95 kDa were observed in six animals.

Our results showed 51% (26/51) of animals with anti-OPXV antibodies by ELISA. On the basis of our definition of a true positive, 47.1% (24/51) of the animals were suspected to have had previous exposure or a current infection by a circulating OPXV. Of these 24 positive samples, all had at least two immunodominant bands present, 33% (8/24) had only one ELISA dilution positive, and the remaining 67% (16) had both ELISA dilutions positive. Species-specific seroprevalence rates were calculated (Table 1). Raccoons displayed the highest seroprevalence rates (75%), followed by coyotes (67%) and kit foxes (55%). Seroprevalence rates in each habitat type sampled are as follows in descending order: forest and grassland ecotone (60%), grassland (59%), shrubland (36%), and forest (20%). One kit fox from the grassland showed high OD values in both ELISA dilutions, but was negative by WB. One coyote from the grassland was positive by ELISA only in the 1:100 dilution, and showed the 62-kDa band. This animal was considered negative because it did not meet the requirements to be considered a true positive according to our definition, even though it presented low positive values in both assays. Four wild carnivores (one bobcat, one raccoon, and two gray foxes) were negative by ELISA but presented one or more immunodominant bands. However, because of our definition of a true positive, these animals were considered negative.

The 62-kDa band is likely a protein encoded by the gene A10 (VV-Cop ortholog), the most abundant major core protein important for viral assembly and the gene responsible for stimulation of memory B-cell production. The 49-kDa band may be encoded by the A36R gene that codes for proteins from 43 to 50 kDa that accumulate late during infection, present on the surface of the extracellular viral envelope. The 39-kDa band is a protein encoded by the G9R gene, highly conserved throughout the Poxvirus family and is required for efficient viral replication (Ojeda et al. 2006).

All samples were negative (<0.5 IU/mL) for rVNAs by RFFIT. World Organisation for Animal Health guidelines stipulate that values >0.5 IU/mL represent an adequate response to rabies vaccination (World Organisation for Animal Health 2013).

The observed prevalence of anti-OPXV antibodies and immunodominant bands consistent with OPXV infection within the carnivores we captured in northwestern Mexico suggests that OPXV infections in this area are widespread and relatively common. Ecological studies with OPXV elsewhere in the world show that rodents are the primary reservoir for this genus of virus (Crouch et al. 1995; Begon et al. 1999; Chantrey et al. 1999; Hazel et al. 2000; Doty et al. 2017). Our results indicate either that circulation of OPXV exists within many carnivore species in northwestern Mexico, or that OPXV infection may potentially have been transmitted to these carnivore species by interaction with host reservoirs (e.g., ingestion of prey), particularly rodents.

The exposure of OPXV in carnivores of our sampling sites may be a result of the dietary habits of these species and trophic ecology. Some ecoepidemiologic disease models have demonstrated that predators may indirectly promote epidemics of infectious diseases, and disease epidemics may taper off in an environment that becomes free of predators (Morozov 2011). Given that rodents are one of the primary prey items for these predators, predator–prey–pathogen interactions may play a role in the natural disease transmission linkages of OPXV in North America. In a population of infected prey, the presence of a predator has been shown to enhance the success of a pathogen (Baker and Smith 1997; Caceres et al. 2009; Morozov 2011). This predator-dependent dynamic may exist in northwestern Mexico; however, further research into the ecologic aspects of OPXV transmission in this region is needed. The seroprevalence found in the forest and grassland ecotone and grassland habitat was comparatively higher than the seroprevalence of the forest and shrubland; however, this result may potentially be due to the majority of animals being captured in these two habitats (62%, 32/51). Additional research is needed to delineate the role that habitat may play in the circulation of OPXV in the natural environment.

The USDA Animal and Plant Health Inspection Service has conducted ORV bait drops in Arizona as well as in far-west Texas, which is approximately 210 km from our study site (Blanton et al. 2009). On rare occasions, coyotes have been documented traveling long distances; the longest recorded distance was 310 km by a male coyote in the Rocky Mountains (Kolbe and Squires 2004). Given the small likelihood that these animals had a potential exposure to the ORV bait in the US, all samples were additionally tested for presence of rabies antibodies by RFFIT. All samples were negative for rabies virus antibodies, which indicated that all the OPXV positives we observed in this study are not a result of exposure to ORV baits.

Antibodies to OPXV have been identified in previous studies with rodents, skunks (Mephitis spp.), and three genera of Procyonidae from central and southeast Mexico (Regnery 1987; Emerson et al. 2009; Martínez-Duque et al. 2013; Gallardo-Romero et al. 2016; Hodo et al. 2018). Our findings supported the previous studies on OPXV in Procyonidae, as we found raccoons to harbor the highest seroprevalence rates in this region of northwestern Mexico. Our results also introduced new evidence of OPXV infection in other carnivores, including the coyote, gray fox, kit fox, and American badger, and indicated that OPXVs can infect or circulate within widely diverse mammalian taxa in Mexico, and should prompt an increased awareness for OPXV in North America and the potential risks to human health that these viruses may pose. Apart from assessing previous OPXV exposure from serologic studies of carnivores, further studies would benefit from additional utilization of molecular and phylogenetic techniques to determine infection status and taxonomy of OPXV.