Serosurvey of Coyotes (Canis latrans), Foxes (Vulpes vulpes, Urocyon cinereoargenteus), and Raccoons (Procyon lotor) for Exposure to Influenza a Viruses in the USA

Wild birds are the primary reservoir of influenza A viruses (IAVs; Clark and Hall 2006). However, mammalian species are increasingly found infected by IAVs. Seals, whales, horses, felines, mustelids, rodents, lagomorphs, swine, as well as humans, are known hosts of IAVs and the host range continues to expand (Veldhuis Kroeze and Kuiken 2017).

Wild mesopredator species, such as coyotes (Canis latrans), gray foxes (Urocyon cinereoargenteus), red foxes (Vulpes vulpes), and raccoons (Procyon lotor), play unknown roles in influenza ecology. These species prey on and scavenge wild birds that could be infected with IAVs, they have widespread distributions, and increasingly have contact with human activity, urban dwelling species (such as rats, dogs, etc.), as well as other wild species (Kowalski et al. 2015). Free-ranging mesopredator species have also been reported to be infected with IAV (Robertson et al. 2006; Thiry et al. 2007; Hall et al. 2008).

There have only been a few studies investigating IAVs in wild mesopredator species (Hall et al. 2008; Root et al. 2014; DiGeronimo et al. 2019). To address the question of whether influenza viruses naturally infect these species, we acquired sera from across the US and analyzed them for evidence of exposure to IAVs. It is critical to investigate and characterize influenza biology in mesopredator species to create accurate risk assessments for agricultural and human health. Our data provided insight into influenza ecology and the potential roles of wildlife species in IAV transmission cycles.

We obtained sera from live-captured coyotes, red and gray foxes, and raccoons from multiple places, including Dane County, Wisconsin; Putnam County, Georgia; Tooele and Cache counties, Utah; Albany County, New York; Aransas and Webb counties, Texas; Fulton County, Georgia; and San Francisco County, California. The Wisconsin, California, and Fulton County, Georgia samples came from urban environments, and the rest were from wild environments or a captive breeding facility (Cache County, Utah). The samples represented a wide range of years (2009–18), and some samples from New York did not have known dates of sampling. We received sera at the US Geological Survey, National Wildlife Health Center, Madison, Wisconsin.

We screened the sera for the presence of IAV antibodies using the MultiS-Screen AI Virus Antibody Kit (IDEXX Laboratories, Westbrook, Maine, USA), an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The Cornell University Animal Health Diagnostic Center (Ithaca, New York, USA) performed canine influenza virus hemagglutination inhibition (HI) assays for H3N8/H3N2 on antibody-positive samples using their established procedures.

We performed HI on ELISA-positive samples, looking for exposure to likely avian strains of IAVs (Beard 1970). Sera taken from a Texas feral pig sampled in 2010 that tested negative by ELISA for IAV antibodies served as our negative control. The representative avian influenza viruses (AIV) used were A/ Long-tailed duck/ME/295/2011 (H3N8), and A/Mallard/New Brunswick/2010-00339 (H1N1). The human influenza A viruses were A/CA/04/2009 (H1N1) and A/TN/1-560/2009 (H1N1), both isolated during the 2009/2010 H1N1 human influenza pandemic. The human seasonal H1N1 virus was A/TX/H1N1/ 2007. St. Jude Children's Research Hospital (Memphis, Tennessee, USA) kindly provided the human IAVs.

We obtained 139 sera samples, from 112 coyotes, 18 foxes, and 9 raccoons (Table 1). The ELISA analysis revealed 137 of the sera were negative for influenza antibodies. Two sera (1.4%), a coyote (2010-1473) and a raccoon (2010-1455), both sampled in south Texas in early 2010, showed serological evidence of exposure to influenza viruses (Table 1).

We sent a portion of each positive serum for HI analyses against two isolates (H3N8 and H3N2) of canine influenza viruses. Both ELISA positive sera were negative for the presence of antibodies against the canine influenza viruses. To determine exposure histories to AIV, we conducted HI analyses using AIV (H1N1 and H3N8) that were isolated in the same time frames as the sera were collected and are common in wild birds. Both sera tested negative for antibodies to these avian viruses (Table 2).

We also conducted HI using two isolates of the 2009/2010 human H1N1 pandemic virus, as well as a seasonal human influenza virus. As shown in Table 2, both the raccoon 2010-1455 and coyote 2010-1473 sera contained antibodies to the human pandemic viruses, with HI titers of 1:40 to the California pandemic isolate and 1:10–1:20 to the Tennessee pandemic isolate. Both sera were also HI positive to the 2007 human seasonal H1N1 virus: the raccoon at 1:80 and the coyote with a titer of 1:20.

Our serosurvey showed that wild mesopredators have a low incidence of exposure (1.4%, 2/139) to IAVs. Previous surveys had similar results. An eight-state survey in the US of wild raccoon sera had a 2.3% seroprevalence (Hall et al. 2008) and in Japan, the seroprevalence in wild raccoons was 1.89% (Yamaguchi et al. 2014). Those studies showed exposures to only avian influenza viruses, often multiple subtypes of AIV, but we found no evidence of exposure to AIV.

Our study is notable in that both a coyote and a raccoon were exposed to the human H1N1 pandemic influenza virus. Although skunks have been found infected with the pandemic IAV (Britton et al. 2010), our findings are novel for coyotes and raccoons. Both sera were collected in south Texas during the pandemic. Interestingly, both animals were also HI positive to a human seasonal H1N1 influenza virus. We do not know the amount of serological cross-reactivity between the pandemic and seasonal viruses, but it is clear that the two animals were exposed to one or more human influenza viruses. It is most likely that the wild animals were exposed to the virus from human sources and highlights the risks of virus cross-transmission.

It is clear that the epidemiology of influenza is dynamic, and the wildlife-urban interface is increasingly important to monitor. As new IAVs arise, it is critical to monitor this disease in wild mammals, both as risks to natural wildlife populations and as potential health threats to humans.

We thank the many people who assisted with this project. We are grateful to the Boehringer Ingelheim-NIH Veterinary Summer Scholars Program. This work was funded by the US Geological Survey, and the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract HHSN272201400006C. The use of trade, product, or firm names does not imply endorsement by the US Government.