Coinfection of Canine Distemper Virus and Rabies Virus in Wildlife Samples Submitted for Routine Rabies Testing Open Access

Canine distemper virus (CDV), also known as Canine morbillivirus, is a multihost pathogen that causes significant infection in both wild carnivores and unvaccinated domestic animals globally (Kapil and Yeary 2011; Viana et al. 2015; Pope et al. 2016; Gilbert et al. 2020; Taylor et al. 2021). Taxonomically, CDV belongs to the order Mononegavirales, family Paramyxoviridae, and genus Morbillivirus. Typically, viral particles are shed in respiratory secretions, urine, and feces of infected animals (Kapil and Yeary 2011). Clinical signs of canine distemper include fever, coughing, sneezing, respiratory discharge, vomiting, and diarrhea. Advanced illness manifests neurologically, including seizures, paralysis, deteriorating motor skills and senses, and encephalitis (Vila Nova et al. 2018). Unstable in the environment, CDV relies on host contagion to persist and replicate. Vaccinations against CDV are widely used and successful in controlling viral transmission in domestic animals, but the disease continues to be a risk among unvaccinated domestic animals and wildlife (Kapil and Yeary 2011).

Whereas canine vaccination against RV is legally mandated in many jurisdictions within the US and Canada, vaccination against CDV is not required, although it is highly recommended by veterinary advocacy groups such as the American Animal Hospital Association (AAHA; Ellis et al. 2022). The AAHA's vaccination guidelines suggest that the core vaccination series, including vaccination against CDV, be completed in puppies as early as 6 wk of age. Their revaccination protocols recommend booster administration within 1 yr after completion of the initial series, with subsequent boosters to be administered at intervals of 3 yr or longer depending on individual immunogenicity (Ellis et al. 2022). Despite AAHA recommendations, CDV vaccinations may be overlooked or ignored, especially in the absence of routine veterinary care, posing a risk of infection to other susceptible domestic animals and wildlife.

Infections with rabies virus (RV) and CDV may present similar clinical signs, and both are untreatable once illness manifests. Unlike RV, CDV is not a reportable disease, and there are few records regarding the number of CDV-positive animals in the US. However, some municipalities record and publish data on outbreaks in wildlife (New York City Department of Health and Mental Hygiene 2018). Such reports provide some background epidemiological data, but overall CDV morbidity and mortality remains imprecise, or it is based on modeling of smaller outbreaks. In the US, rabies is endemic among raccoons, Procyon lotor, striped skunk, Mephitis mephitis, and red fox, Vulpes vulpes, populations, with at least 2,587 individuals of these three species diagnosed as rabid during 2020 (Ma et al. 2022). Given the public health significance of rabies, animals showing clinical signs of canine distemper, even in areas where the risk is presumed low, may require further diagnostic differentiation to rule out RV infection.

Clinical similarities between rabies and canine distemper present challenges when outbreaks of either occur. In the US, throughout areas where the risk of rabies among carnivores is believed low, such as in parts of the Midwest and Pacific Northwest (Ma et al. 2022), one might assume CDV is the cause of neurological illness among mesocarnivores. Elsewhere, such as in the eastern US, the converse may be true. For example, between 2009 and 2011, surveillance in New York City's Central Park detected 133 rabid raccoons, and this outbreak was managed effectively with a trap-vaccinate-release effort (Slavinski et al. 2017). However, as with rabies, canine distemper is endemic to New York State, and periodic outbreaks occur. During 2018 a canine distemper outbreak was identified in New York City's Central Park, with more than 170 raccoons testing positive for CDV (New York City Department of Health and Mental Hygiene 2018). These outbreaks prompted an investigation by the New York State Rabies Laboratory (NYSRL) to determine how often CDV might occur concurrently with RV infection. Once rabies has been diagnosed in an animal, follow-up testing rarely occurs, since RV is assumed to be the cause of death. Our objective was to determine the passive co-occurrence of CDV infection among a subset of animals submitted for rabies testing to the NYSRL.

Most animals submitted for such surveillance are negative for detection of RV. A search of the 2017–19 NYSRL database yielded 1,302 specimens, representing 17 species from 17 US states: Procyon lotor (n=777), Mephitis mephitis (n=230), Vulpes vulpes (n=153), Felis catus (n=86), Marmota monax (n=17), Canis latrans (n=7), Lynx rufus (n=7), Equus ferus caballus (n=6), Bos taurus (n=5), Canis familiaris (n=3), Dicotyles tajacu (n=3), Ovis aries (n=2), Odocoileus virginianus (n=2), Capra hircus (n=1), Didelphis virginiana (n=1), Enhydra lutris (n=1), and Pekania pennanti (n=1). These convenience specimens were received either for rabies diagnostic testing in New York (n=1,028) or for confirmation and RV variant typing from state or federal agencies from across the US (n=274).

At the time of necropsy, tissue smear slides were made of the brainstem and cerebellum for RV detection using the direct fluorescent antibody (DFA) test. Subsamples of tissue homogenates of brainstem and cerebellum (approximately 50 mg) of RV-positive specimens were suspended in 1 mL of Dulbecco's Modified Eagle Medium for nucleic acid extraction and confirmatory testing by real-time qualitative reverse transcriptase PCR (qRT-PCR). Rabies diagnosis was confirmed with a triplexed assay, targeting two conserved regions of the nucleoprotein gene and an exogenous transcript control to check for inhibition and extraction efficiency (Dupuis et al. 2015). Similarly, CDV was diagnosed with qRT-PCR with a previously published assay, using the qScript One-Step qRT-PCR Kit, Low-ROX (Quantbio, Beverly, Massachusetts, USA) according to the manufacturer's instructions and using oligonucleotides modified and optimized to target the nucleoprotein, due to its low genetic variability among genotypes (Elia et al. 2006). The NYSRL tested all 1,302 rabies-positive samples for concurrent CDV infection with qRT-PCR, including several terrestrial mammalian species not generally considered susceptible to canine distemper, to increase the breadth of surveillance. Seventy-three of the 1,302 specimens (5.6%) were both CDV and RV positive (Fig. 1).

The cycle threshold (Ct) values for CDV were high, with a mean of 34.06, median 34.99, and standard deviation of 4.32. All samples under our Ct cutoff of 40 were repeated from extraction of primary brain tissue to confirm results. Animals confirmed with both reproducible rabies and neurological canine distemper were striped skunks (n=1), red foxes (n=3), and raccoons (n=69), as shown in Fig. 2. By species, coinfections were approximately 9% in raccoons, 2% in foxes, and 0.4% in skunks. We hypothesized that the high Ct values were due mostly to early onset, subclinical, or resolving neurological involvement of CDV, near the limit of detection of our assay. In one report, animals with active CDV infection had mean Ct values of 16.28, while animals without clinical signs had a mean of 28.75, although those researchers performed RT-PCR on a homogenate of various tissues, while we used brain exclusively (Pope et al. 2016). Furthermore, it was not possible to determine if any of the animals in our study would have succumbed due to CDV in the absence of RV infection.

In the US, cross-species transmissions of CDV from domestic dogs to wildlife have been reported. Although canine distemper and rabies are both controlled in domestic animals by vaccination in North America, wildlife remains as intermediary disease hosts or reservoirs (Kapil and Yeary 2011). Accidental or purposeful translocations of rabid animals pose a threat to global prevention programs. Such occurrences resulted in a large-scale outbreak of raccoon rabies in the northeastern US (Cartter et al. 1992) and establishment of mongoose rabies throughout the Caribbean (Styczynski et al. 2017). In 2018, a juvenile dog from South Korea imported into western Canada was found to be infected with a CDV variant (Asia-1) not previously identified in North America (Waldron 2019). Although this example remained an isolated case, infectious disease emergence risks are concerning, and mitigation efforts are intensive and costly. As exemplified by both canine distemper and rabies, management of such diseases in wildlife remains challenging.

Parental rabies vaccination is highly effective among domestic animals, while oral rabies vaccination programs may reduce the disease burden among wildlife, as exemplified by the use of a licensed vaccinia-rabies glycoprotein recombinant vaccine (Maki et al. 2017). Unlike oral rabies vaccination, oral CDV vaccination has not shown the same immunogenicity as vaccines administered parenterally, limiting opportunities for wildlife administration (Connolly et al. 2013), and large-scale vaccination of free-living animals against CDV remains unfeasible (Gilbert et al. 2020). One large-scale analysis showed that vaccination of wild canids did not significantly eliminate the threat of CDV transmission (Viana et al. 2015).

Both CDV and RV may spread rapidly during breeding seasons, especially in suburban areas, where shelter and food are readily available (Taylor et al. 2021). Since clinical signs of rabies and canine distemper may be similar, laboratory testing is necessary for a definitive diagnosis. Public health and agricultural departments should consider that when local outbreaks of either RV or CDV occur, these viruses may co-circulate. A study in Ontario, Canada, found that 69% (n=32) of rabies positive raccoons and 21% (n=34) of rabies-positive striped skunks were positive for CDV using real-time RT-PCR of conjunctival swabs during a concurrent outbreak in the greater Toronto and Hamilton metropolitan area (Jardine et al. 2018). Our passive surveillance across the entire state of New York over a 3-yr period suggests that these concurrent outbreaks may be rare, especially regarding simultaneous neurological involvement of CDV and RV. Additional research should incorporate both histopathology and serology for CDV detection, as neurological involvement is not guaranteed. Ideally, recognizing the holistic utility of both passive and active surveillance systems, future studies should consider the prevalence of CDV infection and other pathogens among RV-negative samples; the potential pathobiological role of concomitant agents in neurological or other conditions for inclusion within open differential diagnostic systems; and any subtle epidemiological impacts played by such concurrent infections at the environmental interface of humans, domestic animals, and wildlife within a One Health context.

We thank the US Department of Agriculture (USDA) for providing out-of-state samples as part of their enhanced rabies surveillance and the Wadsworth Center Applied Genomics Technology Core and Bio-informatics Cores. This work was supported by funding from USDA (17005119).

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