Suburban Population of Bobcats (Lynx rufus) in Connecticut, USA, Tested Negative for SARS-CoV-2, November 2021–February 2022 Open Access

When SARS-COV-2 rapidly spread across the globe in March and April 2020, the potential of wildlife as a virus reservoir was not immediately evident, although human-to-animal transmission of the virus was reported in pets and in farmed, zoo, and wild animals (Delahay et al. 2021; Hosie et al. 2021). Many complex interactions between human and animal health are based in towns and cities (Murray et al. 2022). Thus, a One Health perspective that addresses human-wildlife interactions is vital (Messmer 2020).

Wild carnivores were once restricted to places with low human density, but enforcement of wildlife laws in the US has resulted in population increases of free-ranging wild felids and canids, such as bobcats (Lynx rufus) and coyotes (Canis latrans). Furthermore, these populations are becoming established in areas with medium (i.e., exurban) to high (i.e., urban) housing densities (Young et al. 2019; Henger et al. 2020). For example, bobcat occupancy in Connecticut was estimated at nearly 100% for areas with 0–700 buildings/km² (Beattie 2020), indicating that bobcats live in rural, exurban, and suburban neighborhoods. Because bobcat occupancy rates are high throughout Connecticut, the likelihood of bobcats having direct or indirect contact with household cats (Felis cattus) and dogs (Canis familiaris) is presumed to be high.

The susceptibility of free-range bobcats to SARS-CoV-2, and whether these animals might play any significant role in the spread or as reservoirs of the virus is unknown. Because the World Health Organization has urged monitoring potential reservoirs, we took advantage of an existing bobcat project to assess this species for SARS-CoV-2 shedding and exposure.

We assessed the exposure of 38 free-living bobcats from Connecticut, US, to SARS-CoV-2 with a serologic and virus detection survey. Our goal was to determine the exposure of bobcats to SARS-CoV-2 such that geographic risk zones for wildlife could be established within a medium-sized city.

Bobcats were trapped and released as part of an ongoing project that happened to span the SARS-C0V-2 pandemic. The focal study area was the greater Hartford area of Hartford County, Connecticut. Hartford is a medium-sized city with a population of >121,000 people. This area includes both public (<30%) and private (>70%) land. Summarizing landcover with the National Oceanic and Atmospheric Administration Office of Coastal Management (2016) 1-m raster (University of Connecticut 2022; Fig. 1) for the towns of Hartford, West Hartford, Avon, Blooming, Simsbury, and Windsor, the study area was mostly mixed forest (45.9%), impervious (20.4%), and Developed Open Space (17.8%) such as urban parks and golf courses. The mixed forest is oak and hickory, with black oak (Quercus velutina), red oak (Quercus rubra), shagbark hickory (Carya ovata), red maple (Acer rubrum), and sugar maple (Acer saccharum). Cultivated land and pasture for agriculture, although not abundant (3.6%), was present principally as tobacco, corn, apples, and livestock production.

Bobcats (n=38) were captured from 19 November 2021 through 11 February 2022 with cage traps (Camtrip Cages, Caging Bobcats, Barstow, California, USA; NB Bobcat Trap, Wildlife Control Supplies, East Granby, Connecticut, USA) and foot-hold traps (MB-550 padded, Minnesota Trapline Products, Pennock, Minnesota, USA). All bobcats were immobilized with a combination of 10 mg/kg ketamine hydrochloride (ketamine hydrochloride 100 mg/mL, Covetrus, Portland, Maine, USA) and 1.5 mg/kg xylazine hydrochloride (AnaSed [xylazine] 100 mg/mL, Akorn, Gurnee, Illinois, USA) by intramuscular injection with a jab pole or dart gun (Belsare and Athreya 2010). Once immobilized, bobcats were sexed, weighed, and given unique identification ear tags. Bobcats were classified as adults by weight, reproductive status, and tooth wear (Serieys et al. 2015). Nasal swabs (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were taken (n=36). Blood samples (n=38) were collected from the medial saphenous vein with a 22-gauge, 2.5-cm needle into Vacutainer (Becton Dickinson, Franklin Lakes, New Jersey, USA) serum tubes (Table 1). Sera and swabs were refrigerated at 4 C before testing. All bobcats were held overnight for anesthetic recovery and released at the original capture site the following day.

We extracted RNA from nasal swab samples with the RNeasy Plus Mini Kit (Qiagen, Germantown, Maryland, USA) according to manufacturer instructions. Two sets of primers and TaqMan (Thermo Fisher) probes (Table 2) were used to target sequences within the N1 and N2 genes of SARS-CoV-2 (CDC 2019). A TaqMan Fast Virus-1-Step Master Mix (Thermo Fisher) was used for reverse transcription quantitative PCR assays. The reaction was performed on the ABI 7500 Fast platform (Thermo Fisher) with a temperature profile of 50 C for 5 min, 95 C for 2 min, followed by 45 cycles of amplification (95 C for 3 s and 55 C for 30 s). As reverse transcription quantitative PCR species control, primers and probe that targets the feline MC1R gene were used to confirm the feline origin of the samples (Kanthaswamy et al. 2012). We did not detect SARS-CoV-2 genomic RNA in any of the nasal swabs obtained from trapped bobcats. All the samples tested contained cat DNA, confirming the feline origin of the samples.

Testing to detect antibodies against SARS-CoV-2 was performed on serum samples obtained from bobcats with the GenScript cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript, Piscataway, New Jersey, USA). The assay was performed, and ELISA cut-off values were established according to manufacturer instructions. The kit contains positive and negative controls. As external control, rabbit sera anti–SARS-CoV spike (S) protein (NR-4569; BEI Resources, Manassas, Virginia, USA), anti–SARS-CoV-2 spike rabbit polyclonal antibody (NR-52947; BEI Resources), and non–human primate convalescent serum to SARS-CoV-2 (NR-52401; BEI Resources) were used. Antibodies against the virus were detected in the kit positive control and the positive serum samples used as external controls. Antibodies against SARS-CoV-2 were not detected in any of the bobcat serum samples.

Our results suggest that the sampled bobcat population in Connecticut had not yet been exposed to a source of the SARS-CoV-2 virus at the time of this study. From an animal and public health perspective, the coexistence of wildlife, pets, and people in urbanized landscapes, which includes medium-sized cites, increases the potential exposure of both humans and animals to infectious agents (Buttke et al. 2015; Rulli et al. 2017; Wilkinson et al. 2018). There is evidence that domestic cats transmit pathogens to bobcats either directly or indirectly (Bevins et al. 2012). Additionally, experimental infection of cats with SARS-CoV-2 resulted in virus transmission to other cats by respiratory droplets (Bosco-Lauth et al. 2020; Shi et al. 2020) or direct contact (Barrs et al. 2020; Halfmann et al. 2020). Variants of SARS-CoV-2 with increased transmissibility, such as delta or omicron, represent additional risks (Rowe et al. 2022). The virus has also been detected in wild and captive felids, including tigers (Panthera tigris), lions (Panthera leo), leopard cats (Prionailurus bengalensis euptilurus), fishing cats (Prionailurus viverrinus), snow leopards (Panthera uncia), and cougars (Puma concolor; Islam et al. 2021).

The solitary nature of bobcats may possibly insulate them, at least partially, from both contracting and spreading pathogens such as SARS-CoV-2; other than mating and capturing prey, both intraspecific and interspecific interactions are rare for adult bobcats (Allen et al. 2015). Further work involving more bobcats and additional locations will be needed to confirm the susceptibility or otherwise of bobcats to SARS-Cov-2 infection and any role they may play in spread or maintenance of the virus.