Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA

Abstract

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American's wildlife populations.

Figure 1

Geographic distribution of specimen harvest. Geographic distribution of wildlife sampled for SARS-CoV-2 in Vermont during the 2021 and 2022 hunting seasons is shown. Specimens are shown based on the reported town where the harvest occurred and colored according to the number of samples collected from each location. Graphs were generated using QGIS version 3.28.2 (Firenze) https://qgis.org/.

Figure 2

Environmental Swabs Show Contamination of SARS-CoV-2 Nucleocapsid DNA in Laboratory Environment. Samples from residual clinical SARS-CoV-2 positive specimens (pos #1–3), laboratory surfaces and equipment used to process/aliquot field samples (BSC = Biosafety Cabinet) and a selection of deer that initially tested positive for SARS-CoV-2 nucleic acid with the N1 primer set were analyzed by RT-PCR (to detect either RNA or DNA) and PCR (to detect RNA). The difference in cycle threshold (CT) between PCR and RT-PCR for each sample is shown. Positive control clinical samples shown in orange circles, environmental swabs shown in blue squares, deer samples shown in gray triangles.

Figure 3

Case Counts and Prevalence of COVID-19 in Vermont and New York. Geographic distribution of COVID-19 cases (reported by the Vermont Department of Health and New York State Department of Health) during the during the surveillance period for the 2022 season (Oct 15th–Nov 15th 2022) at the county level. Raw case counts are shown on left and prevalence (case counts/county population) is shown on right (population counts are an estimate based on US Census 2022 data). Graphs were generated using QGIS version 3.28.2 (Firenze) https://qgis.org/.