Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, is capable of infecting a variety of wildlife species. Wildlife living in close contact with humans are at an increased risk of SARS-CoV-2 exposure and if infected have the potential to become a reservoir for the pathogen, making control and management more difficult. Objective To conduct SARS-CoV-2 surveillance in urban wildlife from Ontario and Québec, Canada, increasing our knowledge of the epidemiology of the virus and our chances of detecting spillover from humans into wildlife. Methods Using a One Health approach, we leveraged activities of existing research, surveillance, and rehabilitation programs among multiple agencies to collect samples from 776 animals from 17 different wildlife species between June 2020 and May 2021. Samples from all animals were tested for the presence of SARS-CoV-2 viral RNA, and a subset of samples from 219 animals across 3 species (raccoons, Procyon lotor ; striped skunks, Mephitis mephitis ; and mink, Neovison vison ) were also tested for the presence of neutralizing antibodies. Results No evidence of SARS-CoV-2 viral RNA or neutralizing antibodies was detected in any of the tested samples. Conclusion Although we were unable to identify positive SARS-CoV-2 cases in wildlife, continued research and surveillance activities are critical to better understand the rapidly changing landscape of susceptible animal species. Collaboration between academic, public and animal health sectors should include experts from relevant fields to build coordinated surveillance and response capacity.

SARS-CoV-2 wildlife surveillance in Ontario and Québec, Canada