SARS-CoV-2 wildlife surveillance in Ontario and Québec

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 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. The objective of this study is to conduct SARS-CoV-2 surveillance in urban wildlife from Ontario and Québec, 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 ribonucleic acid, and a subset of samples from 219 animals across three 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 ribonucleic acid 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.


Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global coronavirus disease 2019 (COVID- 19) pandemic and has been maintained through human-to-human transmission. However, humans are not the only species susceptible to infection. Over the course of the current pandemic, a range of domestic and wild animal species have been reported to either be naturally infected with SARS-CoV-2 or susceptible to the virus in experimental infections (1)(2)(3)(4). As of April 30, 2022, 36 countries have reported positive SARS-CoV-2 cases in 23 different animal species to the World Organisation for Animal Health (5). Other species have been identified as potential hosts based on sequence analysis of the host cell receptor of SARS-CoV-2, angiotensin 1 converting enzyme 2 and predicted binding affinity (6,7).

SURVEILLANCE
Many wild animal species, such as raccoons, skunks and bats, thrive in the ecological overlap with humans and are thus at an increased risk of being exposed to SARS-CoV-2 (8). Several peri-domestic species have been experimentally shown to become infected with and shed SARS-CoV-2 (9,10). SARS-CoV-2 infection has also been reported in wild or free-ranging animals that have been naturally exposed, including American mink (Neovison vison) in Spain (11) and, more recently, white-tailed deer (Odocoileus virginianus) in multiple locations across North America (12)(13)(14)(15)(16). In Ontario, this includes identification of a probable case of deer-to-human viral transmission (16). Infection in animals can result in mild to severe symptoms of respiratory disease up to and including death via interstitial pneumonia (e.g. mink) (17, 18). Other species do not show clinical signs of infection (e.g. skunks) (9,10) or show only mild and transient symptoms in some individuals, such as elevated temperature (e.g. white-tailed deer) (19).
The concept of One Health recognizes that human and animal health are interdependent (20). The spillover of virus from humans or domestic animals into wildlife is concerning not only due to the possible deleterious effects on wildlife, but because these wild populations have the potential to act as reservoirs for SARS-CoV-2. Pathogens that have an animal reservoir are inherently more difficult to control and the spread of SARS-CoV-2 through animal populations could further contribute to the development of variants of concern (VoCs), potentially undermining the efficacy of countermeasures such as antivirals and vaccines (21,22). As such, there have been calls for increased surveillance at the human-wildlife interface (23). Urban areas around the world have been a particular area of concern and focus (24-26). The higher density of both human and some periurban wildlife species populations in urban centres can lead to more frequent human-animal contact and increased potential for disease transmission. Additionally, people who have close contact with wildlife, such as biologists, rehabilitators, and hunters and trappers, may be at higher risk of being exposed to the virus and of facilitating its spread among wildlife. The impact of SARS-CoV-2 infection on wildlife health is not fully understood. Early detection of any spillover is therefore critical to preventing and addressing these concerns.
Given the risk of reverse-zoonotic SARS-CoV-2 transmission and our lack of knowledge of the virus in local wildlife, there was an urgent need to elucidate the epidemiology of the virus at the human-wildlife interface to help wildlife management and public health officials better communicate risk and plan management strategies. We therefore conducted SARS-CoV-2 surveillance in wildlife across Ontario and Québec, with a major focus on the southern regions of both provinces. These areas have high human population densities and include major urban centres such as Toronto and Montréal. Between spring 2020 and spring 2021, incidences of COVID-19 peaked in Montréal and the surrounding regions in early January 2021, with rates exceeding 400 cases per 100,000 population in Montréal and Laval (27).
Incidences between spring 2020 and spring 2021 in the Greater Toronto Area peaked in April 2021, with case rates in the City of Toronto and Peel also exceeding 400 per 100,000 population (27).

Methods
Many experts have recommended a One Health approach for animal SARS-CoV-2 testing, which balances concerns for both human and animal health and is based on knowledge of experts in both fields (28,29). As such, our work was conducted through consultation and cooperation among a wide variety of agencies:

Raccoons and skunks
Raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) are peri-domestic species that are good candidates for reversezoonotic disease surveillance due to their high density in urban areas and their frequent close contact with people, pets and refuse. They are also subject to ongoing rabies surveillance operations in both Ontario and Québec, making them easy to sample. In Ontario, wildlife rabies surveillance and testing are conducted by the NDMNRF on roadkill, animals found dead for other reasons, and wildlife that were sick or acting strangely. Submissions are received mainly from southwestern Ontario, and most animals received by the program and subsequently sampled and tested for SARS-CoV-2 came from urban centres within this region or had a case history of close contact with people ( Figure 1). In Québec, a similar wildlife rabies surveillance program is coordinated by le Ministère des Forêts, de la Faune et des Parcs du Québec and testing and other post-mortem examinations are performed by the Québec CWHC. As was the case in Ontario, animals sampled by the Québec CWHC for SARS-CoV-2 testing came mainly from urban areas ( Figure 1). The Ontario CWHC laboratory also contributed a small number of raccoon and skunk samples from animals submitted to them for post-mortem examination. Carcasses were sampled using a combination of oral, nasal, and rectal swabs, respiratory tissue and intestinal tissue (Table 1). Swabs were stored in individual 2 mL tubes with ~1 mL of universal transport medium (UTM; Sunnybrook Research Institute) and 30-60 mg tissue samples were stored dry in tubes.

Big brown bats
Bats are known carriers of coronaviruses (35)(36)(37). As such, concerns have been raised over the possible susceptibility of North American bats to SARS-CoV-2 (38). Species such as the big brown bat (Eptesicus fuscus) frequently roost in buildings, which brings them into close contact with people and increases the likelihood of SARS-CoV-2 exposure. Big brown bat oral swabs and guano samples for SARS-CoV-2 PCR testing were collected by staff at the Granby Zoo, which runs a rehabilitation program over the winter to care for bats that have been disturbed during their hibernation. Guano samples were stored dry in 2 mL tubes.

Other species
Other samples for SARS-CoV-2 PCR testing were obtained opportunistically through the Ontario and Québec regional CWHC laboratories, which receive a wide variety of wildlife species for post-mortem examination ( Table 1). Animals were selected for sampling based on potential for SARS-CoV-2 infection. This could be due to urban habitat, human contact or to predicted species susceptibility based on prior research. The number and type of samples collected varied by carcass and depended on carcass condition (Table 1). Severe acute respiratory syndrome coronavirus 2 polymerase chain reaction analysis  SURVEILLANCE at 60°C for 10 minutes then 95°C for 2 minutes followed by 44 amplification cycles of 95°C for 10 seconds and 60°C for 15 seconds. Quantstudio 3 software (Thermo Fisher Scientific Inc., Waltham, Massachusetts, US) was used to determine cycle thresholds (Ct). All samples were run in duplicate and samples with Cts less than 40 for both gene targets in at least one replicate were considered positive.

Antibody testing
Antibody testing was performed on cardiac blood, chest cavity fluid and serum samples at the NML in Winnipeg, Manitoba. All samples were stored at -20°C prior to testing. Cardiac blood samples were collected onto Nobuto filter strips by saturating the length of the strip with 100 µl of blood. To obtain the 1:9 dilution required for testing, saturated Nobuto strips were cut into 4-5 pieces and placed into a 2 mL tube containing 360 µl phosphate buffered saline pH 7.4 containing 0.05% Tween 20 and eluted overnight at 4°C. Nobuto strips collected from chest cavity fluid were processed in the same way, whereas serum samples were diluted 1:9 with Sample Dilution Buffer. Samples were mixed by vortexing and tested using the GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript US, Inc. Piscataway, New Jersey, US) according to the manufacturer's protocol.
Briefly, 60 µl of a sample was added to 60 µl HRP-conjugated RBD solution and incubated at 37°C for 30 minutes. A 100 µl aliquot of the mixture was transferred to the ELISA microwell test plate and incubated at 37°C for 15 minutes. Microwells were washed four times with 260 µl wash buffer then 100 µl TMB substrate was added to each well. Following a 20-minutes incubation in the dark at room temperature, 50 µl of Stop Solution was added to each well. Absorbance was read immediately at 450 nm.
Each assay plate included positive and negative controls that met required quality control parameters. Percentage inhibition was calculated for each sample using the following equation: % inhibition = (1-optical density sample / optical density negative control) x 100% Samples with greater than or equal to 30% inhibition were considered positive for SARS-CoV-2 neutralizing antibodies.

Results
We tested 776 individual animals from 17 different wildlife species for SARS-CoV-2. These animals were collected primarily from urban areas in southern Ontario and Québec between June 2020 and May 2021 (Table 1). We found no evidence of SARS-CoV-2 viral RNA in any of the tested samples and no evidence of neutralizing antibodies in a subset of 219 individuals (141 raccoons, 36 striped skunks, 42 mink).

Discussion
Our study did not detect any spillover of SARS-CoV-2 to wildlife in Ontario and Québec. Raccoons and skunks were the most commonly tested species. Results from experimental studies have suggested these species may be susceptible to SARS-CoV-2, but the lack of and low quantity of infectious virus shed by raccoons and skunks, respectively, suggest they are an unlikely reservoir for SARS-CoV-2 in the absence of viral adaptations (9,10). Similarly, a challenge study with big brown bats found that they are resistant to SARS-CoV-2 infection and do not shed infectious virus (40). Conversely, minks are susceptible to SARS-CoV-2 infection, but no evidence of SARS-CoV-2 was detected in any of the mink sampled. While this could be attributed to low effective sample size, to date SARS-CoV-2 has been infrequently detected in wild mink populations globally. It should be noted, however, that these experimental studies on raccoons, skunks and big brown bats (9,10,40) were conducted using parental SARS-CoV-2. The susceptibility of these species to VoCs is presently not known and may differ from susceptibility to the parental strain (41). Additionally, challenge studies assessing susceptibility tend to be conducted on small numbers of young, healthy individuals, so results may not be reflective of the full range of possible responses to infection in the wild.
As the pandemic progresses, new evidence is emerging on susceptible wildlife that may act as competent reservoirs for the virus. For example, white-tailed deer are now considered a highly relevant species for SARS-CoV-2 surveillance in light of their experimentally determined susceptibility as well as evidence of widespread exposure to the virus via antibody and PCR testing across North America (12)(13)(14)(15)(16)19). Continued surveillance efforts should be adaptive and include targeted testing of highly relevant species as they are identified. In Ontario and Québec, these would include mink, white-tailed deer and deer mice (Peromyscus maniculatus) (9,42). Continuing to include less susceptible species remains important given ongoing viral genomic plasticity and changing host range of VoCs.

Limitations
There are several limitations for this study that need to be acknowledged. First, the majority of our SARS-CoV-2 testing was done by RT-PCR, which is only capable of detecting active infection. Antibody testing, which identifies resolved infection or exposure, is more likely to find evidence of SARS-CoV-2 in surveillance studies since results are less dependent on timing of sample collection. Antibody testing typically requires samples from live animals or fresh carcasses, which limited our ability to use it; however, the testing performed allowed for test validation in raccoons, skunks and mink, which may facilitate more antibody testing in future. Second, we relied on different kits for RNA extraction due to logistical challenges. Based on our extraction controls, the QIAamp RNA mini kit performed slightly better compared to the Nuclisens EasyMag (~2 Cts) for swab samples. Conversely, the Nuclisens EasyMag performed slightly better (~2 Cts) compared to the RNeasy mini plus kit for tissue samples. Third, the type of samples we collected may also have limited our ability to detect SARS-CoV-2 infection. Viral replication can vary among tissue types and therefore some tissues are more optimal for viral RNA detection than others (1). In the present work, animals were sampled opportunistically as a part of preexisting programs, and we were not able to consistently collect the same sample sets. Additionally, the sample types were from live animals and carcasses and not optimized; certain sample types were sometimes unavailable (e.g. tissue samples from live animals) or were not sufficient for collection.

Conclusion
A One Health approach is critical to understanding and managing the risks of an emerging zoonotic pathogen such as SARS-CoV-2. We leveraged activities of existing research, surveillance, and rehabilitation programs and expertise from multiple fields to efficiently collect and test 1,690 individual wildlife samples. The absence of SARS-CoV-2-positive wildlife samples does not exclude spillover from humans to Canadian wildlife, given the limitations cited above. Continued research in this area is both important and pressing, particularly as novel VoCs emerge. Public and animal health sectors should continue to work collaboratively with academic and government partners to help prevent the spread of SARS-CoV-2 from people to wildlife, monitor for spillover, and address any issues should they arise. There is an urgent need for a coordinated wildlife surveillance program for SARS-CoV-2 in Canada. This approach will help protect the health of both Canadians and wildlife, now and in the future.