Investigations on the Potential Role of Free-Ranging Wildlife as a Reservoir of SARS-CoV-2 in Switzerland

Abstract

Amid the SARS-CoV-2 pandemic, concerns surfaced regarding the spread of the virus to wildlife. Switzerland lacked data concerning the exposure of free-ranging animals to SARS-CoV-2 during this period. This study aimed to investigate the potential exposure of Swiss free-ranging wildlife to SARS-CoV-2. From 2020 to 2023, opportunistically collected samples from 712 shot or found dead wild mustelids (64 European stone and pine martens, 13 European badgers, 10 European polecats), canids (449 red foxes, 41 gray wolves, one golden jackal) and felids (56 Eurasian lynx, 18 European wildcats), as well as from 45 captured animals (39 Eurasian lynx, 6 European wildcats) were tested. A multi-step serological approach detecting antibodies to the spike protein receptor binding domain (RBD) and N-terminal S1 subunit followed by surrogate virus neutralization (sVNT) and pseudotype-based virus neutralization assays against different SARS-CoV-2 variants was performed. Additionally, viral RNA loads were quantified in lung tissues and in oronasal, oropharyngeal, and rectal swabs by reverse transcription polymerase chain reactions (RT-qPCRs). Serologically, SARS-CoV-2 exposure was confirmed in 14 free-ranging Swiss red foxes (prevalence 3.1%, 95% CI: 1.9-5.2%), two Eurasian lynx (2.2%, 95% CI: 0.6-7.7%), and one European wildcat (4.2%, 95% CI: 0.2-20.2%). Two positive foxes exhibited neutralization activity against the BA.2 and BA.1 Omicron variants. No active infection (viral RNA) was detected in any animal tested. This is the first report of SARS-CoV-2 antibodies in free-ranging red foxes, Eurasian lynx, and European wildcats worldwide. It confirms the spread of SARS-CoV-2 to free-ranging wildlife in Switzerland but does not provide evidence of reservoir formation. Our results underscore the susceptibility of wildlife populations to SARS-CoV-2 and the importance of understanding diseases in a One Health Concept.

Keywords: RBD-ELISA; S1-ELISA; fox; indirect immunofluorescence test; lynx; one health; pseudotype-based virus neutralization assay; spillover; surrogate virus neutralization test; wildcat; zoonosis.

Figure 1

Distribution of all sampled animals in the different cantons of Switzerland and in Liechtenstein. Grey shades show land features, blue indicates rivers and lakes, and grey lines mark canton boundaries on the QGIS-designed map. Each dot on the map represents one sampled animal. Visualization of the data was performed using the Quantum Geographic Information System (QGIS 3.28.3—Firenze version, Open Source Geospatial Foundation Project (http://qgis.org, accessed on 1 May 2024).

Figure 2

Timeline of serological analyses. Samples with ODs above the cutoff values (≥mean + 3SD) and those exhibiting high ODs under the cutoff values (≥mean + 2SD) in ELISA underwent further testing with sVNT, iIFT, and PVNA.

Figure 3

SARS-CoV-2 serological results for foxes tested with RBD-, S1-, and Omicron S1-ELISA, as well as with sVNT and Omicron sVNT. The OD values of the different assays for the pre-COVID and post-COVID populations are represented. The boxes cover the 25th to 75th percentiles, while the whiskers represent the upper and lower 25th percentiles. For populations with less than 10 animals, values were plotted as points with a small jitter effect on the y-axis to prevent overplotting. When the population exceeded 10 animals, values were depicted using both dot plots and box plots. A cutoff, defined as the sum of the mean values for the pre-COVID population and three times the standard deviation, is denoted by a horizontal red line on the figure.

Figure 4

Distribution of serologically positive, suspect positive, and negative foxes in the different cantons of Switzerland and Liechtenstein. Grey shades show land features, blue indicates rivers and lakes, and grey lines mark canton boundaries on the QGIS-designed map. Each dot on the map represents one sampled animal.

Figure 5

SARS-CoV-2 serological results for lynx tested with RBD-, S1-, and Omicron S1-ELISA, as well as with sVNT and Omicron sVNT. The OD values of the different assays for the pre-COVID and post-COVID populations are represented. The boxes cover the 25th to 75th percentiles, while the whiskers represent the upper and lower 25th percentiles. For populations with less than 10 animals, values were plotted as points with a small jitter effect on the y-axis to prevent overplotting. When the population exceeded 10 animals, values were depicted using both dot plots and box plots. A cutoff, defined as the sum of the mean values for the pre-COVID population and three times the standard deviation, is denoted by a horizontal red line on the figure.

Figure 6

Distribution of serologically positive, suspect positive, and negative lynx in the different cantons of Switzerland and Liechtenstein. Grey shades show land features, blue indicates rivers and lakes, and grey lines mark canton boundaries on the QGIS-designed map. Each dot on the map represents one sampled animal.

Figure 7

SARS-CoV-2 serological results for wildcats tested with RBD-, S1-, and Omicron S1-ELISA, as well as with sVNT and Omicron sVNT. The OD values of the different assays for the pre-COVID and post-COVID populations are represented. The boxes cover the 25th to 75th percentiles, while the whiskers represent the upper and lower 25th percentiles. For populations with less than 10 animals, values were plotted as points with a small jitter effect on the y-axis to prevent overplotting. When the population exceeded 10 animals, values were depicted using both dot plots and box plots. A cutoff, defined as the sum of the mean values for the pre-COVID population and three times the standard deviation, is denoted by a horizontal red line on the figure.

Figure 8

SARS-CoV-2 serological results for wolves tested with RBD-, S1-, and Omicron S1-ELISA, as well as with sVNT and Omicron sVNT. The OD values of the different assays for the pre-COVID and post-COVID populations are represented. The boxes cover the 25th to 75th percentiles, while the whiskers represent the upper and lower 25th percentiles. For populations with less than 10 animals, values were plotted as points with a small jitter effect on the y-axis to prevent overplotting. When the population exceeded 10 animals, values were depicted using both dot plots and box plots. A cutoff, defined as the sum of the mean values for the pre-COVID population and three times the standard deviation, is denoted by a horizontal red line on the figure.

Figure 9

SARS-CoV-2 serological results for mustelids tested with RBD-, S1-, and Omicron S1-ELISA, as well as with sVNT and Omicron sVNT. The OD values of the different assays for the pre-COVID and post-COVID populations are represented. The boxes cover the 25th to 75th percentiles, while the whiskers represent the upper and lower 25th percentiles. For populations with less than 10 animals, values were plotted as points with a small jitter effect on the y-axis to prevent overplotting. When the population exceeded 10 animals, values were depicted using both dot plots and box plots. A cutoff, defined as the sum of the mean values for the pre-COVID population and three times the standard deviation, is denoted by a horizontal red line on the figure.