Wildlife Sentinel: Development of Multispecies Protein A-ELISA for Detection of SARS-CoV-2 Antibodies in Zoo Animals as a Proof of Concept for Wildlife Surveillance.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first emerged in Wuhan in 2019 and rapidly spread worldwide. During the course of the COVID-19 pandemic, numerous reports highlighted infections of wild animals by SARS-CoV-2. Nevertheless, further research is required to understand the virus potential to infect various animal species, which is crucial for evaluating its future evolution and the potential reemergence of SARS-CoV-2. The total concentration of immunoglobulin G (IgG) represents a valuable yet underused diagnostic parameter for health assessments in wild animals, primarily due to the absence of effective diagnostic tools. A protein A-based indirect ELISA can serve as an efficient method to identify IgG antibodies against different pathogens in wildlife surveillance programs. To develop a multispecies protein A-ELISA for IgG detection against SARS-CoV-2, we used 44 animal species serum samples to ascertain the protein A binding affinity, and 88 serum samples, chosen for the strong binding affinity to protein A, were used to identify IgG antibodies against SARS-CoV-2. The serum samples were obtained from animals housed in Safari Park Dvůr Králové, Czech Republic. The zoo animals are in proximity to humans, facilitating the exploration of potential reverse transmission events of SARS-CoV-2 from humans to animals. Also, they undergo routine veterinary examinations, providing convenient access to blood samples. Therefore, they can be easily used for the development of protein A-based indirect ELISA for wildlife disease surveillance programs. Based on the ELISA results, antibodies to SARS-CoV-2 were detected in the sera of 16 animals. To further confirm these findings, the ELISA-positive samples were subjected to virus neutralization assays. This additional testing revealed the presence of SARS-CoV-2 neutralizing antibodies in the serum of two white rhinoceros (Ceratotherium simum simum) and one Persian leopard (Panthera pardus tulliana).