Virus neutralization test for confirmation of ELISA-positive SARS-CoV-2 antibodies in dogs

Constant antigenic changes, new variants and easy transmission of SARS-CoV-2 virus should acquire greater zoonotic attention and need to remain alert. In this retrospective study the aim was to analyze seropositivity to SARS-CoV-2 in dogs by commercial ELISA. The Virus neutralization test (VNT) was modified for the purpose of confirmation of SARS-CoV-2 antibodies in ELISA-positive dog sera. The sera were collected from 204 dogs from different veterinary clinics across Vojvodina Province, Serbia, during COVID-19 pandemic. For the screening of antibodies a commercial double multi-species antigen ELISA was used, followed by the VNT modified with SARS-CoV-2 as a confirmatory test. VNT was modified as "one step" test using local isolate of SARS-CoV-2 and the results were checked by cytopathic effect in cell culture on the 96-well microtiter plate. Obtained data have shown that 9 out of 204 dogs were positive by ELISA (4.4%), while 2 (0.97%) sera were doubtful. VNT confirmed 9 positive dogs, but 2 doubtful samples were negative, exhibiting the seroconversion to SARS-CoV-2 in 4.4% dogs from Vojvodina region during pandemic of COVID-19. VNT with SARS-CoV-2 helped to elucidate ELISA ambiguous results. The occurrence of antibodies to SARS-CoV-2 in dogs in this study during COVID-19 pandemic suggested the possibility of viral transmission to dogs, implicating the potential for zoonotic transmission. This was the first research on seroconversion to SARS-CoV-2 in dogs from the Province of Vojvodina, the northernmost part of Serbia.