The emergence of SARS-CoV-2 raised concerns about the potential for interspecies transmission, particularly among domestic animals. We evaluated the seroprevalence of SARS-CoV-2 antibodies in domestic cats from various sites in North America. A total of 216 serum samples collected between December 2019 and February 2022, were analyzed using four different enzyme-linked immunosorbent assays (ELISAs), including a commercial surrogate virus neutralization test (sVNT), a commercial double antigen test (dN ELISA), and two in-house developed indirect ELISAS based on receptor-binding domain (RBD iELISA) and the nucleocapsid (N iELISA) proteins, respectively. Seropositive samples in the commercial ELISAs were subject to virus neutralization test (cVNT) employing the Wuhan-like USA-WA1/2020 SARS-CoV-2 isolate. Our findings revealed that, 6 % (12/216) of the cat serum samples tested positive by the sVNT, while 4 % (9/216) tested positive for the dN-ELISA. Interestingly, the N iELISA showed a higher seroprevalence, with 31 % of the samples testing positive, possibly due to cross-reactive antibodies against the N protein of other coronavirus commonly found in cats. There was a high concordance between sVNT, cVNT, and RBD iELISA. Among positive sVNT cat serum samples, 75 % (9/12) exhibited neutralizing titers with all samples also being positive by RBD iELISA. Notably, the RBD iELISA and sVNT demonstrated high sensitivity and specificity in detecting SARS-CoV-2 antibodies (100 and 79 %
100 and 90 %, respectively). In conclusion, our study provides important insights into the seroprevalence of SARS-CoV-2 antibodies in domestic cats, highlighting the potential for interspecies transmission and the need for continued monitoring of SARS-CoV-2 in animal populations.