Cross-Sectional Serosurvey of Companion Animals Housed with SARS-CoV-2–Infected Owners, Italy

A fter emerging in Wuhan, China, in December 2019, coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly became a serious threat to human health worldwide (1-3).Italy has experienced one of the highest rates of human deaths in the world (4).Questions concerning the role of companion animals in the COVID-19 pandemic arose after a dog in Hong Kong reportedly tested positive for SARS-CoV-2 (5).In addition, the World Organisation for Animal Health defi ned COVID-19 as an emerging disease in animals and began promoting surveys on the prevalence of SARS-CoV-2 infections among animals (6).In this context, serologic tests are essential for rapid and accurate screening of animal populations.Few studies have been conducted to clarify the effects domestic animals have in sustaining the SARS-CoV-2 transmission cycle (5,7-9; Q. Zhang et al., unpub.data, https://www.biorxiv.org/content/10.1101/2020.04.01.021196v1).Because Italy suffered high COVID-19 incidence rates and the country has >32 million companion animals, health authorities were interested in examining virus transmission between humans and animals.We conducted a cross-sectional serologic survey among domestic dogs and cats in Italy to identify a possible association between SARS-CoV-2 infection in humans and animals.We used serologic tests to detect specifi c antibodies from animals living in close contact with SARS-CoV-2-positive human patients. The StudyBlood was collected from pets during routine activities performed by veterinary practitioners, who shared serum samples with us.Owners provided written consent for research purposes.We used 198 samples, 130 from dogs and 68 from cats, collected during the March-June 2020 COVID-19 epidemic in Italy and 100 serum samples, 65 from dogs and from 35 cats, collected in different regions of Italy before 2019 as prepandemic controls.A recombinant antigen corresponding to the nucleocapsid (N) protein of SARS-CoV-2 has been expressed in human embryonic kidney 293T cells, which have been used to develop Eradikit COVID19-IgG (IN3diagnostic, https://www.in3diagnostic.com) a sensitive and specifi c ELISA to detect SARS-CoV-2 antibodies in human serum samples.However, our initial attempts to validate the specifi city of this ELISA on pet serum samples were unsuccessful.We switched the reaction from solid-phase to solution-phase kinetics using the same antigen and the specifi city improved.Thus, we used a novel immunoassay, xMAP (Luminex Corp., https://www.luminexcorp.com),which is based on paramagnetic beads.We developed a fl ow cytometry-based system and applied it to serum samples from cats and dogs.