The SARS-CoV-2 pandemic, originating from animals and primarily spreading through human-to-human transmission, raises concerns about animal susceptibility and potential roles in the pandemic's continuation. Sporadic SARS-CoV-2 infections have been reported in dogs and cats, with both RNA detection in samples and antibody presence in sera from pets in affected areas. Experimental infections have shown varying susceptibility across species, with cats exhibiting more severe infection than dogs. This study aimed to investigate the prevalence of SARS-CoV-2 infection in a large sample of companion animals (dogs and cats) in northern Italy during a period of widespread human infection to better understand the extent of animal involvement under natural conditions.

Previous studies have shown evidence of SARS-CoV-2 infection in dogs and cats, both through detection of viral RNA in respiratory and fecal samples and the presence of neutralizing antibodies in their sera. These studies, however, were limited in sample size. Experimental infection studies revealed that cats are more susceptible to SARS-CoV-2 than dogs, exhibiting respiratory pathology and higher viral shedding. The need for large-scale studies to assess the extent of animal infection under natural conditions was highlighted. This large-scale study aimed to address this gap.

Between March 15 and May 11, 2020, 603 dogs and 316 cats were sampled from various Italian regions, primarily Lombardy. Samples included oropharyngeal, nasal, and/or rectal swabs from 494 animals and sera from 188 dogs and 63 cats with complete information, plus additional sera from diagnostic laboratories. Swab samples were tested for SARS-CoV-2 RNA using real-time RT-PCR targeting the nucleoprotein and envelope protein genes. Sera were analyzed using plaque reduction neutralization tests (PRNTs) to detect SARS-CoV-2 neutralizing antibodies. Data analysis included Fisher’s exact test to compare antibody detection across households with known COVID-19 status and between sexes, and Spearman’s correlation to analyze the relationship between human COVID-19 case numbers and antibody detection in animals. The study was approved by the Ethics Committee of the Department of Veterinary Medicine, University of Bari, Italy.

Despite all 494 animals with swab samples testing negative for SARS-CoV-2 RNA, including those with respiratory symptoms, neutralizing antibodies were detected in 15 dogs (3.3%) and 11 cats (5.8%). Antibody titers ranged from 1:20 to 1:160 in dogs and 1:20 to 1:1280 in cats. Among samples from households with known COVID-19 status, seropositivity was higher in dogs (12.8%) and cats (4.5%) from COVID-19-positive households compared to those from negative households (1.5% and 2.6%, respectively). Dogs from COVID-19-positive households were significantly more likely to test positive for SARS-CoV-2 neutralizing antibodies (Fisher’s exact test, *p* = 0.004). Male dogs showed a significant association with seropositivity (*p* = 0.045). In Lombardy, there was a positive trend (though not statistically significant) between the proportion of seropositive dogs and the human disease burden (Spearman’s *r* = 0.771, *p* = 0.103). A similar, non-significant trend was observed for cats.

This study provides further evidence of SARS-CoV-2 exposure in companion animals in areas with high human infection rates. The absence of positive PCR results despite the presence of neutralizing antibodies in some animals suggests that shedding periods might be relatively short. The study highlights the higher susceptibility of cats compared to dogs, consistent with prior research. The findings' similarity to human seroprevalence rates in other studies indicates that companion animal infection is not uncommon. Although animal-to-human transmission is unlikely, it may be more probable under certain conditions like those found in high-density animal populations, such as mink farms. The study suggests that continued serological surveillance of pets could offer valuable insights into community disease dynamics.

This large-scale study confirms SARS-CoV-2 exposure in companion animals living in areas with high human infection rates. While PCR tests were negative, the presence of neutralizing antibodies in a subset of dogs and cats points towards past exposure. Dogs showed a greater likelihood of infection from COVID-19-positive households. Future research should focus on refining detection methods, understanding risk factors, and investigating the potential for animal-to-animal and animal-to-human transmission.

The study's cross-sectional design prevents determining the precise timing of infection in seropositive animals. The reliance on owner-reported COVID-19 household status might introduce some inaccuracy. The smaller number of seropositive animals limited the statistical power of some analyses. The absence of complete information for all animals also impacted the ability to investigate associations with certain factors fully.