Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy

SARS-CoV-2 was first detected in late 2019 and rapidly spread worldwide. Although transmission is predominantly human-to-human, concerns remain about the role of companion animals in the pandemic. Sporadic natural infections in dogs and cats and experimental infections indicating susceptibility in cats (and limited susceptibility in dogs) motivated an assessment of infection and exposure in pets living under natural household conditions. This study aimed to determine the prevalence of active infection and prior exposure to SARS-CoV-2 among cats and dogs in Italy during a period of high human case incidence, and to evaluate risk factors such as household COVID-19 status, sex, age, and clinical signs.

Prior reports identified SARS-CoV-2 RNA and antibodies in pets from COVID-19-affected areas and showed, experimentally, that cats can develop respiratory disease and transmit to in-contact cats, whereas dogs show poor susceptibility and minimal shedding. Small-scale field studies and case reports exist, but comprehensive, population-level data under natural ownership conditions have been limited. This study builds on those findings by providing a large-scale serological and molecular survey in Italian household pets during the early pandemic.

Study design and sampling: Companion animals (603 dogs, 316 cats) from various Italian regions—predominantly Lombardy—were sampled during routine veterinary visits between 15 March and 11 May 2020. Ethics approval was granted by the University of Bari Department of Veterinary Medicine (approval number 15/2020). Clinical and household data (breed, sex, age, recent exposure to COVID-19-positive or suspected-positive humans, and presence of respiratory signs) were collected for 340 dogs and 188 cats. Pets from known COVID-19-positive households included 47 dogs and 22 cats for serology and 64 dogs and 57 cats for PCR, with one animal per positive household. Specimen collection: Oropharyngeal, nasal, and/or rectal swabs were collected from 494 pets using synthetic fiber swabs: oropharyngeal (303 dogs, 173 cats), nasal (183 dogs, 78 cats), rectal (66 dogs, 30 cats). Swabs were placed in 1.5 mL viral transport medium after collection. Molecular testing: RNA extraction and preparation were performed in BSL-3 containment. Two real-time RT-PCR assays targeting SARS-CoV-2 nucleoprotein (N) and envelope (E) genes were used (Corman et al.). One-step RT-PCR (Superscript III One-Step with Platinum Taq) with specified primer/probe concentrations and cycling (reverse transcription at 55 °C for 10 min; 95 °C for 3 min; 45 cycles of 95 °C for 15 s and 58 °C for 30 s). Positive controls were human SARS-CoV-2 RNA extracts. Serology: Plaque reduction neutralization tests (PRNT) were performed using SARS-CoV-2/human/Liverpool/REMRQ0001/2020 on Vero E6 cells. Sera were heat-inactivated (56 °C, 1 h), serial twofold diluted in DMEM with 2% FBS and gentamicin, incubated with virus (800 PFU/mL) at 37 °C for 1 h, then inoculated onto Vero E6 cells with standard overlay. After 48 h at 37 °C and 5% CO2, cells were fixed and stained. PRNT80 titers were defined as the highest serum dilution achieving ≥80% plaque reduction; positive samples were repeated for confirmation. Specificity controls included reference sera/ascites positive for canine enteric coronavirus, canine respiratory coronavirus, and feline coronavirus, which were tested by PRNT against SARS-CoV-2. Data analysis: Associations between seropositivity and household COVID-19 status or sex were assessed with two-sided Fisher’s exact tests. Correlations between provincial human COVID-19 incidence and animal seropositivity were assessed with Spearman’s correlation. Analyses used GraphPad Prism 6.

- PCR results: All 494 swabbed animals (314 dogs, 180 cats) tested negative for SARS-CoV-2 RNA, including 38 dogs and 38 cats with respiratory signs at sampling. Among these, 64 dogs and 57 cats were from confirmed COVID-19-positive households. - Serology: Neutralizing antibodies were detected in 15/451 dogs (3.3%) and 11/191 cats (5.8%). Titer ranges: dogs 1:20–1:160; cats 1:20–1:1280. None of the seropositive animals had respiratory signs at the time of sampling. - Household exposure: Dogs from COVID-19-positive households were significantly more likely to be seropositive (6/47; 12.8%) than dogs from COVID-19-negative households (2/133; 1.5%) (Fisher’s exact p = 0.004). Cats showed 1/22 (4.5%) seropositive in COVID-19-positive households vs 1/38 (2.6%) in COVID-19-negative households (p = 1.000). Suspected-positive households: dogs 1/7 (14.3%); cats 0/3 (0%). - Sex: Male dogs had higher seropositivity than females (7/83; 8.4% vs 2/105; 1.9%; p = 0.045). In cats, male 2/31 (6.5%) vs female 0/30 (0%); p = 0.492. - Age: Of 423 animals with age recorded, 0/30 under 1 year were seropositive. Positivity by age group: 1–3 years 6.5% (6/92), 4–7 years 2.9% (3/102), ≥8 years 3.0% (6/199). - Geographic correlation (Lombardy provinces with ≥10 samples): Positive trends between animal seropositivity and human COVID-19 burden: dogs Spearman r = 0.771 (p = 0.103); cats r = 0.696 (p = 0.125). - Specificity: Reference sera/ascites positive for other canine/feline coronaviruses tested negative by SARS-CoV-2 PRNT, supporting assay specificity.

The study demonstrates that while active SARS-CoV-2 infection (by PCR) was not detected in a large cohort of Italian household pets during March–May 2020, a measurable fraction had neutralizing antibodies, indicating prior exposure. Seropositivity was associated with household human infection for dogs and showed higher titers in cats, aligning with experimental data that cats are more susceptible and can develop higher antibody responses. The absence of PCR positivity suggests short windows of viral shedding in pets, consistent with experimental timelines where cats cease shedding by ~10 days post-infection and seroconvert by 7–13 days. The observed correlation trends between human infection burden and animal seropositivity in Lombardy support that pet exposure reflects community transmission dynamics. These findings indicate that pets may serve as sentinels of household/community exposure but are unlikely to be major drivers of human transmission under typical pet ownership conditions. Inclusion of pets in sero-epidemiological surveillance could enhance understanding of transmission patterns, particularly as human case numbers decline and contact tracing improves.

This large-scale field study of Italian household cats and dogs found no active SARS-CoV-2 infection by PCR but identified serological evidence of exposure in 3.3% of dogs and 5.8% of cats, with increased seropositivity among dogs from COVID-19-positive households. Results corroborate higher susceptibility and antibody responses in cats observed in experimental studies and suggest that pet exposure is not uncommon in high-burden areas. Pets likely play a minimal role in human transmission under normal ownership conditions, though specific high-density animal settings may differ. Future research should clarify risk factors for pet infection (including behavioral and physiological sex differences), define shedding duration and transmission potential under natural conditions, and integrate pet serosurveillance into broader public health monitoring.

- Cross-sectional field sampling precluded determination of infection timing relative to seroconversion and potential shedding windows. - All swab PCRs were negative; if shedding is brief, infections may have been missed due to sampling timing. - Limited metadata: many sera from diagnostic labs lacked detailed history, household status, and clinical data. - Small numbers of seropositive animals reduced power for subgroup analyses (e.g., sex effects in cats, neuter status in dogs). - Movement restrictions during the pandemic may have delayed veterinary visits, introducing sampling bias. - Provincial correlation analyses were limited to areas with ≥10 samples and did not reach statistical significance.