key: cord-0730971-36ie1kfr authors: Decaro, Nicola; Grassi, Andrea; Lorusso, Eleonora; Patterson, Edward I.; Lorusso, Alessio; Desario, Costantina; Anderson, Enyia R.; Vasinioti, Violetta; Wastika, Christida E.; Hughes, Grant L.; Valleriani, Fabrizia; Colitti, Barbara; Ricci, Dominga; Buonavoglia, Domenico; Rosati, Sergio; Cavaliere, Nicola; Paltrinieri, Saverio; Lauzi, Stefania; Elia, Gabriella; Buonavoglia, Canio title: Long‐term persistence of neutralizing SARS‐CoV‐2 antibodies in pets date: 2021-09-12 journal: Transbound Emerg Dis DOI: 10.1111/tbed.14308 sha: 4e1f4e29908b2de91f868c96ead87253d4747c79 doc_id: 730971 cord_uid: 36ie1kfr We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS‐CoV‐2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID‐19 human patients. TA B L E 1 Serological follow-up for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in positive dogs and cats Abbreviations: ELISA, enzyme-linked immunosorbent assay; ND, not done; Neg, negative; Pos, positive; PRNT 80 , plaque reduction neutralization test; >QR, above the quantification range; VN, virus neutralization. a Values in brackets represent the ratio between the optical densities of the tested serum and the positive control (cut-off value = 50%). b Values in brackets represent the ratio between the optical densities of the tested serum and the positive control (cut-off value = 20%). c Antibody titre is expressed as the highest serum dilution with 80% reduction in plaques in inoculated VERO-E6 cells compared to the control, with 1:20 being the lowest serum dilution tested. d Antibody titre is expressed as the highest serum dilution giving 100% reduction of cytopathic effect in inoculated VERO-E6 cells, with 1:10 being the lowest serum dilution tested. we report the results of a longitudinal study in SARS-CoV-2 seropositive pets demonstrating the persistence of neutralizing antibodies for up to 10 months in some animals. The pets included seven dogs and two cats, which had SARS-CoV-2 neutralizing antibodies according to previous studies (Decaro, Vaccari, et al., 2021; Patterson et al., 2020) or at a first screening (Table 1) . The age of the tested animals ranged from 1.5 to 11 years and from 7 to 17 years for dogs and cats, respectively. All seven dogs and one of two cats were from COVID-19 positive households, but none of the sampled animals had developed COVID-19 clinical signs. Only one dog (Dog 7) had been found to shed SARS-CoV-2 RNA by real-time PCR (Decaro, Vaccari, et al., 2021) . For pets living in COVID-19 households, sera collection was initially carried out between 7 and 60 days after showed that four of seven dogs and two of two cats had SARS-CoV-2 neutralizing antibodies at 8 months or more after the first positive testing (Table 1 ). For one dog that had tested positive for SARS-CoV-2 by real-time RT-PCR (Decaro, Vaccari, et al., 2021) , sera were available only for the first 3 months after infection and displayed antibodies through PRNT and VN at all time points. The remaining two dogs tested positive by PRNT and VN only at the first sampling, which may account for a shorter duration of the humoral immunity rather than for an older infection, since these animals were infected during the first wave of the COVID-19 pandemic . A great discrepancy was observed between serological tests based on ELISA and neutralization tests. Five dogs that were seropositive by PRNT and/or VN at one or more time points were completely negative by both commercial ELISA tests. The dog (Dog 7) that had been found positive for SARS-CoV-2 RNA (Decaro, Vaccari, et al., 2021) was constantly seropositive by both PRNT and VN, but invariably negative by the ID.vet ELISA and positive at two out of three time points by the In3Diagnostic ELISA. In contrast, the discrepancy between PRNT and VN was less evident, being generally restricted to few sera with low neutralizing antibody titres. The discrepancy between ELISA and neutralization tests may be related to a lower sensitivity of ELISA or, alternatively, to a lack of specificity of neutralization assays. However, the latter hypothesis could be ruled out since previous experiments have demonstrated that pre-pandemic sera that were antibody positive for endemic coronaviruses of dogs and cats test negative by SARS-CoV-2 neutralization assays (Decaro, Vaccari, et al., 2021; Patterson et al., 2020; Zhang et al., 2020) . The lower sensitivity of ELISA compared to VN or PRNT may be due to a different kinetic between the antibody response raised against the viral nucleoprotein (the antigen used in both ELISA tests) and that directed against the spike protein (the main target of neutralizing antibodies). Interestingly, for Cat 1 there was no evidence for exposure to COVID-19 positive human patients. Therefore, it is likely that this cat was infected by an asymptomatic owner with undiagnosed SARS-CoV-2 infection or, alternatively, it acquired the virus from other infected people or animals. Despite the increasing number of reports of SARS-CoV-2 infection in dogs and cats, no long-term monitoring has been carried out so far to evaluate the persistence of specific antibodies in pets. To our knowledge, the antibody response in pets has been monitored for a maximum of 2-3 months after infection, displaying relatively stable or increasing titres and no evidence of seroreversion (Hamer et al., 2021) . Zhang et al. (2020) followed two cats for about 4 months, finding that neutralizing antibodies peaked after 10 days from the first sampling and then decreased to detection limit in 110 days. Our study, which was conducted using four different serological assays, demonstrates that similar to humans, dogs and cats may develop a long-term neutralizing antibody response against SARS-CoV-2. At which extent the presence of neutralizing antibodies is able to protect these animals from SARS-CoV-2 reinfection is currently unknown, thus requiring further studies. Dynamics of SARS-CoV-2 neutralising antibody responses and duration of immunity: A longitudinal study Cross-sectional serosurvey of companion animals housed with SARS-CoV-2-infected owners SARS-CoV-2 infection in dogs and cats: Facts and speculations Possible human-to-dog transmission of SARS-CoV-2, Italy Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival SARS-CoV-2 infections and viral isolations among serially tested cats and dogs in households with infected owners in Texas, USA. Viruses, 13, 938 Persistence of humoral response upon SARS-CoV-2 infection Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy Maintenance of neutralizing antibodies over ten months in convalescent SARS-CoV-2 afflicted patients A serological survey of SARS-CoV-2 in cat in Wuhan Long-term persistence of neutralizing SARS-CoV-2 antibodies in pets The authors are grateful to Maria Stella Lucente, Cristiana Catella, Carlo Armenise, Arturo Gentile (University of Bari), Liana Teodori and Alessandra Leone (Istituto Zooprofilattico Sperimentale dell'Abruzzo e Moro within the CRUI-CARE Agreement. The authors declare no conflict of interest. The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The study was authorized by the Ethics Committee of the Department of Veterinary Medicine, University of Bari (approval number 15/2020). The data that support the findings of this study are available from the corresponding author upon reasonable request.