A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs

Abstract

IntroductionCoronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available.MethodsHerein, the first comparative study of six “in house” and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT–qPCR results) as a reference. Four “in house” tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera).Results and discussionELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (>90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs.

Prevalence of SARS‐CoV‐2 in domestic cats (Felis catus) during COVID‐19 pandemic in Latvia

“…however, in the same study, no cross-reactions were seen when feline infectious peritonitis-positive cat sera were tested using N proteinbased ELISA (Diezma-Díaz et al, 2023). The limitation of our study, however, is that antibody positive serum samples detected by double antigen ELISA were not confirmed by VNA expressing concerns regarding a potential for false positive results.…”

“…( 2022 ) showed absence of anti‐SARS‐CoV‐2 antibodies in 99 stray cats when double antigen ELISA was used. In contrast, in a more recent study specifically focused on the comparison of SARS‐CoV‐2 serological tests in cats showed stronger correlation between tests based on S protein and virus neutralisation assay (VNA) than those based on protein N; however, in the same study, no cross‐reactions were seen when feline infectious peritonitis‐positive cat sera were tested using N protein‐based ELISA (Diezma‐Díaz et al., 2023 ). The limitation of our study, however, is that antibody positive serum samples detected by double antigen ELISA were not confirmed by VNA expressing concerns regarding a potential for false positive results.…”

Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia

“…Animals and humans are susceptible to a large number of different coronaviruses, in fact, it has been shown that all pathogenic human coronaviruses have their origin in animals, which is why studies should focus on their role in the transmission of SARS-CoV-2. 49 In the present study, a human-animal transmission was considered based on the results of Smith et al 50 in the United Kingdom, who ruled out that dogs and cats were reservoirs of infection for humans. However, we cannot be sure about the trasmission direction, which will only be confirmed through further studies.…”

Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia

“…Previous papers used the ID Screen ® kit in their studies. [33][34][35][36] Each plate contained 96 microwells sensitised with recombinant antigen of purified N protein of SARS-CoV-2, to which the following items were added: two negative controls (NC); two positive controls (PC); and 92 problem sera previously homogenised by vortexing. The optical density (OD) reading was performed using the Cytation 3 multimodal microplate reader (BioTek Instruments, Inc. Winooski, VT, USA) with a wavelength of 450 nm.…”