Development of the test kit for detection of SARS-CoV-2 antibodies in sera of susceptible animals

Abstract

The novel coronavirus infection COVID-19, caused by the SARS-CoV-2, has triggered a pandemic, and has also been reported in animal populations – in farm minks, dogs and felines: domestic cats, lions and tigers. The susceptibility of some animal species to the SARS-CoV-2 has been proven by experimental infection. Serological methods are effectively used to detect the infection in animals. Currently, methods such as neutralization test, immunofluorescence assay and enzyme-linked immunoassay are used to detect antibodies to coronaviruses. Thanks to these studies, a test kit was developed based on an indirect enzyme-linked immunoassay to detect the SARS-CoV-2 antibodies in sera of susceptible animals. The use of a purified concentrated inactivated virus as an antigen allows the detection of antibodies to various SARS-CoV-2 immunodominant proteins (S and N). The reaction conditions were optimized, and a positive-negative threshold was established by testing of 154 negative sera from animals of six species (ferrets, minks, foxes, arctic foxes, cats and dogs). The method reproducibility analysis showed that the average value of the variation coefficient did not exceed 7%, which is an acceptable value. The specificity and sensitivity of the neutralization test, when testing 30 sera from ferrets was 100 and 92.6%, respectively. The high diagnostic sensitivity and specificity shown by testing of 50 serum samples from minks, foxes, cats and dogs with different immune status, allow us to recommend the developed test kit for screening and monitoring tests and post-vaccination immunity control.

The Luciferase Immunoprecipitation System (LIPS) Targeting the Spike Protein of SARS-CoV-2 Is More Accurate than Nucleoprotein-Based LIPS and ELISAs for Mink Serology

“…Tis fnding suggests that N antibodies are less abundant and less persistent in naturally exposed mink. Tis would support the previous study on vaccinated cats and dogs [18] and in naturally exposed companion animals [31] and similar observations in humans where a faster waning of anti-N antibodies levels compared to anti-RBD antibodies has been reported [32,33].…”

“…In contrast, a recent study in humans showed that LIPS using antibodies against the N protein is more sensitive than the detection of antibodies against the S protein and that N antibodies generally appear earlier than spike antibodies [23]. Sera analysed in [23] derived from patients with positive PCR results only indicated an early stage of infection, whereas samples from animals analysed by other authors and the ones included in this study were derived from a later stage of infection [ 13,18,27]. Diferent results obtained in [23] on humans compared to published data on animals might be due to diferent sera origins or that anti-N antibodies appear earlier but also decrease faster than anti-S antibodies.…”

“…However, several in-house ELISA assays targeting the S protein have been developed and deemed ft for serological screening in animals. A recent study reported that antibody titres against the N protein in sera of vaccinated cats and dogs were too low to be detected using the IDV assay [18]. In contrast, their in-house indirect ELISA, targeting both S and N proteins, detected SARS-CoV-2 antibodies in vaccinated animals.…”

“…Furthermore, a recent study demonstrated that a correlation between VNT and N-targeted serological assays is mediated by the concomitant presence of RBD or S1/S2 proteins on the N protein [14]. However, currently, the only commercially available serological assays for animals target the N protein [15][16][17][18].…”

Coronavirus Infections in Animals: Risks of Direct and Reverse Zoonoses

“…The results of testing diagnostic preparations proved their high efficiency. Thus, the specificity and sensitivity of the developed enzyme immunoassay method with respect to the neutralization reaction for postvaccination sera amounted to 100 and 92.6%, respectively [31]. With the assistance of the Rosselkhoznadzor, monitoring programs were implemented in the populations of fur animals (mink, sable, polecat) at 34 fur farms, 36 livestock farms (cattle and small cattle, pigs, poultry), 274 private households, and petting zoos.…”