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. 2021 Dec;12(1):1597-1609.
doi: 10.1080/21505594.2021.1936433.

Seroprevalence of SARS-CoV-2 (COVID-19) exposure in pet cats and dogs in Minnesota, USA

Mythili Dileepan  1 , Da Di  1 , Qinfeng Huang  1 , Shamim Ahmed  1 , Daniel Heinrich  2 , Hinh Ly  1 , Yuying Liang  1
Affiliations

Seroprevalence of SARS-CoV-2 (COVID-19) exposure in pet cats and dogs in Minnesota, USA

Mythili Dileepan et al. Virulence. 2021 Dec.

Abstract

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is continuing to spread globally. SARS-CoV-2 infections of feline and canine species have also been reported. However, it is not entirely clear to what extent natural SARS-CoV-2 infection of pet dogs and cats is in households. We have developed enzyme-linked immunosorbent assays (ELISAs) using recombinant SARS-CoV-2 nucleocapsid (N) protein and the receptor-binding-domain (RBD) of the spike protein, and the SARS-CoV-2 spike-pseudotyped vesicular stomatitis virus (VSV)-based neutralization assay to screen serum samples of 239 pet cats and 510 pet dogs in Minnesota in the early phase of the COVID-19 pandemic from mid-April to early June 2020 for evidence of SARS-CoV-2 exposures. A cutoff value was used to identify the seropositive samples in each experiment. The average seroprevalence of N- and RBD-specific antibodies in pet cats were 8% and 3%, respectively. Among nineteen (19) N-seropositive cat sera, fifteen (15) exhibited neutralizing activity and seven (7) were also RBD-seropositive. The N-based ELISA is also specific and does not cross react with antigens of common feline coronaviruses. In contrast, SARS-CoV-2 antibodies were detected at a very low percentage in pet dogs (~ 1%) and were limited to IgG antibodies against SARS-CoV-2 N protein with no neutralizing activities. Our results demonstrate that SARS-CoV-2 seropositive rates are higher in pet cats than in pet dogs in MN early in the pandemic and that SARS-CoV-2 N-specific IgG antibodies can detect SARS-CoV-2 infections in companion animals with higher levels of specificity and sensitivity than RBD-specific IgG antibodies in ELISA-based assays.

Keywords: COVID-19; Elisa; SARS-CoV-2; cat; dog; feline coronaviruses; neutralization antibodies; seroprevalence; zoonoses.

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Figures

Figure 1.
Figure 1.
Serological tests of pet cat sera by ELISA. (a) Purified recombinant SARS-CoV-2 N and RBD proteins shown in SDS-PAGE gel after Coomassie blue staining. (b) A representative SARS-CoV-2 N IgG ELISA with pet cat sera. Normal cat serum purchased from a commercial source, the positive control (SARS-CoV N-specific mAb 1C7C7), and two seropositive samples (#29 and #11) are shown. (c) Pet cat sera tested by RBD IgG ELISA. The positive control (mAb 1C7C7), and seropositive samples are shown. (d) A batch of pet cat sera were tested with both N and RBD IgG ELISA. None of the RBD-positive sera are N-negative. The ID# of N seropositive samples are shown. (e) Evaluation of pet cat sera with IgG ELISA against feline infectious peritonitis virus (FIPV) antigens. Each serum was tested pairwise in uncoated and coated wells in technical duplicates. The adjusted OD450 value was calculated by subtracting OD450 value of uncoated well from that of the coated well. The cutoff OD450 value was calculated as described in Materials and Methods and shown as a red dash line
Figure 2.
Figure 2.
Quantification of SARS-CoV-2 neutralizing antibodies (nAb) in pet cat sera. The SARS-CoV neutralizing assay was conducted using a SARS-CoV-2 S pseudotyped replication-defective VSV expressing the firefly luciferase (FLUC) reporter gene. The FLUC activity was measured at 24 h post-infection and normalized to control wells (and set as 1). Each sample was measured in technical duplicates. (a) The pseudotyped virus entry is dependent on SARS-CoV-2 host receptor hACE2. The neutralization assay was conducted with increasing concentrations of recombinant hACE2. (b) A representative neutralization assay of pet cat serum samples. Two independent experiments were conducted
Figure 3.
Figure 3.
Serological tests of pet dog sera. (a) Validation of the pooled N-based ELISA test. A pool of 5 pet dog samples were tested in the standard dog N-based ELISA as described in Materials and Methods. The negative control (Neg) consists of all 5 N seronegative samples confirmed by previous individual ELISA test. The positive control (1 pos) consists of 4 N seronegative samples and 1 seropositive sample. The cutoff OD450 value is shown in a red dashed line. (b) Testing of pet dog sera by the pooled N-based ELISA. The positive control (pos ctrl) consists of one confirmed N seropositive serum and four confirmed N seronegative sera, and a seropositive pool (#45) are shown. (c) Identification of the seropositive pet dog sample in pool #45 by individual N-based ELISA. A positive control (pos ctrl) and the seropositive sample #432 are shown. Each sample was measured in technical duplicates
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