key: cord-0873726-ka0dpack
authors: Udom, Kitikhun; Jairak, Waleemas; Chamsai, Ekkapat; Charoenkul, Kamonpan; Boonyapisitsopa, Supanat; Bunpapong, Napawan; Techakriengkrai, Navapon; Amonsin, Alongkorn
title: Serological survey of antibodies against SARS‐CoV‐2 in dogs and cats, Thailand
date: 2021-07-08
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.14208
sha: 4b3178a87d7a9bb5c5388708daf12237c9c4f36a
doc_id: 873726
cord_uid: ka0dpack

Coronavirus disease of 2019 (COVID‐19) caused by severe acute respiratory syndrome virus type 2 (SARS‐CoV‐2) is an emerging severe acute respiratory disease affecting global human health. In this study, a large‐scale serological survey of antibodies against SARS‐CoV‐2 in dogs and cats was conducted during the first and second waves of COVID‐19 outbreaks in Thailand, from April to December 2020. A total of 3215 serum samples were collected from dogs (n = 2102) and cats (n = 1113) living in Bangkok and in the vicinities. Serum samples were tested for SARS‐CoV‐2 antibodies by using an indirect multispecies enzyme‐linked immunosorbent assay (ELISA). Positive and suspected samples were additionally tested for neutralizing antibodies by the surrogate virus neutralization test (sVNT). The indirect ELISA results showed that 1.66% (35 out of 2103) of dogs and 0.36% (four out of 1112) of cats were positive for SARS‐CoV‐2 antibodies. The sVNT results showed that all ELISA‐positive and suspected samples were negative for neutralizing antibodies. Positive serum samples (35 dogs and four cats) were obtained from clinically healthy animals and animals with mild respiratory signs aged <1–13 years living in Bangkok and Samutprakarn Provinces. In summary, a serological survey revealed evidence of anti‐N‐IgG antibodies suggesting SARS‐CoV‐2 exposure in both dogs and cats during the first and second COVID‐19 outbreaks in Thailand.

and cats in close contact with SARS-CoV-2-infected persons has been reported in Belgium, China, France, Hong Kong, Spain, the UK and the USA (Abdel-Moneim & Abdelwhab, 2020; Newman et al., 2020; Ruiz-Arrondo et al., 2020; Sailleau et al., 2020) . With respect to antibody detection, a serological survey of SARS-CoV-2 in cats in China reported that 14.7% of cats were found to be positive by using commercial enzyme-linked immunosorbent assays (ELISA) based on receptor binding domain (RBD) . In Italy, a serological study of dogs and cats living in COVID-19-positive households showed that 3.4% and 3.9% of dogs and cats developed neutralizing antibodies against SARS-CoV-2, respectively (Patterson et al., 2020) . In Germany, 0.69% (six out of 920) of cats were found to show antibodies against SARS-CoV-2 by ELISA and immunofluorescence tests (Michelitsch et al., 2020) . These reports emphasized evidence of natural infection by SARS-CoV-2 in dogs and cats. In this study, we conducted a large-scale serological survey of SARS-CoV-2 antibodies in 3215 serum samples from domestic dogs and cats in Bangkok and in the vicinity during the period encompassing the first and second waves of COVID-19 outbreaks in Thailand, from April 2020 to December 2020.

In this study, we collected 3215 serum samples from dogs (n = 2102) and cats (n = 1113) during routine health care visits at the Chulalongkorn University Small Animal Hospital between April and December 2020. These animals were from six zones of Bangkok and nearby provinces (Nakhon Pathom, Nonthaburi, Pathum Thani, Samut Sakhon 

To detect SARS-CoV-2 antibodies in serum samples, the ID Screen® SARS-CoV-2 Double Antigen Multi-species ELISA kit (ID VET, Montpellier, France) was used. This indirect ELISA was based on the detec-tion of anti-SARS-CoV-2 nucleocapsid antibodies (IgG) in the tested animal serum (Sailleau et al., 2020) . Indirect ELISA tests were performed according to the manufacturer's instructions. Briefly, 25 µl of each serum sample and positive and negative control samples were transferred to separate wells, diluted with 25 µl of dilution buffer and incubated at 37 • C for 45 min. Each well was washed with 300 µl of washing buffer. After washing, 100 µl of horseradish peroxidase (HRP)conjugated N protein recombinant antigen was added and incubated at 25 • C for 30 min. Then, each well was washed three times with 300 µl washing buffer. After washing, 100 µl of the substrate was added to each well, and samples were incubated at 25 • C for 20 min. Then, 100 µl of stop solution was added to stop the reaction. After the addition of stop solution, the optical densities (ODs) at 450 nm were read. The OD of each sample was calculated as the S/P percentage (S/P%). Serum with S/P% > 60% was defined as positive, while serum with S/P% 50−60% was considered 'suspect' . Indirect ELISA with the ID Screen® SARS-CoV-2 Double Antigen Multi-species ELISA kit shows 100% sensitivity (10 positive samples tested) and 100% specificity (47 negative samples tested) which can be used with individual samples from susceptible species, including dogs and cats.

To detect the presence of neutralizing SARS-CoV-2 antibodies, in addition to the ELISA tests, both positive and suspect sera (n = 50) together with randomly selected sera (n = 15) were subjected to surrogate virus neutralization tests (sVNT). The VNT was performed by using the cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (Gen-Script Biotech, Jiangsu, China) which detects neutralizing antibodies against the interaction between the virus RBD and the ACE2 cell surface receptor . In brief, 50 µl of each 1:10 diluted serum sample was mixed with 50 µl HRP conjugated to the SARS-CoV-2 spike RBD (HRP-RBD) and incubated at 37 • C for 30 min. After that, each mixture was added to a plate that was precoated with ACE2 protein and incubated at 37 • C for 15 min. Then, 260 µl of washing buffer was added to each well, and they were washed four times. After washing, 3,3' ,5,5'-tetramethylbenzidine solution was added, and the plate was incubated at 25 • C for 15 min. Then, the ODs at 450 nm were read by using a microplate reader. The OD of each sample was calculated as the inhibition percentage (% inhibition); serum with % inhibition above 20% was considered as positive, and serum within % inhibition not exceeding 20% was considered negative (Meyer et al., 2020) . The cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit features 95-100% sensitivity and 99.93% specificity .

The chi-square test was used for statistical analysis of the variation in SARS-CoV-2 seropositivity with demographic variables (sex, age and 

In this study, we collected 3125 serum samples from dogs and cats (Table S2 ). In terms of location, we found positive dogs in all zones of Bangkok and in one province (Samut Prakarn), while cats were found to show low positivity in zones 1, 2 and 5 (Table 2) 

Thailand has faced at least three waves of COVID-19 outbreaks. The first wave took place from March to May 2020, and the second wave of the outbreak started in late March 2021, and the number of confirmed SARS-CoV-2 cases is still rising ( Figure S1 ). Although SARS-CoV-2 mainly circulates in the human population, causing a significant impact on human health and socioeconomic conditions, there are some reports of SARS-CoV-2 infection in domestic animals. A study on host receptors for SARS-CoV-2 showed that many animal species, including cats, ferrets, orangutans and monkeys, have angiotensin-converting enzyme-2 receptors identical or similar to those of humans. Therefore, the transmission of SARS-CoV-2 from humans to those animals is theoretically possible (Hossain et al., 2020) . A field survey on domestic animals in known COVID-19-positive areas and households showed that natural infection with SARS-CoV-2 can occur in dogs and cats living in these locations, resulting in seroconversion against SAR-CoV-2 in the animals (Patterson et al., 2020) . In this study, our results showed that domestic dogs (1.66%) and cats (0.36%) in Bangkok and vicinity tested positive for anti-N-IgG antibodies against SARS-CoV-2. The seropositivity in this study is comparable to that observed in several countries.

For example, in Germany, only 0.69% (six out of 920) of cat sera from a veterinary diagnostic laboratory were seropositive by indirect multispecies ELISA and indirect immunofluorescence tests (Michelitsch et al., 2020 Zhao et al., 2021 ) . Similarly, in France, 15.4% of dogs (two out of 13) and 23.5% of cats (eight out of 34) from COVID-19-positive households exhibited high seropositivity by microsphere immunoassay and neutralization tests (Fritz et al., 2021) .

Serological diagnosis is an essential tool to determine viral exposure in populations. To date, several serological assays have been developed and employed to facilitate diagnosis of SARS-CoV-2 infections. The spike (S) and nucleocapsid (N) structural proteins of SARS-CoV-2 display high immunogenic activity, making these proteins attractive candidates for the development of serological diagnostic assays . Both ELISA and neutralization assays are typically considered the gold standards for the detection of antibodies against SARS-CoV-2 Tan et al., 2020; Tehrani et al., 2020 ) . The ELISA and sVNT assays used in this study feature high sensitivity and specificity . Spike-based assay, especially with the S1 subunit, is reportedly the most specific for the diagnosis of COVID-19, whereas N-based assays offer a higher sensitivity (Kontou et al., 2020; Liu et al., 2020; Okba et al., 2020; Tehrani et al., 2020 ) . Nevertheless, a comparable sensitivity and specificity of at least 98% were reported for both the S-and N-based commercially available assays in a recent head-tohead comparative study (National, 2020) . Even though similar diagnostic performance was observed for both assays, detection of antibodies against S protein is advantageous as it correlates well with neutralization test (Folegatti et al., 2020; Ni et al., 2020; Okba et al., 2020 ) .

Therefore, it was not unexpected that the 50 sera tested positive on Nbased ELISA in this study lacked neutralizing activity. One limitation of our study is the discrepancy between the results of indirect ELISA and sVNT, which deserves further investigation. This discrepancy is consistent with findings of some previous studies. For example, a serological study of cats in Germany showed six positive serum samples by indirect multispecies ELISA, and only two out of six were VNT positive (Michelitsch et al., 2020) . In China, a serological study in dogs and cats showed discrepancies between results of ELISA and VNT assays, which might have been related to the background of the animals. The animals from pet hospitals and stray dogs and cats had low neutralizing antibody levels when compared with animals owned by COVID-19 patients Zhao et al., 2021 ) . Another explanation for the discrepancy is that the timing of animal infection and serum sample collection may affect the ELISA and VNT test results. Antibody response to SARS-COV-2 has been found to be relatively high within 10 days of infection, but the duration of the peak titre was short . N is immunodominant, inducing an earlier and higher immune response (Bond et al., 2020; Hachim et al., 2020 ) . While the neutralizing antibody response develops later and peaks at 7-13 days after infection (Bosco-Lauth et al., 2020; Schlottau et al., 2020; Shi et al., 2020 ) . It should be noted that the structure of the nucleocapsid protein is conserved, cross-reactivity with antibodies against other betacoronavirues, such as OC43 and HKU1, is another concern regarding the use of N-based ELISA in human cases. However, CRCoV, a betacoron-avirus, shares only 45.8-46.2% nucleotide similarity with SARS-CoV-2.

Also, FCoV and CECoV, the most common coronavirus in cats and dogs, belong to Alphacoronavirus-1 and considerably different from SARS-CoV-2 (Sharun et al., 2020) . Thus, cross-reactivity with other CoVs by ELISA is possible, even less likely, and requires further investigation.

In conclusion, our results demonstrated that dogs and cats living in COVID-19-affected areas in Thailand with unknown SARS-CoV-2 exposure developed IgG antibodies against the virus. The role of dogs and cats in SARS-CoV-2 maintenance and transmission within species or across species from animals to humans remains unclear. Serosurveys for SARS-CoV-2 infection in animals should be routinely conducted. Therefore, awareness should be raised of SARS-CoV-2 infection in pets, and recommendations on the potential risks of the humanpet interface should be provided for high-risk occupations and the general public.

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We would like to thank the staff at the Chulalongkorn University Small Animal Hospital for collaboration in sample collection. 

This study was conducted under the approval of the Institute for Animal Care and Use Committee protocol of the Faculty of Veterinary Sciences, Chulalongkorn University (IBC#2031022 and IACUC#2031050).

The data that support the findings of this study are available in the supplementary material for this article.

All authors declare no conflict of interest.

Alongkorn Amonsin https://orcid.org/0000-0001-6769-4906