Low Prevalence of SARS-CoV-2 Antibodies in Canine and Feline Serum Samples Collected during the Covid-19 Pandemic in Hong Kong and Korea

1. Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease (COVID-19) pandemic, belongs to the genus of Betacoronaviruses in the family Coronaviridae. Coronaviruses can infect a wide range of birds and mammals, and manifest high genetic diversity due to mutations and recombination of their genomes [1,2,3]. Several members of the family Coronaviridae circulate continuously in human and animal populations and mostly cause mild symptoms in humans, such as the common cold or mild enteric disease [4,5,6,7]. In contrast, the SARS-CoV and the Middle East respiratory syndrome-associated coronavirus (MERS-CoV) are highly pathogenic in humans and cause severe lower respiratory tract infections that can further develop into acute respiratory distress syndrome (ARDS) and extrapulmonary manifestations [4,8,9,10,11]. SARS-CoV and MERS-CoV which originated in bats and infected humans via intermediate hosts are well-known for cross-species transmission. Similarly, it is believed that the novel SARS-CoV-2 that emerged in late 2019 in Wuhan, China, has a zoonotic origin from bats, while the intermediate host has not yet been identified [12,13,14].

While the current global pandemic is driven by human-to-human transmission, concern has arisen regarding disease transmission between humans and susceptible animals as evidenced by sporadic cases of SARS-CoV-2 infection in companion, farmed, wild and captive zoological animals that have been in close contact with infected humans [15,16,17,18,19,20]. Knowing the zoonotic origin of the disease and the potential capacity of the virus to adapt to other species, it is important to understand the extent of natural SARS-CoV-2 infection of animals, in particular cats and dogs in households that are in direct contact with their owners and other household members. To date, natural SARS-CoV-2 infection has been sporadically detected in companion animals where human-to-animal transmission was suspected in almost all cases [16,21,22,23,24]. In line with this, serological investigations in cats and dogs have been conducted in different countries since the early phases of the COVID-19 pandemic. Molecular and antibody prevalence studies revealed that dogs and cats living in areas of high human infection had been occasionally infected by SARS-CoV-2, thus confirming previous reports of natural infections of companion animals [25,26,27]. Specifically, the seroprevalence of SARS-CoV-2 in cats with an unknown history of exposure to the virus was 0.69% (6/920) in Germany [28], 0.76% (1/131) in Croatia [29], 0.4% (2/500) in the Netherlands [30], and 3.3% (15/451) in Italy [31]. In dogs, the prevalence of neutralizing antibodies was as low as 0.31% (2/654) in Croatia [29], 0.2% (1/500) in the Netherlands [30], and 3.3% (15/451) in Italy [31]. Not surprisingly, the seroprevalence of animals from COVID-19-affected households was higher, ranging from 10% to 12.8% in dogs and 4.5% to 43.8% in cats [25,26,27,31].

Hong Kong and Seoul are two of the most densely-populated urban cities in Asia, where companion animals often live in close contact with humans. It is estimated that 241,900 (9.4%) households in Hong Kong keep dogs and/or cats [32], whereas 1 in 3 South Korean households have companion animals, with the number of pet dogs and cats reaching 8.6 million (6.02 million dogs and 2.58 million cats) [33]. In this study, we used an ELISA that detects the SARS-CoV-2 receptor binding domain (RBD) to assess the prevalence of anti-SARS-CoV-2 antibodies in 1,040 cats and 855 dogs with an unknown history of exposure to the disease during the early phase of the pandemic in Hong Kong and Seoul. Positive results were confirmed using a surrogate virus neutralization (sVNT) and plaque reduction neutralization test (PRNT). In addition, we investigated the prevalence of SARS-CoV-2 RNA shedding in oronasal swabs from a subset of 301 healthy free-roaming cats presented to a trap-neuter release desexing clinic in Hong Kong using RT-PCR.

2. Materials and Methods

2.1. Sample description

Seoul, Korea 

Residual diagnostic sera from 344 dogs and 488 cats collected from April to July of 2020 were obtained from three veterinary practices in different districts of Seoul, Republic of Korea. All animals were sampled by veterinarians during routine healthcare visits for various reasons. For each sample, the following information was available: species, gender, age at sampling, and date of sampling.

Hong Kong, China 

Residual diagnostic sera were collected from 511 dogs (from January 2021 until August 2021) and from 251 cats (from March 2020 to April 2021) presenting for routine healthcare visits at a large veterinary primary care and referral hospital in Sham Shui Po, Hong Kong. In addition, serum samples, nasal swabs and oropharygneal were collected from 301 healthy free-roaming colony cats desexed in a trap-neuter release (TNR) program at a large animal shelter in Wan Chai, Hong Kong, from January 2021 to May 2021.

Positive control sera were obtained from a dog and a cat admitted to the Agriculture, Fisheries, and Conservation Department (AFCD) quarantine facility because their owners were diagnosed with COVID-19. These two animals had been confirmed to be shedding SARS-CoV-2 in respiratory and fecal secretions and tested positive for serum-neutralizing antibodies using plaque reduction neutralization 90 (PRNT₉₀) test, with titers of 1:160 for the dog and ≥1:320 for the cat [16,34].

For use as negative controls and ELISA optical density (OD) cut-off determination, 46 feline sera and 23 canine sera were collected before COVID-19 outbreaks, before January 2020, that had been stored at -80°C were used. Animal ethics approval for this study was granted by the Animal Ethics Committee of the City University of Hong Kong, approval no. A-0478. Research licenses for sampling colony cats were granted by The Government of the Hong Kong SAR, Department of Health (20-164 to 20-179).

2.2. Assay description and optimisation

All serum samples were heat-inactivated at 56^oC for 30 min before use. The sera were tested using an ELISA to detect anti-SARS-CoV-2 spike glycoprotein receptor binding domain (RBD) antibodies. The protocol was based on a previously described assay with modifications [35]. Briefly, 96-well plates (Nunc MaxiSorp, Thermo Fisher Scientific, cat. no. 44-2404-21) were coated with 100 ng per well of SARS-CoV-2 Spike RBD-mFc Recombinant Protein (Sino Biological, Beijing, China, Sinobiological, cat. no. 40592-V05H) diluted in PBS buffer and refrigerated overnight at 4^oC. The next day, coated plates were blocked using 100 μl of Chonblock blocking/sample dilution ELISA buffer (Chondrex Inc, Redmon, US, cat. no. 9068) for 1 h at room temperature. Heat-inactivated serum samples were diluted 1:100 in Chonblock blocking/sample dilution ELISA buffer, with a 15 min incubation at room temperature. After 3 rounds of washing the plate with PBS containing 0.2 % Tween 20, the diluted sera samples were transferred to the coated plate and incubated in a humidified chamber at 37°C for 2 h. Washing was performed before adding horseradish peroxidase (HRP)-conjugated goat anti-feline or canine IgG antibodies (Thermo Fisher Scientific, cat. no PA1-84673 or A18763 respectively) diluted 1:2000 in PBS. The plates were incubated in a humidified chamber for 1 h at 37°C, then washed. Subsequently, 100 μl of 3,3',5,5' tetramethylbenzidine (TMB) substrate (Ncm TMB One; New Cell and Molecular Biotech Co. Ltd, Suzhou, China, cat. no. M30500) was added into each well. After a 6 min (feline) or 5 min incubation (canine), the reaction was stopped by adding 50 μl of 2 M H₂SO₄ solution and analyzed on an absorbance microplate reader at 450 nm wavelength (SpectraMax ABS, Molecular Devices). Test samples were run in duplicate; each plate contained a duplicate positive control, negative control, and blank control (PBS only). For cut-off determination, the mean of the OD₄₅₀ of negative sera collected pre-COVID-19 plus 3× and 6× the SD of the negative control readings for cats and dogs were used, respectively.

2.3. Confirmatory Serological Tests: Surrogate Virus Neutralization and Plaque Reduction Neutralization tests

Canine and feline sera that tested positive on ELISA were evaluated for SARS-CoV-2 specific-neutralizing antibodies using a commercially available (sVNT; GeneScript Inc. Piscataway, USA) according to the manufacturer's instructions. The OD values were read on a spectrophotometer (Molecular Devices SpectraMax ABS, San Jose, CA USA) at 450 nm. Positive and negative controls provided by the kit were included in duplicate in every run. The quality control and validation of the results were based on the OD values for positive and negative controls falling in the recommended values. The results were interpreted as positive for SARS-CoV-2 neutralizing activity when the sVNT inhibition was calculated to be >20%, while <20% was regarded as a negative result.

Subsequently, canine and feline serum samples with positive results in sVNT were further tested with a plaque reduction neutralization test (PRNT) in a BSL-3 laboratory at the University of Hong Kong, as previously described [34].

2.4. Detection of SARS-CoV-2 RNA shedding in oronasal secretions of healthy-free roaming cats in Hong Kong by RT-PCR

To investigate the prevalence of active shedding of SARS-CoV-2 RNA in oronasal secretions collected from the 301 healthy free-roaming cats in Hong Kong, RT-PCR was performed to detect the E gene and RdRp genes as previously described [16].

2.5. Statistical Analyses

Data analysis was performed using GraphPad Prism software v.9.3.1 (GraphPad Software, LLC, CA, USA). The coefficient of variation (CV) was calculated as the ratio of the standard deviation to the mean multiplied by 100. Intra-assay CV ≤10% and inter-assay CV ≤15% were considered acceptable [36]. A Pearson correlation coefficient was calculated for ELISA-positive serum samples that were further analyzed by sVNT.

3. Results

3.1. Intra- and inter-assay coefficients of variation (CV)

The in-house SARS-CoV-2 RBD ELISA showed intra-assay CV values within acceptable limits, with CV <10 % for the canine and feline sera. The inter-assay CV of the canine ELISA was 5.66 % for the positive control serum and 10.94 % for the negative control serum. The inter-assay CV of the feline ELISA was 9.93 % for the positive control serum and 13.28 % for the negative control serum.

3.2. SARS-CoV-2 seroprevalence in domestic cats

The positive control cat serum (PRNT₉₀ ≥1:320) showed a mean OD₄₅₀ value of 1.918 by RBD-ELISA. The mean OD₄₅₀ value of 46 pre-COVID-19 feline sera was 0.196 ± 0.123, resulting in a cut-off OD₄₅₀ of 0.564 for positive samples. Of the 488 feline serum samples from Korea, 22 samples (4.51%) were deemed to be positive for anti-S RBD antibodies by ELISA with OD₄₅₀ values ranging from 0.606 to 2.336, while of the 552 feline sera collected in Hong Kong, 14 samples (2.54 %) were positive (OD₄₅₀ range 0.580 to 1.388). Specifically, 13 out of 251 sera (5.18%) and only one out of 301 sera (0.33%) sampled in veterinary primary care hospitals and animal shelters in Hong Kong, respectively, tested positive. In addition, nasal and oropharyngeal swabs collected from 301 cats of the animal shelter tested negative for the presence of SARS-CoV-2 RNA by RT-PCR. The 36 feline sera that tested ELISA-positive were evaluated using the sVNT. Nine samples, including eight from Korea (1.64 %) and one from Hong Kong (0.18%), tested positive, with an inhibition ranging from 30.46 to 38.75% (Figure 1). One feline serum sample obtained from Hong Kong tested ELISA negative (OD₄₅₀ 0.512) but was positive by sVNT with an 35.71% inhibition. The Pearson correlation coefficient analysis confirmed the lack of correlation between RBD ELISA and sVNT.

Subsequently, the nine positive samples by sVNT were further evaluated for the presence of neutralizing antibodies by PRNT₉₀ and PRNT₅₀, of which two from Korea (H-C262 and H-C266) were positive. Specifically, H-C262 had a mean OD₄₅₀ of 2.336 and 32.35% inhibition, whereas H-C266 showed a mean OD₄₅₀ of 0.7659 and 30.10% inhibition by RBD-ELISA and sVNT, respectively. One sample (ID 155961) obtained from a veterinary primary care hospital in Hong Kong with an OD450 value of 0.5121 tested positive by sVNT with 35.71 % inhibition. However, this sample was determined negative by PRNT₉₀. Both PRNT₉₀ and PRNT₉₀ gave comparable results. A summary of the SARS-CoV-2-specific antibodies detected from feline sera by three serological assays is shown in Table 1.

3.3. SARS-CoV-2 seroprevalence in domestic dogs

The positive control dog serum (PRNT₉₀ 1:160) showed a mean OD₄₅₀ value of 1.762 by RBD-ELISA. The mean OD value of the negative control pre-COVID sera was 0.223 ± 0.065, yielding a cut-off of 0.610 for positive samples. Of the 344 sera from Korea, 17 (4.94 %) were positive with OD values ranging from 0.632 to 2.479, while of the 511 sera collected in Hong Kong, 33 (6.46 %) were positive with an OD range of 0.611 to 2.135. When the positive sera by RBD-ELISA were tested with sVNT, only one sample from Korea (H-D303; OD₄₅₀ 0.8879) tested positive with a 23.92 % inhibition (Figure 2).

A summary of the SARS-CoV-2-specific antibodies detected from canine sera by three serological assays is shown in Table 2.

3.4. Prevalence of SARS-CoV-2 RNA shedding in oronasal secretions of healthy-free roaming cats in Hong Kong-

SARS-CoV-2 RNA was not detected in any of the samples from the 301 healthy free-roaming cats in Hong Kong, using RT-PCR to detect the E gene and RdRp genes.