key: cord-0715639-952rd88y
authors: Jairak, Waleemas; Charoenkul, Kamonpan; Chamsai, Ekkapat; Udom, Kitikhun; Chaiyawong, Supassama; Bunpapong, Napawan; Boonyapisitsopa, Supanat; Tantilertcharoen, Rachod; Techakriengkrai, Navapon; Surachetpong, Sirilak; Tangwangvivat, Ratanaporn; Suwannakarn, Kamol; Amonsin, Alongkorn
title: First cases of SARS‐CoV‐2 infection in dogs and cats in Thailand
date: 2021-11-19
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.14383
sha: 35a5674d125371dd567adeac0c04ef54104d05f1
doc_id: 715639
cord_uid: 952rd88y

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the coronavirus disease 2019 (COVID‐19) pandemic in humans since late 2019. Here, we investigated SARS‐CoV‐2 infection in dogs and cats during COVID‐19 quarantine at private veterinary hospitals in Thailand. From April to May 2021, we detected SARS‐CoV‐2 in three out of 35 dogs and one out of nine cats from four out of 17 households with confirmed COVID‐19 patients. SARS‐CoV‐2 RNA was detected from one of the nasal, oral, rectal and environmental swabs of dog‐A (15 years old, mixed breed, male dog), cat‐B (1 year old, domestic shorthair, male cat), dog‐C (2 years old, mixed breed, female dog) and dog‐D (4 years old, Pomeranian, female dog). The animals tested positive for SARS‐CoV‐2 RNA from 4 to 30 days after pet owners were confirmed to be COVID‐19 positive. The animals consecutively tested positive for SARS‐CoV‐2 RNA for 4 to 10 days. One dog (dog‐A) showed mild clinical signs, while the other dogs and a cat remained asymptomatic during quarantine at the hospitals. SARS‐CoV‐2 specific neutralizing antibodies were detected in both the dogs and cat by surrogate virus neutralization tests. Phylogenetic and genomic mutation analyses of whole genome sequences of three SARS‐CoV‐2 strains from the dogs and cat revealed SARS‐CoV‐2 of the Alpha variant (B.1.1.7 lineage). Our findings are suggestive of human‐to‐animal transmission of SARS‐CoV‐2 in COVID‐19‐positive households and contamination of viral RNA in the environment. Public awareness of SARS‐CoV‐2 infection in pet dogs and cats in close contact with COVID‐19 patients should be raised.

Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a pandemic disease.

As of 16 June 2021, more than 175 million confirmed human cases have been reported, with over 3.81 million deaths (WHO, 2021c) . Evidence of SARS-CoV-2 spillover from humans to animals has been reported in dogs, cats, tigers, lions, gorillas and minks (McAloose et al., 2020; Newman et al., 2020; Sit et al., 2020) . SARS-CoV-2 infection in domestic dogs and cats has been reported in 22 countries in America, Europe and Asia (Decaro, Balboni et al., 2021; OIE, 2021b) . Cats are susceptible to SARS-CoV-2 infection and can show mild-to-moderate respiratory symptoms, while dogs developed no or mild respiratory symptoms (McAloose et al., 2020; Sailleau et al., 2020; Segalés et al., 2020) .

In Thailand, the current outbreak (3rd wave) of COVID-19 started in late March 2021, and the number of confirmed human cases of SARS-CoV-2 infection is still rising (WHO, 2021a) . During the outbreak, pet dogs and cats of COVID-19-positive patients were optionally quarantined at university and private veterinary hospitals. In this study, we collected swab samples (nasal, oral and rectal swabs) from 35 dogs and nine cats from COVID-19-positive households and examined them for SARS-CoV-2 infection in those animals. We identified SARS-CoV-2 infection in three dogs and one cat by virological testing, serological testing and viral genome analysis. This study is the first to report dogs and cats infected with SARS-CoV-2 in Thailand.

In this study, we investigated SARS-CoV-2 infection in domestic dogs and cats quarantined at private animal hospitals during the third wave of the COVID-19 outbreak reported by Thailand's Centre for COVID-19 Situation Administration (CCSA) (WHO, 2021a) . From March to May 2021, we collected samples from dogs (n = 35) and cats (n = 9) from 17 households located in Bangkok and the vicinity (Table S1 ).

It is noted that all animals lived indoor. The sample collection was conducted under the approval of the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Science, Chulalongkorn University, Thailand (approval No. 2031035) . The sampling of dogs and cats was conducted according to the convenience or willingness for COVID-19 testing of owners and animal hospital staff. In total, nasal swabs (n = 58), oral swabs (n = 61) and rectal swabs (n = 93) from 35 dogs and nine cats were collected. Serum samples (n = 9) were collected from SARS-CoV-2-positive animals. Nasal, oral and rectal swabs were collected by using flocked nylon swabs (Copan, California, USA).

Environmental samples including hair/body swabs (n = 11), water container swabs (n = 11) and floor swabs (n = 11) were collected from cages of COVID-19 positive pets at the animal hospitals. Environmental sampling was conducted before animal sampling and daily cleaning with disinfectant. Each swab was placed in 1 ml of RNAprotect® Tis- 

The swab samples, including nasal swabs (n = 58), oral swabs (n = 61) and rectal swabs (n = 93), were subjected to RNA extraction by using the magnetic bead-based automatic purification equipment of a GENTi™ 32 -Automated Nucleic Acid Extraction System (GeneAll®, Seoul, South Korea). In brief, the swab sample was vigorously vortexed for at least 15 s before removing the swab. Next 200 µl of supernatant was mixed with 7 µl of RNA carrier and then added to an extraction tube. The RNA extraction process was performed according to the manufacturer's instructions. Finally, 50 µl of viral RNA was obtained from the RNA extraction process.

For the detection of SARS-CoV-2, real-time RT-PCR based on primers and probes specific to the E and RdRp genes following WHO recommendations was used , and primers and probes specific to the N1 and N2 genes following the Centers for Disease Control and Prevention recommendations were also used (CDC, 2020) (Table S2) . A one-step real-time RT-PCR assay was performed by using a Superscript III One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen™, California, USA). In brief, a total 25 µl reaction contained 2 µl of RNA, 12.5 µl of 2X reaction buffer of the SuperScript® III Platinum® One-Step Quantitative RT-PCR System (Invitrogen™, California, USA), 1 µl of reverse transcriptase/Platinum Taq, 0.8 mM MgSO4, 0.8 µM each primer and probe and RNase-free water. Thermal cycling was performed at 50 • C for 15 min for reverse transcription, followed by 95 • C for 2 min and then 45 cycles of 95 • C for 15 s, and 60 • C for 30 s for the N1 and N2 genes. For the E and RdRP genes, thermal cycling was performed at 55 • C for 10 min for reverse transcription, followed by 95 • C for 3 min and then 45 cycles of 95 • C for 15 s and 58 • C for 30 s. Samples with a Ct value of <36 were considered positive, while samples with a Ct value of 36-40 were considered suspected and those with a Ct value > 40 were considered negative (CDC, 2020) . In this study, we used the World Organisation for Animal Health (OIE) definition for a confirmed case of animal SARS-CoV-2 infection, in which at least two specific targets (genomic regions) tested positive, indicating SARS-CoV-2 positivity (OIE, 2021a).

We used an ID Screen® SARS-CoV-2 Double Antigen Multispecies ELISA Kit (ID VET, Montpellier, France) to detect SARS-CoV-2 antibodies in serum samples. This indirect ELISA was based on the detection of anti-SARS-CoV-2 nucleocapsid antibodies (IgG) in the tested animal serum and was performed according to the manufacturer's instructions (Sailleau et al., 2020) . 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, incubated at 37 • C for 45 min and washed five times 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, the wells were washed five times with 300 µl of washing buffer. After washing, 100 µl of the substrate was added to each well and incubated at 25 • C for 20 min. Then, 100 µl of stop solution was added to stop the reaction.

The optical density (OD) at 450 nm of each sample was 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 suspected.

To detect the presence of SARS-CoV-2-neutralizing antibodies, sera of dogs and cats were subjected to sVNTs by using a cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript Biotech, Jiangsu, China). The assay detects neutralizing antibodies for the interaction between the virus receptor-binding domain (RBD) and the ACE2 cell surface receptor . Briefly, 50 µl of each 1:10-diluted serum sample was mixed with 50 µl of horseradish peroxidase conjugated to the SARS-CoV-2 spike RBD (HRP-RBD) and incubated at 37 • C for 30 min. After dilution, each mixture was added to each well precoated with ACE2 protein and incubated at 37 • C for 15 min. Then, the wells were washed 4 times with 260 µl of washing buffer. After washing, TMB solution was added and incubated at 25 • C for 15 min. Then, 50 µl of stop solution was added. The OD at 450 nm of each well was read. The OD of each sample was calculated as the inhibition percentage (% inhibition); serum with % inhibition above 20% was considered positive, and serum with % inhibition not exceeding 20% was considered negative (Meyer et al., 2020) .

We performed whole-genome sequencing of 3 SARS-CoV-2 strains by Oxford Nanopore sequencing. All gene segments of SARS-CoV-2 were amplified by ARTICS nCoV-2019 sequencing protocol V3 (LoCost).

Briefly, 8 µl of undiluted RNA was mixed with 2 µl of LunaScript® RT SuperMix (NEB, Ipswich, MA, USA) and incubated at 25 • C for 2 min, 55 • C for 10 min and 95 • C for 1 min for cDNA synthesis. The SARS-CoV-2 primer scheme was used to perform two pools of multiplex PCRs by using Q5® Hot Start High-Fidelity DNA polymerase (NEB, MA, USA) according to the ARTIC protocol. For the ARTIC multiplex PCR, thermal cycling was set at 98 • C for 30s and then 35 cycles of 98 • C for 15s and 65 • C for 5 min. After ARTIC multiplex PCR, library preparation was performed following the Oxford Nanopore rapid sequencing kit (SQK-RAD004) manufacturer's instructions and Midnight SARS-CoV-2 genome sequencing protocol. In brief, PCR products of pools 1 and 2 were mixed (10 µl of pool 1 and 10 µl of pool 2) and 7.5 µl of the mixture was used for binding with 2.5 µl of fragmentation mix from an Oxford Nanopore rapid sequencing kit. After incubation at 30 • C for 1 min, 80 • C for 1 min and 4 • C for 30 s, the product was cleaned up by AMPure XP Bead Cleanup (Beckman Coulter, CA, USA) in a 1:1 ratio and eluted with 10 mM Tris-HCl pH 8.0. One microliter of rapid adapter was added, and the mixture was loaded into a flow cell (Oxford Nanopore MinION device) and run under MinKNOW (6) (v19.12.5) software (Baker et al., 2021) . The output reads from the Oxford Nanopore MinION device were filtered using the sequencing summary file under the following parameters: minimum read length ≥ 500 nt and read quality ≥7. The reads that passed the parameters were converted from "Fast5" into "Fastq" format using the GPU version of the Nanopore Guppy basecaller (v3.4.4) tool. Genome assembly was conducted by using the genome detective program (Vilsker et Phylogenetic analysis of SARS-CoV-2 was performed by comparing with nucleotide sequences of 942 genomes (at least 29,000 base pairs in length) isolated from Thailand from January 2020 to May 2021. The genome sequences were selected and downloaded from the GISAID database. The 5′ and 3′ untranslated regions were trimmed with at least 95% reference genome coverage and retained (Wuhan-Hu-1).

The dataset was aligned using the MAFFT FFT-NS-2 algorithm and default parameter settings (Katoh et al., 2002) . A neighbour-joining tree was constructed by using MEGA program v7.0 (Tempe, AZ, USA) with the maximum composite likelihood substitution model and bootstrapping with 1,000 replicates. Lineage classification was performed by using the Pangolin tool (Rambaut et al., 2020) . Genome mutation analysis of SARS-CoV-2 was performed based on variant classifications and definitions (CDC) (CDC, 2021; Kumar et al., 2016) . Genome positions were based on the reference genome sequence of Wuhan-Hu-1 (MN908947).

Descriptive statistics were used to describe demographic information, locations and types of samples from dogs and cats in this study. Frequencies and percentages were used to report SARS-CoV-2 infection in animals and households. Phylogenetic analysis was performed by the neighbour-joining algorithm with the maximum composite likelihood substitution model and bootstrapping with 1,000 replicates by the MEGA program. Figure 1 and (Table S5) (Table 3) .

+ ( 3 5 . 7 1 ) - - - - - - - 19 May 21 - - - - - - - - - - - -

We reported SARS-CoV-2 infection in three dogs and one cat in Thailand. One animal (dog-A) showed illness with mild respiratory signs, but the other animals did not display any clinical symptoms and did not show any important blood chemistry abnormalities (Table S6) . Similar to other studies, the infected dogs and cat showed non-specific and mild respiratory signs such as nasal discharge, sneezing, coughing and inappetence (Calvet et al., 2021; Klaus et al., 2021) . ( Bosco-Lauth et al., 2020; Shi et al., 2020) . It should be noted that our results showed a high viral load (low Ct value) in environmental samples from dog-A during quarantine at the hospital. Thus, contamination of SARS-CoV-2 in the environment and possible transmission from contaminated areas should not be ignored. Contamination of SARS-CoV-2 from animals to the environment, such as the fur and floor, has been reported in some studies (Klaus et al., 2021; Oreshkova et al., 2020 Calvet et al., 2021; Gaudreault et al., 2020; Musso et al., 2020; Newman et al., 2020; Ruiz-Arrondo et al., 2020; Sailleau et al., 2020; Segalés et al., 2020; Sit et al., 2020) .

In this study, the frequency of SARS-CoV-2 positivity in dogs (8.6%), cats (11.1%) and households (23.5%) was lower than that in previous studies in Brazil, China and the United States based on similar diagnostic assays Calvet et al., 2021; Hamer et al., 2020) , but higher than that reported in some countries (Ruiz-Arrondo et al., 2020; Sailleau et al., 2020; Sit et al., 2020) . There was a limitation that only rectal swabs could be collected from some house- (Calvet et al., 2021; Hamer et al., 2020; Shi et al., 2020; Sit et al., 2020) . The dogs and cat in this study tested positive for SARS-CoV-2 RNA from 5 to 30 days after the index COVID-19 owner tested positive, which is comparable to that in studies in Brazil (11 to 51 days), China (28 days) and the United States (32 days) (Calvet et al., 2021; Hamer et al., 2020; Shi et al., 2020; Sit et al., 2020) .

Infected dogs and cats develop antibodies against SARS-CoV-2 as early as 7 to 14 days post infection. The seropositivity of dogs in a previous study varied but was higher than that in cats Hamer et al., 2020; Patterson et al., 2020) . In this study, we used the parameter of the first pet owner positive by real-time PCR as day 1 Hamer et al., 2020; Patterson et al., 2020) . It should be noted that the discrepancy between the result of ELISA and sVNT had been observed. It has been reported that the N-proteinbased ELISA is less correlated with the neutralization assay (Decaro, Grassi et al., 2021; Decaro, Vaccari et al., 2021; Folegatti et al., 2020; Ni et al., 2020; Okba et al., 2020) . Therefore, it was not unexpected that 

Additional supporting information may be found in the online version of the article at the publisher's website.

How to cite this article: Jairak, W., Charoenkul, K., Chamsai, E., Udom, K., Chaiyawong, S., Bunpapong, N., Boonyapisitsopa, S., Tantilertcharoen, R., Techakriengkrai, N., Surachetpong, S., Tangwangvivat, R., Suwannakarn, K., & Amonsin, A. (2021).

Thailand. Transboundary and Emerging Diseases, 1-13.

https://doi.org/10.1111/tbed.14383

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We would like to thank the Chulalongkorn University animal hospi-