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SARS-CoV-2 transmission from infected owner to household dogs and cats is associated with food sharing

Open AccessPublished:May 25, 2022DOI:https://doi.org/10.1016/j.ijid.2022.05.049

      Highlights

      • A high rate of anthropogenic transmission to dogs and cats was found.
      • SARS-CoV-2 infection of pets was confirmed by whole genome sequencing.
      • Food sharing with owner is a risk factor for SARS-CoV-2 transmission.

      Abstract

      Objectives

      Several cases of reverse transmission of SARS-CoV-2 from human to pets were reported during the first year of the COVID-19 pandemic. Accordingly, the World Organization for Animal Health has recommended to improve SARS-CoV-2 surveillance on household animals to assess the risk of transmission between species. After such recommendation, we studied the potential SARS-CoV-2 infection in household dogs and cats in the city of Guayaquil, the most populated city in Ecuador.

      Methods

      Oral and nasal swab samples were collected from dogs and cats within 10 days of a positive SARS-CoV-2 test result of their owners. Total ribonucleic acid was extracted and detection of viral gene targets N and ORF1ab was performed by quantitative reverse transcription polymerase chain reaction.

      Results

      From the 50 cats and dogs tested, 12 were SARS-CoV-2 positive, giving a total positivity rate of 24%. A total of 1 of 8 cats tested positive, whereas 11 of 42 dogs were positive, yielding a positivity rate of 12.5% and 26.2%, respectively. SARS-CoV-2 was confirmed by whole genome sequencing. In addition, we also found a statistically significant association between SARS-CoV-2 pet positivity and food sharing with infected owners.

      Conclusion

      This study is the second active surveillance of SARS-CoV-2 in household dogs and cats in Latin America. Moreover, it is the first study to address the risk factors associated with potential anthropogenic SARS-CoV-2 transmission to domestic cats and dogs. Given the high presence of free-roaming dogs and cats in rural and urban areas in Latin American countries and the high capacity shown by coronaviruses for interspecies transmission, our findings support the view that SARS-CoV-2 surveillance in pets is necessary to better understand the role that pet-human interaction plays in the COVID-19 spread.

      Keywords

      Introduction

      The rise of SARS-CoV-2 in the Chinese province of Hubei in December 2019 lead to the SARS-CoV-2 outbreak that prompted the World Health Organization to declare a pandemic on March 11, 2020 (
      • Gorbalenya AE
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      ;
      • Zhou P
      • Yang X-L
      • Wang X-G
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      • Zhang L
      • Zhang W
      • et al.
      A pneumonia outbreak associated with a new coronavirus of probable bat origin.
      ;
      • Ortiz-Prado E
      • Simbaña-Rivera K
      • Gómez-Barreno L
      • Rubio-Neira M
      • Guaman LP
      • Kyriakidis NC
      • et al.
      Clinical, molecular and epidemiological characterization of the SARS-CoV2 virus and the coronavirus disease 2019 (COVID-19), a comprehensive literature review.
      ). By the end of March 2022, more than 470 million cases and 6 million deaths have been reported worldwide (https://coronavirus.jhu.edu/map.html). The Americas is one of the most affected regions, with millions of cases and deaths. In Ecuador, more than 700,000 cases and 32,000 deaths were reported by March 2022 (https://www.salud.gob.ec/actualizacion-de-casos-de-coronavirus-en-ecuador/).
      SARS-CoV-2 is a zoonotic virus whose origins and intermediate hosts have been associated with bats and pangolins (
      • Tiwari R
      • Dhama K
      • Sharun K
      • Iqbal Yatoo M
      • Malik YS
      • Singh R
      • Michalak I
      • Sah R
      • Bonilla-Aldana DK
      • Rodriguez-Morales AJ
      COVID-19: animals, veterinary and zoonotic links.
      ). Furthermore, few months after the COVID-19 pandemic outbreak, the reverse transmission of SARS-CoV-2 from humans to domestic and wild animals have been reported worldwide (

      World Organisation for Animal Health (OIE), Infection with SARS-Cov-2 in Animals. https://www.oie.int/fileadmin/Home/MM/A_Factsheet_SARS-CoV-2__1_.pdf, 2020 (accessed 05/10/2022).

      ;
      • Hobbs EC
      • Reid TJ.
      Animals and SARS-CoV-2: species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission.
      ;
      • Mahdy MAA
      • Younis W
      • Ewaida Z.
      An overview of SARS-CoV-2 and animal infection.
      ) According to the World Organization for Animal Health (OIE), more than 20 countries have reported natural infections of SARS-CoV-2 in domestic animals (

      World Organisation for Animal Health (OIE), Infection with SARS-Cov-2 in Animals. https://www.oie.int/fileadmin/Home/MM/A_Factsheet_SARS-CoV-2__1_.pdf, 2020 (accessed 05/10/2022).

      ). In this scenario, the OIE recommends implementing active epidemiologic surveillance and report of SARS-CoV-2 infections in other animal species, especially for those in close contact with humans, such as pets (

      World Organisation for Animal Health (OIE), Infection with SARS-Cov-2 in Animals. https://www.oie.int/fileadmin/Home/MM/A_Factsheet_SARS-CoV-2__1_.pdf, 2020 (accessed 05/10/2022).

      ;
      • Hobbs EC
      • Reid TJ.
      Animals and SARS-CoV-2: species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission.
      . Although both in silico and experimental studies have revealed that SARS-CoV-2 transmission could be less frequent in canines than in felines (
      • Martínez-Hernández F
      • Isaak-Delgado AB
      • Alfonso-Toledo JA
      • Muñoz-García CI
      • Villalobos G
      • Aréchiga-Ceballos N
      • Rendón-Franco E
      Assessing the SARS-CoV-2 threat to wildlife: potential risk to a broad range of mammals.
      ;
      • Shi J
      • et al.
      Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS- coronavirus 2.
      ), naturally-infected household cats and dogs have been reported worldwide, with similar prevalence values, ranging from 0-17.65% for cats and 0-13.33% for dogs (

      World Organisation for Animal Health (OIE), Infection with SARS-Cov-2 in Animals. https://www.oie.int/fileadmin/Home/MM/A_Factsheet_SARS-CoV-2__1_.pdf, 2020 (accessed 05/10/2022).

      ;
      • Hobbs EC
      • Reid TJ.
      Animals and SARS-CoV-2: species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission.
      ;
      • Shi J
      • et al.
      Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS- coronavirus 2.
      ;
      • Barua S
      • Hoque M
      • Adekanmbi F
      • Kelly P
      • Jenkins-Moore M
      • Torchetti MK
      • Chenoweth K
      • Wood T
      • Wang C.
      Antibodies to SARS-CoV-2 in dogs and cats.
      ;
      • Sánchez-Montes S.
      • et al.
      No molecular evidence of SARS-CoV-2 infection in companion animals from Veracruz, Mexico.
      ;
      • Mahdy MAA
      • Younis W
      • Ewaida Z.
      An overview of SARS-CoV-2 and animal infection.
      ) Although most of these cases are asymptomatic, the presence of respiratory symptoms has been previously described (Garigliany et al., 2021;
      • Barrs VR
      • et al.
      SARS-CoV-2 in quarantined domestic cats from COVID-19 households or close contacts, Hong Kong, China.
      ).
      Most of the current reports of SARS-CoV-2 infection in household cats and dogs come from high-income countries (

      World Organisation for Animal Health (OIE), Infection with SARS-Cov-2 in Animals. https://www.oie.int/fileadmin/Home/MM/A_Factsheet_SARS-CoV-2__1_.pdf, 2020 (accessed 05/10/2022).

      ;
      • Hobbs EC
      • Reid TJ.
      Animals and SARS-CoV-2: species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission.
      ;
      • Mahdy MAA
      • Younis W
      • Ewaida Z.
      An overview of SARS-CoV-2 and animal infection.
      ) However, as free-roaming dogs are more frequent in middle- and low-income countries, the potential role of pets on SARS-CoV-2 transmission may represent a major public health threat (

      Orlando NA et al., Determinación de perros que deambulan en las ciudades de Guayaquil y Quito. https://www.investigacionsalud.gob.ec/determinacion-de-perros-que-deambulan-en-las-ciudades-de-guayaquil-y-quito/, 2019 (accessed 05/10/2022).

      ; Charlotte et al., 2021). For instance, the free-roaming dog population in Guayaquil (the most populated city of Ecuador), is greater than 30,000 (

      Orlando NA et al., Determinación de perros que deambulan en las ciudades de Guayaquil y Quito. https://www.investigacionsalud.gob.ec/determinacion-de-perros-que-deambulan-en-las-ciudades-de-guayaquil-y-quito/, 2019 (accessed 05/10/2022).

      ). However, SARS-CoV-2 surveillance studies in cats and dogs in low- and middle-income countries like those in Latin America are still scarce, and only a few reports from Chile, Argentina, and Mexico have been published to date (
      • Neira V
      • et al.
      A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.
      ;
      • Pecora A
      • et al.
      Anthropogenic infection of domestic cats with SARS-CoV-2 alpha variant B.1.1.7 lineage in Buenos Aires.
      ).
      The aim of this work was to study the SARS-CoV-2 infection rate in household cats and dogs owned by patients with COVID-19 in the city of Guayaquil, in the coastal region of Ecuador, where COVID-19 community transmission has been present since the initial outbreak in February 2020.

      Material and methods

      Study design

      A cross-sectional surveillance study was carried out in the city of Guayaquil, a province of Guayas in the coastal region of Ecuador, which included 50 pets (42 dogs and 8 cats). A prospective sampling was carried out in cats and dogs owned by individuals who were diagnosed with COVID-19 and confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) testing within 10 days before pet sample collection. In addition, a risk factor survey was performed for each household included in the study. The sample collection was carried out during the months of March to May 2021. Households with one or more pets were included in the study and all the pets in each household were tested.
      The variables included for the risk factors survey were sex and species of pet, and the following binary variables with yes or no as answers were included: bed sharing between pet and owner, food sharing with pet, pet COVID-19–related symptoms, pet access to trashed food, isolation of the pet from the owner after COVID-19 diagnosis, presence of other animals in the household, and pet-free access to the street.

      Sample collection and RNA extraction

      Household animals were physically restrained for nasal and oral swab sample collection in 1 ml of saline. The samples were preserved in a cold chain until processing in Instituto Nacional de Investigación y Salud Publica laboratory in Guayaquil (Ecuador). The viral ribonucleic acid (RNA) extraction was carried out using a column-based commercial kit from Biomiga Inc. (San Diego, California, USA), following manufacturer's protocol and as has been previously done in our laboratory (
      • Freire-Paspuel B
      • Garcia-Bereguiain MA.
      Poor sensitivity of AccuPower SARS-CoV-2 Real Time RT-PCR kit (Bioneer, South Korea).
      ;
      • Freire-Paspuel B
      • Garcia-Bereguiain MA.
      Analytical and clinical evaluation of "AccuPower SARS-CoV-2 Multiplex RT-PCR kit (Bioneer, South Korea)" and "Allplex 2019-nCoV Assay (Seegene, South Korea)" for SARS-CoV2 RT-PCR diagnosis: Korean CDC EUA as a quality control proxy for developing countries.
      ;
      • Santander-Gordon D
      • Iturralde GA
      • Freire-Paspuel B
      • Zambrano-Mila MS
      • Morales-Jadan D
      • Vallejo-Janeta PA
      • et al.
      The crucial contribution of the universities on the SARS-CoV-2 surveillance in Ecuador: lessons for developing countries.
      ).

      SARS-CoV-2 detection by RT-qPCR

      RNA extracted samples were subjected to RT-qPCR for SARS-CoV-2 detection using the United States Food and Drug Administration emergency use-authorized certified commercial kits, 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech, China), including two viral gene targets: N and ORF1ab. The RT-qPCR reactions were performed, following the manufacturer's instructions. For RNA quality control, human RNAaseP amplification was considered valid as a proxy for extraction quality control, and spectrophotometric measurement of RNA extraction quality was also done.

      Whole genome sequencing

      Four of the SARS-CoV-2 RT-qPCR positive samples were submitted to whole-genome sequencing in a MinION device (Oxford Nanopore Technologies), following the ARTIC V2 protocol (

      Artic Network, SARS-CoV-2. https://artic.network/ncov-2019, 2021 (accessed 05/10/2022).

      ). The genomes were processed using the Artic bioinformatics pipeline and the reads aligned using the Burrows-Wheeler Aligner tool (
      • Li H
      • Durbin R.
      Fast and accurate long-read alignment with Burrows Wheeler Transform.
      ). Genomes annotated with this tool were downloaded and aligned in BioEdit (
      • Hall T
      • Carlsbad CA.
      BioEdit: an important software for molecular biology.
      ) with SARS-CoV-2 as the reference genome sequenced early in the pandemic in Wuhan, “isolate Wuhan-Hu-1” GenBank ID: NC_045512.3. Genome alignment using CLUSTAL-W and phylogenetic analysis using maximum likelihood method with a GTR substitution model and 1000 bootstrap iterations was conducted using IQ-tree 2.2.0 (
      • Minh BQ
      • Schmidt HA
      • Chernomor O
      • Schrempf D
      • Woodhams MD
      • von Haeseler A
      • Lanfear R.
      IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era.
      ) to attempt identifying the SARS-CoV-2 lineages infecting dogs that tested positive.

      Statistical analysis

      Inferential analysis was performed using RStudio statistical software. Significant difference between the presence of SARS-CoV-2 and risk factors were determined using chi-square analysis, whereas significant factors in the presence of SARS-CoV-2 were determined using a logistic regression model.

      Results

      A total of 50 samples from household animals were collected at the city of Guayaquil, including 42 samples from dogs and 8 samples from cats. Regarding sex, 24 samples were collected from male pets and 26 from female pets. The ages of the sampled animals ranged from 4 months to 16 years. Eleven samples from dogs (11/42 = 26.2%) and one sample from cats (1/8 = 12.5%) were positive for N1 and/or ORF1ab SARS-CoV-2 viral genes, with cycle threshold (Ct) values ranging from 30.11-39.36 (see Table 1). Overall, 12 pets of the 50 tested were positive for SARS-CoV-2 as assessed by RT-qPCR, yielding an overall SARS-CoV-2 infection rate of 24%.
      Table 1Ct values for the viral gene targets for pets who were SARS-CoV-2 RT-qPCR-positive included in the study.
      Sample codeSpeciesSex:Ct values
      N viral targetORF1ab viral target
      1DF8dogF30,1132,04
      2DF10dogM35,4739,36
      3DF12dogF37,2136,52
      4DF17dogM34,3036,25
      5DF26catF36,53NA
      6DF33dogM37,07NA
      7DF37dogM33,5738,11
      8DF39dogM37,26NA
      9DF41dogM32,4635,10
      10DF42dogM37,95NA
      11DF44dogF30,8633,51
      12DF45dogM31,7635,08
      Ct: cycle threshold; F: female; M: male; NA: not amplified.
      The bivariant and multivariant analysis for the risk factor included in our survey is detailed in Table 2. For the bivariant analysis, male pets were statistically associated with a higher risk of infection (P = 0.027). For the multivariant analysis, both being male and sharing food with the owner were statistically associated with SARS-CoV-2 infection, with a regression coefficient of 0.26 and P-values of 0.029 and 0.025, respectively.
      Table 2Bi- and multivariate analysis of association of risk factor in relation to SARS-CoV-2 infection in pets (42 dogs and 8 cats) included in the study.
      Risk factorSARS-CoV-2 (+)SARS-CoV-2 (-)Bivariate analysisMultivariate analysis
      OR (IC 95%)P-valueOR (IC 95%)P-value
      SpecieDog11311.92 (0.06-60.59)0.712.07 (0.09-46.8)0.79
      Cat17
      SexFemale42214.46 (0.92-228.2)0.027
      statistically significant association. IC: confidence interval; OR: odds ratio.
      14.46 (0.92-228.2)0.029
      statistically significant association. IC: confidence interval; OR: odds ratio.
      Male816
      Bed sharing with ownerYes9212.02 (0.23-17.81)0.522.3(0.27-19.6)0.37
      No317
      Food sharing with ownerYes8202.62 (0.04-151.9)0.616.17 (0.22-167.4)0.025
      statistically significant association. IC: confidence interval; OR: odds ratio.
      No418
      SymptomsYes4102.19 (0.18-27.1)0.540.89(0.10-7.4)0.91
      No828
      Trash accessYes3107.33 (0.13-428.5)0.332.08(0.06-69.2)0.84
      No928
      Pet isolationYes11341.22 (0.13-12.83)0.320.44(0.00-13.7)0.82
      No14
      Other petsYes7290.22 (0.01-9.51)0.430.07(0.00-1.9)0.08
      No59
      Free street accessYes180.05 (0-6.65)0.210.13(0.00-8.4)0.61
      No1130
      a statistically significant association.IC: confidence interval; OR: odds ratio.
      Whole genome sequencing from four positive samples confirmed SARS-CoV-2 infection in the pets. Three sequences have enough quality and were submitted to GenBank: accession numbers MZ322504, MZ322502, and MZ322409. The genome coverage obtained ranged between 45% and 85%. Sequences were aligned and visualized in BioEdit, showing 98-100% similarity to the viral reference genome isolated in Wuhan early in the pandemic. Alignment analysis conducted with the online tool Basic Local Alignment Search Tool yielded a SARS-CoV-2 identity up to 99.58%. The phylogenetic analysis with the sequences included, clustered all sequences with the early lineages of SARS-CoV-2 circulating in Ecuador during 2020-2021: two into the cluster containing lineage 19 A and 19 B and one with lineage Iota (Figure 1).
      Figure 1
      Figure 1Phylogenetic placement of SARS-CoV-2 sequences obtained from RT-qPCR positive dogs in Guayaquil (Ecuador) in comparison with selected genomes of different viral lineages detected in human samples in Ecuador from 2020-2021. The tree was obtained using maximum likelihood method with a GTR substitution model and 1000 bootstrap iterations was conducted using IQ-tree 2.2.0. Dog samples are presented in red with GenBank accession number. Lineage name and GISAID accession number is presented for all other sequences.GTR: general time reversible; RT-qPCR: quantitative reverse transcription polymerase chain reaction.

      Discussion

      This study is the second active surveillance of SARS-CoV-2 infection in populations of household dogs and cats in Latin America, and the first one to report dogs who were SARS-CoV-2 positive . The other surveillance study involving cats and dogs in Latin America was carried out in Mexico, and no infected animals were found (
      • Sánchez-Montes S.
      • et al.
      No molecular evidence of SARS-CoV-2 infection in companion animals from Veracruz, Mexico.
      ). In addition, two case reports of transmission from human to cats in households in Chile and Argentina have recently been published (
      • Neira V
      • et al.
      A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.
      ;
      • Pecora A
      • et al.
      Anthropogenic infection of domestic cats with SARS-CoV-2 alpha variant B.1.1.7 lineage in Buenos Aires.
      ).
      A SARS-CoV-2 infection rate of 24% was found by RT-qPCR. Moreover, the infection rate was higher in dogs (26.2%) than in cats (12.5%), although this result should be taken with caution because potential bias due to the small sample size for cats cannot be ruled out. In our study, the samples from pets were collected within 10 days of the pet's owner positive RT-qPCR test for SARS-CoV-2. Previous reports in household cats have shown RT-qPCR SARS-CoV-2 detection in nasal and rectal samples that were collected within 2 weeks of their owners’ COVID-19 diagnosis (
      • Barrs VR
      • et al.
      SARS-CoV-2 in quarantined domestic cats from COVID-19 households or close contacts, Hong Kong, China.
      ;
      • Garigliany M
      • et al.
      SARS-CoV-2 natural transmission from human to cat, Belgium, March 2020.
      ). Two additional surveillance studies including cats and dogs in Italy and Spain detected no RT-qPCR SARS-Co-2–positive results in dogs and only a single positive result in cats (
      • Patterson E.
      • Elia G
      • Grassi A
      • Giordano A
      • Desario C
      • Medardo M
      • Smith SL
      • Anderson ER
      • Prince T
      • Patterson GT
      • Lorusso E
      • Lucente MS
      • Lanave G
      • Lauzi S
      • Bonfanti U
      • Stranieri A
      • Martella V
      • Solari Basano F
      • Barrs VR
      • Radford AD
      • Decaro N
      Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.
      ,
      • Ruiz-Arrondo I
      • Portillo A
      • Palomar AM
      • Santibáñez S
      • Santibáñez P
      • Cervera C
      • Oteo JA.
      Detection of SARS-CoV-2 in pets living with COVID-19 owners diagnosed during the COVID-19 lockdown in Spain: a case of an asymptomatic cat with SARS-CoV-2 in Europe.
      ). However, in these studies, the time window used to collect samples after the owners’ positive COVID-19 result was longer. Different time frames between the pet owners COVID-19 diagnosis and the pet sample collection could explain the discrepancy among the available reports. To date, the effective time window for SARS-CoV-2 detection by RT-qPCR in pets have not been assessed, and negative results should be taken with caution.
      Finally, those reports in which SARS-CoV-2 has been detected in pets show that close contact with an infected owner poses a risk to pets, although no risk factor analysis has been carried out yet (
      • Barrs VR
      • et al.
      SARS-CoV-2 in quarantined domestic cats from COVID-19 households or close contacts, Hong Kong, China.
      ;
      • Garigliany M
      • et al.
      SARS-CoV-2 natural transmission from human to cat, Belgium, March 2020.
      ;
      • Patterson E.
      • Elia G
      • Grassi A
      • Giordano A
      • Desario C
      • Medardo M
      • Smith SL
      • Anderson ER
      • Prince T
      • Patterson GT
      • Lorusso E
      • Lucente MS
      • Lanave G
      • Lauzi S
      • Bonfanti U
      • Stranieri A
      • Martella V
      • Solari Basano F
      • Barrs VR
      • Radford AD
      • Decaro N
      Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.
      ;
      • Ruiz-Arrondo I
      • Portillo A
      • Palomar AM
      • Santibáñez S
      • Santibáñez P
      • Cervera C
      • Oteo JA.
      Detection of SARS-CoV-2 in pets living with COVID-19 owners diagnosed during the COVID-19 lockdown in Spain: a case of an asymptomatic cat with SARS-CoV-2 in Europe.
      ). To the best of our knowledge, our study is the first to address a risk factor analysis for SARS-CoV-2 transmission from human to domestic cats and dogs. We found that food sharing between infected owners and their pets was associated with SARS-CoV-2 infection. A limitation in our study was that this risk factor association was based on a survey done retrospectively and no samples from food were taken at the time of pet sample collection. Nevertheless, this result would suggest a potential important role of fomites for human-to-pet SARS-CoV-2 transmission in addition to human-to-human transmission that has been mainly associated with aerosols (
      • Onakpoya IJ
      • Heneghan CJ
      • Spencer EA
      • et al.
      SARS-CoV-2 and the role of fomite transmission: a systematic review.
      ).
      A strong limitation in our study is lack of information regarding SARS-CoV-2 variants infecting cat and dog owners. Our phylogenetic results also need to be interpreted with caution because phylogenies depend on the quality of sequences and the Ct values found in dogs were not optimal for getting better genome coverage, particularly of the Spike gen. Taking this into account, we inferred two dogs that tested positive were infected with early lineages of SARS-CoV-2 and one dog with the Iota variant.
      • Shi J
      • et al.
      Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS- coronavirus 2.
      showed that early SARS-CoV-2 lineages are able to infect dogs in a study conducted in Hong Kong, whereas
      • Miró G
      • Regidor-Cerrillo J
      • Checa R
      • Diezma-Díaz C
      • Montoya A
      • García-Cantalejo J
      • et al.
      SARS-CoV-2 infection in one cat and three dogs living in COVID-19-positive households in Madrid.
      demonstrated a dog's infection with the Alpha variant in a survey in Spain. Unfortunately, we did not have the institutional review board approval to obtain SARS-CoV-2 samples from pet owners by the time this study was done, although a positive RT-qPCR result for the owner done at a Ministry of Health–certified laboratory was mandatory as part of the inclusion criteria. Nevertheless, the pets included in this surveillance were permanently staying within the household during the time of the owners’ isolation due to COVID-19 diagnosis and also close exposure to owners due to food sharing was a risk factor. Therefore, although we cannot totally rule out other sources of infection for the SARS-CoV-2–positive pets because no sequences from the owners were available, we believe that anthropogenic infection of the pets is the most plausible explanation for our results.
      Overall, our results support the need of SARS-CoV-2 surveillance studies in companion animals. Also further research with One Health approach is needed to understand SARS-CoV-2 transmission dynamics in pets in different socioeconomic scenarios, such as in middle- and low-income countries, where household crowding is usually bigger than in high-income countries, and practices like sharing human food leftovers with pets may be more usual.

      Declaration of Competing Interest

      The authors have no competing interests to declare.

      Authors’ contributions

      - All authors contributed toward sample collection and data analysis.
      - MAGB and AO composed the manuscript.
      - All authors read and approved the final manuscript.

      Acknowledgments

      We thank the pet owners, Dr Javier Bravo, and Dr Paulina Bravo for their involvement in this process. We also express our gratitude to the Universidad de Las Américas (Quito, Ecuador) for partially funding this project. We thanks Centro Nacional de Referencia en Genmica from Instituto Nacional de Salud Publica e Investigacion Leopoldo Izquieta Perez for their support in SARS-CoV-2 sequencing.

      Ethical approval

      This study was approved by the Scientific Board of the Instituto Nacional de Salud Pública en Investigación. Nevertheless, according to Ecuadorian regulations, infectious disease diagnosis and surveillance in domestic animals do not require institutional review board approval. All the pet owners signed an informed consent.

      Data availability statement

      The data supporting the findings of this study are available from the corresponding author upon request.

      Appendix. Supplementary materials

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