Edited by: Maureen T. Long, University of Florida, United States
Reviewed by: Ariful Islam, EcoHealth Alliance, United States; Ellen Sparger, University of California, Davis, United States
This article was submitted to Veterinary Infectious Diseases, a section of the journal Frontiers in Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The emergence of the Omicron variant (B.1. 1.529) has brought with it an increase in the incidence of SARS-CoV-2 disease. However, there is hardly any data on its incidence in companion animals. We have detected the presence of this new variant in domestic animals (dogs and cats) living with infected owners in Spain. None of the RT-qPCR positive animals (10.13%) presented any clinical signs and the viral loads detected were low. In addition, the shedding of viral RNA lasted a short period of time in the positive animals. Infection with this variant of concern (VOC) was confirmed by RT-qPCR and sequencing. These outcomes suggest a lower virulence of this variant in infected cats and dogs. They also demonstrate the transmission from infected humans to domestic animals and highlight the importance of active surveillance as well as genomic research to detect the presence of VOCs or mutations associated with animal hosts.
The pandemic associated with the Corona Virus Disease 2019 (COVID-19), produced by the SARS-CoV-2 virus, and has been active for almost 2 years now. To the date, more than 400 million cases have been confirmed in the world with more than 6 million deaths according to the last World Health Organization (WHO) report (
Due to the increased ability of RNA viruses to accumulate mutations, it has been undergoing changes such as the D614G mutation which has been associated with enhanced infectivity (
On November 26th 2021, a new variant was determined by the WHO as the 5th VOC, named Omicron (B.1.1.529). The first sample identified as this variant was taken in the South Africa's Gauteng province on the 9th of November 2021, while the first sequenced case was from a sample collected in Botswana on the 11th of November 2021 (
In Spain, according to data published by the Ministry of Health, the cumulative incidence of SARS-CoV-2 rose from 77 cases per 100,000 inhabitants on 15th November 2021 to 465 on 15th December 2021, showing a significant increase. It continued growing until reaching 3,418 on January 20th, 2022. This growth in cases coincided with the introduction of the Omicron variant in Spain, around mid-December 2021. As could be expected, sequencing since that time demonstrated the increasing dominance of this VOC in the country. In December 13th 2021, a 5.38% of the samples sequenced in the country belonged to the Omicron variant while in March 7th 2022 it raised to 99.13% (
Until now, the Omicron variant is the VOC with the largest number of mutations detected, with 34 of them accumulated in the spike protein. Several of these mutations in the spike protein have been related to increased viral antibody neutralization evasion capacity or higher affinity between the spike/angiotensin-converting enzyme 2 (ACE2) receptor binding (
Within the Omicron variant, five lineages or subvariants are distinguished so far: BA.1, BA.2, BA.3, BA.4 and BA.5. A total of 18 BA.1 and 27 BA.2 central mutations (frequency >99%) were identified, of which 15 are specific of the variant Omicron. Indeed, BA.2 lineage has 32 mutations shared with BA.1, but 28 mutations distinct from BA.1, and BA.3 spike protein is a combination of BA.1 and BA.2 with no new mutations. BA.2 has been observed to reinfect patients previously infected with BA.1, being more prevalent in Denmark (
Shortly after the SARS-CoV-2 virus entered our lives, field studies on the incidence of this virus in animals, as well as experimental studies, began to be carried out to learn about their role in this new disease (
Despite these results suggesting a lower virulence of this variant in infected animals, very different results were observed in an experimental study in wild carnivores (mink,
Samples from domestic animals including cats (
Total RNA was extracted using the column-based High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. Total RNA was suspended in RNase/DNase-free water and stored at−80°C. The detection of the RNA of SARS-CoV-2 was carried out using a diagnostic RT-qPCR, hereafter “Diagnosis PCR”, based on the detection of the envelope protein (E)-encoding gene (Sarbeco) and two targets (IP2 and IP4) of the RNA-dependent RNA polymerase gene (RdRp) in an RT-qPCR protocol established by the World Health Organization according to the guidelines that can be found at
Absolute quantification was carried out by generating a standard curve. For this purpose, a standard stock was provided by the Pasteur Institute corresponding to a load of 109 copies/μl. Subsequently, serial dilutions were performed and tested in triplicate in a RT-qPCR assay to generate a standard curve with a calculated R2 value of 0.9983 for Sarbeco, 0.9994 for IP2 and 0.9928 for IP4.
A specific RT-qPCR was used for the identification of the SARS-CoV-2 Omicron variant, hereafter “Omicron PCR,” targeting both the envelope protein (E) - encoding gene as well as an Omicron-specific spike insertion-deletion mutation (indel_211-214) found in the B.1.1.529/BA.1 lineage and BA.1.1 sublineage, so in the case of the BA.2 and BA.3 Omicron lineages would only be detected by the gen E target. The kit used was the SuperScript III Platinum One-Step qRT-PCR kit (Invitrogen) according to the protocol described in (
Positive samples for RT-qPCR were subjected to attempts of viral isolation using the previously described methods in (
Whole-genome sequences were obtained from the two positive oropharyngeal swabs samples with the higher viral loads based on copies/μl (2.82 x 103and 1.31 x 104) by both “Diagnosis” and “Omicron” RT-qPCRs, following the protocol described by (
Phylogenetic analysis was performed using MEGA X software (
Virus detection in canine and feline patients.
Cat_2, 20th December, 2021 | Rectal swab | 2 DPI | 9.34 x 102 | 1.76 x 102 | NA | Negative |
Dog_8, 1st January, 2022 | Oropharyngeal swab | 3 DPI | 2.82 x 103 | 1.68 x 102 | B.1.1.529 | Negative |
Cat_7, 16th January, 2022 | Oropharyngeal swab | 2 DPI | 2.06 x 102 | 83.4 | NA | Negative |
Cat_19, 22th January, 2022 | Oropharyngeal swab | 3 DPI | 1.31 x 104 | 2.29 x 103 | B.1.1.529 | Negative |
Cat_13, 27th January, 2022 | Oropharyngeal swab | 4 DPI | 3.63 x 102 | 1.05 x 102 | NA | Negative |
Cat_26, 18th March, 2022 | Oropharyngeal swab | 2 DPI | 7.74 x 102 | 30.01 | NA | Negative |
Cat_27, 18th March, 2022 | Oropharyngeal swab | 3 DPI | 3.86 x 102 | 43,82 | NA | Negative |
Cat_28, 19th March, 2022 | Oropharyngeal swab | 4 DPI | 5.64 x 102 | 92,85 | NA | Negative |
A total of 31 additional representative sequences were used for the analysis, including sequences from cats and dogs, the reference genome from Wuhan, as well as variants of concern such as the B.1.1.7 variant from the United Kingdom, variant B.1.35 from South Africa, variant B.1.617.2 from India, variant B.1.1.248 from Brazil and lineages BA.1 and BA.2 of the B.1.1.529 Omicron variant.
The final alignment involved 35 whole-genome sequences with an average amino acid p-distance (1-amino acid identity) lower than 0.001, which is considered adequate since it is within the acceptance threshold of <0.8 (
An analysis of the mutations present in the obtained sequences was carried out by comparing them with the reference strain of the original variant (Wuhan). This analysis was done in GISAID's CoVsurver mutations App.
An indirect ELISA test based on the receptor-binding domain (RBD) of the virus was performed as a screening test (Raybiotech, Georgia, USA). The ELISA was adapted to each species by using a specific anti-species conjugate. Briefly, coated plates were covered with 100 μL of diluted sera (1/40) in PBS containing 0.05% Tween 20 (PBS-T) and incubated at 37° C for 30 min. The plates were then washed four times, 100 μL of the specific anti-species HRP-conjugated IgG (Jackson Immuno Research Laboratories, Cambridgeshire, UK) diluted 1/18,000 in PBS-T was added, and the solution was incubated at 37° C for 15 min. Four washes later, 100 μl of SureBlue Reserve TMB Microwell Peroxidase Substrate (TMB) (KPL, Gaithersburg, MD, USA) were added, and the plates were incubated in the dark, for 10 min. The reaction was stopped by adding 100 μl of 3M H2SO4 to each well. Absorbance at 450 nm was determined using an Anthos 2001 plate reader (Labtec, Salzburg, Austria). The endpoint cut-off was determined by the analysis of a receiver operating characteristic (ROC) curve based on positive divided by negative (P/N) values. Validation of this ELISA test is extensively described in (
Virus neutralization test (VNT) was used to confirm the presence of neutralizing antibodies against SARS-CoV-2 in all the sera collected.
Briefly, the VNT was performed in duplicate in 96-well-plates by incubating 25 μL of two-fold serially diluted sera with 25 μL of 100 TCID50/ml of SARS-CoV-2. The virus-serum mixture was incubated at 37°C with 5% CO2. At 1-h post-incubation, 200 μL of Vero E6 cell suspension were added to the virus-serum mixtures, and the plates were incubated at 37°C with 5% CO2. The neutralization titers were determined at 3 days post-infection. The titer of a sample was recorded as the reciprocal of the highest serum dilution that provided at least 100% neutralization of the reference virus, as determined by the visualization of cytopathic effect (CPE). In addition, at the end of the period (3 days post-infection), cells were fixed with 6% paraformaldehyde and then stained with crystal violet to observe the cytopathic effect.
SARS-CoV-2 RNA was detected by RT-qPCR in seven cats and one dog by both “Diagnosis PCR” and “Omicron PCR.” This represents 10.13% of the total analyzed animals. All of the positive animals were sampled in Madrid and all their positive samples for RT-qPCR were negative for viral isolation (
None of the animals that were part of this study presented any clinical signs at any time either during the quarantine time of their owners or afterwards.
Sera were collected from 15 animals (1 cat and 14 dogs), including Dog_8 and Cat_13 which were also positive for RT-qPCR (15 and 20 days after RT-qPCR positive result, respectively). However, none of the animals showed antibodies.
Among the 15 serum samples collected including both dogs and cat, none of them presented neutralizing antibodies.
The complete genome sequence of SARS-CoV-2 was obtained from the oropharyngeal swabs from both Dog_8 and Cat_19 (GenBank accession numbers:
Phylogenetic analysis of SARS-CoV-2 of the whole-genome sequences from Dog_8, Cat_19, Owner_1, and Owner_2 (gray circle), which were clustered with the SARS-CoV-2 B.1.1.529 (Omicron) and more specifically with lineage BA.1 (gray square). The lineage BA.2 is indicated with a gray triangle. We appreciatively acknowledge the different laboratories and funders of GISAID for offering these SARS-CoV-2 sequences (
After the construction of the phylogenetic tree, we visually verified that the Omicron lineage detected in this study in the dog and cat sequenced as well as the two owners correspond to BA.1 lineage, which it was the dominant in Spain by that date.
Analysis in the CoVsurver mutations app (GISAID) showed that the sequences presented several mutations having as a reference the hCoV-19/Wuhan/WIV04/2019 sequence. The mutations were 37 in the case of Dog_8 and Cat_19 (
List of mutations displayed in the different regions of the genome of SARS-CoV-2 in the sequence obtained in this study of Dog_8.
NSP3 (ORF1a) | K38R, P985S, V1069I, S1265del, L1266I, A1892T |
NSP4 (ORF1a) | T492I |
NSP5 | P132H |
NSP6 (ORF1a) | L105del, S106del, G107del, I189V |
NSP12 (ORF 1b) | P323L |
NSP14 (ORF 1b) | I42V |
Spike | A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE,G339D, S371L, S373P, S375F,K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F |
E | T9I |
M | D3G, Q19E, A63T |
N | P13L, E31del, R32del, S33del, R203K, G204R |
List of mutations displayed in the different regions of the genome of SARS-CoV-2 in the sequence obtained in this study of Cat_19.
NSP3 (ORF1a) | K38R, P985S, V1069I, S1265del, L1266I, A1892T |
NSP4 (ORF1a) | T492I |
NSP5 | P132H |
NSP6 (ORF1a) | L105del, S106del, G107del, I189V |
NSP12 (ORF 1b) | P323L |
NSP14 (ORF 1b) | I42V |
Spike | A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EP, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K,Q954H, N969K, L981F |
NS3 | T14del, L15del |
E | T9I |
M | D3G, Q19E, A63T |
NS7a | ins45EstopLN |
N | P13L, E31del, R32del, S33del, R203K, G204R |
The SARS-CoV-2 B.1.529 (Omicron) variant, the last VOC detected, is nowadays highly disseminated around the world. Definitively in Spain, epidemiological data from the Omicron-associated wave has shown that the transmission rate of this variant is quite superior to other variants such as Beta or Delta. This fact has promoted the rapid spread of this variant, being dominant since November 2021 (
In this study, we detected the Omicron SARS-CoV-2 variant in companion animals, demonstrating that pets are susceptible to the natural infection with this strain. However, the outcomes of this study revealed a relatively low number of positive animals based on RT-qPCR, given that the study involved an active sampling. In all the cases, owners assured high contact with their pets. In addition, the sampling was done at the best time for the detection of the disease (
Another remarkable difference observed in animals infected with the Omicron variant is that viral isolation was not possible from any sample, due to the low viral load of all positive specimens. The fact that viral isolation was not possible could be due, in part, to the lower fusogenicity of this variant with respect to other variants (
All these results may be related to a higher affinity with the human cellular receptor which has been reported in the case of the Omicron variant compared to other variants (
However, these results contrast with those of an experimental study carried out in mink (
Although so far there have been no publications on the presence of the Omicron variant in pets, it has been detected in wildlife, specifically in white-tailed deer (
From what we have observed in this study, it appears that the Omicron variant is less virulent to pets than the previous variants as well as the original isolate. Although 10.13% of the animals analyzed in this field study tested positive for RT-qPCR, low viral loads were detected and none of the infected animals showed any symptomatology according to their owners. This, together with our results previously obtained on other VOCs in animals (
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/
The animal study was reviewed and approved by Complutense University of Madrid's Ethics Committee for Animal Experiments (Project License 14/2020). Written informed consent was obtained from the owners for the participation of their animals in this study.
SB-A and JS-V designed the study. SB-A and LS-M performed the sampling, veterinary inspection, laboratory analysis, and wrote the initial manuscript. LD and JS-V acquired the funds. LD, MP-S, and JS-V reviewed the manuscript. All authors contributed to the article and approved the submitted version.
This research was funded by the Institute of Health Carlos III (ISCIII) Project-Estudio del potencial impacto del COVID-19 en mascotas y linces (reference: COV20/01385) and the REACT ANTICIPA-UCM (reference PR38/21) funded by the Community of Madrid and the European Union through the ERDF (European Regional Development Fund) as part of the Union's response to the COVID-19 pandemic.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The authors would like to thank Belén Rivera, Rocío Sánchez, and Deborah López for their excellent technical support, as well as all the members of the COVID-VISAVET team. The authors are also grateful to all the Veterinary Clinics and owners who participated in this study, especially Begoña Rodero, María del Carmen Sánchez Bernal, Cristina Jurado, Aleksandra Kosowska, and Estefanía Cadenas-Fernández for their major support. The authors would also like to thank the Pasteur Institute for sending us the standard stock for absolute quantification.
The Supplementary Material for this article can be found online at: