Coronavirus disease 2019 (COVID-19) emerged in the Huanan Seafood Wholesale Market in Wuhan, China, in December 2019. ¹ Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causal agent of this disease, which was declared as pandemic by the World Health Organisation (WHO) on 11 ^th March 2020. ² The zoonotic origin of COVID-19 has been evidenced thanks to the high genomic similarity of SARS-CoV-2 with coronaviruses (CoV) recovered from bats ³ and pangolins, the latter considered potential intermediate hosts of the virus. ⁴ Betacoronaviruses such as SARS-CoV-2 belong to the family Coronaviridae. They exhibit linear single-stranded RNA of positive polarity ⁵ and cause respiratory and gastrointestinal diseases in mammals. ⁶ Even though humans are the most frequent route of transmission, it has been reported that cats ( Felis catus) and dogs ( Canis lupus familiaris) are susceptible to SARS-CoV-2 infection. ⁷ This way, reverse zoonosis (anthropozoonosis) is viable through close contact with owners during acute infections. ⁸

Since the beginning of the SARS-CoV-2 outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the virus transmission between species. ⁹ This is the reason why the determination of these hosts has intensified, evidencing a number of reports involving wild, zoo, farm and pet animals. ¹⁰ The zoonotic nature from which the transmission hypothesis has started, determines the importance of investigating animal species considered natural reservoirs of SARS-CoV-2. ¹¹ However, concern for the control and reduction of the spread of the virus has led to more vigorous investigation of the role that pet animals, such as dogs and cats, play in the spread of the disease. Although a cat-to-human transmission case was reported, ¹² it has been clearly defined that domestic canines and felines do not play a relevant role in the virus transmission to humans. ^{8 , 13}

On the other hand, human-animal transmission has been widely reported. ^{14 , 15} This fact generates the need to investigate the implications for public and animal health, taking into account that animals are an epidemiological part of this pandemic. ¹⁶ Various epidemiological and experimental studies, through serological detection of antibodies against SARS-CoV-2, neutralising antibodies, and detection of viral genome by reverse transcriptase polymerase chain reaction (RT-qPCR), have confirmed SARS-CoV-2 in pet animals around the world. ¹⁷ Likewise, the occurrence of emerging variants has been described, as well as their influence on animals, ¹⁸ for example, the Alpha variant (B.1.1.7) in dogs and cats with clinical signs of myocarditis, ¹⁹ and the Delta variant (B.1.617.2) in dogs with clinical digestive and respiratory symptoms. ²⁰ Regarding the Omicron variant (B.1.1.529), concluded that the SARS-CoV-2 virus accumulated mutations within host cells in mice, giving rise to the Omicron variant that was transmitted to humans, indicating a ‘ ping-pong’ (spillover and spillback) evolutionary trajectory between species. ²¹ Transmission of SARS-CoV-2 Delta variant (AY.127) from hamsters to humans ²² and animal-to-human transmission of SARS-CoV-2 within mink farms ²³ have also been reported.

Susceptibility to SARS-CoV-2 is determined by the affinity between the receptor-binding domain (RBD) of the viral spike (S) glycoprotein and the angiotensin-converting enzyme 2 (ACE2) of the host cell. Therefore, since vertebrates have conserved domains of ACE2, transmission of the virus between species becomes possible. ²⁴ Canines have lower susceptibility to SARS-CoV-2 infection in contrast to felines ^{8 , 25} that exhibit greater respiratory pathology and efficient transmission of the virus to other felines through aerosols. ²⁶

In the context of the rapid evolutionary trajectory between species that SARS-CoV-2 has been developing, and taking into account the Report No. 13 of the World Organisation for Animal Health (OIE) of 31 ^st May 2022, which reported 676 outbreaks in animals affecting 23 different species in 35 countries, ²⁷ the need for seroepidemiological monitoring in pet, wild and synanthropic animals becomes essential in order to broadly understand the adaptation, evolution and transmission of SARS-CoV-2. ⁷

In Colombia, in December 2021, a lion exhibited symptoms of infection days after being in contact with a COVID-19 positive keeper. ²⁸ However, seroepidemiological studies of exposure to SARS-CoV-2 by pet animals have not been reported to date in the country. Since the dissemination of SARS-CoV-2 in dogs and cats is weak and short-lived, anti-SARS-COV-2 antibody detection studies are the best choice to determine the circulation of this virus in these companion animals. Of course, the indirect ELISA screening tests that detect immunoglobulin anti-RBD S1 SARS-CoV-2 are called for their greater accuracy in diagnosis. ²⁹ The goal of the present study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (Ig) class G (IgG) in domestic dogs and cats and its epidemiological association with the frequency of COVID-19 patients in Villavicencio city, Colombia.

The overall seroprevalence of anti-SARS-CoV-2 IgG was 4.60% (95% CI = 3.2-7.4). ⁵⁷ Specifically, in canines the results indicated 3.67% (95% CI = 2.1-6.4), and in felines 6.67% (95% CI = 3.6-12.18) ( Table 2). Twenty seropositive individuals (11 canines and 9 domestic felines) were detected through the enzyme immunoassay. In general, 22 animals with a history of respiratory signs (e.g., cough, runny nose, among others) were detected, of which 9.10% (95% CI = 2.53-27.81) were seropositive for SARS-CoV-2 ( Table 3). Additionally, seven immunoassay results were classified as doubtful (gray area). Likewise, all 25 canine pre-pandemic sera were negative ( Figure 2).

Regarding the eight communes, there was a general seroprevalence of 0, 13.63%, 6.67%, 8.06%, 1.02%, 4.76%, 4.82% and 2.5%, respectively ( Table 2). In the map obtained through Kernel density analysis ( Figure 3), it is observed that the density of cases was concentrated mainly in the west of the city. Communes with higher densities of SARS-CoV-2 seropositive animals were 2 and 4 in comparison to COVID-19 cases in humans, with a greater number of positive cases in communes 5, 7 and 8. Other visible sites of concentrations of seropositive animals, though with lower density, corresponded to communes 7 and 8. Finally, communes 1, 3, 5 and 6 had densities ranging from low to zero.

Regarding the SARS-CoV-2 exposure and the risk factors analysed, a statistically significant association between SARS-CoV-2 seropositivity and Commune 2 was found (adjusted OR = 5.84; CI 95% = 1.1-30.88). On the other hand, no significant statistical association was found ( p >0.05) between anti-SARS-CoV-2 IgG seropositivity and the other items assessed ( Table 2). Additionally, among the twenty seropositive animals, only one owner spontaneously confirmed to have positive RT-qPCR result for COVID-19.

Additionally, a Spearman correlation of p = 0.32 was found between the ratio of COVID-19 positive cases (RT-qPCR testing) of humans in November 2020 and domestic dogs and cats from the eight Villavicencio communes, result classified as a positive weak correlation. Finally, a strong positive correlation of 0.81 was found between the feline species and their SARS-CoV-2 seropositivity, as well as a positive correlation of 0.68 between the canine species and their SARS-CoV-2 seropositivity ( Table 4). Table 4.

	Humanos	Caninos	Felinos	Mascotas
Caninos	0.01	1	--	--
Felinos	0.33	0.17	1
Mascotas	0.32	0.68	0.81 *	1

*

p-value = 0.01467.

In the present study, the seroprevalence of SARS-CoV-2 in canines was 3.67% (11/300) and in felines 6.67% (9/135). Felines had more risk of becoming infected with SARS-CoV-2 that dog (adjusted OR = 2.07; 95% CI = 0.78-5.46) ( Table 2) this tendency was no statistically different. In similar studies, Barroso et al. ⁷ found SARS-CoV-2 seroprevalence of 4.7% in dogs and 21.7% in cats in Portugal, determining that, among seropositive animals, 50% had been possibly infected by human-animal transmission. On the other hand, 33.3% of seropositive cats had possibly been infected via the cat-cat route. Colitti et al. ³⁸ found a SARS-CoV-2 seroprevalence of 2.3% in dogs and 16.2% in cats in Italy, and Fritz et al. ¹³ found a SARS-CoV-2 seroprevalence of 15.4% in dogs and 23.5% in cats from France. In all the studies mentioned, SARS-CoV-2 prevalence was higher in cats and its transmission was mostly related to exposure to humans when they were more seropositive and more susceptible to infection. ^{27 , 38 , 39}

As a result of the present study, a positive relationship between seropositivity and the age of the animals was observed.The older animals between 11 and 15 years exhibited this tendency predisposition, but it was not statistically different (adjusted OR = 1.61; 95% CI = 0.16-15.93) ( Table 2). In this sense, a significant trend was found in the fatality and mortality rates of COVID-19 with advanced age in humans, ⁴⁰ given that there is a weakened immune system, underlying chronic diseases, multiple drug therapies, lack of attention and self-care, poor environmental hygiene, loneliness, and lack of adequate support from other family members in this population. ⁴¹ These reasons could be considered with equal value in the case of animals, especially pet animals. ⁴² On the other hand, Shi et al. ²⁵ reported that three-month-old canines exhibited low susceptibility to experimental infection, contrary to the results obtained in cats, since animals aged less than 100 days and up to nine months were highly susceptible to SARS-CoV-2 infection.

In the present study, no significant differences were found for respiratory and digestive symptoms of the animals sampled according to their SARS-CoV-2 seropositivity ( X ² = 0.8206; p = 0.365) ( Table 3). These results are similar to those reported by Pagani et al. ⁴³ and Shi et al. ²⁵ i.e., cats infected with SARS-CoV-2 were asymptomatic or highly susceptible to subclinical infections. Contrarily, in Germany, Keller et al. ⁴⁴ reported animals with mainly respiratory symptoms, describing the case of a cat with unresolved pneumonia, which was associated to the owner positive test for COVID-19. SARS-CoV-2-specific nucleic acid analysis was performed, revealing the complete genome and the presence of infection in that patient.

In both canines and felines, the highest seropositivity occurred in Commune 2 (13.63% [6/44]) ( Table 2), which is located in the southwest of Villavicencio. Despite the fact that it is a commune with a low population (19,491 inhabitants), ⁴⁵ it has been reported with the highest number of inhabitants per house (6 inhabitants) in comparison to the other communes, ⁴⁶ suggesting that having more than one individual infected with SARS-CoV-2 in the same household increased the risk of infection in these pet animals. ³⁸ Likewise, this commune presented a positive association between the seropositivity of the animals sampled (adjusted OR = 5.84; 95% CI = 1.1-30.88) ( Table 2) and the seropositivity of the owners, similarly, Colitti et al. ³⁸ found a positive association between COVID-19 positive owners and their felines’ SARS-CoV-2 seropositivity (OR = 2.5; 95% CI = 1.3-5.2), which may be related to the duration of the pets’ exposure to the infected owners, and the close contact of the felines with their owners, suggesting the development of antibodies in domestic animals as a consequence of viral transmission from owners. ^{38 , 39 , 47} In the present study, the association between positive COVID-19 cases (RT-qPCR testing) in humans versus seropositivity in canines and felines from the eight communes of Villavicencio was weakly positive (Spearman's correlation of p = 0.32, Table 4). This significance may be influenced by the characterization of the survey, where due to social fear or ignorance some owners could indicate that they were not or had not been positive for the disease, while a strong positive relationship was expected, as in the case of the study conducted by Patterson et al. ³⁰ in Italy. On the other hand, the study conducted by Van Aart et al. ⁴⁸ showed that none of the felines had been infected with SARS-CoV-2 despite the fact that these were living with their positive COVID-19 owners. Therefore, these associations between species should be analysed considering different factors.

Animals and humans are susceptible to a large number of different coronaviruses, in fact, it has been shown that all pathogenic human coronaviruses have their origin in animals, which is why studies should focus on their role in the transmission of SARS-CoV-2. ⁴⁹ In the present study, a human-animal transmission was considered based on the results of Smith et al. ⁵⁰ in the United Kingdom, who ruled out that dogs and cats were reservoirs of infection for humans. However, we cannot be sure about the trasmission direction, which will only be confirmed through further studies. Ultimately, successful elimination of SARS-CoV-2 will only be possible by assessing and controlling transmission in all susceptible animal species, a one health approach that could prevent the re-emergence of the virus in the future. ^{51 , 52} Although the 20 canine pre-pandemic sera reacted negatively to the immunodiagnosis, cross-reactions with ancestral coronaviruses in canines and felines are possible, but in low probability when are compared to commercial ELISAs with neutralizing antibody tests. ^{53 , 54} The possible cross-reactivity and the need to verify if the reactive antibodies are neutralizing for SARS-CoV-2 are the main limitations of our study; likewise, it is advisable to carry out studies of “SARS-CoV-2 virus neutralization test from the serum bank obtained, since this is the gold-standard test for the Centers for Disease Control and Prevention (CDC). ⁵⁵

The present study provides the first positive results of anti-SARS-CoV-2 serological tests (ELISA) in domestic dogs and cats in Colombia, with information about the dynamics of virus transmission in Latin America and the world during the COVID-19 pandemic. As mentioned above, cats were more susceptible to natural SARS-CoV-2 infection than dogs, following similar dynamics described in other studies. ^{7 , 38 , 56} The present study does not provide evidence that domestic canines and felines are sources of infection for humans; however, further studies focused on one health should not be ruled out ⁵² in order to improve our knowledge about transmission, epidemiology and dynamics of SARS-CoV-2 and promote a better response to possible future pandemics.