High seroprevalence of SARS-CoV-2 antibodies in household cats and dogs of Lebanon

Abstract

The COVID-19 pandemic has been declared in late 2019. It is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Flu-like symptoms and acute respiratory illnesses are the main manifestations of the disease. Recent studies have confirmed the susceptibility of domestic animals to SARS-CoV-2 infection. However, the seroprevalence of SARS-CoV-2 in household pets and the importance of pets in the epidemiology of this infection remain unknown.

In Lebanon, there is no epidemiological data regarding SARS-CoV-2 infection in companion animals. Thus, this investigation aimed to determine the seroprevalence of SARS-CoV-2 antibodies in household pets of Lebanon during the COVID-19 pandemic.

A cross-sectional study was carried out between April 2020 and February 2021. Blood samples from 145 cats and 180 dogs were collected from 12 veterinary clinics located in the North, Mount, and Beirut governorates. A validated ELISA assay was used to detect the anti- SARS-CoV-2 in the sera of the tested animals.

An overall seroprevalence of 16.92% (55/325) was reported; 13.79% seroprevalence was found in cats (20/145) and 19.44% (35/180) in dogs.

The young age and the cold season were significantly associated with an increased seropositivity rate to SARS-CoV-2 infection (P < 0.01).

These results confirm the circulation of SARS-CoV-2 in household pets, in various geographical regions in Lebanon. Although, there is a lack of evidence to suggest that naturally infected pets could transmit the SARS-CoV-2 infection. Yet, owners diagnosed with COVID-19 should limit their contact with their animals during the course of the disease to curb the risk of transmission.

1. Introduction

A worldwide spread of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has been reported since its emergence in Wuhan China, in December 2019. Although human-to-human spread is believed to be the main route of transmission, some animal species are thought to contribute to the SARS-CoV-2 pandemic (McNamara et al., 2020). Interestingly, experimental models have shown a remarkable susceptibility of cats, ferrets, minks, and dogs to SARS-CoV-2 infection (Bosco-Lauth et al., 2020; Shi et al., 2020). Furthermore, anti-SARS-CoV-2 antibodies were detected in pets in Italy, China, Germany, France, Spain, and the Netherland, suggesting a possible viral ability to cross the inter-species barriers (Michelitsch et al., 2020; Patterson et al., 2020; Segalés et al., 2020; Zhang et al., 2020; Fritz et al., 2021; Zhao et al., 2021). Even though, a human-to-mink and mink-to-human transmission has been postulated (Boklund et al., 2021; Oude Munnink et al., 2021), yet, there is no data confirming SARS-CoV-2 natural transmission between human and domestic animals such as cats and dogs (Pomorska-Mól et al., 2021), and further investigations are required in this area.

The assessment of infection in companion animals seems to be interesting to evaluate the extent of contamination in household pets under natural conditions. Thus, we conducted the first epidemiological survey in Lebanon, to determine the seroprevalence of SARS-CoV-2 antibodies in cats and dogs of Lebanon. In addition, this study aimed to evaluate the risk factors associated with this infection and to provide a basis for a better understanding and management of SARS-CoV-2 infections in pets.

2. Materials and methods

2.1. Serum samples

A cross-sectional study was carried out between April 2020 and February 2021. A total of 325 serum samples were randomly collected from 12 veterinary clinics located in different Lebanese regions, including the North, Mount, and Beirut governorates. The study only included household canines and felines. Pregnancy was an exclusion criterion. All animals were sampled by veterinarians during routine healthcare checks. No permission from the ethical committee was required to collect the specimens. Out of 325 serum samples, 145 originated from cats and 180 from dogs. Sera samples were stored at −80 °C until further serological testing. Informed consent was obtained from the owners for the inclusion of their pets in this study. Data including species, age, gender, collection season, and health conditions have been obtained for all the included animals in our investigation (Table 1 ).

Table 1.

SARS-CoV-2 seroprevalence and associated risk factors in cats and dogs of Lebanon.

Variables	Category	No. examined	No. positive	Seroprevalence (%)	95% CI	P value
Species	Cats	145	20	13.79	9.11–20.35	0.18
	Dogs	180	35	19.44	14.33–25.84
Age (years)	< 1 year	75	17	22.66	14.66–33.34	< 0.01†
	1–3	80	20	25	16.81–35.48
	4–8	120	15	12.5	7.72–19.60
	9+	50	3	6	2.06–16.22
Gender	Male	134	17	12.68	8.07–19.38	0.09
	Female	191	38	19.89	14.85–26.13
Geographical area	North Lebanon	90	21	23.33	15.80–33.05	0.08
	Mount Lebanon	120	14	11.66	7.08–18.63
	Beirut	115	20	17.39	11.55–25.34
Health condition	Healthy	112	23	20.53	14.09–28.93	0.09
	Respiratory symptoms	96	19	19.79	13.05–28.86
	Gastrointestinal symptoms	85	12	14.11	8.26–23.07
	Other	32	1	3.12	0.55–15.74
Collection Season	Fall	93	17	18.27	11.74–27.34	< 0.01†
	Winter	70	21	30	20.54–41.54
	Spring	78	9	11.53	6.19–20.50
	Summer	84	8	9.52	4.91–17.68

^{†Statistically significant.}

2.2. Serological assay

Serum samples were screened for antibodies against SARS-CoV-2 using a validated commercial ELISA assay (ID Screen® SARS-CoV-2 Double Antigen Multi-species) as described previously (Pomorska-Mól et al., 2021). The test is intended to detect antibodies to the SARS-CoV-2 nucleocapsid in the serum of diverse species. According to the manufacturer, the assay had a sensitivity and specificity of 100%. Positive and negative controls supplied by the kit were included in each testing plate.

The signal was measured at an optical density (OD) of 450 nm by microplate photometer (Thermo Scientific™ Multiskan™ FC, Inc., Waltham, MA, USA). The sample to positive ratio percentage (S/P%) has been calculated for each sample using the following formula:

$$\mathrm{S}/\mathrm{P}\%=\left(\mathrm{OD}\text{sample}-\mathrm{OD}\text{negative control}\right)/\left(\mathrm{OD}\text{positive control}-\mathrm{OD}\text{negative control}\right)\mathrm{x}100.$$

S/P% ≥ 60 was regarded as positive.

2.3. Statistical analysis

Statistical analyses were performed using IBM Statistical Packages for Social Sciences (IBM SPSS, version 22.00, IBM Corp, Armonk, N.Y, USA). Confidence intervals were calculated using the Wilson score method. The Chi-square or Fisher exact test was used to assess the significance of SARS-CoV-2 seroprevalence with specific risk factors. P-value <0.05 was considered to be statistically significant.

3. Results

Fifty-five out of the 325 examined pets showed seropositivity to SARS-CoV-2, leading to an overall prevalence of 16.92% (55/325) (95% CI: 13.24–21.38).

As shown in Table 1, no significant difference has been reported in the seroprevalence of SARS-CoV-2 in dogs 19.44% (35/180) and in cats 13.79% (20/145) (P = 0.18).

Similarly, Risk did not differ by gender as the seroprevalence was estimated at 12.68% (17/134) (95% CI: 8.07–19.38) for males and 19.89% (38/191) (95% CI: 14.85–26.13) for females (P = 0.09).

Regional seroprevalence was the highest in North Lebanon (23.33%, 95% CI: 15.80–33.05), and varied from 11.66 to 17.39% in Mount Lebanon and Beirut, respectively (Table 1). However, the difference in the seroprevalence reported was not statistically significant (P = 0.08).

At the time of sampling, 20.53% (25/55) of animals with anti-SARS-CoV-2 antibodies were healthy, 19.79% (19/55) displayed respiratory symptoms, 14.11% (12/55) presented with gastrointestinal disturbances, and 3.12% (1/55) suffered from liver disease. These differences were not statistically significant (P = 0.09).

Regarding the age, the seroprevalence was significantly higher among the <1 and 1–3 years olds (22.66%, 95% CI: 14.66–33.34 and 25%, 95% CI: 16.81–35.48, respectively), as compared to pets between 4 and 8 and > 9 years of age (12.5%, 95% CI: 7.72–19.60 and 6%, 95% CI: 2.06–16.22, respectively) (P < 0.01).

In addition, a significantly higher seropositivity to SARS-CoV-2 has been observed in the fall and winter seasons (18.27, 95% CI: 11.74–27.34 and 30%, 95% CI: 20.54–41.54, respectively) as compared to spring and summer seasons (11.53%, 95% CI: 6.19–20.50 and 9.52%, 95% CI: 4.91–17.68, respectively) (P < 0.01).

4. Discussion

Experimental animal models and reports on natural exposure have proven that some animal species are susceptible to SARS-CoV-2 infections (Stout et al., 2020). Yet, little is known about the prevalence of SARS-CoV-2 infection in household pets. Overall, 145 and 180 serum samples collected from household cats and dogs, respectively, were analyzed. Our findings demonstrated that both cats and dogs are susceptible to SARS-CoV-2 infection and that there is no significant difference in the seroprevalence of SARS-CoV-2 in dogs and cats (P = 0.18). Interestingly, our results did not support previous studies that suggested dogs were less susceptible to SARS-CoV-2 infections (Bosco-Lauth et al., 2020; Shi et al., 2020) or that dogs in Italy, China, and the United States had higher seroprevalence of SARS-CoV-2 antibodies (Barrs et al., 2020; Hamer et al., 2021; Patterson et al., 2020). The discrepancy in the reported prevalences could be due to the use of different diagnostic methods with varying degrees of sensitivity and specificity.

The similar susceptibility of cats and dogs to SARS-CoV-2 infection reported in our study could be attributed to the fact that both animal species share the same Angiotensin-converting enzyme 2 receptor (ACE2) as humans (Stout et al., 2020).

The seroprevalence of SARS-CoV-2 antibodies in cats from Lebanon (13.79%, 20/145) was considerably higher than the seroprevalence reported at the beginning of the COVID-19 pandemic in Germany (0.69%, 6/920) (Michelitsch et al., 2020), Italy (4.2%, 14/333) (Patterson et al., 2020), Spain (6.4, 23/360) (Segalés et al., 2020), and Poland (1.79%, 5/279) (Pomorska-Mól et al., 2021), but lower than the seroprevalence reported in France (23.5%, 8/34) and the United States (43.8%, 7/16), which were targeting cats living with COVID-19 positive owners (Hamer et al., 2021; Fritz et al., 2021).

The seroprevalence of SARS-CoV-2 antibodies in dogs from Lebanon (19.44%, 33/180) was notably higher than the seroprevalence reported in Poland (1.17%, 4/343) (Pomorska-Mól et al., 2021), Croatia (7.56, 13/172) (Stevanovic et al., 2021), France (15.4%, 2/13) (Fritz et al., 2021), and Italy (12.8%, 6/47) (Patterson et al., 2020).

In the current investigation, serum samples were randomly collected during different pandemic stages from household pets with no regard to the owners' COVID-19 status, and the reported seroprevalence was impressively high compared to other countries for similar conditions.

The seroprevalence of SARS-CoV-2 antibodies varies between countries and between different regions of a country. The reasons for these variations are many; for instance, there are differences in the inclusion and exclusion criteria, the diagnostic methods used, and the SARS-CoV-2 status of the pet owners. As a result, reliable comparisons cannot be made.

On the other hand, the seroprevalence of SARS-CoV-2 antibodies was not significantly different between male and female pets (P = 0.0.9) suggesting that gender is not a risk factor for SARS-CoV-2 infections in pets. Our data support a previous report showing no evidence of an increased risk of SARS-CoV-2 infection based on human gender, although a higher risk of severe disease has been seen in males (Petersen et al., 2020).

Furthermore, our study has shown a significant association between the age of pets and the seropositivity to SARS-CoV-2 antibodies; suggesting that younger animals (< 1 year; 22.66%), and (1–3 years; 25%) are considered at higher risk of acquiring SARS-CoV-2 infection as compared to the older groups (4–8 years; 12.5%), and (>9 years; 6%) (P < 0.01).

In contrast to our study, a previous report did not show any association between the age of pets and SARS-CoV-2 seropositivity (Fritz et al., 2021) or an interrelation between an increased risk of SARS-CoV-2 seropositivity and older age (Patterson et al., 2020), making this association inconclusive and requiring further investigations.

During the study period, the seropositivity to SARS-CoV-2 antibodies was significantly higher in the fall (18.27%, 17/93) and winter (30%, 21/70) seasons as compared to spring (11.53%, 9/78) and summer (9.52%, 8/84) (P < 0.01), which was in agreement with a previous study where an increased incidence of SARS-CoV-2 infections has been reported in the cold weather (Pomorska-Mól et al., 2021). Indeed, it has been shown that low environmental temperatures and limited sunlight favor the survival of SARS-CoV-2 (Liu et al., 2020; Nichols et al., 2021).

Surprisingly, there was no significant difference in SARS-CoV-2 seropositivity of companion animals between the studied Lebanese governorates (P = 0.08), suggesting the absence of variation in SARS-CoV-2 transmission and the application of preventive measures across Beirut, north and mount Lebanon.

Additionally, no significant association has been found between SARS-CoV-2 seropositivity and the health status of the examined pets, supporting previous findings that asymptomatic animals could still spread the virus (Shi et al., 2020; Neira et al., 2021; Pagani et al., 2021). However, respiratory and gastrointestinal signs have been reported as a consequence of SARS-CoV-2 infections in cats (Leroy et al., 2020; Natale et al., 2021).

Experimental studies have confirmed the susceptibility of cats and other carnivores including dogs to SARS-CoV-2 infection, with the possibility of spillage to other species (Bosco-Lauth et al., 2020; Halfmann et al., 2020; Shi et al., 2020), yet no evidence of zoonotic transmission or host-specific mutations has been reported in cats (Barrs et al., 2020; Hosie et al., 2021a; Hosie et al., 2021b). These findings contrast other reports where mink to human transmission and mink-specific mutations have been documented (European Centre for Disease Prevention and Control, 2020).

Previous serological surveys and reports of mild infections detected in cats and dogs of COVID-19 positive owners, pointed to the possible risk of household pet contamination by guardians (Andersen et al., 2020; Leroy et al., 2020).

Nevertheless, evidence continues to suggest that the risks of spillage from infected owners to pets, and pet implications in COVID-19 transmission are trivial (Fritz et al., 2021).

Interestingly, no association has been found between the prevalence of anti-SARS-CoV-2 antibodies and the number of pets in the house, highlighting that pet infection occurs following contact with an infected human rather than animal-to-animal transmission (Fritz et al., 2021).

However, experimental models have shown a potential transmission of SARS-CoV-2 infection between cats (Gaudreault et al., 2020; Halfmann et al., 2020). These conflicting data highlight the importance of examining the infection course in the condition of the natural transmission, in terms of viral load and duration of viral shedding to achieve a better understanding of the viral transmission dynamic from animal to animal and from human to animal and vice versa (Fritz et al., 2021).

5. Conclusions

To the best of our knowledge, this is the first study to investigate the seroprevalence of SARS-CoV-2 antibodies in cats and dogs of Lebanon, and a remarkably high seroprevalence has been reported. This study confirms the circulation of SARS-CoV-2 infection in household pets of Lebanon. Although there is a lack of evidence suggesting the involvement of household pets in the epidemiology of SARS-CoV-2 infection in humans (Schulz et al., 2021), preventive measures should be taken to avoid the contact between the infected owners and their pets, thus limiting the potential risk of transmission of the infection.

Animal welfare statement

The authors declare that they have followed the European standards (Directive 2010/63/EU) on the protection of animals used for scientific purposes.

The ethical approval was waived, since we have used diagnostic data collected during routine procedures.

A consent form has been completed by the owners to include their animals in this study.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Sara Khalife, Marwan Abdallah: Conceptualization, Methodology.

Sara Khalife: Data curation, Writing- Original draft.

Sara Khalife: Visualization, Investigation.

Sara Khalife, Marwan Abdallah: Writing- Reviewing and Editing.

All authors read and approved the final manuscript.

Declaration of Competing Interest

The authors declare no conflict of interest.