Serological Survey of SARS-CoV-2 in Wild Canids in Serbia: First Report in Red Foxes and Golden Jackals

1. Introduction

Members of the family Coronaviridae are ubiquitous worldwide and comprise a group of enveloped, positive-sense single-stranded RNA viruses. Coronaviruses (CoVs) are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Alphacoronaviruses and betacoronaviruses have been confirmed primarily in bats, whereas birds are recognized as the main reservoirs of gamma- and deltacoronaviruses [1]. Coronaviruses are also characterized by a high mutation rate, which contributes to their genetic diversity and adaptive potential. It has long been recognized that coronaviruses can cause respiratory and gastrointestinal diseases in domestic and wild animals. Well-known representatives include canine coronavirus (CCoV), which causes enteric disease in dogs and related species, and canine respiratory coronavirus (CRCoV), which is associated with mild to severe respiratory disorders in dogs [2,3].

In December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China, causing coronavirus disease 2019 (COVID-19) [4,5]. The clinical presentation of COVID-19 ranges from mild symptoms such as fever, dry cough, fatigue, and loss of taste or smell to severe disease and fatal outcomes [6]. The rapid spread of the disease overwhelmed healthcare systems, caused substantial economic losses, and led to the implementation of unprecedented preventive measures worldwide, including travel and trade restrictions. Globally, COVID-19 spread at an exceptional pace and resulted in approximately 676 million confirmed human infections and about 6.9 million deaths [7].

Genetically, SARS-CoV-2 is closely related to earlier zoonotic coronaviruses responsible for the severe acute respiratory syndrome (SARS) outbreak in 2002–2003 and the Middle East respiratory syndrome (MERS) outbreak in 2012 [8,9]. However, the epidemiological characteristics of COVID-19 differ considerably from those of previous outbreaks, which were geographically limited. In contrast, COVID-19 exhibited rapid global spread, reaching pandemic proportions with high morbidity and mortality. It is widely assumed that SARS-CoV-2 originated from wildlife and spilled over to humans at a live animal market in the Chinese province of Hubei. Bats are considered the natural reservoir of the virus, while the intermediate host has not been definitively identified, although the Asian palm civet (Paradoxurus hermaphrodites) has been suggested as a potential candidate [10].

SARS-CoV-2 is a single-stranded RNA virus belonging to the genus Betacoronavirus. The viral genome encodes four main structural proteins: membrane (M), envelope (E), spike (S), and nucleocapsid (N). The N and S proteins play a key role in inducing the humoral immune response and antibody production [6]. Betacoronaviruses, including SARS-CoV-2, utilize the angiotensin-converting enzyme 2 (ACE2) receptor for host cell entry, a receptor that is present in many animal species [11]. Nevertheless, susceptibility varies among species; for example, experimentally challenged coyotes did not become infected, suggesting that they are not competent hosts for SARS-CoV-2 [12]. Since the beginning of the pandemic, several variants of concern have emerged, including Alpha, Beta, Delta, and Omicron. These variants arise from viral mutations, particularly in the spike protein, and differ in transmissibility, pathogenicity, and their impact on diagnostics and vaccine performance [13].

SARS-CoV-2 is a novel zoonotic virus with significant zoonotic and reverse zoonotic potential. It is assumed that the virus initially spilled over from wildlife to humans and subsequently spread predominantly through human-to-human transmission via close contact and respiratory droplets. However, the virus has also been confirmed in companion animals (dogs and cats), ferrets, and deer, indicating its ability to cross species barriers and cause reverse zoonotic transmission [14,15]. Situations involving close contact between humans and animals, such as pet ownership, hunting activities, and farming, are of particular concern. Notably, SARS-CoV-2 has been detected in white-tailed deer living in proximity to urban areas, suggesting human-to-deer spillover followed by deer-to-deer transmission [16]. Of special interest are minks that are highly susceptible to COVID-19 and develop severe respiratory illness with 35-55% of mortality. Outbreaks on mink farms in the Denmark further demonstrated efficient human-to-animal transmission, and viral mutations observed in mink were subsequently associated with spillback and transmission of infection back to humans, which resulted in culling of minks and closing of mink farms [17].

Numerous studies have investigated the presence of SARS-CoV-2 in domestic, wild, and zoo animals to better understand viral origin and identify potential animal reservoirs. According to reports from the World Organization for Animal Health (WOAH), since March 2020 the virus spread to 36 countries and was detected in at least 29 animal species across 775 reported events [18]. Carnivores are considered to be of a higher risk to acquire SARS-CoV-2 infection. Studies in domestic and wild carnivores indicate that infection is often asymptomatic or associated with mild and transient viremia, although mild gastrointestinal and respiratory signs have been reported, particularly in felids. SARS-CoV-2 has been confirmed in wild felids such as lions, tigers, and pumas, as well as in wild canids, including foxes and jackals. In contrast, pigs, cattle, and sheep appear to be resistant to SARS-CoV-2 infection [19,20].

Serological investigation plays a crucial role in detecting past exposure to SARS-CoV-2 in animal population, particularly because active infection is transient and virus is difficult to detect by molecular methods after a week whereas antibodies persist for longer periods. IgM antibodies are detectable for a limited time, followed by the development of IgG antibodies, which may be detected for up to one year, although titers decline significantly after approximately six months [21,22].

In Serbia, the first COVID-19 case was registered on 6 March 2020. According to official data from the Institute of Public Health of the Republic of Serbia “Dr Milan Jovanović Batut” and the Ministry of Health, a total of 2,459,432 confirmed COVID-19 cases and 17,606 deaths were recorded by January 2023 [23]. Data on SARS-CoV-2 infection in animals in Serbia remain scarce. Nevertheless, several studies have confirmed the presence of SARS-CoV-2 RNA and antibodies in owned dogs and cats [24,25]. Detection of SARS-CoV-2 in domestic carnivores suggests that wild carnivores may also be susceptible to infection [12].

Wild carnivores in Serbia are of particular interest as potential viral sentinels due to increasing population sizes, mostly resulting from the absence of natural predators and the implementation of oral rabies vaccination programs since 2010. According to the Statistical Yearbook of the Republic of Serbia 2023 [26], the estimated population size of red foxes is 53,999 individuals, while the golden jackal population numbers approximately 22,596. Red foxes are widely distributed throughout Serbia, whereas golden jackals are more densely populated in lowland areas, particularly in northern and central regions of the country.

The aim of this study was to assess the exposure of wild canids, specifically red foxes (Vulpes vulpes) and golden jackals (Canis aureus), to SARS-CoV-2 in Serbia and to evaluate their potential role in virus circulation at the wildlife–human interface. Blood samples from 165 free-living foxes and jackals were tested for the presence of anti-SARS-CoV-2 antibodies using a commercial ELISA and a parallel in-house ELISA assay.

2. Materials and Methods

2.1. Sample Collection and Geographic Distribution

A total of 165 blood samples from red foxes (Vulpes vulpes) and golden jackals (Canis aureus) were collected for serological testing. Of these, 83 samples originated from foxes and 82 from jackals. The samples were randomly selected based on sample quality, ensuring balanced geographical representation across all study areas. Samples from red foxes and golden jackals were collected from the beginning of August 2024 to the end of March 2025 within the framework of the annual national program for monitoring the effectiveness of oral rabies vaccination in foxes and other wild carnivores, which has been implemented in Serbia since 2011. The program is established by the Veterinary Directorate of the Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia and is conducted in accordance with the Rulebook on the Program of Animal Health Protection Measures for 2024 [27]. All sampled animals were older than one year, while data on animal sex were not available. Samples were collected from ten administrative districts including the City of Belgrade, covering a total of 26 municipalities (Table 1).

Blood specimens from red foxes and golden jackals were collected directly from the heart of carcasses by trained hunters. Blood was collected into sterile vacuum blood collection tubes without anticoagulant. The samples were transported to the Virology Department of the Institute of Veterinary Medicine of Serbia (IVMS) in the portable fridges with ice packs to maintain the cold chain during transport. In the laboratory, blood samples were kept at room temperature to allow spontaneous coagulation and clot retraction. After serum separation, samples were centrifuged for 10 min at 2500 rpm. The sera were subsequently heat-inactivated at 56 °C for 30 min. Aliquots of the processed sera were transferred into sterile microtubes and stored at −20 °C until testing.

2.2. Serological Screening by Enzyme-Linked Immunosorbent Assay (ELISA)

Serological screening for specific anti-SARS-CoV-2 IgG antibodies was performed using a commercially available and validated ELISA kit, while an in-house ELISA developed at Institute for the Application of Nuclear Energy (INEP) was applied to selected samples as the comparative method.

2.2.1. Commercial ELISA

Laboratory analyses were conducted with a commercial ELISA kit “ID Screen® SARS-CoV-2 Double Antigen Multi-Species” (IDVet, Grabels, France), designed for the detection of antibodies against the nucleocapsid (N) protein of SARS-CoV-2. The assay is based on an indirect ELISA format and is suitable for use in multiple animal species. All procedures were carried out in accordance with the manufacturer’s instructions.

Briefly, serum samples were diluted 1:2 in the provided dilution buffer, and 100 µL of diluted samples, positive control, and negative control were added to microplate wells pre-coated with recombinant SARS-CoV-2 N antigen. Plates were incubated for 45 min at 37°C. After incubation, wells were washed five times with 300 µL of wash buffer to remove unbound material. Subsequently, 100 µL of horseradish peroxidase (HRP)-conjugated multispecies secondary antibody was added to each well, followed by incubation for 30 min at room temperature. Plates were then washed five times as described above. Next, 100 µL of tetramethylbenzidine (TMB) substrate solution was added to each well and, incubated for 20 min at room temperature in the dark. The enzymatic reaction was stopped by adding 100 µL of stop solution, and optical density (OD) of each sample was measured at 450 nm using a spectrophotometer Victor 3V (PerkinElmer 1420 multilabel counter).

Results were expressed as S/P percentage (S/P,%) and interpreted according to the manufacturer’s criteria. Serum samples with S/P values below 50% were considered negative, samples with S/P values between 50% and 60% were classified as doubtful, and samples with S/P values equal to or above 60% were considered positive.

2.2.2. In-House ELISA

An indirect ELISA (iELISA), originally developed for the detection of anti–SARS-CoV-2 IgG antibodies in human COVID-19 patients [28,29] and registered by the Medicines and Medical Devices Agency of Serbia (ELISA SARS-CoV-2 IgG; Certificate of registration No. 515-02-02373-20-003, dated 14 July 2020), was subsequently modified for serological testing of wild canid blood samples. The assay was adapted by replacing the original anti-human IgG secondary antibodies with anti-dog IgG, allowing the quantification of the immune response in wild canids.

The in-house ELISA procedure applied in this study is described below:

High-binding 96-well polystyrene microplates were coated overnight at 4 °C with 100 µL per well of recombinant SARS-CoV-2 nucleocapsid (N) protein (ACRO, USA; CAT# NUN-C5227) at a final concentration of 1 µg per well in carbonate–bicarbonate coating buffer (pH 9.0). Plates were washed twice with phosphate-buffered saline (PBS, pH 7.5), stabilized with 200 µL of 1% sucrose per well for 15 min at room temperature, air-dried overnight, and stored at 4 °C until use.

Serum samples were diluted 1:100 in PBST supplemented with 1% bovine serum albumin (BSA), and 100 µL of each diluted sample was added per well. Plates were incubated for 1 h at 37 °C and washed five times with PBST. Horseradish peroxidase–conjugated rabbit anti-dog IgG secondary antibody (CAT No. ab112828, Abcam, UK), diluted 1:1000 in PBST with 1% BSA, was added at 100 µL per well and incubated for 1 h at 37 °C, followed by washing.

Color development was achieved by adding 50 µL of TMB substrate solution A followed by 50 µL of TMB substrate solution B per well, incubating for 20 min at 37 °C, and stopping the reaction with 100 µL of sulfuric acid (H₂SO₄). Optical density (OD) was measured at 450 nm using a microplate reader (Victor 3V, PerkinElmer, USA).

Positive and negative control sera from red foxes and golden jackals were selected based on their results obtained with the commercially ELISA kit and pooled accordingly. Blank wells containing 100 µL PBST with 1% BSA were included. The cut-off value was defined as the mean OD of negative sera plus three standard deviations. Samples with OD values above the cut-off were considered positive.

2.3. Statistical Analyses

Differences in SARS-CoV-2 seroprevalence among species and regions were analyzed by Pearson’s chi-squared test, with 95% confidence intervals (CI). Ninety-five percent confidence intervals (95% CI) were calculated to assess the precision of seroprevalence estimates across species and regions. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using Microsoft Excel 2007 (Microsoft Corp., Redmond, WA, USA) and SPSS Statistics version 19.0 (IBM Corp., Armonk, NY, USA).

Agreement between the commercial ELISA and the adapted assay was evaluated using Cohen’s kappa (κ) coefficient [30]. The strength of agreement was interpreted according to commonly accepted criteria (poor: κ < 0.20; fair: 0.21–0.40; moderate: 0.41–0.60; substantial: 0.61–0.80; almost perfect: > 0.80).

For data visualisation was used QGIS Development Team (2026). _QGIS Geographic Information System. Open Source Geospatial Foundation Project. Available at: https://qgis.org.

3. Results

Serological screening conducted by commercial ELISA revealed that 17 (10.3%, 95% CI: 5.6–14.9) of 165 tested red foxes and golden jackals were seropositive for SARS-CoV-2. Among 83 red foxes, antibodies against SARS-CoV-2 were detected in 11 animals (13.3%, 95% CI: 6.8–22.5), whereas reactive sera were identified in 6 of 82 jackals (7.3%, 95% CI: 2.7–15). Despite the higher antibody detection rate in red foxes, no statistically significant difference between species was observed (p = 0.21).

Samples were collected across 10 regions, including the City of Belgrade, but seropositive animals were found in only four districts. The highest seroprevalence was observed in the Južnobanatski District, with 11 positive animals (35.5%, 95% CI: 19.2–54.6), including 8 red foxes and 3 jackals. In the Mačva District, 3 animals had anti-SARS-CoV-2 antibodies (15.8%, 95% CI: 3.4–39.6; 2 foxes, 1 jackal), followed by the Kolubara District with 2 positive (13.3%, 95% CI: 1.7–40.5; 1 fox, 1 jackal) and the Braničevo District with a single positive jackal (3.6%, 95% CI: 0.1–18.3) (Figure 1). Differences in seroprevalence among regions were statistically significant (p < 0.001).

Notably, Južnobanatski District accounted for 72.7% of all seropositive red foxes and 50% of jackals with detectable antibodies, highlighting this district as the main hotspot of SARS-CoV-2 exposure. Overall, the highest seroprevalence was observed in the northern part of Serbia, in Vojvodina region, indicating both species-specific and regional patterns of exposure among wild canids.

All samples were additionally tested with the adapted ELISA. Antibodies against SARS-CoV-2 were detected in 12 of 165 animals (7.3%; 95% CI: 3.8–12.4%), including 8 of 83 red foxes (9.6%; 95% CI: 4.3–18.1%) and 4 of 82 golden jackals (4.9%; 95% CI: 1.3–12.0%). The adapted ELISA showed substantial agreement with the commercial assay (Cohen’s κ = 0.66), suggesting comparable performance between the two methods under the conditions of this study.

4. Discussion

Surveys describing cases of SARS-CoV-2 infection in animals have so far been widely reported in pets, particularly domestic cats and dogs, across North America, Europe, Asia, and South America [31,32]. However, data on SARS-CoV-2 exposure in wild canids remain scarce, despite the fact that the wildlife species are well-known reservoirs of viral pathogens.

In the present study, we assessed SARS-CoV-2 exposure in free-ranging wild canids, specifically red foxes (Vulpes vulpes) and golden jackals (Canis aureus) in Serbia. Our results revealed that 17 out of 165 tested animals were seropositive, corresponding to an overall seroprevalence of 10.3%. Comparable studies conducted in other European countries have demonstrated heterogeneous results. In Croatia, ELISA reactivity was detected in 2.9% of red foxes and 4.6% of tested golden jackals [20]. In Switzerland, serological exposure to SARS-CoV-2 was confirmed in free-ranging red foxes, with an estimated seroprevalence of 3.1% [33]. In another Swiss study, only one red fox was found SARS-CoV-2 positive by qRT-PCR among 246 samples collected from 153 animals housed in zoos [34]. Conversely, a study from Poland did not identify any positive red foxes in either molecular or serological investigations involving 292 hunted animals [35]. We assume that published findings vary significantly depending on the species studied, methodological approaches, sample sizes, and SARS-CoV-2 variants circulating during the pandemic, which was consistent with previous observations [31,36,37].

Furthermore, data from investigations involving other wild canids are similarly limited. Goldberg et al. (2024) [38], during surveillance of wildlife communities across Virginia and Washington D.C., USA, detected SARS-CoV-2 antibodies in 36.4% of tested raccoons. In contrast, several studies failed to confirm SARS-CoV-2 infection in wolves, either in free-ranging populations or zoo-housed animals [39]. Similarly, no SARS-CoV-2-positive coyotes or eastern wolves were detected by RT-PCR in surveillance studies conducted in Ontario and Québec, Canada [3,40].

The fox and jackal samples included in this study originated from ten districts, including the City of Belgrade, while positive samples were detected in only four districts (Južnobanatski District, Mačva District, Kolubara District, and Braničevo District). The observed distribution of seropositive animals reflects the epidemiological situation regarding SARS-CoV-2 in the human population in Serbia. Following several waves of COVID-19 between March 6, 2020 and 2022, a period of reduced infection rates was recorded. However, starting from May 2024, SARS-CoV-2 showed moderate fluctuations throughout the season, with a noticeable maximum (103.1 cases per 100,000 population) at the end of September 2024 [41]. Since animal samples in our study were collected from summer 2024 to spring 2025, we assumed that there is a positive relationship between the proximity of urban areas and seropositivity in wild canids, and these results coincide with the highest peak in the post-COVID-19 season in 2024 in Serbia. Similar associations between urbanization and seropositivity in wildlife have been reported previously [38].

An exceptionally high incidence rate of SARS-CoV-2 in humans was recorded in the Južnobanatski District (2646.55 per 100,000 inhabitants) which is consistent with our findings in wild canids from the same region. Significantly, 72.7% of seropositive foxes and 50% of seropositive jackals originated from the same district. Our data are in agreement with earlier studies indicating that animals living in close proximity to humans may face an increased risk of exposure [32]. Jackals and foxes are known to approach human habitations, farms, and zoos and likely mix with domestic animals and humans [35,42]. Foxes and jackals are known to transmit numerous pathogens, many of which have zoonotic potential [43]. Red foxes live and move in small groups while jackals generally live in pairs and have territorial behavior. Jackals are omnivores and scavengers. Both species feed on rabbits, skunks, rodents, and are in close contact with other potential sources and reservoirs of SARS-CoV-2 [12,20].

In the present study, red foxes showed a higher seroprevalence than golden jackals, with antibodies detected in 11 of 83 foxes (13.3%) and 6 of 82 jackals (7.3%). This is in line with findings reported by Porter et al. (2022) [12], who showed that experimentally infected foxes developed mild clinical signs and were capable of viral shedding, whereas infected coyotes remained asymptomatic. Such results highlight the importance of further investigations in red foxes, which may represent a potential wildlife host. Additionally, it has been that red foxes are more susceptible to infection because they possess the ACE2 receptor, which facilitates SARS-CoV-2 binding and entry [36,44].

Serological assays are important diagnostic tools for SARS-CoV-2 surveillance, particularly in wildlife studies. Although RT-PCR remains the reference method for confirming active SARS-CoV-2 infection, its application in wildlife surveillance presents several practical limitations. The persistence of viral RNA is transient, the success of detection is highly dependent on the timing of sampling, and the quality of the sample is often inadequate. For these reasons, serological approaches are often more suitable for population-based surveillance and retrospective assessment of viral circulation [21].

Among serological techniques, ELISA assays offer several practical advantages. They are technically less demanding, cost-effective, suitable for high-throughput screening, and do not require biosafety level-3 (BSL-3) facilities, unlike virus neutralization tests, which are considered the gold standard but are time-consuming and require trained personnel [21,45].

Many commercial ELISA assays for SARS-CoV-2 are based on the nucleocapsid (N) protein. The N protein is highly immunogenic, abundantly expressed during infection, and elicits a strong antibody response, making it a suitable target for serological detection [46,47]. In the present study, we used a commercial multi-species ELISA from Innovative Diagnostics (IDVet), France based on the N protein. This assay has been widely applied across a broad range of animal species, including cats, dogs, rabbits, bats, wild boars, ferrets, goats, lions, cattle, and horses [48,49,50,51,52]. This test has shown high diagnostic performance, with reported sensitivity and specificity of 99.1%, as indicated by the manufacturer and supported by previous evaluations [49,51].

All samples were additionally tested using an adapted in-house ELISA. The lower number of seropositive results obtained with the adapted assay (12/165; 7.3%) compared to the commercial ELISA (17/165; 10.3%) can be explained by methodological differences between the assays. Variations in antigen presentation, assay design, and cut-off determination can influence the classification of samples, particularly those with low antibody levels [53]. Commercial assays are typically validated using large panels of well-characterized samples. In contrast, in-house assays are often developed on smaller sample sets, which may result in reduced sensitivity. Despite these differences, the substantial agreement observed between assays (Cohen’s κ = 0.66) indicates that the two approaches were broadly comparable. Similar levels of concordance between commercial and in-house serological assays have been reported previously [54].

As far as we know, this study provides the first evidence of SARS-CoV-2 exposure in wild canids in Serbia. Certain limitations should also be considered. The geographic distribution of sampled areas was not fully balanced, and the number of collected samples varied among districts. A more uniform sampling strategy would allow a clearer interpretation of the spatial distribution of SARS-CoV-2 exposure.

Finally, further optimization and validation of serological assays for wildlife samples remain essential. As emphasized by Jemeršić et al. (2021) [20], particular attention should be given to sampling methodology, species tested, and sample quality. Wildlife samples are frequently affected by hemolysis and variable storage conditions, which can affect the accuracy of results.