High Prevalence of Antigens of and Specific Antibodies Against Various Viral Pathogens in European Wildcats (Felis silvestris) from Southwest Germany, 2020–22 Open Access

The European wildcat (Felis silvestris) population in Europe suffered a significant decline until the early 20th century because of extensive hunting and habitat fragmentation caused by roads (Piechocki 1990). Recent years have seen a remarkable recovery in the European northwestern metapopulation (Breitenmoser et al. 2021), thanks to enhanced conservation measures (Gerngross et al. 2023). In Germany and Switzerland there has been a rapid increase in recent years, but this is by no means representative of the situation in Europe (Breitenmoser et al. 2021). Populations in Bulgaria are currently considered stable, but the Iberian population is declining (Breitenmoser et al. 2021). In Scotland, the wildcat population is thought to be functionally extinct (because of genetic mixing with domestic cats). There is a lack of data for other areas of distribution (Breitenmoser et al. 2021).

Overall, the wildcat population still faces several threats, with road traffic remaining the primary cause of death (Bastianelli et al. 2021). Because of the further spread in some areas, there is an increased contact with domestic cats, especially in new distribution areas or at the edges of ranges (Nussberger et al. 2014, 2023). Hybridization with domestic cats (Felis catus) therefore represents a significant threat (Bastianelli et al. 2021; Nussberger et al. 2023). In addition, diseases pose a threat to the slowly recovering wildcat population (Vogt 1984; Piechocki 1990; Krone et al. 2008; Bastianelli et al. 2021). There is also the possibility that wildcats act as a reservoir of pathogens for domestic cats (Bisterfeld et al. 2022). Common viruses in cats, such as feline herpesvirus (FHV), feline calicivirus (FCV), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline parvovirus (FPV), and feline coronaviruses (FCoV), are prevalent among both unvaccinated feral domestic cats, and vaccinated domestic and outdoor cats (Maruyama et al. 2003; Beatty and Hartmann 2021). These viruses can be transmitted to wildcats through close contact during mating, bites, or exposure to feces. Previous studies have shown the presence of viral pathogens in wildcat populations in Europe (Table 1). The highest prevalence rates for FeLV (52.9%) and FCoV (47.1%) have been detected in areas where hybridization rates are high (Heddergott et al. 2018). Looking at FIV only, Fromont et al. (2000) were able to detect antibodies (Ab) against FIV with a serum prevalence of 8% in 38 wildcats in France after analyzing blood samples using Ab-ELISA. All other studies, in various countries, have yielded negative results (Daniels et al. 1999; Leutenegger 1999; Račnik et al. 2008; Millán and Rodríguez 2009; Duarte 2012; Steeb 2015, Heddergott et al. 2018). Most of the analyses so far have been based on very small sample sizes or used samples collected over a longer period of time and in larger areas. Much larger sample sizes limited in time and space need to be examined in order to make concrete statements about the health status of the European wildcat and the influence of the domestic cat on it.

Analyzing large sample sizes for a rare and protected species such as the European wildcat is challenging. The elusive lifestyle of the wildcat and the small population size make sampling even more difficult (Piechocki 1990). A valuable way of obtaining sample material from endangered species is from dead specimens (Richini-Pereira et al. 2010; Schwartz et al. 2020; Rohner et al. 2021). In addition to collecting data such as sex, nutritional status, and age class, further investigations can be initiated, for example, to obtain an overview of the health status of the species.

The aim of our present study was therefore to analyze a large sample size from a short time period and a restricted area for the presence of antibodies or antigens of typical feline viral diseases in European wildcats. We hypothesized the large sample size limited in time and space would enable more accurate conclusions to be drawn about the health status of the wildcat population studied. We also hypothesized that the data would show influences of age group and sex, with males and adult animals being more likely to have antibodies against or antigen of the viruses studied, for example, through transmission during ranking fights (Hellard et al. 2011). However, we also considered that sample material from found-dead and previously frozen animals might lead to false-negative results or lower titers in some cases, because of poorer sample quality, such as an excessive dilution caused by water released during thawing.

The German Federation for the Environment and Nature Conservation, Friends of the Earth Germany, state association of Rhineland-Palatinate (Bund für Umwelt und Naturschutz Deutschland [BUND], Landesverband Rheinland-Pfalz) and the state of Rhineland-Palatinate performed a comprehensive dead animal monitoring program (Leonhardt et al. 2021). The program involved dissecting 102 European wildcats, found dead between 2020 and 2022. Necropsies were carried out in the Working Group for Wildlife Research at the Clinic for Birds, Reptiles, Amphibians and Fish at Justus Liebig University Giessen (Giessen, Germany). The wildcats were stored at −18 C until examination. After collecting morphological and morphometric data (e.g., pelage patterns, body measurements, gut lengths), additional information on sex, age class, and nutritional status was gathered according to a protocol (Eskens et al. 2015). Nutritional status was evaluated based on the presence of subcutaneous and visceral fat, as well as the presence or absence of fat in the coronary furrow and the fatty tissue surrounding the kidneys. Nutritional states were scored as very good, good, moderate, poor, very poor, and cachectic. Age classes were determined based on x-ray images of canines and categorized as juvenile, immature, subadult, and adult (Götz 2015).

Necropsies were performed following standard pathological practice (Eskens et al. 2015; Freie Universität Berlin [FU Berlin] 2015). A large amount of sample material was collected during the necropsy, including the effusion fluid that leaked into the body cavities. The effusion fluid comprises not only partially hemolyzed blood but also thawed water and organ components. Because of the absence of blood and serum samples, effusion fluid was preserved in SafeSeal micro tubes (Sarstedt AG & Co. KG, Sarstedtstr. 1, D-51588 Nümbrecht, Germany) at −18 C for serological testing for antibodies against FHV, FCV, FIV, FPV, and FCoV or antigen (Ag) of FeLV.

Individuals of F. silvestris were distinguished from the domestic cat based on their distinct morphological characteristics and intestinal length (Vogt 1984; Piechocki 1990; Kitchener et al. 2005; Müller 2005, 2011; Krüger et al. 2009). To ensure accurate results, in cases where hybridization was suspected during dissection because of an intestinal length over 200 cm, or for example because of a continuous dorsal line, a 0.5 × 0.5–cm piece of muscle was stored in ethanol and sent to the Senckenberg Institute (Conservation Genetics Group, Senckenberg Research Institute and Natural History Museum, Frankfurt, Germany) for genetic testing, following the protocol established by Steyer et al. (2016) and von Thaden et al. (2020).

For serologic testing, 2-mL vials of effusion fluid were packed in an insulated box with ice packs at −18 C and sent to the Laboklin veterinary laboratory (Bad Kissingen, Germany), arriving there at ≤4 C. Commercial ELISAs or indirect fluorescent antibody tests (IFATs) were used to investigate the samples after thawing and resuspending. The assays were internally validated for serum samples according to Deutsche Akkreditierungsstelle (German accreditation) guidelines at Laboklin. The same sample volumes were used for effusion fluids as listed for serum samples by the manufacturers, as an experimental approach for this study. All remaining steps were performed according to the manufacturers' protocols for the respective assays. Antibody detection assays (NovaTec Immundiagnostica GmbH, Dietzenbach, Germany) were used to detect the presence of antibodies against FIV and FCoV. The presence of FeLV was confirmed using an antigen-ELISA (NovaTec Immundiagnostica GmbH). Antibodies against FHV, FCV, and FPV were detected serologically using IFATs (Megacore Diagnostik GmbH, Hörbanz, Austria) and positive results were reported as titers.

A logistic regression was performed for each virus with the dependent variable presence or absence of antigens of and/or antibodies against viral pathogens, and the independent variables age, sex (0: female, 1: male), and nutritional status (Heddergott et al. 2018). For simplification, the age classes juvenile and immature were summarized as animals in their first year of life (0: juvenile + immature) and the subadult and adult animals formed a further age class (1: subadult + adult). The nutritional status was summarized in three classes: 1 = poor, very poor, or cachectic; 2: moderate; 3: good or very good. The two-way interactions between the independent variables were also tested; these were removed from the model if they were not significant. Multiple pairwise comparisons were adjusted using the Bonferroni correction. The results are given as odds ratios, including 95% confidence intervals (CI). The statistical analysis was carried out using SAS 9.4 (SAS Institute 2013).

All 102 specimens were confirmed to be wildcats. The identification of 62 individuals as wildcats was based on the analysis of morphological and morphometric data. The remaining 40 animals were confirmed as wildcats by genetic testing. Of the wildcats analyzed, 60 (58.8%) were male and 40 (41.8%) were female. The majority of the animals were adult (61.9%), in good to very good nutritional condition (58.8%; Table 2), and died because of polytrauma (86.3%). In the remaining wildcats, the underlying cause of death was determined as follows: five cases were attributed to soft tissue trauma, one case was associated with a single trauma, and four cases were attributed to cachexia. One of the wildcats was shot illegally, and the underlying cause of death of three wildcats remained unclear.

Antibodies against, or antigens of, all viral pathogens examined, except FIV, were detected in the wildcat population analyzed. Antigens of FeLV were detected in 29/102 (28.4%) of the samples. Anti-FHV antibodies were found in 23/102 (22.5% of samples) at titers between 1:20 and 1:80, and anti-FCV antibodies were found in 21/102 (20.6%) samples at titers between 1:20 and 1:80. In 18/102 (17.6%) wildcat samples we detected antibodies against FCoV, whereas 14/102 (13.7%) had antibodies against FPV at titers between 1:20 and 1:640 (Table 3).

Overall, 61/102 (59.8%) wildcats tested positive for antigens of or specific antibodies against at least one virus, with 26/102 (25.5%) of the animals testing positive for antigens of or antibodies against more than one virus. A single wildcat showed antigens of FeLV as well as specific antibodies against all viruses tested except for FIV. The remaining 25 (24.5%) wildcats had combinations of antibodies and antigens associated with 2–4 viruses (Table 4).

Statistical analysis revealed only significant influences of sex and age group on the presence of antibodies against FCV. Males had a 3.64-fold higher chance of having an antibody titer against FCV compared to females. Additionally, adult animals had a 9.7-fold higher chance of having an antibody titer against FCV than juvenile and immature animals. For all other results, there was no significant influence of sex, age class, or nutritional status (Table 5).

To our knowledge, the sample analyzed in this study is the largest sample size (102) ever tested for viral pathogens in wildcats in a close geographical context and within a relatively short period of 2 yr. In the wildcat population analyzed, contact with all tested pathogens except FIV was detected and more than half (59.8%) of the wildcats examined had contact with at least one of the examined pathogens, but no pathological lesions associated with a viral infection or a fatal outcome resulting from such infection were detected. We were unable to determine the impact of the pathogens on the wildcat population, as most of the samples were collected from wildcats found next to roads. In most cases, the cause of death was polytrauma, probably as a result of a vehicular collision. Thus, it is still uncertain how many wildcats die because of infectious diseases, as except for road-killed animals they are usually not found and, even those carcasses are not always investigated further.

The prevalence of FeLV antigen that we detected, 28.4%, was similar to prevalence detected in some other studies analyzing dead wildcats: 25% (n=32) by Duarte et al. (2012) and 24% (n=38) by Fromont et al. (2000). In other studies, the prevalence was lower (18%, n=80, Steeb 2015) or higher (53%, n=34; Heddergott et al. 2018), but in all studies the sample size was considerably smaller or cats were sampled over a longer period of time and in larger areas, which only allows a limited comparison.

In comparison with the prevalence of antibodies against FCV that we found (20.6%), other studies using dead wildcats have showed lower prevalences (Leutenegger et al. 1999; Steeb 2015), whereas those sampling live wildcats found higher prevalences (Millán and Rodríguez 2009). This may indicate that the samples from dead wildcats are of poorer quality, which could result in false-negative outcomes. Nevertheless, this approach cannot be universally applied and is therefore open to question. This is also shown by the evidence of the highest prevalence of antibodies against FHV to date (22.6%) in this study compared to previous studies, regardless of whether the samples came from live or dead animals (Daniels et al. 1999; Leutenegger et al. 1999; Millán and Rodríguez 2009; Steeb 2015; Heddergott et al. 2018).

We detected antibodies against FCV and FHV with relatively high prevalences in this study. However, no macroscopic changes were found, such as glossitis, stomatitis or ulcerative changes, that would indicate the occurrence of feline upper respiratory tract disease; such lesions would have been expected if the infection had occurred recently.

Antibodies against FIV were not detected. This is consistent with the results of previous studies (Daniels et al. 1999; Leutenegger et al. 1999; Račnik et al. 2008; Millán and Rodríguez 2009; Duarte et al. 2012; Steeb 2015; Heddergott et al. 2018). Only Fromont et al. (2000) were able to detect antibodies against FIV in samples from three live-caught wildcats in France, although the proof of the presence of the pathogen in the form of virus isolation is still lacking in this study. All three wildcats in that study had been caught in an area where wildcats had been absent for a long time and had only just re-established themselves. It is reasonable to assume that these wildcats had come into contact with the pathogen through contact with domestic cats. Nevertheless, the question remains open as to whether the virus is really transmitted from domestic cats to European wildcats, as this is the only evidence to date, or whether the virus originated in the wildcat population and is endemic to it (Fromont et al. 2000).

Feline immunodeficiency virus is a lentivirus belonging to the Retroviridae family. Other nondomesticated cats, such as lions (Panthera leo), pumas (Puma concolor), leopards (Panthera pardus), and cheetahs (Acinonyx jubatus), have species-specific strains of FIV that largely cross react with the strain found in domestic cats (Olmsted et al. 1992). However, there are differences, such as puma lentivirus in the puma, which require different detection methods (Kania et al. 1997; Franklin et al. 2007). It is possible that the European wildcat may have its own strain of FIV that cannot be detected using conventional testing methods designed for the domestic cat strain. Our study only indicated absence of FIV infections that are closely related to the domestic cat strain. To increase the probability of detection, future tests should use multiple viral strains (Franklin et al. 2007).

In both our study (17.6%) and the majority of others, FCoV could not be detected or was present at a low prevalence (Daniels et al. 1999; Leutenegger et al. 1999; Millán and Rodríguez 2009; Steeb 2015). With the exception of one case (47%, Heddergott et al. 2018), prevalence in wildcats is quite low compared with domestic cats. In multicat households and animal shelters, the prevalence ranges from 31.8% to 100% (Fehr et al. 1996; Klein-Richers et al. 2020). This discrepancy is most likely due to the mode of transmission, because FCoV is mainly transmitted through feces (Lutz 2019), and wildcats, which are solitary animals (Piechocki 1990), are likely to have less exposure to infected feces compared to domestic cats living in close proximity to one another.

All studies that detected antibodies against FPV, whether in both living and dead wildcats, have reported higher prevalences than in our study (Leutenegger et al. 1999; Millán and Rodríguez 2009; Steeb 2015; Heddergott et al. 2018). There is no obvious reason for this difference.

Comparing our study and previous studies, it remains unclear whether detection rates are consistently higher in living or dead wildcats, or if the material used for investigations has an impact. It is important to note that material obtained from deceased animals may be of lower quality. Previous studies have been conducted on smaller sample sizes, making it challenging to compare with the present study and draw conclusions about the health of the studied wildcat populations. To address this, future studies need to analyze significantly larger samples from other populations and compare different sample materials. We used established serological methods on nonserum sample material—effusion fluid. The tests reacted in the same way as serum samples in the positive case. However, effusion fluid is not an optimal sample material for the test systems used; clearer reactions might be seen if the systems were adapted to the sample material. Additionally, effusion fluid contains thawing water and other components in addition to blood, which dilutes the sample to an unknown extent. Therefore, it can be assumed that some tests may have produced false-negative results or lower titers because of the sample being too diluted.

Data on age class, nutritional status, and sex were available for all the wildcats analyzed. However, a statistical analysis of the influence of these three parameters on the presence of antibodies or antigens was only possible to a limited extent, as there were too few individuals in the individual categories.

It can be assumed that adult animals generally have a higher probability of having antibodies, because of their longer lifespan, which increases their chances of coming into contact with infected cats, whether domestic or wild, and becoming infected themselves (Wobeser 2007). It is hypothesized that males may have a higher risk of contracting pathogens transmitted through saliva or bites (Lutz et al. 2019) because of hierarchy battles (Hellard et al. 2011). Nevertheless, a previous study has failed to demonstrate a significant influence of age group, sex, or nutritional status (Steeb 2015), and despite the large sample size of our study, only the influence of sex and age group on the presence of antibodies against FCV was significant. It remains unclear whether this lack of significance is due to a small sample size or if there is genuinely no significant influence of these three factors.

Close contact with domestic cats in new distribution areas could potentially lead to reciprocal transmission of viral pathogens. There are currently an estimated 15.2 million domestic cats living in Germany (Zentralverband Zoologischer Fachbetriebe Deutschlands 2022) and 7,000–10,000 wildcats (Ellwanger et al. 2021). Especially in areas characterized by open land, there are high hybridization rates that exceed 60% in some areas (Streif et al. 2022), which indicates close contact between the two species, despite the Germany-wide hybridization rate currently estimated to be very low at 3.9% (Steyer et al. 2018). Given the significantly higher number of domestic cats compared to wildcats, it is reasonable to assume that transmission primarily originates from domestic cats to wildcats. Domestic cats not only pose a risk to wildcats in terms of the typical feline pathogens, they are also carriers of zoonotic diseases, most notably toxoplasmosis, and can transmit Toxoplasma gondii not only to other domestic cats, but also to wild animals and humans (Maruyama et al. 2003; Gerhold and Jessup 2013).

The prevalence of FeLV antigens in domestic cats has decreased in recent years and is currently 1–9% in Germany (Gleich et al. 2009; Studer et al. 2019; Giselbrecht et al. 2023). This is significantly lower than the prevalence of 28.6% that we detected in wildcats. This might indicate that FeLV is mainly transmitted between wildcats, with little or no reciprocal transmission with domestic cats. Studies on domestic cats often fail to include stray animals, which are more frequently in contact with wildcats, are often unvaccinated, and may therefore transmit not only FeLV but also FIV or other pathogens (Maruyama et al. 2003; Gerhold and Jessup 2013; Candela et al. 2022). However, there are a few studies dealing with viral pathogens in stray domestic cats (Nájera et al. 2021; Candela et al. 2022; Alves et al. 2023) and these also show lower prevalences for FeLV than our study (Nájera et al. 2021). Wildcats may therefore form a reservoir for pathogens that have been gradually eliminated from the domestic cat population thanks to vaccination recommendations (Ständige Impfkommission Veterinärmedizin 2023) and strict hygiene measures.

The high prevalence of FeLV antigens detected in our study is cause for concern, as FeLV can be associated with serious secondary diseases (Lutz 2019) that are often fatal. Additionally, the sampling method used leaves unanswered the question of how many wildcats actually die from FeLV-associated diseases.

The origin of the viral pathogens in the analyzed wildcat population remains unknown. It is important to consider the potential impact of domestic cats on the wildcat population. The increasing spread of wildcats may lead to more contact with domestic cats and wildcats from other populations, potentially increasing the transmission of pathogens. Therefore, it is crucial to implement strict monitoring programs to detect, and if possible prevent, any population collapse caused by viral pathogens at an early stage.

We thank the German Federation for the Environment and Nature Conservation, Friends of the Earth Germany, state association of Rhineland-Palatinate (Bund fuer Umwelt und Naturschutz Deutschland [BUND], Landesverband Rheinland-Pfalz) for collecting and providing the wildcats. All carcasses of F. silvestris were collected within the framework of the project “Monitoring of dead wildcats in Rhineland-Palatinate (Totfundmonitoring Wildkatze in Rheinland-Pfalz)” of the German Federation for the Environment and Nature Conservation, Friends of the Earth Germany, state association of Rhineland-Palatinate BUND. Collection of dead found F. silvestris was permitted by the responsible Nature Conservation Agencies. It was not an animal experiment (JLU_kTV_14_2022). At the time the study was conducted, Simon F. Müller and Nicole Nagler were employed at Laboklin GmbH & Co. KG (Bad Kissingen, Germany). The measurement of samples of this study was discounted by Laboklin GmbH & Co. KG to support local wildlife research.