Almost three quarters of the wildcats (70.3%) were infected with at least one cardio-pulmonary nematode species, confirming previous reports on the presence of A. abstrusus, T. brevior, A. chabaudi and Capillaria spp. in F. silvestris in Germany [24, 25].
In Europe, the prevalences of lungworm species vary between different studies. While the present study revealed a prevalence of 42.2% for A. abstrusus, Steeb  detected nematode stages in 27 of 85 (31.8%) histologically examined lungs of F. silvestris from different parts of Germany and Luxembourg, of which 21 (24.7%) were infected with larval stages of A. abstrusus. In Greece, infection rates were comparable with a prevalence of 43.5% in necropsied wildcats , whereas the prevalence in Romania was lower with 4.3% . In Italy, prevalences ranged from 0.0 to 62.5% [19, 20]. This high variance in Italian prevalences and the low prevalence in Romania may be explained by methodological differences. Veronesi et al.  dissected damaged lung tissue with forceps resulting in 62.5% A. abstrusus-positive lungs, while Falsone et al.  and Deak et al.  examined rinses of squeezed lung tissue with no or only low detection of A. abstrusus, respectively.
Although T. brevior has already been detected in European wildcats in Germany, data on its prevalence have not been available so far . The T. brevior prevalence of 31.3% found in the present study was higher than expected, as this parasite was previously assumed to play a more important role in southern rather than in more northern European countries . In Southern Europe, lower, similar or higher prevalences ranging between 14.9 and 71.4% were observed for F. silvestris in Romania, Greece and Italy, respectively [19, 20, 22, 23]. In domestic cats, lower rates of 1.4–14.2% were reported in Bulgaria, Spain and various regions of Italy based on faecal examination . The rather high prevalence detected in the current study could be due to an increased occurrence in Germany in recent years due to climate change, as T. brevior develops most rapidly at 22–27 °C in intermediate hosts such as Helicella spp. . Moreover, the increasing distribution of T. brevior’s natural host, namely F. silvestris, might be positively related to the occurrence of this parasite. Another possible hypothesis is that T. brevior might have already been endemic in Germany for a longer period, but was usually misdiagnosed in routine faecal diagnosis of domestic cats, probably mainly in clinical settings, because of the high similarity of L1 to those of A. abstrusus [8, 16].
Compared to other feline cardio-pulmonary nematodes, A. chabaudi has been neglected for a long time. More than 50 years after its first description in a European wildcat , this parasite was “rediscovered” in a domestic cat in Italy in 2014 . Since then, further cases of infected wildcats in Romania and Bosnia and Herzegovina have been reported [36, 37]. Moreover, prevalences of 6.3–56.5% in dissected wildcats were found in Italy, Romania and Greece, respectively [20, 22, 23]. The prevalence of 53.1% determined in the present study may be slightly underestimated because the hearts of five wildcats negative for A. chabaudi in their lungs were not available for examination. However, the prevalence rate is similar to that observed in Greece and higher than that in Italy. In both the present and the Greek study , A. chabaudi was the most common cardio-pulmonary parasite and was correspondingly detected more frequently than its much better known relative A. abstrusus. Due to its frequent occurrence, more attention should be paid to A. chabaudi, and further studies on its life cycle are needed to unravel potential transmission cycles to wildcats (and domestic cats [35, 38]) and its pathogenicity.
The determined prevalence of Capillaria spp. in the examined wildcats was 3.1%. In contrast, Krone et al.  detected C. aerophila in 13.3% (2/15) F. silvestris from the German federal states North Rhine-Westphalia, Rhineland-Palatinate and Saarland. In Italy, Greece and Romania, C. aerophila prevalence ranges from 18.8 to 34.0% [19, 20, 22, 23]. A possible explanation for the low prevalence in the present study could be the examination method, where additional rinsing of the opened trachea and bronchi might have resulted in higher recovery, as Capillaria specimens may have been missed in the mucosa.
Unfortunately, species identification of Capillaria spp. by sequencing 18S rRNA and Cox 1 amplicons was not feasible. While the 18S rRNA shows a high degree of conservation, allowing reliable genus determination , the mitochondrial Cox 1 gene harbours high intraspecific polymorphisms as observed in C. aerophila isolated from dogs and cats in Italy, where eight C. aerophila haplotypes were observed with differences ranging from 0.4 to 5.5% . The three Cox 1 sequences obtained in the present study differed by 14.9–16.5% from the above-mentioned C. aerophila sequences, indicating that the found Capillaria sp. represents at least a new haplotype, if not a new species. The parasites’ worldwide spread and wide range of different hosts including cats, dogs and many different wild mammals could explain the existence of highly diverse haplotypes. Unfortunately, the haplotypes of C. aerophila are unknown in both wildcats and domestic cats in Europe, with the exception of Italy [18, 20]. However, the observed high genetic distance to C. aerophila, comparable to that to C. boehmi, rather implies that the species found here is not C. aerophila. Capillaria boehmi, another capillariid of low host specificity  that occurs in the respiratory tract of carnivores (mainly foxes, mustelids and dogs ), can not only be deferred because of its low nucleotide identity, but furthermore excluded because of its specific localisation in the host . While C. boehmi parasitises the upper respiratory tract, i.e. nasal cavities and sinuses , the Capillaria specimens in this study were detected exclusively in the trachea and bronchi. Interestingly, these detected specimens showed a high degree of Cox 1 nucleotide identity (98.5–100%) with a Capillaria specimen from an Australian cat (GenBank acc. no. MH665363), which merits further investigations.
About one-third of the infected wildcats showed single infections, whereas approximately two-thirds harboured two or three cardio-pulmonary nematode species. The most frequently detected coinfection was that of A. abstrusus and A. chabaudi, which affected about a quarter of the positive wildcats. Thus, this infection was also the most frequent overall, regardless of whether single or coinfections were considered. The second most common infection was a single infection with A. chabaudi and a triple infection with A. abstrusus, A. chabaudi and T. brevior, each with equal percentages. However, unlike the coinfection of A. abstrusus and A. chabaudi, these were not statistically significantly associated. In other studies from Italy and Romania, coinfections with 2–4 different species accounted for about one-third of all infections. The most frequent coinfections were those of A. abstrusus and T. brevior and of A. chabaudi and C. aerophila in 20.0 and 13.8% of positive animals, respectively [20, 23].
Besides coinfections, the intensity of infection can have various effects on the health of a wildlife population . In this study, infection intensities ranged from a single to up to 167 specimens. In most cases, however, the wildcats suffered only from mild infections with a few specimens of each parasite species. However, it has to be considered that experimental factors may have contributed to the low infection intensity, such as limited detection of partially decomposed worms or their outright loss due to decomposition. Furthermore, the A. chabaudi intensity may have been higher in the 12 animals in which the lungs were positive but no hearts were available for examination. Nevertheless, also other studies report comparably low infection intensities, with three T. brevior individuals found in a wildcat from Romania  and 9–12 A. chabaudi in F. silvestris from Bosnia and Herzegovina, Greece and Romania [12, 36, 37].
The prevalence of parasites in wildlife is influenced by many factors . In the present study, sample condition as an indirect factor and all intrinsic factors tested (sex, age, nutritional condition) influenced the prevalence of at least one cardio-pulmonary nematode species, whereas the extrinsic factor ‘month of finding’ had no effect. The significantly higher prevalence of A. abstrusus and total cardio-pulmonary nematodes in fresh than in moderate fresh or rotten carcasses is attributable to the corresponding decomposition of the parasites, with the very thin A. abstrusus in particular becoming lytic and therefore difficult to detect. In contrast, T. brevior was detected significantly more often in moderate fresh/moderate rotten than in fresh individuals, indicating that a mild decomposition of host tissue does not affect the detection of larger parasites.
Regarding intrinsic factors, the significantly higher T. brevior prevalence in male than female wildcats may be explained by the immunosuppressive effect of testosterone  or behavioural differences. Compared to female F. silvestris, males require more prey and have a wider prey spectrum due to their larger size and home ranges , making them more likely to have higher infection rates. However, higher infection rates in males were not observed for other parasite species, indicating that sex-dependency for T. brevior might rather be a random effect. Moreover, the occurrence of not only male- but also female-biased parasitism, being favoured by e.g. immunosuppression during pregnancy and lactation, is frequently reported; therefore, the effect of sex on parasite infection is often controversially discussed . Host age had a significant effect on the prevalence of A. abstrusus and total cardio-pulmonary parasite infections, which were more common in adult wildcats than in younger age groups. This observation was already reported for A. abstrusus in domestic cats . An opposite age dependency was observed regarding T. brevior infection, where immature (5–10 months) animals were significantly more often infected than adults (> 25 months). In Italy, the same observation was made for domestic cats, with younger individuals (≤ 6 months and < 1 year, respectively) being more frequently infected than cats in older age groups [48, 49]. Age dependency might be mediated by differing lifespans of and immunity against the respective parasites. Clinical studies with experimentally infected domestic cats show that infections with A. abstrusus last for at least 25 weeks , whereas infections with T. brevior start to resolve after 6–9 weeks . The long lifespan of A. abstrusus and the potentially impeded development of immunity may favour the accumulation of this parasite in the host, whereas the short lifespan of T. brevior and a presumed development of efficient immunity counteracts accumulation and reinfection, thus limiting its occurrence mainly to younger animals. Although the lifespan of the parasites and/or the immune response might differ between domestic cats and wildcats, the effect of nutritional condition, which was negatively correlated with the infection rate of T. brevior, with animals in bad/cachectic condition being more frequently infected than those in good/very good condition, also supports this hypothesis. For instance, a poor condition may favour parasite persistence due to a weakened immune response, while parasite infection itself can lead to the deterioration of body condition, for instance by damaging host tissue [42, 52]. To confirm this hypothesis, further studies on feline immunity to cardio-pulmonary nematode infections are needed.
Unfortunately, it is difficult to relate the determined parasite infection intensities to the wildcats’ health status as no further examinations could be performed and only few comparative data are available. Histological lung examination of single or coinfected wildcats revealed mild to severe pulmonary lesions and (broncho-)pneumonia attributed to A. abstrusus, T. brevior and/or A. chabaudi [20, 37]. Affected animals may also suffer clinically as shown for a wildcat infected with A. abstrusus, T. brevior, A. chabaudi and C. aerophila, which was hospitalised in very poor general condition with respiratory symptoms and severe lung lesions on radiograph . Such reports, although rare, demonstrate the potential impact of cardio-pulmonary parasites on the health of individual European wildcats, especially when animals suffer from coinfections, which may exhibit increased pathogenicity compared to single infections . In the present study, however, about two-thirds of the infected wildcats were coinfected with two to three cardio-pulmonary nematode species, but only a small proportion was in bad or cachectic condition, suggesting that such multiple infections alone are usually not causative of poor health and that other factors contribute to this condition.
While knowledge on wildcats is rather poor, the clinical impact of lungworm infections in domestic cats is more comprehensively described. In these, A. abstrusus and T. brevior can cause severe respiratory symptoms and T. brevior infections of kittens in particular can be fatal [11, 13, 55, 56]. Infections with C. aerophila may also result in bronchial disease with cough and wheezing . In contrast, the effects of A. chabaudi infections in domestic cats are unknown to date. In one of the two published cases, no pathohistological examination of the road-killed cat was reported , and in the other case, the cat suffered from a mixed infection with A. abstrusus and T. brevior . The necropsy of this cat, which showed severe respiratory distress and died shortly after admission, revealed no parasitological or pathological findings consistent with cat angiostrongylosis. Nevertheless, no conclusions about the (lack of) pathogenic potential of A. chabaudi in domestic cats can be drawn from a case with only two adult nematodes detected. Therefore, a potential emergence in domestic cats poses an unpredictable health impact .
Overall, there is a risk of spillover of feline cardio-pulmonary parasites at the wildlife-domestic animal interface. Not only do the different parasites share the same intermediate and paratenic hosts, e.g. small rodents, birds and other vertebrates [7,8,9], but these hosts, especially small rodents, are also preyed upon by both wild and domestic cats. Thus, indirect transmission from European wildcats to domestic cats and vice versa can be assumed [20, 24, 34]. The high prevalence of cardio-pulmonary nematodes in wildcats observed in the present study may favour their indirect transmission to domestic cats and pose a serious health threat; therefore, awareness of lungworm infection in domestic cats should be raised.