A cross-sectional study of Trichinella spp. infection in wolves (Canis lupus) reveals first evidence of T. spiralis in the species in Poland

Trichinella spp. are zoonotic nematodes which have been confirmed in more than 150 species of animals (15). Wild carnivores are considered the main natural reservoir, which include the Canidae, Felidae, Ursidae, Mustelidae and Procyonidae families as they are exposed to Trichinella infecting their prey (26, 42, 59). However, omnivorous and herbivorous animals also participate in sylvatic circulation of these parasites, and it is these which are believed to be mainly responsible for human exposure (12, 15, 41, 62, 63). The simple life cycle of this pathogen allows for frequent host changes, especially in the natural environment. The higher the number of different hosts infected with Trichinella, the higher the probability of carnivore infection. Wild carnivores being the largest harbour for Trichinella, it follows that the parasite biomass is observed to be higher in wildlife populations than in farm or domestic animals. Although, the most common human infections were caused by the consumption of pork meat containing live Trichinella larvae in the past, wild boar meat has taken on significance as a source of infection for human and domestic animals. With the increase of the size of the population wolves (Canis lupus), the largest extant member of the Canidae family, and a species native to Europe, America and Asia, their role as a possible link in the transmission of pathogens of public health concern is growing. According to the recent report of Boitani et al. (17), the total number of wolves in Europe (excluding Belarus and the Russian Federation) increased significantly over the five years reaching the estimated level of 21,500 animals in 2022. In Poland, wolves were exterminated until 1998, when the species became legally protected; therefore, access to samples originating from this species was limited and except for inhabitants of the Polish Carpathian Bieszczady Mountains, their abundance underrepresented the population (10, 18, 62). However, the strict protection policy in place for this carnivore, its lack of natural enemies and the availability of wild ungulate prey caused the rise of the number of these animals from 770 in 2010 to 4,328 in 2022 (72). Poland is inhabited by one of the largest wolf populations in Europe, only Romania and Italy having comparable populations (17). The European wolf population consists of nine subpopulations, of which three (Carpathian, Baltic and Central-European) cohabit Poland (17). The range of the wolf in Poland, which a few years ago was limited to the eastern part of the country, has expanded to western regions, creating a transboundary Central-European population spreading further into north-eastern Germany (17, 50). As a result, the access to grey wolf carcasses for diagnostic and epidemiological purposes is increasing, which enables researchers to obtain more information about the circulation of Trichinella spp. in these wild animals (43).

Grey wolves play an important role in keeping ecosystems in balance (31). Wolves hunt in packs, and the same sociality which prompts this behaviour and makes them excellent predators also makes them good facultative scavengers (43, 77). In Europe, the grey wolf diet consists mainly of medium-sized wild ungulates such as roe deer (Capreolus capreolus), wild boar (Sus scrofa) and chamois (Rupicapra rupicapra) but they also hunt larger animals, e.g. red deer (Cervus elaphus) and elk (Alces alces), and small mammals, e.g. beavers, rodents and hares (51, 61). Wolves occasionally may also prey on smaller carnivorous species including foxes, badgers, dogs and cats, or even demonstrate cannibalism associated with their agonistic behaviour and territorialism (43). The feeding habits of wolves predispose them to being frequently exposed to Trichinella infection. The presence of Trichinella spp. in grey wolves has been previously reported in many European countries. Trichinella britovi dominated in south, central and eastern European countries, e.g. Lithuania (31), Latvia (22), Estonia (47) and Italy (4, 35); and T. nativa did in northern Europe, e.g. Finland (1) and Russia (60). So far, T. spiralis has only been detected in grey wolves occasionally, e.g. in Croatia (8), Germany (39) and Finland; and T. pseudospiralis has been detected only once in these carnivores in Italy. None of the previous studies have confirmed species other than T. britovi in wolves in Poland. The most recent data on the infestation of these parasites in wolves date from a decade ago (10). Since then, trichinellosis in wolves in Poland has been understudied, while the population of this predatory species has at least doubled and wolves have come to be widely reported in urbanised areas in most of the country.

The aim of this work was to update the data on the prevalence of Trichinella spp. in grey wolves in Poland. This article is the first study reporting the presence and molecular identification of T. spiralis in this species in the country.

The high level of forestation, the abundance of game which is the basic food supply for wolves, the lack of natural enemies in the wild and the complete protection of the species have created ideal conditions for the expansion of the wolf population in Poland. As an excellent predator but also a scavenger, every wolf is likely to be exposed to feed that may contain Trichinella larvae during its lifetime. The prevalence of these parasites in wild carnivores therefore depends on the population size of and Trichinella species prevalence in infected animals from other host groups in a given area. Wolves are not only considered an important reservoir and carrier of Trichinella, but also an indicator species (74) for parasite environmental contamination and human risk exposure (44). The overall prevalence of Trichinella spp. of 44.8% in the tested wolves in this study was comparable with those in previous reports in the country (Table 3) (10, 18, 51). However, while the studies revealed Trichinella infection in more than half of wolves, they only sought to identify T. britovi larvae in a rather limited group of animals in terms of numbers and origin (10, 18, 26, 51). Similar infection rates were recorded in Serbia (46.5–49.5%), Bosnia and Herzegovina (38.89%) and Finland (33–39.2%) (Table 3). Interestingly, in the countries where the wolf population size is as large as in Poland, such as Italy, Romania or neighbouring Germany, the Trichinella prevalence in the species is in general lower, especially in the studies that used a representative number of animals. The Italian prevalence ranged from 8.9% to 31.0% (3, 4, 24, 43, 61, 69, 71), the Romanian from 30.5% to 31.0% (16, 25) and the German from 3.70% to 4.04% (30, 39). Notably higher prevalence was recorded in wolves in Latvia, where it was 69%–100% (6, 7, 22); in Estonia, where researchers noted 63.2–79.4% (29, 54); and in Russia, where up to 97.5% of wolves carried Trichinella (20, 54, 60) (Table 3). In some other part of Europe, Spain being an example, the incidence of Trichinella in wolves was low; however, these reports are quite dated and based on limited numbers of wolves (Table 3). A fairly recent report from Sweden, where the wolf population is quite small and still regulated by culling (36), showed a very low prevalence of Trichinella (5.6%) (40) (Table 3). In our study, the prevalence differed significantly between different regions of Poland, which may be connected to the different prey structure, biology and parasite circulation in the relevant local environments. Attention should also be paid to climatic variables, as increasing temperatures and reduction of environmental humidity may lower the survival time of larvae in host carcasses (58). On the other hand, environmental changes may influence the biology and ecology of the main host species, reducing their number and changing their age composition, the changes having the possible forms of habitat fragmentation due to the expansion of the wildlife–human interface or shortening of trophic chains, inducing stress, which may lead to host–parasite disequilibrium. Parasitic invasions are flagship examples of climate-sensitive infections, which can trigger outbreaks or epidemics caused by opportunistic and neglected parasites (34).

The overall number of Trichinella larvae in the muscle tissue of 43 wolves varied between 0.020–5.92 per gram of tissue, with a mean of 0.42, which suggested less extensive invasion than was found in previous studies, among others in those of Bień et al. (0.009–27 lpg) in Poland (10), Omeragić et al. (mean 6.9, range 0.6–33 lpg) in Bosnia and Herzegovina (55) and Martinez-Carrasco et al. (range 0.8–45 lpg) (43), Scancerlli et al. (range 1–1,070 lpg) (68) and Ricchiutti et al. (mean 4.5 lpg) in Italy (65). However, each of these studies reported T. britovi or T. pseudospiralis infections but not any of T. spiralis. Pozio (61) pointed out that the extent of infection with T. britovi was higher than that of infection with T. spiralis and explained that by the different characteristics of these nematode species (57), which was confirmed in the studies of Omeragic et al. (55) and Blaga et al. (16). The findings of our study do not fit this hypothesis, because the mean lpg for T. spiralis- infected wolves was slightly higher than that for T. britovi infection (although the difference was not statistically significant). We found, however, statistical significance in the difference between the larval burdens in three different muscles of wolves infected with any Trichinella species, higher potential to be the predilection site for Trichinella larvae in wolves emerging for the forelimb and tongue muscles. However, the frequency of parasite detection in the latter might have been unrepresentatively low because of the impossibility of testing the some animal heads because they had already been removed for rabies testing and because of some technical difficulties in sample processing to those encountered by Sharma et al. (70). Our results correspond to findings by Ricchiutti et al. (65), who reported a higher larval burden in tibial muscle, with a mean of 36.9 lpg (39–1,070), than in diaphragm pillars, in which the mean burden was 8.7 lpg (1–174). Also Sharma et al. (70) found that wolverine (Gulo gulo) diaphragm samples showed much lower larvae burdens than tongue samples. Similarly to them, we also did not find any correlation of the age or sex of the animal with the Trichinella prevalence or larvae load, except for in the pups. This is also confirmed in the investigation among Alaskan wolves by Zarnke et al. (80). Several researchers have tried to determine the relevance of the differences in location of larvae, and Kapel et al. (32) concluded that it may be dose-dependently related to the way a species obtains food from another species which is a Trichinella host, which implies a lifestyle factor or the actual involvement of particular muscles as an animal acquires and consumes food. Kozar and Kozar (33) tried to confirm the greater affinity of the Trichinella larvae for the muscle parts which work more intensely by initiating increased exercise in mice. The results obtained have important diagnostic significance, indicating that the appropriate muscle should be selected for the detection of Trichinella larvae. Despite the detection of the highest number of larvae in the tongue, it seems that a better choice for the researcher should be the forelimb muscles because of the consistently higher lpg level and lack of problems with its digestion; they offer greater ease of preparation than the wolf tongue observed in the study which was not digested easily.

This is the first report on the occurrence of T. spiralis in wolves in Poland, and one of few in Europe (Table 3). The infection of almost half (45%) of Polish wolves with T. spiralis suggested changes occurring in the wolf-trichinella-environment triad. Such a high T. spiralis prevalence in wolves had never been reported before, and this is the first evidence of this parasite species in wolves in Poland (10, 18). Ricchiuti et al. (65) summarised extant studies and found that in a total of 488 wolves from European countries, 67.8% were identified as infected with T. britovi, while only 2.5% were found to be infected with T. spiralis. These latter originated from Croatia, Finland, Germany, Serbia and Spain (Table 3). In these countries (except Finland), T. spiralis is constantly circulating domestically or has only been eradicated quite recently, and more importantly, it is also actually detected in the sylvatic environment in a higher percentage of wild boars than T. britovi is (5, 23, 30, 76). In Poland, Trichinella has been almost eradicated from pigs, leaving the prevalence at 0.000088% (14). It is, however, still at 0.3% prevalence in wild boars, with the predominant species being T. spiralis (13). The high burden of the larvae in wild boar biomass and high number and density of the wild boar population in Poland factor into the maintenance of the circulation of T. spiralis in the environment. In the study, the high prevalence of T. spiralis in wolves discovered in north-western Poland coincided with the endemic occurrence of this parasite species in wild boar in the area and adjacent areas of Germany (11, 30). The prevalence of T. spiralis in the wild boar population in this region certainly has an impact on the spread of this nematode species in wildlife, as evidenced by the detection of T. spiralis in the foxes from this area (13). The occurrence of T. britovi in wolves in north-eastern (Podlaskie) and south-eastern (Podkarpackie) provinces is also consistent with the higher ratio of T. britovi to T. spiralis in wild boar and foxes in these provinces in relation to the north-western part of the country (13). Therefore, not only the prevalence but also the nematode species identified in wolves will depend on which host species are available prey and on the prevalence and larvae load in that prey.

The significance of wolves in Trichinella transmission is not fully understood. As an apex predator often preying on wild boar, they should be considered as a good indicator of the Trichinella presence in the environment, especially in Poland, where the nematode’s circulation is now concentrated mostly in the sylvatic cycle. However, while wild boar, red foxes and rodents are considered the main reservoirs responsible for the maintenance of Trichinella spp. in the environment, wolves may contribute to its long-distance dispersal as they may migrate hundreds of kilometres to establish new packs or populations or in search of an adequate food base (48, 56, 73). Also because of their growing numbers in the country, wolves are increasingly approaching human settlements, which may be an increasing public health concern (44, 65). The wolves included in the study mostly died in traffic accidents; therefore, they may mainly represent migratory individuals in dispersal, often referred to as compromised. Wolves generally live and prey in packs, and their survival is dependent on their social skills and certain behaviour such as evasion of humans. Therefore, we should consider that the wolves studied did not accurately represent the entire population, and that the noted prevalence of Trichinella may correspondingly be unrepresentative, because such individuals usually did not hunt on their own when they had a pack and now they are outside it, more often feed on smaller mammals or carrion. They are observed preying and scavenging on wild boar, which, in our opinion, may be the explanation for the high percentage of these wolves infected with T. spiralis. While wolf exposure to Trichinella is associated with the prevalence in their prey, there may be a possibility of spill-back from the predator to the parasite maintenance host, albeit probably more incidental than regular in occurrence (43). No data is available on wild boar scavenging on wolves, and some reports suggested that wild boar would not feed on wolves because of their fear of the large predator, contrasting this with boars’ ready scavenging on foxes (64). However, a wolf carcass may undoubtedly be consumed by other scavengers such as ravens, small rodents or other predators like foxes, badgers, raccoon dogs, bears and even other wolves, from which a risk derives for further Trichinella transmission and new outbreaks of trichinellosis. In the situation when almost 45% of wolves are infected by either T. britovi or T. spiralis, such transmission is quite likely to occur. Another possible reservoir of T. spiralis and other species, albeit one not yet reported, are beavers (66), which increasingly fall prey to wolves and constitute as much as 45% of their diet in some areas of the country (51). The last question is the impact of Trichinella infection on wolves’ health. Wolves are still protected species under the Bern Convention, despite their status having been downgraded from “strictly protected” to “protected” quite recently. Unlike humans, for whom trichinellosis has serious clinical consequences, animals rarely show any clinical signs of the disease (59). The parasite may be associated with abnormal behaviour because of the pain or discomfort caused by larvae migrating trough the muscle tissue, as was suspected for aggressive grizzly bears (78). Such impact was also suggested of exposure to another parasite, Toxoplasma gondii, which altered the behaviour of wolves so that they took more risks, including dispersing from their pack or seeking to become pack leaders (45). Hence, the ecological effect of Trichinella infection in wolves needs further studies.

Since the carcasses of wolves and other carnivores may be a source of Trichinella larvae which are relatively resistant to environmental conditions (57, 66), a recommendation to remove and dispose of them should be made as a prevention measure to limit the pathogen circulation and the further exposure of humans and animals to infection.