Trichinella spp. are ubiquitous nematode parasites with a broad host spectrum. In humans, there is trichinellosis, a foodborne zoonoosis affecting a massive number of people worldwide (Dupouy-Camet, 2000; Pozio, 2007). Modern taxonomic studies indicate that Trichinella includes 8 valid species and 4 genotypes (Pozio, Hoberg et al., 2009). The genus has also been proposed to form 2 clades, i.e., encapsulated and non-encapsulated (Pozio and Murrell, 2006). All species of Trichinella can infect humans (Dupouy-Camet, 2000). Trichinella spiralis has typically been associated with pork in a domestic ( = synanthropic) cycle, while other species are more often linked with wildlife in a sylvatic cycle (Kozar and Kozar, 1965; Chadee and Dick, 1982; Kjos-Hanssen, 1984; Kapel et al., 1998; Webster et al., 1999; Murrell and Pozio, 2000). The red fox and wild boar have been identified as typical reservoir animals for Trichinella spp. in the European Union (EU) (Murrell and Pozio, 2000; Nöckler et al., 2006; Pozio, Rinaldi et al., 2009). Although human infection with Trichinella spp. has been increasing in some EU countries, such as Romania and Bulgaria (Cuperlovic et al., 2005), in many countries no autochthonous human cases have been diagnosed for decades; moreover, no meat inspection findings from industrialized pork production have been made (Anonymous, 2005a). Therefore, new EU regimens allow countries to request derogations for mandatory Trichinella sp. testing in “areas with negligible Trichinella risk” (Anonymous, 2005a). This has intensified the need for regular surveys and a deeper understanding of the epidemiology of sylvatic Trichinella spp. infections in Europe (Anonymous, 2005b).

In Finland, meat inspection has revealed a small to worrisome number of swine infections starting in the early 1980s and peaking in 1996; more than 200 swine were found to be infected at the maximum point, after which the number of cases decreased until 2004, when the last infected swine was diagnosed. The reduction was probably associated with the swine industry's modernization process (Oivanen and Oksanen, 2009). As a human disease, trichinellosis is very rare in Finland, with only 8 human infections reported since the late 1800s and the last one more than 3 decades ago (Oivanen, 2005). Nonetheless, several wildlife surveys have indicated a high prevalence in lynx (Oksanen et al., 1998; Oivanen et al., 2002), foxes, raccoon dogs, and other carnivores (Freeman, 1964; Hirvelä-Koski et al., 1985; Oivanen et al., 2002). In older studies, no molecular methods for species-specific identification of Trichinella spp. were available. Moreover, previous surveys have had representative sample sizes from southern parts of the country, but only a limited number of samples from the northernmost part, i.e., Finnish Lapland. In the present survey, distribution of Trichinella spp. in host species was analyzed, with an emphasis given to the northern part of the country.

The aims of this study were: (1) to describe the distribution of Trichinella spp. infection in Finnish carnivorous wild mammals; (2) to analyze how infection intensity varies among animal hosts and Trichinella species; and (3) to determine if, and if so, how the infection probabilities depend on the population density of animal species.

MATERIALS AND METHODS

Finland is located between the latitudes of 60° and 70°. It is bordered by Russia in the east, Sweden in the west, and Norway in the north. Approximately half of the western border to Sweden is across the Baltic Sea. In the south lies the Gulf of Finland, across which Estonia is located. To manage game animals, Finland is divided into 15 administrative units, i.e., game management districts, or GMDs,  http://www.riista.fi (Fig. 1). The southern part of the country has a northern temperate climate, whereas the northern part has sub-Arctic conditions. During a normal winter, the whole country is snow-covered, with the mean monthly temperature in winter (December–February 1971–2000) varying from –2 C to –14 C for the south and north, respectively ( http://www.fmi.fi/saal/), (Fig. 2).

Figure 1

Sample prevalence of Trichinella spp. in 15 Finnish game management districts. All 5 host species with sample sizes of over 100 individuals are shown, i.e., lynx (Felis lynx), brown bear (Ursus arctos), wolf (Canis lupus), raccoon dog (Nyctereutes procyonoides), and red fox (Vulpes vulpes).

Figure 2

Trichinella spp. distribution by municipality with mean winter temperature isotherms in Finland (based on mean monthly temperature from December to February during 1971–2000). One symbol for each species in a given municipality is indicated irrespective of the number of positive samples observed.

Analyzed hosts

The carnivore sample consisted of 2,483 animals from 9 species (Table I), including 1,010 red foxes (Vulpes vulpes), 662 raccoon dogs (Nyctereutes procyonoides), 402 lynx (Felis lynx), 125 brown bears (Ursus arctos), 102 wolves (Canis lupus), 75 pine martens (Martes martes), 53 badgers (Meles meles), 31 otters (Lutra lutra), and 23 American minks (Mustela vison).

Table I

Sampled carnivores in Finland during 1995–2005, discovered Trichinella-infected animals, sample prevalence with ranges between different game management districts (GMDs), number of observed carnivores, median infection densities with ranges, and estimated population sizes of the studied carnivores.

RESULTS

Of the 2,483 animals analyzed, Trichinella species were found in 617. Different host species showed varying sample prevalences (range 0–46%; Table I) Almost half (46%) of the lynx (Table I) harbored Trichinella spp., followed by wolves (39%), raccoon dogs (28%), and red foxes (19%). Prevalences of less than 10% were detected in pine martens, badgers, bears, and otters. No larvae were detected in American mink samples.

It is noteworthy that, in a given animal species, prevalence varied markedly between GMDs, e.g., the range for red fox was 0–62% and the range for lynx 8–80% (Table I). The overall Trichinella spp. prevalence from all sampled host species was also unequally distributed (Wald's P < 0.001), varying from 2.6% in northern Finland (Lapland) to 67% in the middle of the country. However, only 6 of the sampled animals were from the district of the highest prevalence. The 3 northernmost GMDs (Fig. 1) had the lowest sample prevalence (a total of only 6% for all Trichinella spp. infection cases, but 37% of all studied samples). Sample prevalence of all individual host species in the GMD showed a clear tendency to be higher in the south (Fig. 1). Moreover, the prevalence showed variation within each GMD also in common host species, such as the raccoon dog (data not shown), indicating patchy distribution at the local level.

Variable autocorrelation caused fundamental obstacles when analyzing geographical and host species–specific differences in prevalence by multivariable logistic regression, i.e., most of the independent variables intended for exploring the effect showed high correlation (Table II). The most abundant and ubiquitous carnivores in the country are red foxes and raccoon dogs; to simplify the correlation matrix, these 2 species were combined. The combined Trichinella spp. prevalence in red foxes and raccoon dogs was positively correlated with the abundance of raccoon dogs and negatively correlated with the north-south coordinate. This indicates that the prevalence of positive raccoon dogs and red foxes decreases toward the north. In addition, the prevalence of Trichinella spp. infection was high when there were high numbers of raccoon dogs in the area. Red foxes, raccoon dogs, and the WRI for hosts, i.e., the abundance of raccoon dogs, red foxes, lynx, and wolves, also decreased toward the north (Table II).

Table II

Correlating associations (Rs) between the regional abundance of Trichinella hosts, regional sample prevalence, and geographic gradient in Finland based on data from 1999 to 2005. Spearman's rho correlation matrix with correlation coefficient, and 2-tailed P value (in parentheses). Measurements are at game management district level (15 game management districts, GMDs, except 11 for proportion of Trichinella infection*).

Trichinella species were successfully identified in 303 animals by multiplex PCR; for 25 animals, amplification did not yield a specific reaction (7.6% of all 328 animals analyzed). Four species were identified, i.e., T. spiralis, T. nativa, T. britovi, and T. pseudospiralis. Single Trichinella species were found in 281 (93%) of the host animals successfully analyzed, with mixed infections in 22 (7%). Trichinella nativa was the most common single species (80.1%), followed by T. spiralis (12.8%), T. britovi (6.0%), and T. pseudospiralis (1.1%), the last of which was found as a single infection in only 3 animals, and in a mixed infection in 4 more individuals. From mixed infections, more than 2 different species were never found, but all possible 2 species combinations of the 4 species were observed. The most common combination was T. nativa with T. britovi (9/22), followed by T. nativa with T. spiralis (8/22), and T. nativa with T. pseudospiralis (2/22). Other possible combinations (T. spiralis with T. britovi or T. pseudospiralis and T. britovi with T. pseudospiralis) were seen only once.

All 4 Trichinella species were present in the southern part of the country (Fig. 2); in the northern part, only T. nativa and T. spiralis were found. Moreover, all 4 species were sympatric in areas where the mean winter temperature is from –2 C to –10 C (monthly average from December to February during 1971–2000;  http://www.fmi.fi/saa/).

The Trichinella species were not equally distributed in the different host species (Pearson chi-square P < 0.05). All 4 Trichinella species were found as single or mixed infections in the lynx, red foxes, and raccoon dogs (Fig. 3). In all host species, the most common Trichinella species was T. nativa, which was found alone or in mixed infection in 80.5% of the 303 positive samples (Fig. 3). Of the 5 Trichinella-positive bears, 4 harbored T. nativa alone (1 could not be typed), as did the only positive otter in this material. Trichinella spiralis was recovered in single or mixed infections from 46 sylvatic animals of 303 samples (15%); the host species involved were the raccoon dog, red fox, lynx, and wolf (Fig. 3). Trichinella pseudospiralis was found in a single infection only 3 times, twice in the raccoon dog and once in the lynx, but in mixed infections twice in the lynx, once in a red fox, and once in a raccoon dog. Of all 28 animals with T. britovi, 46% were lynxes, 21% were raccoon dogs, and 21% were red foxes. Interestingly, lynxes were relatively more often infected with T. britovi than other species (Fig. 3; Wald's P < 0.05), and when compared with the raccoon dog, the latter had 3 times higher odds of being infected with T. spiralis.

Figure 3

Relative distribution of the outcome of Trichinella-specific multiplex-PCR by host species with 5 or more larval isolates (n), red fox (Vulpes vulpes), raccoon dog (Nyctereutes procyonoides), lynx (Felis lynx), brown bear (Ursus arctos), and wolf (Canis lupus).

The parasite burden was unequally distributed. Different hosts showed variations in infection density, and different Trichinella species also had different burdens (Fig. 4). One raccoon dog had the highest burden, 760 lpg, in a mixed infection of T. nativa with T. britovi. Only 3 cases with T. pseudospiralis alone were observed, but it also exhibited the highest single species burden (median 488 lpg; 569 lpg in 2 raccoon dogs and 15 lpg in 1 lynx). The burden was second highest in mixed infections (median 19 lpg), followed by single infections of T. spiralis, T. nativa, and T. britovi (medians 9.75, 8.50, and 3.00 lpg, respectively). Linear regression analysis showed that interaction of host species and Trichinella species has a significant (P  =  0.013) effect on infection density (lpg) when the red fox, raccoon dog, or wolf is used as a host in the model. To balance variation of sampling host species at different times, year was used as a covariate in the model. The interaction between animal species and Trichinella species is reflected in, for instance, T. spiralis having a higher larval burden in the raccoon dog than in other animals. However, in the raccoon dog, infection intensities did not differ significantly between Trichinella species (F-test, P > 0.05). In lynxes, T. spiralis was found less often than T. britovi or T. nativa (Fig. 3), and the intensity also differed from that of other Trichinella species, although both T. britovi and T. nativa had an equal burdens (median 0.40 lpg with T. spiralis vs. 2.0 and 1.4 lpg in T. britovi and T. nativa, respectively; log-transformed lpg, 1-way ANOVA without T. pseudopspiralis, P  =  0.04; Fig. 4).

Figure 4

Box and leaf plot of infection density as larvae per gram (lpg) of muscle by different Trichinella species in host species with 5 or more isolates in Finland during 1999–2005. The few “only pseudospiralis” cases are omitted.

A clear north-south gradient in both prevalence and animal population densities led us to compare reservoir animals at the regional level (Table I). The most abundant and ubiquitous host species were red foxes and raccoon dogs. These 2 host species were compared at the national level as reservoir species based on summarized data from the GMD level (Table III). At the national level, comparison of species-specific prevalence (Fig. 1) showed that the red fox was more often infected than the raccoon dog. When calculated as a proportion of estimated infected host animals of the estimated total population size at the GMD level (Table III), the range of the national prevalence was 19–47% for the red fox and 16–49% for the raccoon dog (Table III). When the numbers of estimated infected individuals were compared, there were 1.3–1.5 times more infected raccoon dogs than red foxes in the country. When infected tissue mass was compared, there were 1.4–1.7 times more Trichinella spp.–infected raccoon dog muscle. Raccoon dogs had 3.8 times higher average infection intensity than red fox (100.3 lpg vs. 26.4 lpg, respectively). Therefore, one can estimate that, at the national level, the raccoon dog as a host species carried a 5.4–6.3 times heavier Trichinella spp. burden than the red fox.

Table III

Estimated number of the population and infected individuals in Finnish carnivore hosts and Trichinella burden in the species calculated based on observed Trichinella prevalence in each GMD and population estimated based on annual bag in GMDs.

DISCUSSION

Sylvatic animals acquire Trichinella spp. infection by consumption of prey, by scavenging, or by cannibalism. The red fox has been identified as an important reservoir host, and by being ubiquitous in its distribution, it is a good indicator species for continent-wide comparisons. Davidson et al. (2006) have reviewed the literature and summarized European studies on parasite prevalence in red fox. Data from northern Europe show Trichinella spp. prevalence to vary from 0% in Denmark to 4.5% in Sweden and 4.8% in Norway. In the Baltic countries, prevalence in red fox is 28.9–40.6% (Malakauskas et al., 2007), 4.4% in northwestern Poland (Balicka-Ramisz et al., 2007), 16% in Slovakia (Hurníková et al., 2006), and 1.8% in Hungary (Széll et al., 2008). Earlier studies from Finland have reported varying prevalence, i.e., 16% (6/38) (Rislakki, 1956), 4% (4/105) (Freeman, 1964), 33% (5/15) (Hirvelä-Koski et al., 1985), and 37% (58/158) (Oivanen et al., 2002). In our investigation, the red fox prevalence varied in different districts from 0% to over 60% (Table I, Fig. 1). Thus, in Finland, conclusions based on infected individuals and the total number of analyzed animals from various parts of the country are problematic. This indicates that the uneven distribution of host species in the country must be considered before any national-level conclusions regarding the national prevalence of host species can be made. Although sylvatic Trichinella spp. infection is very common, large geographical differences in prevalence were found with all host species studied (Fig. 1). The southern part of the country exhibited very high sample prevalence, but in Lapland only moderate prevalence occurred, e.g., for the red fox, 53% in the south versus 1.4% in Lapland. Oivanen et al. (2002) made similar observations in the red fox, with limited numbers of positive animals from the northern part of the country and a prevalence of 4% (2/54) in Lapland, 52% (35/68) in the southwest, and 62% (21/34) in the southeast. Of reports from Europe, only the prevalence in Baltic countries is similarly intense to the southern part of Finland (Malakauskas et al., 2007). Interestingly, data from the Baltic countries indicate that wildlife prevalence has been increasing during the last few decades (Malakauskas et al., 2007). In other parts of the world, intranational geographical differences have also been identified (Prestrud et al., 1993; Széll et al., 2008). Therefore, representativeness of the sampling schemes should be considered when comparing different studies of Trichinella spp. prevalence intranationally or between countries. In Norway, Davidson et al. (2006) reported, based on red fox data, that in the southeastern part of the country, the prevalence was higher than in other parts, decreasing toward the north, similar to the pattern observed here.

Interestingly, combined Trichinella spp. prevalence in the red fox and raccoon dog was strongly and positively correlated with the population density of raccoon dogs in the GMD (Table II). This finding supports the earlier observation of a high raccoon dog population in an area being a risk factor for Trichinella spp. infection in Finnish lynxes (Oksanen et al., 1998).

When evaluating the importance of host species by estimating Trichinella spp. burden in the host species, the raccoon dog was shown to have a higher burden than the red fox, although the prevalence was higher in red foxes in most districts (Fig. 1). The raccoon dog population is relatively more concentrated in the southern part of the country, where the overall prevalence of Trichinella spp. is higher and the raccoon dog had a higher infection density (Fig. 4). Therefore, the Trichinella spp. burden is 5–6 times higher in raccoon dogs than in red foxes (Table III). The total mass of infective material from red foxes and raccoon dogs in the country is remarkably high, being 109,800–649,700 kg yearly (Table III); one may estimate (Kauhala, 2007) that half of this amount will be circulating in nature. This should be considered when informing hunters on how to deal with small carnivorous carrion. Hunting practices in Russia, where skinned wolves and other carnivore carcasses are commonly used as bait, are associated with an exceptionally high local prevalence of 97.5% (Pozio et al., 2001). Similar behavior may partially explain the patchy prevalence distribution in Finland, with “hot spots” in some GMDs. However, even without hunters, raccoon dog carcasses are commonly available for scavenging animals in the Finnish countryside. Raccoon dogs reproduce in good numbers, but many young animals die during the winter (compare autumn and spring data, Table I). Road kill, e.g., raccoon dogs and foxes, is commonly found along the sides of the motorways.

Trichinella nativa was the most common species identified among all sampled host species. This is congruent with other wildlife studies, indicating that T. nativa dominates in the Palearctic and Arctic, while T. britovi does so in more temperate regions (Handeland et al., 1995; Pozio and Murrell, 2006). The southern part of Finland, up to a latitude of 62°, is an area in which both T. nativa and T. britovi co-exist. In Estonia, the –6 C January isotherm has been speculated to be the thermal limit of T. britovi and –4 C is the limit for dispersal of T. nativa in more temperate areas (Pozio et al., 1998). Nevertheless, in the southern part of Finland, both T. nativa and T. britovi existed in an area where the average temperature from December to February drops to –8 C to –10 C. We found all 4 Trichinella species in the southern part of the country (latitude 62°; Fig. 2). All 4 Trichinella species have also been reported in sympatric Baltic areas (Malakauskas et al., 2007).

The domestic species, T. spiralis, was remarkably identified in 15% of the sylvatic isolations and was recovered from all parts of Finland. Intriguingly, T. spiralis was found in a fox (Fig. 2) from an area in Lapland with no previous reports of any domestic infection outbreaks. To our knowledge, this is the northernmost isolation of T. spiralis (see the map published in the review by Pozio and Murrell, 2006). In Lithuania and Latvia, T. spiralis was reported in wildlife only in areas with pig infections (Malakauskas et al., 2007). The latter authors concluded that a domestic source is needed to contaminate wildlife with disposed offal from an infected pig. Our finding of T. spiralis in Lapland contradicts their conclusion, indicating that T. spiralis may exist in a sylvatic cycle without external sources from synantropic animals or swine offal. A similar pattern was seen in an epidemiological survey in Germany, which revealed T. spiralis larvae in 0.07% of sampled foxes (Wacker et al., 1999), although the German pork industry has been deemed virtually free of Trichinella spp. infection and human outbreaks have been associated with imported meat products (Jansen et al., 2008).

Host-parasite interaction was a significant factor in parasite intensity, indicating that certain species may reproduce better in particular hosts. The raccoon dog exhibited the highest parasite intensity for all Trichinella species, but burdens caused by different species of Trichinella did not differ in raccoon dogs, although the few detected T. pseudospiralis cases exhibited the highest burden. This is in accordance with earlier experimental studies where both T. nativa and T. spiralis were found to be equally well adapted to this host species (Näreaho et al., 2000; Mikkonen et al., 2001). It is noteworthy that T. spiralis was both relatively and absolutely most often found in the raccoon dog and, moreover, that the parasite reproduces with higher intensities in raccoon dogs than in other host species. The raccoon dog has a high prevalence both in Baltic countries (Malakauskas et al., 2007) and in Finland, where it is an invasive alien species. However, in areas where it is native, reports do not show such a high prevalence, e.g., only 1.6% in Japan (Kobayashi et al., 2007). Data from Lithuania indicate that foxes are more often T. spiralis carriers than are raccoon dogs (8.2% of all isolates vs. 4.3% in raccoon dogs; Malakauskas et al., 2007). Although found less frequently than in raccoon dogs in the present study, T. spiralis was also commonly detected in the red fox; both animal species are closely associated with farm houses and barns. In the Finnish epidemiologic situation, they more likely act as donors of the infection to the domestic cycle rather than as recipients.

Interestingly, lynxes were relatively more frequently infected with T. britovi than with other species. Trichinella spp. prevalence in lynxes varied in different GMDs, from 8% to 80% (Table I). A study of Swiss lynxes has shown a prevalence of 27% (15/55), with variation between cantons (Frey et al., 2009). The same study reported a prevalence of 1.6% in foxes, with obvious geographical differences. The study reported only T. britovi in both foxes and lynxes, but the investigators did not provide infection intensities. In our data, both T. nativa and T. britovi had similar infection intensities in lynxes, but T. spiralis exhibited lower infection intensities and infected lynxes relatively less often. This may indicate a host-parasite adaptation that does not favor T. spiralis in lynxes.

Trichinella pseudospiralis was identified in only 7 animals, 4 times in different combinations of mixed infections and 3 times as single infections, twice in a raccoon dog and once in a lynx. Therefore, the low numbers of identification do not allow any conclusions to be drawn about host adaptations. However, the highest burden of T. pseudospiralis within the host species in both raccoon dogs and lynxes is in accordance with earlier studies showing a high reproduction index of this species in sylvatic hosts, but a low reproduction index in domestic animals (Pozio and Murrell, 2006).

We found 1 otter to harbor T. nativa. Reports of Trichinella spp. in otters are very rare. Data summarizing 20 yr of discoveries in the International Trichinella Reference Centre report only 2 otters infected by T. britovi (Pozio et al., 2009). The otter is mentioned, however, as a host species in Campbell's (1983) comprehensive review.

The high parasite burden with all Trichinella species identified in the raccoon dog shows that the raccoon dog is an adaptive host for Trichinella spp. High Trichinella spp. reproduction in naturally infected raccoon dogs has also been reported in other studies (Oivanen et al., 2002; Malakauskas et al., 2007). In Finland, the risk of the red fox or raccoon dog being infected with Trichinella spp. is strongly dependent on the sampling area. Their combined prevalence showed a significant association with the north-south coordinate and with raccoon dog abundance (Table II).

Although sylvatic infection is common in Finland and T. spiralis, a typically domestic species, is commonly associated with the sylvatic cycle, there are very few reports of human trichinellosis in Finland (see references in Oivanen, 2005). This reflects effective transmission barriers. Pig production has been industrialized and meat inspection is efficient. Backyard pork production and consumption are uncommon. Hunters know the risk of sylvatic Trichinella infection and, in Finnish cuisine, foods are well cooked.