The Wolf (Canis lupus) as an Indicator Species for the Sylvatic Trichinella Cycle in the Central Balkans

Trichinellosis is still a zoonosis of worldwide socioeconomic significance. In Europe, the Balkan region is endemic for Trichinella infections in domestic pigs (Sus scrofa) and humans (Pozio 2007) and a resurgence of trichinellosis occurred in the 1990s (Čuperlović et al. 2005). Of 1,100 documented cases of human trichinellosis across Europe in 2004, almost 90% (984) were reported from Balkan countries including Bulgaria, Croatia, Romania, and Serbia (Dupouy-Camet 2006). In Serbia, 144 outbreaks with 2,257 cases were reported between 2001 and 2010 (Sofronić-Milosavljević et al. 2013).

The main source of infection for humans in the domestic cycle are domestic pigs, with backyard pigs being the most important source of infection in the Balkans (Živojinović et al. 2013). However, wildlife is generally the most significant reservoir of Trichinella spp. (sylvatic cycle), and game meat (wild boars; Sus scrofa) may be an infection source for humans, but even species that are not used for human consumption play an important epidemiologic role as a potential source of infection for domestic pigs (Pozio and Murrell 2006). Therefore, monitoring wildlife is needed for the risk assessment of Trichinella spp. transmission to the pig population (Pozio et al. 2009). Although the red fox (Vulpes vulpes) is a natural reservoir of Trichinella spp., and is considered to be an indicator species in many countries, the wolf (Canis lupus), with a prevalence of Trichinella spp. infection of 31%, has recently been described as an indicator species for assessment of Trichinella infection risk in Croatia (Beck et al. 2009).

The wolf is at the top of the food chain and its population in the Central Balkan region has been estimated at >2,000 individuals, of which 700–800 are in Serbia, >1,000 in Macedonia, and 400 in Bosnia and Herzegovina (B&H) (Boitani 2000; Milenković et al. 2007). The International Union for Conservation of Nature lists the wolf as vulnerable in Serbia (Savić et al. 1995); national evaluations have not been performed in Macedonia and B&H. However, little is known about Trichinella spp. infection in wildlife in this area, especially in wolves, of which only a few have been examined (Cvetković et al. 2011; Živojinović et al. 2013). To assess the importance of wolves as a reservoir of sylvatic trichinellosis in the Central Balkans, we studied the prevalence, species identification, and spatial distribution of Trichinella spp. in the wolf population of this region.

In cooperation with local hunters, carcasses of legally hunted wolves were collected during 2006–13 in the Central Balkans (Serbia and neighboring areas of Macedonia and B&H). Carcasses (only heads in a few cases) of 116 specimens (105 from Serbia, three from B&H, and eight from Macedonia) from 31 localities were collected (Fig. 1). For each animal, the hunting date, sex (62 males, 51 females, three unidentified), and locality of origin were reported. The tongue was removed in the field and frozen at −18 C until testing for Trichinella. With the use of the protocol of Gamble et al. (2000), 50 g of muscle tissue from the tongue base was digested individually. Trichinella larvae were microscopically counted and worm burden was expressed as number of larvae per gram of muscle tissue (LPG). Larvae were stored in 90% ethyl alcohol at −18 C until molecular identification. The DNA was extracted from a pool of 10 larvae (or less: five, six, and eight, for three isolates) per sample, with the use of the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. Extracted DNA was resuspended in 150 µL of nuclease-free water and stored at −20 C. Identification of Trichinella larvae species was performed by multiplex PCR according to a published protocol (Pozio and La Rosa 2009), modified to increase the sensitivity of the PCR reaction to detect mixed-species infections in DNA extracted from pools of larvae. The method was first validated with pools of 10 larvae containing T. spiralis and T. britovi in different proportions, and the results clearly showed amplification of both targets (band of 173 base pairs [bp] for T. spiralis, two bands of 127 bp and 253 bp for T. britovi). The PCR reaction consisted of 12.5 µL PCR Master Mix (2X) (Fermentas; Thermo Fisher Scientific), 2 µL 10 µM ESV forward and reverse primer and 1 µL 10 µM ITS1 forward and reverse primer, and 1.5 µL nuclease-free water and 5 µL DNA extracted from the sample in a 25-µL reaction volume. Amplification was performed over 40 cycles in an automated thermocycler (The Veriti® Thermal Cycler, Applied Biosystems, Life Technologies, Foster City, California, USA), under the following cycling conditions: initial denaturation at 95 C for 4 min, followed by 40 cycles of denaturation at 95 C for 20 sec, annealing at 55 C for 30 sec, elongation at 72 C for 30 sec, and a final elongation step at 72 C for 3 min. PCR products were visualized by electrophoresis in 2.5% agarose gel stained with Midori green (Nippon Genetics Europe GmbH, Dueren, Germany). Pools of 10 larvae from three Trichinella reference strains: Trichinella spiralis (code ISS3), Trichinella britovi (code ISS2), and Trichinella pseudospiralis (code ISS13) (all stains from International Trichinella Reference Center, Rome, Italy), representing the species circulating in the Balkan region (Pozio and Zarlenga 2013), were used as positive controls. Estimation of fragment size was based on comparison by a 50-bp DNA ladder (Fermentas).

The differences in prevalence among the 8 study years were analyzed with the use of the G-test (Sokal and Rohlf 1995). The difference in prevalence between males and females was analyzed by chi-square test. All data were analyzed with the use of Statistica 5.1 (Statsoft, Tulsa, Oklahoma, USA). The level of significance was P<0.05.

We found Trichinella larvae in 54 of the 116 (46.5%) wolves, most (90.5%) of which were from Serbia, where 52 were positive (49.5%, 95% CI = 39.9–59.1%). In B&H we found Trichinella larvae in one of the three examined animals; in Macedonia, one of the eight (12%) was infected. This is the first report of Trichinella in wolves of B&H and Macedonia.

There was no difference in prevalence between sexes (40.3% in males and 54.9% in females [χ² = 2.39, P<0.122]) or among study years (G = 3.876, P<0.365; Table 1). Most Trichinella-infected wolves in Serbia originated from the central regions of the country (Fig. 1) characterized by forested mountainous areas, and along the border with Bulgaria and Romania; no infected animals were found in the westernmost localities examined.

The average LPG was 11.4±14.9 (range, 0.95–76). The lower and higher LPG values originated from animals collected in localities that were close together; even in animals from the same territory, there was high LPG variability. All larvae were identified as T. britovi. This finding is in agreement with reports from Serbia, which identified only this species in a total of seven wolves (Cvetković et al. 2011; Živojinović et al. 2013), and a Europe-wide investigation that showed that 98% of Trichinella infections in wolves were due to T. britovi and only 2% to T. spiralis (Pozio et al. 2009). However, in neighboring Croatia, even 10% of T. spiralis were reported among a small sample of Trichinella-positive wolves (n = 21; Beck et al. 2009). The absence of T. spiralis in our study is unexpected, as this species has been observed in wild boars, red foxes, wild cats (Felis sylvestris), and golden jackals (Canis aureus) in Serbia (Cvetković et al. 2011; Živojinović et al. 2013), some of which were collected in the same localities as the wolves in this study. This may in part be attributed to the majority of the sample coming from mountainous, less-inhabited areas that are remote from pig farms. The wolves collected in the central-east region of the country live closer to human settlements and accordingly spillover between the domestic and the sylvatic cycles may be expected.

The high prevalence of T. britovi in wolf populations of the Central Balkans may be explained by scavenging behavior and hunting practices. A preliminary investigation of wolf feeding habits in Serbia showed that scavenging at unregulated garbage dumps is the dominant feeding strategy (Ćirović et al. 2010). The lack of sanitary disposal of hunted animal carcasses increases the prevalence of Trichinella in wolves (Pozio et al. 2001), which may be particularly important for the sylvatic cycle of Trichinella in the Central Balkans because of the hunters' practice of removing the head and skin (as a trophy) from carnivores and leaving the carcass in the field, making it available to scavengers. Scavenging may also explain our finding of a higher frequency of infected wolves in mountainous and forested areas than in lower areas characterized by river plains, where other food sources may be available. Pozio (1998) found that sylvatic trichinellosis was more prevalent in forested than in human-inhabited areas.

The red fox is an indicator species for the assessment of sylvatic trichinellosis in Europe, but has not been investigated systematically in Serbia. Data from a few animals in a small area show a prevalence of Trichinella infection of 5% (1/20) and 12.3% (7/57), respectively (Petrović et al. 2012; Živojinović et al. 2013). The high prevalence of Trichinella infection we found suggests that wolves are important in the sylvatic cycle of T. britovi in the Central Balkans. Because of its abundance, the wolf may be considered an indicator species for risk from the sylvatic Trichinella cycle in this region. Another potentially important reservoir of infection is the golden jackal, whose abundance in this region is increasing and is being studied.

We express sincere gratitude to Edoardo Pozio (International Trichinella Reference Center, Istituto Superiore di Sanita, Rome, Italy) for critical review of the manuscript and useful suggestions. The study was supported by grants (projects TR 31034, III 41019, and TR 31009) from the Ministry of Education, Science and Technological Development of Serbia.