Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

Molecular identification and phylogenetic analysis of Trichinella isolates from different provinces in mainland China.

Polymerase chain reaction (PCR) and sequencing are useful for species identification of Trichinella spp., especially when their morphological characteristics useful for identifying taxa are lacking. In the present study, nine Trichinella isolates from different provinces in mainland China were identified by the PCR-based method using the 5S ribosomal DNA intergene spacer region (5S ISR) and the mitochondrial large subunit ribosomal DNA genes as molecular markers. The results indicated that eight isolates originating from domestic pigs and one isolate originating from civet cat (Paguma larvata) showed identical DNA banding pattern to Trichinella spiralis. Sequence analysis of the 5S ISR gene further confirmed that the nine Trichinella isolates were T. spiralis and revealed the intraspecies genetic variation within T. spiralis.

[Efficacy of tribendimidine against three isolates of Trichinella spiralis in mice].

OBJECTIVE: To evaluate the efficacy of tribendimidine (TBD) against 3 geographical isolates of Trichinella spiralis in mice. METHODS: Isolates of T. spiralis from Henan (hereinafter referred to as HnT.s), Yunnan (referred to as YnT.s) and Heilongjiang (referred to as HIjT.s) were used in the study. 144 Kunming strain mice were divided into 2 groups: 72 mice in group A (adult stage, treatment at 5 d after infection), and 72 mice in group B (encapsulated larva stage, treatment at 53 d after infection). Group A was further divided equally into 12 sub-groups. Mice in every 3 sub-groups were each infected orally with 200 T. spiralis larvae of the 3 isolates respectively, and the remained 3 subgroups served as untreated control. Mice in the 3 sub-groups infected with one isolate were orally treated with TBD at a single dose of 10, 20, and 30 mg/kg, respectively. Group B was treated as group A but with a course of TBD once daily at a dose of 100, 200, and 300 mg/(kg x d) for 7 d, respectively. Mice in group A were sacrificed 2 d post-treatment and adult worms were recovered from the small intestine and counted. Those in group B were sacrificed 10 d after completion of 7 d treatment. The intact diaphragm was removed and digested for collecting larvae. Worm burden and worm reduction of each treated sub-group were calculated and statistically compared with the respective control. RESULTS: In group A, the mean worm burden in the treated sub-groups infected with HnT.s and YnT.s were all significantly lower than that of the controls (P < 0.01), with a mean worm reduction rate of 39.0%, 57.9%, and 86.0% in HnT.s sub-groups, and of 34.9%, 69.3%, and 92.2% in YnT.s sub-groups, respectively, showing an increase with the dosage, 2 mice in each of the 30mg/kg sub-groups were cured. The worm burden in the 10 mg/kg of HljT.s subgroup was similar to that of the control (P > 0.05), but was significantly lower in the other 2 sub-groups than that of the controls (P < 0.01). The worm reduction rate in the 3 sub-groups was 27.9%, 57.4%, and 60.7%, respectively. In all treated sub-groups of group B, the mean worm burden was significantly lower than that of the controls (P < 0.05), with a mean worm reduction rate of 57.8%, 75.4%, and 87.5% in HnT.s sub-groups, of 74.5%, 92.4%, and 99.1% in YnT.s sub-groups, and of 50.5%, 53.3%, and 61.6% in HljT.s sub-groups, respectively, with the 3 dosages. CONCLUSION: Tribendimidine shows adequate efficacy on Trichinella spiralis adults and on encapsulated larvae of the 3 geographical isolates in mice, with better effect on Yunnan isolate.

Molecular identification of a Trichinella isolate from a naturally infected pig in Tibet, China.

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.

Genetic evidence substantiates transmission of Trichinella spiralis from one swine farm to another.

BACKGROUND: Trichinella spiralis ranks seventh in the risk posed by foodborne parasites. It causes most human cases of trichinellosis and is the most frequent cause of Trichinella outbreaks on pig farms and in wild boar, worldwide. Veterinary inspectors seek the source of outbreaks in hopes of limiting the spread. Established molecular tools are inadequate for distinguishing among potential T. spiralis infection sources because genetic variability in these zoonotic pathogens is limited in Europe. Microsatellite markers proved successful in tracing an outbreak of T. britovi, a related parasite harboring much more genetic variation. Here, we successfully employed microsatellite markers to determine the genetic structure of T. spiralis isolates from two pig outbreaks, discovering notable uniformity among parasites within each farm and discovering an epidemiological link between these two outbreaks. METHODS: The individual larvae from five isolates of T. spiralis from two pig farms and from ten wild boars were genotyped using nine microsatellite markers to examine their genetic structure. RESULTS: Notably uniform parasite populations constituted each farm outbreak, and the parasites from the first and second outbreaks resembled each other to a notable degree, indicating an epidemiological link between them. Wild boar harbored more genetically variable larval cohorts, distinguishing them from parasites isolated from domestic pigs. CONCLUSIONS: Microsatellite markers succeeded in distinguishing isolates of the highly homogeneous T. spiralis, aiding efforts to track transmission. Each outbreak was composed of a homogenous group of parasites, suggesting a point source of contamination.

Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo).

Understanding parasite diversity and distribution is essential in managing the potential impact of parasitic diseases in animals and people. Imperfect diagnostic methods, however, may conceal cryptic species. Here, we report the discovery and phylogeography of a previously unrecognized species of Trichinella in wolverine (Gulo gulo) from northwestern Canada that was indistinguishable from T. nativa using the standard multiplex PCR assay based on the expansion segment 5 (ESV) of ribosomal DNA. The novel genotype, designated as T13, was discovered when sequencing the mitochondrial genome. Phylogenetic analyses of the mitochondrial genome and of 15 concatenated single copy orthologs of nuclear DNA indicated a common ancestor for the encapsulated clade is shared by a subclade containing Trichinella spiralis and Trichinella nelsoni, and a subclade containing T13 and remaining taxa: T12 + (T2 + T6) + [(T5 + T9) + (T3 + T8)]. Of 95 individual hosts from 12 species of mammalian carnivores from northwestern Canada from which larvae were identified as T. nativa on multiplex PCR, only wolverines were infected with T13 (14 of 42 individuals). These infections were single or mixed with T. nativa and/or T6. Visual examination and motility testing confirmed that T13 is encapsulated and likely freeze-tolerant. We developed a new Polymerase Chain Reaction-Restriction Fragment Length Polymorphism which unequivocally distinguishes between T13 and T. nativa. We propose Trichinella chanchalensis n. sp. for T13, based on significant genetic divergence from other species of Trichinella and broad-based sampling of the Trichinella genome. Exploration of Alaskan and Siberian isolates may contribute to further resolution of a phylogeographically complex history for species of Trichinella across Beringia, including Trichinella chanchalensis n. sp. (T13).

Trichinellosis survey in the wild boar from the Toledo mountains in south-western Spain (2007-2008): molecular characterization of Trichinella isolates by ISSR-PCR.

In Spain, trichinellosis represents a public health problem, with an average of five outbreaks per year, wild boar meat being the main source of infection. A trichinellosis survey (2007-2008 hunting campaign) was carried out on wild boars in the Toledo Mountains (south-western Spain, EU) in the context of a surveillance programme on wildlife diseases. A total of 2216 wild boars from different locations of the region were examined. The examination was carried out by veterinarians in the local abattoir (Matadero Municipal de Toledo). The positive samples were sent to the Department of Parasitology (Facultad de Farmacia, UCM) for experimental isolation and specific identification by inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR). Using this technique we identified 17 isolates as Trichinella spiralis with an electrophoretic profile indistinguishable from the T. spiralis reference strain (ISS48). We confirmed that ISSR-PCR is a robust technique for the molecular identification of Trichinella isolates. According to our results, the prevalence of T. spiralis in wild boars from the Toledo Mountains (>800 m above sea level) during the hunting season was approximately 0.77%. The prevalence of T. spiralis (100% of our observations) is a good example of the persistence of this species in sylvatic conditions (coming from the domestic cycle), if a good wild host is abundant. Our observations confirm the major prevalence of T. spiralis over T. britovi in this region, as well as the risk to human health represented by the consumption of uninspected wild boar meat.

Human dispersal of Trichinella spiralis in domesticated pigs.

To investigate the human impact on the evolutionary ecology of animal pathogens, we compared genetic diversity of severe foodborne parasites contracted by eating infected pork or wild game. In particular, we characterized Trichinella spp. from twenty-eight countries and four continents by genotyping nine microsatellite loci and sequencing one-fifth of the mitochondrial genome. All specimens of Trichinella spiralis, a swine parasite that can infect many species of wildlife, were remarkably uniform across Europe, North Africa, and the Americas. Far greater diversity characterized a comparable sample of Trichinella britovi, which parasitizes various sylvatic mammals endemic to Eurasia and North-Western Africa. A limited sample of T. spiralis in Asia, where swine were first domesticated, encompassed greater genetic variability than those in the West, as did small samples of Trichinella nativa and Trichinella murrelli, which parasitize wildlife hosts. We conclude that European lineages of T. spiralis originated several thousand years ago, approximately when pigs were first domesticated there. These data also imply that Europeans inadvertently introduced T. spiralis to the Americas via infected pigs and/or rats. Despite evidence that early hominid hunters ingested foodborne parasites by hunting wild game millions of years earlier, swine husbandry has governed the subsequent transmission, dissemination, and evolutionary diversification of T. spiralis. Where viable parasites have been eliminated from their diet, the residual risk posed to swine by exposure to wildlife or rats should be more precisely defined because breaking the cycle of transmission would confer enduring economic and health benefits.

Mixed infection, Trichinella spiralis and Trichinella britovi, in a wild boar hunted in the Province of Cáceres (Spain).

The etiological agents of human trichinellosis are distributed worldwide in domestic and wild animals. In Spain, two morphologically indistinguishable Trichinella species have been described--Trichinella spiralis and Trichinella britovi--that are perpetuated in both domestic and sylvatic cycles. The present work reports a double natural infection involving these species in a wild boar killed by hunters in the Province of Cáceres, Spain. After artificial digestion of the boar's muscles, nine larvae/g were collected. These were characterized by multiplex-PCR and Western-blotting using the Trichinella-specific monoclonal antibodies US5 and US9, and both T. spiralis and T. britovi were detected. The mechanism by which this wild boar came to acquire a mixed infection remains unclear.

Differentiation of Trichinella species (Trichinella spiralis/Trichinella britovi versus Trichinella pseudospiralis) using western blot.

BACKGROUND: Trichinellosis is a meat-borne zoonotic disease caused by parasites of the genus Trichinella. To date, 12 taxa have been described. The identification of Trichinella species is crucial in order to identify the possible source of infection, the geographical origin of the parasite and to assess risk of infection for domestic pigs and humans. Specific identification of the etiological agent is not always feasible using direct methods since the source of infection can be untraceable. The aim of this study was to develop a diagnostic tool to infer the causative Trichinella species using western blot patterns of sera derived from infected animal and human hosts. METHODS: Sera from mice experimentally infected with Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis and Trichinella papuae were tested by western blot using homologous and heterologous crude worm extracts (CWE) and a highly sensitive detection system based on chemiluminescence. In addition, sera from pigs experimentally infected with T. spiralis, T. britovi and T. pseudospiralis and from patients with confirmed T. spiralis, T. britovi and T. pseudospiralis infections, were also included. RESULTS: Sera from mice infected with one Trichinella species reacted with CWE proteins from all four investigated species. Likewise, sera derived from pigs and humans infected with one Trichinella species reacted with CWE proteins from all the three investigated species. Using T. spiralis CWE, sera from T. pseudospiralis-infected hosts yielded a characteristic pattern of reactivity using Wb, which differed to that produced by T. spiralis/T. britovi- or T. papuae-infected host sera. CONCLUSIONS: The present study suggests that western blot using T. spiralis CWE may be a useful tool to distinguish Trichinella infections caused by T. pseudospiralis from those caused by T. spiralis or T. britovi. This method may support epidemiological investigations, particularly when the source of infection is not traceable.

[Study on 5S rDNA sequence of two isolates of Trichinella from Guangxi].

OBJECTIVE: To analyze 5S ribosomal DNA (5S rDNA) sequences of two Trichinella isolates from Guangxi. METHODS: The fragments of 5S rDNA were obtained by PCR from the isolates of Debao and Nandan, and sequencing was made for the PCR products. Homogeneity, genetic distance matrix and phylogenetic tree were analyzed by related software. 5S rDNA sequences of the two isolates were compared separately with those of Trichinella species in GenBank. RESULTS: 5S rDNA sequences of three Trichinella isolates (Debao, Nandan and T. spiralis) showed the same length at 695 bp. There were 4 variable positions. The homogeneities of Debao and Nandan isolates with T. spiralis were 99.0% and 99.1% respectively. The homogeneities between Debao isolate and Nandan isolate was 98.8%. Compared with other Trichinella isolates in GenBank, they were all less than 94.2%. The evolutionary distance among isolates of Debao and Nandan and T. spiralis was 0.014. Meanwhile, the evolutionary distances between the Guangxi isolates and other Trichinella isolates in GenBank were more than 0.056. Phylogenetic tree analysis revealed that two isolates of Guangxi and T. spiralis located at the same node, revealing a close relationship. Bootstrap confidence values in two phylogenetic trees were 96 and 99, respectively. CONCLUSION: The two Trichinella isolates of Guangxi show a high homogeneity with T. spiralis, locate at the same nodes in phylogenetic tree,suggesting that the Debao and Nandan Trichinella isolates be identified as T. spiralis.

High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis.

Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors.

Rediscovery of Trichinella spiralis in red foxes (Vulpes vulpes) in Ireland after 30 years of oblivion.

OBJECTIVE: To determine whether or not Ireland can be considered as Trichinella-free, after more than 30 years of no reported infections in domestic and sylvatic animals and in humans. METHODS: Samples of muscle tissue from the tongue, masseter, and foreleg of red foxes (Vulpes vulpes) were subjected to artificial digestion and to multiplex-PCR analysis for identifying Trichinella larvae at the species level. RESULTS: Four of 454 examined foxes were positive for larvae (overall prevalence 0.9%). The positive foxes had been collected in Donegal County (one fox; prevalence of 7.7%), Cork County (two foxes; 3.1%), and Waterford County (one fox; 4.2%). All larvae were identified as Trichinella spiralis. CONCLUSIONS: The detection of Trichinella-positive foxes in Ireland is an example of how the lack of infections among domestic animals and humans does not suffice for establishing Trichinella-free status. The results also confirm that the sylvatic cycle can last for tens of years, independently of the existence of a domestic cycle.

Characterisation of Trichinella isolates from Bulgaria by molecular typing and cross-breeding.

Trichinella spp. larvae were collected from domestic and wild-life animals in association with 15 human trichinellosis outbreaks registered between 1999-2002 in Bulgaria. Furthermore, Trichinella spp. isolates were obtained from 62 naturally infected wild animals and of a rat. All isolates were subjected to speciation by both multiplex PCR and cross-breeding experiments. Epidemiological and clinical data were collected and analysed using standard protocols for epidemiological surveillance and control of outbreaks. Only two species were identified-Trichinella britovi and Trichinella spiralis. Results obtained by molecular typing fully matched those of cross-breeding. More specifically, parasite isolates obtained upon 15 epidemic outbreaks revealed the predominance of T. britovi (n = 10) when compared to T. spiralis (n = 5). With regard to host origin, the predominant species detected among wild boar was T. britovi (n = 4), and T. spiralis was identified in one wild boar sample only. Among the isolates obtained from domestic pig products, T. britovi was found in five cases and T. spiralis in four cases, respectively. In the naturally infected wild animals not related to epidemics, only T. britovi was demonstrated. The present results provide a strong indication that both T. britovi and T. spiralis operate within domestic and sylvatic cycles in Bulgaria. Geographically, the distribution of T. britovi appears to include Central, Southern, Eastern and Western parts of the country, and wildlife animals from the Mid Balkan Mountains and Mid Sredna Gora Mountains, T. spiralis was found in Western and Southwestern Bulgaria, only.

Identification of Trichinella isolates from naturally infected stray dogs in Egypt.

Larvae of Trichinella species recovered from the diaphragms of 2 stray dogs killed during a governmental antirabies campaign in Cairo, Egypt, were fed to white mice for production of adult worms and larvae for morphological and isoenzyme studies. Comparisons were made with reference species of Trichinella spiralis, Trichinella nelsoni, Trichinella nativa, and Trichinella pseudospiralis. Results indicated that the 2 Trichinella specimens from the dogs were morphologically and biochemically identical with T. spiralis.

Arbitrarily primed polymerase chain reaction of individual Trichinella specimens.

Recently, 5 sibling species and 3 other phenotypes were identified in the genus Trichinella. Single primers of arbitrary nucleotide sequence were used to produce random amplified polymorphic DNA starting from decreasing amounts of Trichinella spiralis and Trichinella britovi DNA. Reproducible amplification products from 30 pg of DNA were obtained using 1 of 6 examined primers. These fragments distinguish between 2 European Trichinella species, T. spiralis, showing a 1,350-bp band, and T. britovi, showing 400- and 1,100-bp bands. The developed procedure allows the characterization of crude DNA preparations of single muscle-stage larvae, avoiding time-consuming passages of parasites in laboratory animals.

Analysis of Chinese isolates of Trichinella spiralis by molecular biotechnology.

OBJECTIVE: To investigate the relationships among Trichinella spiralis isolates obtained from different hosts and geographical regions in China. MATERIALS AND METHODS: Six isolates of Trichinella spiralis obtained from Changchun, Tianjin, Xi'an, Henan and Yunnan were detected. All the isolates were collected from pigs or dogs. Restriction fragment length polymorphism (RFLP), isoenzyme electrophoresis and soluble protein analysis were used. RESULTS: Distinctive banding patterns in restriction maps were seen between Changchun and other isolates studied. A cloned specific repetitive DNA sequence (1.12 kb) from Changchun isolate was then selected to prepare probe for Southern blotting of DNA fragments. The hybridizing banding patterns among the isolates were different and only the Changchun isolate showed the 1.12 kb band. The Changchun isolate was also distinguishable from the other isolates in isoenzyme patterns of GP1, G6PD, HK, 6PGDH and AK, as well as in isoelectric focusing electrophoresis where the Changchun isolate presented a specific band at 4.1 P1. CONCLUSIONS: There are at least two different biological types of Trichinella spiralis in China and the differences between them may be attributed to different geographical origins and/or different hosts.

Sylvatic trichinellosis in southwestern Spain.

The epidemiology of Trichinella spp. in their main sylvatic hosts, wild boar (Sus scrofa ferus and red fox (Vulpes vulpes), in Extremadura (southwestern Spain) was studied. We examined 88 Trichinella spp.-positive wild boar muscle-tissue samples from a total of 29,333 killed animals, referred to the Veterinary Parasitology Department (University of Extremadura, Spain) by the Extremadura Veterinary Service. Additionally, 227 red foxes killed during the hunting season and thus not subject to veterinary controls were examined for trichinellosis. Trichinella spp. larvae were found in six (3%) of the red foxes. All samples were examined using direct diagnostic techniques, including trichinoscopy and artificial digestion. The mean intensity of infection was 74.8 larvae/g (LPG) of muscle tissue in wild boars, compared to 30.6 LPG in foxes. Trichinella spiralis (sensu stricto) predominated over T. britovi in wild boars. Random amplified polymorphic DNA (RAPD) and alloenzyme typing showed that 74% of infected wild boars had only T. spiralis, 21% had only T. britovi, and 5% showed mixed infections. In contrast, 33% of infected foxes were infected only with T. spiralis, while 67% had T. britovi, suggesting a clear predominance of the latter in foxes. We suspect the existence of a paranthropic or sylvatic cycle in large areas of this region; given the ease of transfer between sylvatic and domestic or semi-domestic animals, this implies a high epidemiological risk.

Restriction fragment length polymorphism (RFLP) analysis of genomic DNA of 5 strains of Trichinella spiralis in China.

Five restriction endonucleases were used to digest genomic DNA from 5 isolates of Trichinella spiralis obtained from Changchun, Tianjin, Xian, Henan and Yunnan. All the isolates were secured from pigs except the Changchun strain which came from dog. The DNA fragments digested by endonuclease were separated by agarose gel electrophoresis. The Changchun isolate had a EcoRI band at 1.12kb and a DraI band at 1.97kb which were unique to this isolate. A cloned specific repetitive DNA sequence (1.12kb) from the Changchun strain was selected to prepare a probe for the Southern blotting of EcoRI restriction DNA fragments for the 5 isolates. The 1.12kb hybridizing band did not appear except in the Changchun isolate. These results seem to indicate that there are differences between the isolates obtained from hosts in different geographical regions.

Estimating the genetic divergence and identification of three Trichinella species by isoenzyme analysis.

Isoenzyme-based approach was applied to compare Trichinella spiralis, T. britovi and T. pseudospiralis species. Among 13 enzyme systems examined, esterase (EST), malic enzyme (ME) and phosphoglucomutase (PGM) have been found as fully diagnostic, with no common allele in species studied. Adenosine deaminase (ADA), adenylate kinase (AK), hexokinase (HK), peptidase leucyl-alanine (PEP-C) and fructose-bis-phosphatase (FBP) have been capable of distinguishing the two species from resulting profiles. In addition, ADA, AK and PGM displayed the enzyme expression in the lowest amounts of muscle larvae in systems tested (100 larvae/100 microliters of extracts). Based on allozyme data, T. pseudospiralis has been found as the most distinct species within the group of taxa. Only a subtle genetic variability was recorded for T. pseudospiralis in which solely phosphoglucomutase exhibited variant patterns. In addition to the study of reference isolates, T. spiralis from lowland fox in Eastern Slovakia has been evidenced by use of genetic markers. This finding has proved that T. britovi is not the exclusive species parasitizing in the sylvatic ecosystem of the Slovak region.