Diversity of Trichinella species in carnivores from Bosnia and Herzegovina.

BACKGROUND: In Bosnia and Herzegovina, domestic and wild carnivores represent a significant driver for the transmission and ecology of zoonotic pathogens, especially those of parasitic aetiology. Nevertheless, there is no systematic research of Trichinella species in animals that have been conducted in Bosnia and Herzegovina, even though trichinellosis is considered the most important parasitic zoonosis. The available results of the few studies carried out in Bosnia and Herzegovina are mainly related to the confirmation of parasitic larvae in the musculature of domestic pigs and wild boars or data related to trichinellosis in humans. The objective of our study was to present the findings of a comprehensive investigation into the species composition of Trichinella among 11 carnivorous species within the territory of Bosnia and Herzegovina, as follows: red fox (Vulpes vulpes), grey wolf (Canis lupus), brown bear (Ursus arctos), wildcat (Felis silvestris), pine marten (Martes martes), European badger (Meles meles), weasel (Mustela nivalis), European polecat (Mustela putorius), Eurasian lynx (Lynx lynx), but also dog (Canis lupus familiaris) and cat (Felis catus). RESULTS: In the period 2013-2023, carnivore musculature samples (n = 629), each consisting of 10 g of muscle tissue, were taken post-mortem and individually examined using the artificial digestion method. In the positive samples (n = 128), molecular genotyping and identification of parasitic larvae of Trichinella spp. were performed using a PCR-based technique up to the species/genotype level. Positive samples were used for basic PCR detection of the genus Trichinella (rrnS rt-PCR technique) and genotyping (rrnl-EVS rt-PCR technique). The Trichinella infection was documented for the first time in Bosnia and Herzegovina among red foxes, grey wolves, brown bears, dogs, badgers and Eurasian lynx, with a frequency rate of 20.3%. Additionally, the presence of T. britovi infection was newly confirmed in Bosnia and Herzegovina, marking the initial documented cases. Furthermore, both T. britovi and T. pseudospiralis infections were observed in the wildcat population, whereas T. britovi and T. spiralis infections were detected in pine martens. Consistent with previous research, our findings align particularly regarding carnivores, with data from other countries such as Germany, Finland, Romania, Poland and Spain, where T. britovi exhibits a wider distribution (62.5-100%) compared to T. spiralis (0.0-37.5%). T. britovi is more common among sylvatic carnivores (89.0%), while T. spiralis prevails in wild boars (62.0%), domestic swine (82.0%) and rodents (75.0%). CONCLUSION: The results of our study represent the first molecular identification of species of the genus Trichinella in Bosnia and Herzegovina. Additionally, our findings underscore the necessity for targeted epidemiological studies to thoroughly assess trichinellosis prevalence across diverse animal populations. Considering the relatively high frequency of trichinellosis infection in investigated animal species and its public health implications, there is an evident need for establishing an effective trichinellosis surveillance system in Bosnia and Herzegovina.

Enhanced Genomic and Transcriptomic Resources for Trichinella pseudospiralis and T. spiralis to Underpin the Discovery of Molecular Differences between Stages and Species.

Nematodes of the genus Trichinella are important pathogens of humans and animals. This study aimed to enhance the genomic and transcriptomic resources for T. pseudospiralis (non-encapsulated phenotype) and T. spiralis (encapsulated phenotype) and to explore transcriptional profiles. First, we improved the assemblies of the genomes of T. pseudospiralis (code ISS13) and T. spiralis (code ISS534), achieving genome sizes of 56.6 Mb (320 scaffolds, and an N50 of 1.02 Mb) and 63.5 Mb (568 scaffolds, and an N50 value of 0.44 Mb), respectively. Then, for each species, we produced RNA sequence data for three key developmental stages (first-stage muscle larvae [L1s], adults, and newborn larvae [NBLs]; three replicates for each stage), analysed differential transcription between stages, and explored enriched pathways and processes between species. Stage-specific upregulation was linked to cellular processes, metabolism, and host-parasite interactions, and pathway enrichment analysis showed distinctive biological processes and cellular localisations between species. Indeed, the secreted molecules calmodulin, calreticulin, and calsyntenin-with possible roles in modulating host immune responses and facilitating parasite survival-were unique to T. pseudospiralis and not detected in T. spiralis. These insights into the molecular mechanisms of Trichinella-host interactions might offer possible avenues for developing new interventions against trichinellosis.

Establishment of a method for detecting Trichinella spiralis in ovine muscle tissues using real-time fluorescence quantitative PCR.

Trichinellosis is caused by Trichinella spiralis, a meat-borne zoonotic disease transmitted to humans through the consumption of infected undercooked or raw meat. Surveillance using safe and precise diagnostic tools to diagnose T. spiralis in sheep is needed to assess the incidence and probability of transmission from sheep to humans. In this study, we developed a real-time PCR assay to detect T. spiralis DNA in ovine muscle samples that can be used as an alternative surveillance tool to ensure food safety using newly designed primers. The assay is specific for the Scfld4 gene of Trichinella (T1) and enables the detection of larvae in ovine muscle tissue samples with high sensitivity and specificity. Trichuris ovis, Oesophagostomum dentatum, Haemonchus contortus, and Bunostomum trigonocephalum showed no nonspecific amplification. The assay could detect Trichinella DNA concentrations as low as 0.0026 ng/µL, equivalent to 0.0064 larvae, indicating a high sensitivity for T. spiralis detection. We used this real-time PCR to detect 73 ovine muscle samples from an ovine abattoir, and five samples tested positive via real-time PCR but negative via microscopy. This assay may provide a more specific and sensitive method for rapidly detecting Trichinella larvae in ovine muscle tissues.

Human Outbreak of Trichinellosis Caused by Trichinella papuae Nematodes, Central Kampong Thom Province, Cambodia.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase-, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

A two-step morphology-PCR strategy for the identification of nematode larvae recovered from muscles after artificial digestion at meat inspection.

To ensure that meat from livestock and game is safe for human consumption, European legislation lays down rules for mandatory testing. Helminth larvae are a category of zoonotic foodborne pathogens that can contaminate meat. Among helminths, the only zoonotic nematode regulated in Europe regarding meat inspection is Trichinella spp.. It is precisely during Trichinella testing that other potentially zoonotic larvae can be found. Due to current lack of tools, their identification is often very complicated. Nematode larvae other than Trichinella, recovered from artificial digestions of pig and wild boar muscles from France and Germany, were subjected to a newly developed two-step identification scheme, which includes both morphological examination and molecular assays. The first step is a general orientation towards a broad taxonomic group; the second step consists of targeted identification based on the results of first step. Different parasites were identified, some of which were not zoonotic such as Metastrongylus spp. and Angiostrongylus vasorum, but others are known to be zoonotic such as Toxocara cati, Ascaris suum, and Uncinaria stenocephala. The strategy is efficient for the identification of nematode larvae recovered from muscles but could also be applied for larvae from other sources.

Synthetic gene as target to assess the sensitivity of PCR to detect Trichinella spp. larvae in meat from a non-endemic region.

Trichinellosis is a zoonotic disease exotic in Brazil but commonly found worldwide including South American countries like Argentina. International trading of swine meat needs an official Trichinella-free diagnosis commonly carried out by pepsin-HCl digestion of diaphragm tissue fragments followed by microscopic examination for the presence or absence of Trichinella larvae. The easiness of this diagnostic method allows it to be performed at slaughtering plants but, in contrast, it lacks sensitivity and does not allow species differentiation, which is fundamental for determining geographical and species distribution of different genotypes. In our study, we aimed to evaluate a highly sensitive diagnostic method based on the polymerase chain reaction (PCR) that would allow us to detect and classify different species of Trichinella. Thus, we designed a synthetic gene and selected five sets of primers targeting specific regions of the Trichinella genome. The synthetic gene was cloned into a plasmid and then used to optimize PCR conditions. Using our PCR, we were able to detect 0.001 pg of the synthetic gene, which corresponded to 0.01 larvae. Then, we collected 175 samples of Suidae (domestic and wild boars) diaphragm fragments that were pooled into groups, digested with pepsin-HCl, and had the DNA extracted for analysis by PCR. The clinical samples evaluated were negative by PCR. Our results indicate that the PCR-based method might be a useful diagnostic method complementary to the pepsin-HCl digestion method currently in use, mostly in non-endemic areas.

Survey of Toxoplasma gondii and Trichinella spp. in hedgehogs living in proximity to urban areas in the Czech Republic.

Hedgehogs (Mammalia: Erinaceidae) are omnivorous nocturnal animals typically living in anthropogenic areas. They may be suitable as sentinels for a wide range of zoonotic infections. Only a few studies have investigated hedgehogs (and then as representative wildlife species) to establish their role in the life cycle of such tissue parasites with zoonotic potential as Toxoplasma gondii or Trichinella spp. Working with frozen hedgehog cadavers, we tested for these parasites using T. gondii DNA-specific magnetic capture isolation plus polymerase chain reaction and Trichinella spp. digestion assay. All of 50 examined hedgehogs were negative for Trichinella spp. larvae in their muscles, but brain tissue from 5 out of 26 Erinaceus europaeus (19.2%) and 4 out of 24 E. roumanicus (16.6%) tested positive for T. gondii DNA. Frequency of T. gondii for both hedgehog species was equal, as was distribution between males and females and across age categories. Although a few studies have suggested the possibility of Trichinella spp. infection in hedgehogs, the zero prevalence in the tested hedgehogs is not surprising in view of the generally low prevalence of Trichinella spp. in Central Europe. Our results show that hedgehogs are susceptible to infection by T. gondii and can be used as indicator wildlife animal species in anthropogenic ecosystems.

Human trichinellosis caused by Trichinella britovi in Greece, and literature review.

Trichinellosis is a cosmopolitan zoonotic parasitic disease caused by the nematodes of the genus Trichinella, through the consumption of raw or semi-raw infected meat from swine, horses and wild animals. This disease has been sporadically reported in Greece since 1946. The aim of the present study was to describe a trichinellosis case in a patient hospitalized in northern Greece, in 2017. A 47-year-old male was admitted to hospital with intense generalized myalgia, periorbital swelling, fever, exhaustion and anorexia. Biochemical and haematological profile showed eosinophilia and elevated creatine phosphokinase (CPK). Anti-Trichinella spp. IgG and IgM antibodies were detected by serology and Trichinella spp. larvae were found in two muscle biopsies by compressorium and histological examination. A larva collected from the muscle biopsy was identified as Trichinella britovi by polymerase chain reaction (PCR). Albendazole (400 mg twice per day × 10 days) was administered and the clinical condition of the patient promptly improved. This is the first identification of T. britovi in a patient in Greece.

Prevalence and molecular identification of Trichinella species isolated from wildlife originating from Limpopo and Mpumalanga provinces of South Africa.

Trichinella species are widely distributed on all continents with the exception of Antarctica, although the full spectrum of Trichinella species found in sub-Saharan African countries, and their hosts, has not been fully documented. This study was conducted to determine the prevalence of Trichinella in wildlife from the Greater Kruger National Park (GKNP) and adjacent areas located in the Limpopo and Mpumalanga provinces of South Africa, and to identify the species and/or genotypes of Trichinella larvae isolated from muscle tissues, using molecular techniques. A review of Trichinella spp. and their wildlife hosts reported during 1964-2011 was also conducted and the results were compared with our current study. Ninety samples representing 15 mammalian, two bird and three reptile species were screened for Trichinella infection during 2012-2016, using artificial digestion. Isolates detected were identified using a multiplex polymerase chain reaction (PCR) amplification of the internal transcriber spacers ITS1 and ITS2, and expansion segment V (ESV) regions of ribosomal DNA, followed by molecular analysis of the sequences. Twenty samples from seven wildlife species were positive for Trichinella spp. larvae, with an overall prevalence of 21.1% (20/90). The prevalence was higher in carnivores (18.9%, 18/90) than in omnivores (2.2%, 2/90). Analysis of sequences showed that eight of the isolates - two from spotted hyaena (Crocuta crocuta) (2/8), three from lion (Panthera leo) (3/13), one from leopard (Panthera pardus) (1/6), one from small spotted genet (Genetta genetta) (1/2) and one Nile monitor lizard (Varanus niloticus) (1/2) - conformed to Trichinella zimbabwensis. One isolate from a hyaena was grouped under the encapsulated species clade comprising T. nelsoni and genotype Trichinella T8 reported to be present in South Africa. This is the first report confirming natural infection by T. zimbabwensis in hyaena, leopard, genet and Nile monitor lizard, adding to the body of knowledge on the epidemiology of Trichinella infections in the Greater Kruger National Park of South Africa. Ten Trichinella-like larval isolates recovered after digestion from four wildlife species in this study (2012-2016) revealed inconclusive results due to DNA degradation resulting from poor storage or too few larvae for analysis, in comparison to 20 unidentified isolates from five wildlife species during the 1964-2011 period.

Outbreak of Trichinella T9 Infections Associated with Consumption of Bear Meat, Japan.

An outbreak of trichinellosis occurred in Japan in December 2016. All case-patients had eaten undercooked bear meat, from which Trichinella larvae were subsequently isolated. DNA sequencing analysis of the mitochondrial genes cytochrome c-oxidase subunit 1 and internal transcribed spacer 2 confirmed that Trichinella T9 had caused the outbreak.

Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues.

Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegansC. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.

Multiplex TaqMan qPCR assay for specific identification of encapsulated Trichinella species prevalent in North America.

BACKGROUND Human trichinellosis is a foodborne parasitic zoonotic disease caused by ingestion of raw or undercooked meat infected with nematode larvae of the genus Trichinella. In the USA, sporadic cases and outbreaks caused by the consumption of wild game meat infected with Trichinella have been reported. The current methods for diagnosis such as serology and microscopy are not specific, may result in false negative results, and cannot differentiate encapsulated Trichinella larvae to species level. The molecular protocols currently available for the differentiation of all encapsulate Trichinella species prevalent in North America have some limitations such as the inability to identify and resolve the presence of several Trichinella species in a single test. OBJECTIVES/METHODS In this study we developed and evaluated a multiplex TaqMan quantitative real-time polymerase chain reaction (qPCR) assay, which can simultaneously detect, identify and differentiate all species of encapsulated Trichinella occurring in North America i.e., T. nativa, T. spiralis, T. murrelli and Trichinella T6, even in cases of multiple infection in a single sample. We investigated two human biopsies and 35 wild animal meat samples considered as having a high likelihood of harboring Trichinella larvae obtained from the United States during 2009-2017. FINDINGS Using the multiplex assay describe here, 22 (59%) samples that tested positive contained Trichinella spp., were identified as: T. nativa (n = 7, including a human biopsy), T. spiralis (n = 9, including a human biopsy), T. murrelli (n = 3), Trichinella T6 (n = 1). Results also included two rare mixed infection cases in bears, a T. nativa/T. spiralis from Alaska and a T. spiralis/Trichinella T6 from California. The species identifications were confirmed using a conventional PCR targeting the rRNA ITS1-ITS2 region, followed by DNA sequencing analysis. The estimated limit of detection (LOD) was approximately seven larvae per gram of meat. MAIN CONCLUSIONS Differentiation of Trichinella spp. is needed to improve efforts on identification of case, optimize food safety control and better understand the geographic distribution of Trichinella species. The Trichinella qPCR multiplex proved to be a robust, easy to perform assay and is presented as an improved technique for identification of all known encapsulated species occurring in North America continent.

Molecular identification of Sarcocystis lutrae in the European otter (Lutra lutra) and the European badger (Meles meles) from the Czech Republic.

Muscular sarcosporidial infections by Sarcocystis lutrae (Apicomplexa: Sarcocystidae) from the otter (Lutra lutra) and badger (Meles meles) (Carnivora: Mustelidae) were found in the Czech Republic. As part of a diversity evaluation of Sarcocystis in wild carnivores during 2016-2017, samples of diaphragm, tongue and hind-limb muscles were collected from nine districts, examined by compression and characterized molecularly. Cyst walls were thin, with no visible protrusions, and histological sections of infected muscle tissue showed no host responses. Fourteen of 17 badgers (82% prevalence) and one otter (100% prevalence) were positive for sarcocysts. Sequence analyses at four loci (18S rRNA, 28S rRNA, ITS1 and cox1) confirmed the identity as S. lutrae. This is also the first report of a co-infection with muscular sarcocystosis and Trichinella in badger. The finding of Trichinella is important from the zoonotic point of view, since badgers are used for meat consumption. Similar and future monitoring of both parasitic taxa are needed.

Trichinella Nativa Outbreak With Rare Thrombotic Complications Associated With Meat From a Black Bear Hunted in Northern Ontario.

BACKGROUND: Although trichinellosis is known to cause thrombotic disease, serious thrombotic events are rare and have not been previously associated with Trichinella nativa infection. METHODS: Patient interviews and medical chart reviews were conducted on 10 men who became ill following consumption of a common source of black bear meat. Trichinella serology on patient sera as well as polymerase chain reaction (PCR) and larval identification of the meat samples was conducted. RESULTS: All 10 exposed individuals developed an acute illness clinically compatible with trichinellosis, characterized by fever, abdominal pain, and diarrhea, along with eosinophilia ranging from 0.9 × 109/L to 6.1 × 109/L. Within 2 weeks of the diarrheal illness, systemic symptoms developed in all exposed individuals characterized by fever, myalgia, periorbital edema, and fatigue. ST-elevation myocardial infarction and sinus venous tract thrombosis occurred as a complication of trichinellosis in 2 patients. Acute serology was nonreactive in all patients, though convalescent serology was reactive in 6 of 8 (75%) patients for whom sera was available. Multiplex PCR identified T. nativa from the bear meat, and was corroborated by microscopic larval identification. CONCLUSIONS: We report a 100% attack rate of T. nativa from bear meat among those who were exposed, and demonstrate that this species can cause serious thrombotic complications of trichinellosis in humans. Education of hunters and the public regarding the importance of proper preparation of wild game prior to ingestion is warranted.

Long-read sequencing improves assembly of Trichinella genomes 10-fold, revealing substantial synteny between lineages diverged over 7 million years.

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host-pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases.

First case of Trichinella nativa infection in wild boar in Central Europe-molecular characterization of the parasite.

The examination of wild boars gained in Poland shows for the first time occurrence of Trichinella nativa, freeze-resistant species of Trichinella in this host from the central Europe region. This finding is not only one of several cases of T. nativa invasion in wild boars all over the world but also one of the very few cases of T. nativa detected so far beyond the known boundary of occurrence of this species. The molecular characterization of discovered larvae based on analysis of partial genes: 5s rDNA-ISR and CO1 confirm the findings. Moreover, the analyzed DNA sequences of both genes present new haplotypes of T. nativa in comparison to that described previously.

The occurrence of Trichinella species in the cougar Puma concolor couguar from the state of Colorado and other regions of North and South America.

Trichinella species are zoonotic nematodes that infect wild carnivores and omnivores throughout the world. We examined the prevalence and species of Trichinella infections in cougars (Puma concolor couguar) from Colorado, United States. Tongues from cougars were examined by pepsin-HCl artificial digestion to detect Trichinella spp. larvae. The species or genotype of individual worms was identified by multiplex polymerase chain reaction (PCR). Trichinella spp. larvae were detected in 17 of 39 cougars (43.6% (28.7-59.5%)). Five of the cougars (12.8%) were infected with T. murrelli, 3 (7.7%) were infected with T. pseudospiralis, and 1 (2.6%) had Trichinella genotype T6. Trichinella spp. larvae from eight cougars were not identified at the species level, due to degraded DNA. The high prevalence of Trichinella spp. in cougars from Colorado and reports of the parasite in other populations of Puma spp. suggest that this large predator is a key mammalian reservoir.

Prevalence and intensity of Alaria alata (Goeze, 1792) in water frogs and brown frogs in natural conditions.

In the last 15 years, the mesocercariae of Alaria alata have frequently been reported in the wild boar during routine Trichinella inspections made compulsory for the trade of venison meat in Europe. If these studies have focused primarily on mesocercariae isolated from meat, few works have been done so far to understand the circulation of the parasite in natural conditions especially in the intermediate hosts. This study focuses on the second intermediate hosts of this parasite assessing the suitability of two amphibian groups-brown frogs and water frogs sensu lato-for mesocercarial infection on an area where A. alata has already been identified in water snails and wild boars. During this study, both groups showed to be suitable for mesocercarial infection, with high prevalence and parasite burdens. Prevalence was higher in the brown frog group (56.9 versus 11.54 % for water frogs) which would indicate that it is a preferential group for infection on the study area, though reasons for this remain to be investigated. No significant difference among prevalences was observed between tadpoles and frogs. This study, the first focusing on A. alata in these amphibians in Europe, provides further information on circulation of this parasite in natura.

Seroprevalence of Trichinella sp. in Wild Boars (Sus scrofa) from Yanggu-gun, Gangwon-do, Korea.

A total 7 outbreaks of trichinellosis have occurred in Korea, mostly as a result of consumption of raw wild boar (Sus scrofa) meat. Since only 1 serological survey on wild boars had yet been performed in Korea, the present study aimed to estimate the prevalence of trichinellosis in wild boars and some species of rodents by artificial digestion and serological examinations in Yanggu-gun, Gangwon-do, the endemic area of trichinellosis. Both the wild boar and rodent muscle samples revealed no Trichinella larvae by direct examination and artificial digestion method. However, serological examinations revealed that 4 wild boar sera samples out of 118 (3.4%) were positive to Trichinella antigen. Although the recovery of Trichinella larvae ended in a failure, it is proved for the first time that the sylvatic cycle of Trichinella has been maintained in wild boars of Gangwon-do, Korea.

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.