Trichinella T6 Research Articles

Identification of Trichinella taxa by ITS-1 amplicon next-generation sequencing with an improved resolution for detecting underrepresented genotypes in mixed natural infections

BackgroundAmplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis.MethodsThe new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR.ResultsNGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada.ConclusionsThis new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten.Graphical

624. Outbreak of Human Trichinellosis Linked to Bear Meat Infected with Multiple Trichinella Species — Arizona, Minnesota, and South Dakota, 2022

Abstract Background In July 2022, the Minnesota Department of Health was notified of a patient with fevers, myalgias, periorbital edema, and eosinophilia; providers suspected trichinellosis. Human trichinellosis is rare in the United States and usually acquired from wild game meat. We investigated to confirm the diagnosis, ascertain additional cases, and identify the source of infection. Methods Trichinellosis was defined as compatible symptoms in a person who consumed an epidemiologically implicated meal (probable) or had Trichinella antibodies (confirmed). Blood samples from exposed persons were tested for Trichinella antibodies and with unbiased sequencing for microbial cell-free DNA. Microscopy and a parasite detection assay based on deep sequencing were performed on suspected source meat. Results One week before symptom onset, the patient and 8 relatives from 3 states shared a meal including meat from a bear harvested in Saskatchewan, Canada. The meat was frozen for 5 weeks until being grilled with vegetables and served rare. There were a total of 6 cases (4 probable and 2 confirmed, attack rate = 75%). Six persons had a trichinellosis-compatible illness, of whom five submitted samples for Trichinella antibody testing and two had positive results. Plasma samples from the two antibody-positive patients were tested for microbial cell-free DNA, and both were positive for Trichinella spp. DNA. Motile Trichinella species larvae ( >800 larvae per gram) were identified in bear meat frozen for 4 months. Deep sequencing detected 3 Trichinella genotypes, 2 of which matched reference sequences for T. nativa and Trichinella T6. The third genotype did not match any reference sequence; the closest match (94%) was Trichinella T9, which has only been detected in Japan. Conclusion We used multimodal diagnostic testing to characterize an outbreak of human trichinellosis and confirm bear meat as the source. Freezing kills Trichinella species commonly implicated in pork-associated outbreaks, but T. nativa and Trichinella T6 are freeze-resistant. Consumers of wild game meat should be informed that adequate cooking is required to kill Trichinella parasites. Further work on identification and classification of Trichinella species in North America is needed. Disclosures David Berman, DO, Precision Health Solutions, St. Petersburg, Florida: Advisor/Consultant

Performance of indirect enzyme-linked immunosorbent assay using Trichinella spiralis-derived Serpin as antigen for the detection of exposure to Trichinella spp. in swine

Indirect enzyme-linked immunosorbent assay (ELISA) utilizing excretory-secretory (E-S) antigens of Trichinella spiralis is currently the method of choice for testing pigs and wild boars for exposure to Trichinella spp. The E-S proteins are released by first-stage larvae (L1) of this parasitic nematode maintained in vitro. However, the production of these antigens is cumbersome and time-consuming. The process requires animals to be experimentally infected with the parasite as the source of L1. Antigen production using recombinant technology would be more time- and cost-effective. In this study, we produced a Serpin of T. spiralis as a recombinant protein secreted by the yeast Pichia pastoris. The diagnostic performance of indirect ELISA with purified Serpin antigen was compared to that of E-S ELISA. Both Serpin ELISA and E-S ELISA demonstrated 98 % diagnostic specificity in testing 1056 pigs from the Canadian Trichinella-free commercial herd. Twenty of 21 pigs with non-negative test results in E-S ELISA tested negative by the confirmatory Western blot (WB) assay. Therefore, the diagnostic specificity of combined E-S ELISA and WB was 99.9 %. Forty-five sera collected at or after six weeks from 34 pigs experimentally infected with various numbers of T. spiralis L1 produced positive results in both E-S and Serpin ELISA, resulting in 100 % diagnostic sensitivity. However, testing of sera serially collected from four pigs experimentally infected with various low doses of T. spiralis L1 demonstrated a delayed Serpin-specific antibody response compared to seroconversion detected by E-S ELISA in three animals. Moreover, Serpin ELISA demonstrated significantly lower sensitivity for detecting antibodies induced by experimental infections of pigs with T. britovi, T. nativa, Trichinella T6 and T. pseudospiralis, suggesting that it will not provide consistent detection of exposure to sylvatic Trichinella spp. The validation data support the application of Serpin ELISA in seroepidemiological surveys for detecting exposure to T. spiralis in swine.

High prevalence, intensity, and genetic diversity of Trichinella spp. in wolverine (Gulo gulo) from Yukon, Canada

BackgroundSpecies of Trichinella are globally important foodborne parasites infecting a number of domestic and wild vertebrates, including humans. Free-ranging carnivores can act as sentinel species for detection of Trichinella spp. Knowledge of the epidemiology of these parasites may help prevent Trichinella spp. infections in northern Canadian animals and people. Previous research on Trichinella spp. in wildlife from Yukon did not identify risk factors associated with infection, or the diversity and identity of species of Trichinella in regional circulation, based on geographically extensive sampling with large sample sizes.MethodsIn a cross-sectional study, we determined the prevalence, infection intensity, risk factors, and species or genotypes of Trichinella in wolverine (Gulo gulo) in two regions of Yukon, Canada, from 2013–2017. A double separatory funnel digestion method followed by mutiplex PCR and PCR-RFLP were used to recover and identify species of Trichinella, respectively.ResultsWe found larvae of Trichinella in the tongues of 78% (95% CI 73–82) of 338 wolverine sampled. The odds of adult (≥ 2 years) and yearling (1 year) wolverine being Trichinella spp.-positive were four and two times higher, respectively, compared to juveniles (<1 year). The odds of Trichinella spp. presence were three times higher in wolverine from southeast than northwest Yukon. The mean intensity of infection was 22.6 ± 39 (SD, range 0.1–295) larvae per gram. Trichinella T6 was the predominant genotype (76%), followed by T. nativa (8%); mixed infections with Trichinella T6 and T. nativa (12%) were observed. In addition, T. spiralis was detected in one wolverine. Out of 22 isolates initially identified as T. nativa in multiplex PCR, 14 were analyzed by PCR-RFLP to distinguish them from T. chanchalensis, a recently discovered cryptic species, which cannot be distinguished from the T. nativa on multiplex PCR. Ten isolates were identified either as T. chanchalensis alone (n = 7), or mixed infection with T. chanchalensis and T. nativa (n = 2) or T. chanchalensis and Trichinella T6 (n = 1)].ConclusionsWolverine hosted high prevalence, high larval intensity, and multiple species of Trichinella, likely due to their scavenging habits, apex position in the food chain, and wide home range. Wolverine (especially adult males) should be considered as a sentinel species for surveys for Trichinella spp. across their distributional range.Graphical

Trichinella nativa and Trichinella T6 in arctic foxes (Vulpes lagopus) from northern Canada

Parasitic zoonotic nematodes of the genus Trichinella circulate in wildlife and domestic hosts worldwide through the ingestion of infected meat. Due to their role as scavengers and predators in terrestrial and marine arctic ecosystems, Arctic foxes (Vulpes lagopus) are ideal sentinels for the detection of Trichinella spp. In this study, we determined the prevalence, larval intensity, and species of Trichinella from 91 trapped Arctic foxes collected around the northern Canadian communities of Sachs Harbour (Ikaahuk) on Banks Island (n = 23), and Ulukhaktok and Cambridge Bay (Ikaluktutiak) on Victoria Island (n = 68). Using pepsin-HCl digestion, larvae of Trichinella spp. were recovered from the left forelimb muscle (flexor carpi ulnaris) in 19 of the 91 foxes (21% prevalence, 95% CI: 14–30%). For the first time in Arctic foxes in Canada, Trichinella species were identified using multiplex PCR that was followed up with PCR-RFLP to distinguish between T. nativa and T. chanchalensis. All infected foxes harbored T. nativa, and one fox was co-infected with Trichinella T6; the latter is a new host record. Age of the fox was significantly associated with Trichinella spp. infection and the odds of being infected were three times higher in foxes ≥2 years of age (p = 0.026), indicating cumulative exposure with age. While Arctic foxes are seldom harvested for human consumption, they serve as sentinel hosts of Trichinella spp., confirming the presence of the parasite in wildlife in the region.

Epidemiology and hypothetical transmission cycles of Trichinella infections in the Greater Kruger National Park of South Africa: an example of host-parasite interactions in an environment with minimal human interactions.

Knowledge on the epidemiology, host range and transmission of Trichinella spp. infections in different ecological zones in southern Africa including areas of wildlife-human interface is limited. The majority of reports on Trichinella infections in sub-Saharan Africa were from wildlife resident in protected areas. Elucidation of the epidemiology of the infections and the prediction of hosts involved in the sylvatic cycles within specific ecological niches is critical. Of recent, there have been reports of Trichinella infections in several wildlife species within the Greater Kruger National Park (GKNP) of South Africa, which has prompted the revision and update of published hypothetical transmission cycles including the hypothetical options based previously on the biology and feeding behaviour of wildlife hosts confined to the GKNP. Using data gathered from surveillance studies and reports spanning the period 1964–2019, confirmed transmission cycles and revised hypothesized transmission cycles of three known Trichinella species (T. zimbabwensis, Trichinella T8 and T. nelsoni) are presented. These were formulated based on the epidemiological factors, feeding habits of hosts and prevalence data gathered from the GKNP. We presume that the formulated sylvatic cycles may be extrapolated to similar national parks and wildlife protected areas in sub-Saharan Africa where the same host and parasite species are known to occur. The anecdotal nature of some of the presented data confirms the need for more intense epidemiological surveillance in national parks and wildlife protected areas in the rest of sub-Saharan Africa to unravel the epidemiology of Trichinella infections in these unique and diverse protected landscapes.

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.

Present status of trichinellosis - a neglected zoonosis in India

Trichinellosis is a meat-borne helminthic zoonosis, caused by different species of the genus Trichinella. The disease is cosmopolitan in distribution and affects around 10,000 people annually around the globe. Based on genetic, biochemical and biological variability, 9 valid species (T. britovi, T. murrelli, T. nativa, T. nelsoni, T. papuae, T. patagoniensis, T. pseudospiralis, T. spiralis and T. zimbabwensis) and 3 genotypes (Trichinella T6, T8 and T9) of the parasite have been recognized. These species infect around 100 mammalian species including domestic and wild pigs, horses, game animals and wild carnivores. The infection starts with consumption of raw or undercooked meat or meat products containing encysted muscle larvae of the parasite. Most of the infections or outbreaks have been associated with the consumption of meat or meat products of pigs, wild boars, horses, crocodiles, walruses and dogs. Trichinoscopy is used in the veterinary inspection of pork in slaughterhouses and meat-packing facilities in many countries. It is a rapid process, but low in sensitivity and fails to detect mild infection. The muscle digestion method using HCl-pepsin is more sensitive and thus preferred. Recent outbreak of trichinellosis in Uttarakhand state opened up issues related to rapid diagnosis and lack of consumer awareness regarding safe cooking habits of meat of pig origin. This status report is an attempt to compile the information on Trichinella spp. infection in animals and humans in India at one place to draw the attention of medical and veterinary personnels involved in disease investigation and active research on zoonotic diseases.

Tongue has higher larval burden of Trichinella spp. than diaphragm in wolverines (Gulo gulo)

Trichinella is an important zoonotic parasite found in a range of wildlife species harvested for food and fur in Canada. We compared larval intensity from tongue and diaphragm, the best predilection sites in other animal species, from naturally infected, wild wolverines (Gulo gulo) (n = 95). Muscle larvae of Trichinella spp. were recovered by the pepsin/HCl artificial digestion method (gold standard) using double separatory funnels, and species were identified using multiplex PCR. Prevalence was 83% (79/95). Of those positive for Trichinella spp. (n = 79), 76 (96.2%) were detected in both tissues, 2 (2.5%) were positive only on diaphragm, and 1 (1.3%) only on tongue. A total of 62 of 79 wolverines (78.5%) had higher larval burden in tongue than in diaphragm, whereas 17 wolverines (21.5%) had higher larval burden in diaphragm. The predilection site (higher larval burden) of Trichinella spp. larvae did not vary significantly between juvenile and adult wolverines (P = 0.2), between male and female wolverines (P = 0.9), and among wolverines classified as having low and high larval intensities overall (P = 0.2). Trichinella T6 was the predominant genotype (63 of 79; 80%), followed by T. nativa (T2) (6 of 79; 8%). Mixed infections of T2 and T6 were observed in 9 of 79 (12%) wolverines. Larval intensity of Trichinella T6 was higher in tongues than diaphragms. No statement can be made for T2 due to insufficient T2 positive samples. In conclusion, tongues are a better site for sampling than diaphragms in future surveys of Trichinella larval intensity in wolverines; however, either tissue is suitable for prevalence studies.

Hybridization is limited between two lineages of freeze-resistant Trichinella during coinfection in a mouse model

Hybridization between two closely related but distinct genetic lineages may lead to homogenization of the two lineages with potentially novel phenotypes, or selective pressure to avoid hybridization if the two lineages are truly distinct. Trichinella nativa and Trichinella T6 are zoonotic nematode parasites which can be distinguished genetically despite occasional hybridization. Here, using an experimental murine model, we attempt to determine whether there are barriers to hybridization when sizeable numbers of each lineage are allowed to coinfect a host. Two mice were independently infected with equal numbers of T. nativa and T6. The offspring of these coinfections were genotyped at two microsatellite loci and one mitochondrial locus capable of distinguishing T. nativa from T6 genotypes. Among larvae in the F1 generation, offspring of every possible mating were encountered. Most larvae (63.6%) derived from T. nativa×T. nativa matings, while 21.1% of offspring were the product of T6×T6 matings, and only 15.3% were hybrid offspring of T. nativa×T6 crosses, differing markedly from null expectations. In this experimental model, T. nativa and Trichinella T6 were able to mate, but ratios of offspring indicated pre- or post-zygotic barriers to hybridization that may include assortative mating, genetic incompatibilities, and/or differences in the fitness of offspring. These barriers would limit gene flow between these two lineages in a natural setting, serving as a barrier to their homogenization and promoting their persistence as distinct and separate entities.

Discernible but limited introgression has occurred where Trichinella nativa and the T6 genotype occur in sympatry

The genetic diversity within and among parasite populations provides clues to their evolutionary history. Here, we sought to determine whether mitochondrial and microsatellite DNA variation could be used to evaluate the extent of differentiation, gene flow and historical reproductive isolation among the freeze resistant parasites Trichinella nativa and the Trichinella T6 genotype infecting wolverines (Gulo gulo) in Nunavut, Canada. To this end, we genotyped Trichinella isolates derived from the diaphragms of 39 wolverines from this locale to reference strains of T. nativa and the Trichinella T6 genotype. Results showed that among a subset of 13 isolates examined, individuals resembled T. nativa in their mitochondrial DNA, but resembled the Trichinella T6 genotype when assayed at expansion segment V and the internal transcribed spacer of the nuclear rDNA. To adjudicate among these conflicting diagnoses, we further characterized each isolate at several nuclear microsatellite loci and again compared these to data from reference strains. Statistical assignment established that the nuclear genomes of most Nunavut isolates corresponded to those of the Trichinella T6 genotype; however, two isolates corresponded to T. nativa, and one isolate exhibited equal similarity to both reference strains. Taken as a whole, the evidence suggests that these isolates derive from the T. nativa matrilineage, but that their nuclear genomes resemble individuals previously designated as Trichinella T6. Assuming distinct lineages, this argues for cross-hybridization among these genotypes. Although introgression has occurred, recognizable genetic distinctions persist. One possibility is that selection disfavors the survival of hybrid offspring in most instances. Alternatively, the recent disappearance of glacial barriers might have increased contact, and therefore introgression. Broader geographic sampling will be required to determine the extent to which hybridization occurs beyond this particular geographic focus.

Prevalence of Trichinella spp. in Black Bears, Grizzly Bears, and Wolves in the Dehcho Region, Northwest Territories, Canada, Including the First Report of T. nativa in a Grizzly Bear from Canada

Samples of muscle from 120 black bears (Ursus americanus), 11 grizzly bears (Ursus arctos), and 27 wolves (Canis lupus) collected in the Dehcho Region of the Northwest Territories from 2001 to 2010 were examined for the presence of Trichinella spp. larvae using a pepsin-HCl digestion assay. Trichinella spp. larvae were found in eight of 11 (73%) grizzly bears, 14 of 27 (52%) wolves, and seven of 120 (5.8%) black bears. The average age of positive grizzly bears, black bears, and wolves was 13.5, 9.9, and approximately 4 yr, respectively. Larvae from 11 wolves, six black bears, and seven grizzly bears were genotyped. Six wolves were infected with T. nativa and five with Trichinella T6, four black bears were infected with T. nativa and two with Trichinella T6, and all seven grizzly bears were infected with Trichinella T6 and one of them had a coinfection with T. nativa. This is the first report of T. nativa in a grizzly bear from Canada. Bears have been linked to trichinellosis outbreaks in humans in Canada, and black bears are a subsistence food source for residents of the Dehcho region. In order to assess food safety risk it is important to monitor the prevalence of Trichinella spp. in both species of bear and their cohabiting mammalian food sources.

A 10-year wildlife survey of 15 species of Canadian carnivores identifies new hosts or geographic locations for Trichinella genotypes T2, T4, T5, and T6

A survey of wild carnivores in Canada was conducted over a 10-year period to determine the prevalence and genotypes of Trichinella. Muscle samples collected from 1409 animals representing 15 hosts species were enzymatically digested to recover Trichinella larvae. Larvae were recovered from a total of 287 (20.4%) animals and PCR identified four genotypes of Trichinella. Trichinella nativa was found in 5 host species and was the most commonly found genotype. Trichinella T6 was present in 7 species of carnivores, and coyote and badger are new host records for this genotype. The recovery of T. pseudospiralis and T. murrelli from cougars is the first documentation of these species in Canada and in cougars. The cougar was also the only host species in which all four genotypes of Trichinella were identified. Black bears and walruses had the highest tissue levels of larvae in this study and are also the species most frequently associated with human trichinellosis in Canada. This work identifies additional host species and expanded geographic ranges for 4 genotypes of Trichinella in North America. Failure to demonstrate T. spiralis in wildlife and continued negative results from ongoing surveillance activities in swine provide additional evidence that T. spiralis is not present in Canada.

Trichinella zimbabwensis in a naturally infected mammal

Trichinella zimbabwensis has been detected in wild and farmed Nile crocodiles (Crocodylus niloticus) and in wild monitor lizards (Varanus niloticus) of several African countries, but it has never been detected in mammals in nature, in spite of its infectivity to rodents, pigs, foxes and monkeys under laboratory conditions. The aim of this work was to describe the first detection of T. zimbabwensis in a naturally infected lion (Panthera leo) of the Kruger National Park (KNP) of South Africa. The sequence of the expansion segment V, a highly variable non-coding sequence of the large subunit ribosomal RNA of the genus Trichinella, of larvae from the lion was identical to that of larvae of T. zimbabwensis collected from a Nile crocodile originating from the same locality as the lion, suggesting a possible transmission of this parasite between mammals and reptiles. The KNP proves to be a very interesting area for parasites of the genus Trichinella since three taxa (Trichinella nelsoni, Trichinella T8 and T. zimbabwensis) circulate among the wildlife of this protected area.

Trichinella nelsoni and Trichinella T8 mixed infection in a lion (Panthera leo) of the Kruger National Park (South Africa)

In South Africa, Trichinella sp. was first discovered in 1966 in the wildlife of the Kruger National Park (KNP). Since then, both Trichinella T8 and Trichinella nelsoni have been detected in the KNP, leading to a debate on the existence of a gene flow between the two taxa. In 2006–2008, four lions were killed in the Manyeleti Game Reserve, the Mthethomusha Nature Reserve, Numbi Gate, and Skukuza, which border the KNP. Larvae were isolated from muscles by artificial digestion. The molecular identification of single larva by multiplex PCR, followed by a specific PCR to distinguish between Trichinella T8 and Trichinella britovi, revealed Trichinella T8 in the lions from Manyeleti and Skukuza, a mixed infection with T. nelsoni and Trichinella T8 in the lion from Mthethomusha, and T. nelsoni in the lion from Numbi. No larva with a hybrid pattern between the two taxa was observed. No hybrid offspring resulted when crossing single males and females of T. nelsoni and Trichinella T8 in both directions, whereas hybrid offspring were obtained when crossing T. britovi and Trichinella T8 in both directions. This is the first report of a mixed infection with two Trichinella taxa in a host from the KNP, where both Trichinella T8 and T. nelsoni circulate among wildlife. Despite the sympatry status of these two taxa, field and laboratory data seem to exclude the possibility of gene flow, confirming their evolutive separation.

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.

Trichinella T6 and Trichinella nativa in Wolverines (Gulo gulo) from Nunavut, Canada

Infection of Trichinella spp. is common among animals in the Canadian Arctic. We determined the prevalence of Trichinella spp. infection in wolverines (Gulo gulo) from Nunavut, Canada. Diaphragms from 41 wolverines were examined by artificial digestion. Trichinella spp. larvae were detected in 36 (87.8%) examined animals. Trichinella T6 was detected in 33 (91.7%), Trichinella nativa in only one (2.8%), and a mixed Trichinella T6 and T. nativa infections were detected in two (5.6%) wolverines. This is the first report of Trichinella spp. infection in wolverines from Nunavut and the first report of sympatric Trichinella T6 and T. nativa in any host. The high prevalence of Trichinella spp. infection in combination with the natural history of wolverines suggests that the mustelid may be a key species in the natural cycle of these parasites in Arctic and Subarctic areas.

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes ( Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex ( T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.

Two-dimensional electrophoresis and mass spectrometry for the identification of species-specific Trichinella antigens

A proteomic approach was utilized for fine antigenic characterization of the closely related Trichinella genotypes Trichinella britovi T3 and Trichinella T8. Crude extract of muscle larvae L1 (LCE) from both isolates were analyzed by 2D-PAGE. Over 500 protein spots were reproducibly separated in both genotypes. These separated proteins were identified in Western blot with IgG1 and IgG3 from homologous and heterologous hyperimmune sera raised in BALB/c mice. A group of 20 and 15 spots migrating at 50–60 kDa and pH 5.5–6.5 in T. britovi and Trichinella T8 maps, respectively, reacted with the IgG1 from heterologous sera whereas a group of minor spots of similar migration patterns did not. Low cross-reactivity occurred for IgG3. MALDI-TOF and MALDI-TOF/TOF analysis of these antigens identified a predicted enolase, the protein P49 and a predicted actin among the cross-reactive proteins and two hypothetical actins among the non cross-reactive proteins.

Nonisotopic single‐strand conformation polymorphism analysis of sequence variability in ribosomal DNA expansion segments within the genus Trichinella (Nematoda: Adenophorea)

A nonisotopic single-strand conformation polymorphism (SSCP) approach was employed to 'fingerprint' sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. In addition, phylogenetic analyses of the D3 sequence data set, employing three different tree-building algorithms, examined the relationships among all of them. These analyses showed strong support that the encapsulated species T. spiralis and T. nelsoni formed a group to the exclusion of the other encapsulated species T. britovi and its related genotypes Trichinella T8 and T9 and T. murrelli, and T. nativa and Trichinella T6, and strong support that T. nativa and Trichinella T6 grouped together. Also, these eight encapsulated members grouped to the exclusion of the nonencapsulated species T. papuae and T. zimbabwensis and the three representatives of T. pseudospiralis investigated. The findings showed that nonencapsulated species constitute a complex group which is distinct from the encapsulated species and supported the current hypothesis that encapsulated Trichinella group external to the nonencapsulated forms, in accordance with independent biological and biochemical data sets.