Trichinella Nativa Research Articles

Epizootiological monitoring of wolf helminths in Northern and Central Kazakhstan.

Wolves (Canis lupus) play a role in nature, including the regulation of the number of ungulates and the use of dead animals. In addition, wolves are a natural link and carrier for the spread of many parasitic invasions. Hence, the main task in preventing the spread of parasitic invasions is to regulate the wolf population. This study aimed to monitor the endoparasitological fauna of wild wolves inhabiting Northern and Central Kazakhstan. Overall, 81 wolves were investigated for parasitic worms using the K. I. Scriabin method. Wolf intestinal materials were collected from the following six regions: North Kazakhstan, Pavlodar, Kostanay, Akmola, Ulytau, and Karaganda. The genetic diversity of the parasites was identified using a polymerase chain reaction with specific primers. After data collection, a comprehensive statistical analysis was performed. Several helminth types were identified in wolves, including Echinococcus granulosus, Taenia hydatigena, Mesocestoides spp., Toxascaris leonina, Trichinella nativa, Alaria alata, and Dirofilaria repens. Based on the results of this study, young male wolves aged 1-4 years were the most vulnerable to helminthiasis. Wolves living in steppe and semi-desert regions are often exposed to helminth infections. The prevalence of T. nativa in the wolves was 20.4%. This study also revealed the presence of echinococcosis among wolf populations in Karaganda and Kostanay, with prevalence rates of 4.1% and 4.7%, respectively. The overall prevalence of tapeworms in wolves was 54.3%. This study highlights the significance of understanding the potential risks associated with helminth infections in wild carnivores because helminths can act as disease reservoirs and pose a threat to humans, livestock, and other wild carnivores. These results can contribute to the development of effective control and management strategies for helminth infections in wolves, which can infect humans and livestock.

A Review of the Importance of Trichinosis in Public Health and the Status of Infection with This Parasite in Ethiopia

Trichinella is one of the most important zoonotic parasitic nematodes with worldwide prevalence. Trichinella larvae are usually found inside the muscle tissue of the animals such as pigs and mice. This parasite enters into human’s small intestine following the consumption of infected pork and, then, excretes the male worms through his/her feces. Eight different species of this parasite have so far been identified including Trichinella spiralis, Trichinella nativa, Trichinella nelsoni, Trichinella britovi, Trichinella murrelli, Trichinella pseudospiralis, Trichinella papuae, and Trichinella zimbabwensis. Other remaining worms are transferred to other parts of the body by the blood, and from there they migrate, especially, through the muscles of the abdominal wall, tongue, and larynx, thereby causing swelling and calcareous cysts inside the muscles. Other mammals such as cats, dogs, foxes, pigs, and boars also usually get this disease after eating the meat of mice or other infected animals. Humans can also develop this disease by consuming pork or boar meat. The spread of this disease is rare in Islamic countries where pork is not consumed due to the religious observance, but its spread is rapid in countries from Europe and South America where the disease afflicts 12% of their human population mainly due to a failure to cook or freeze the meat perfectly.

Parasites of five vagrant Polar bears (Ursus maritimus) swimming to Iceland during 2008 to 2016

During 2008 to 2016, five polar bears (Ursus maritimus) swam from the East Greenland population to Iceland and were shot soon after walking ashore. Each bear was dissected. Ectoparasites were searched for in their ears and fur, helminths in the gastrointestinal tract and Trichinella larvae in muscles. Protozoan cysts, oocysts and helminth eggs were also searched for in faecal samples. No ectoparasites were detected. Two bears (40%) hosted fourth stage larvae of the nematode Contracaecum osculatum (strain B) in the stomach. Characteristic scars, noted as craters in the rectum wall, indicated previous acanthocephalan infection in one bear. Three polar bears were infected by the nematode Trichinella nativa. No protozoan parasites were detected in faecal samples, but cysts of the heterokontophyte Blastocystis sp. were found in faeces of two bears (40%). This is the first report of C. osculatum, Blastocystis sp. and an acanthocephalan infection in free-living polar bears. Keywords: Iceland, parasites, Ursus maritimus, polar bear, vagrant, new host records

SPECIES COMPOSITION OF WOLF (CANIS LUPUS) HELMINTH FAUNA IN KAZAKHSTAN

The article presents data on the study of the species composition of wolf helminth fauna in the period from 2019 to 2021. A total of 39 wolves (Canis lupus) were examined, and their parasite fauna was assessed. The study identified the following types of helminths Echinococcus granulosus, Taenia krabbei, Dypilidium spp., Mesocestoides spp., Toxascaris leonina, Trichinella nativa, Dirofilaria repens. The extensiveness of the invasion wolves by helminths was relatively high in the western part region (96.5%), and towards the north-central part region, this indicator significantly decreased (65.2%). The average number of helminths was high, with some infected animals carrying several types of parasites. Thus, the intensity of invasion by helminths in wolves was 7.6 specimens per infected individual host. The research aimed to study the endoparasitic fauna of wild wolves using complex classical parasitological and molecular genetic methods. Samples of helminths were differentiated by amplification and sequencing using the marker gene cytochrome oxidase COX1 (GenBank accession number: MT877205, MZ505444, MZ669895, MZ724175). There is a need to assess the distribution of helminths in intermediate and final hosts populations and provide appropriate health education to avoid maintaining parasite life cycles and infestations, which will become a public health problem in the future.

PECULIARITIES OF THE NATURAL ORIGIN OF TRICHINELLOSIS IN THE CHUKOT PENINSULA TERRITORY

Trichinellosis is a natural focal nontransmissive zoonotic disease, the causative agent of which is the nematode Trichinella nativa (T2). The collection of biological material was carried out on the territory of the coastal settlements of the Chukot Peninsula. The objects of the presented work were the representatives of the order carnivore (Carnivora) of the following families: canids (Canidae), feline (Felidae), bears (Ursidae), walrus (Odobenidae), seal (Phocidae) and the whale (Сetacea). Larvae detection from the muscle tissue of animals was carried out by standard methods. Infection rates were determined by the extensiveness (EI) and intensity of invasion (II).During the evolution Trichinella have acquired a wide range of hosts with similar trophic relations, and therefore they ensure the long-term functioning of the natural focus. The organism of the animals, which are host species, plays the role of reservoir (the reservation phase). There were the following components of the guest component among the studied host species: 5 species of terrestrials, and 4 species of marine mammals. The animals’ contamination ranged from 60.0% to 100% in terrestrials, and it ranged from 0.9% to 4.3% in marine animals. Successful circulation of Trichinella nativa (dispersion phase, distribution) under the extreme conditions of Arctic region became possible due to several reasons. They are: getting tolerated to low temperatures in the process of evolution, the process of decay and a number of other adaptive properties that allow them to maintain viability and invasive properties in the corpses of terrestrial carnivores and marine mammals during the long winter. The natural focus of trichinellosis on the territory of the Chukot Peninsula is unique and multifaceted in nature, and it is of interest for researchers to learn environmental and general biological laws, taking into account the traditional economic activities of the indigenous population.

HELMINTHS OF WOLF IN THE VORONEZH RESERVE AND ADJACENT TERRITORIES

The wolf is the largest predatory mammal in the Central Black Soil region of Russia. The close trophic relationships of the wolf with wild ungulates and other animals play an important role in the formation of the modern predator helminth fauna. Helminthological materials from the wolf have been collected in the Voronezh Reserve and in adjacent natural areas for a long-term period (1981–2018). The modern fauna of the helminths of the wolf of the Voronezh Reserve contains 16 species. The main taxonomic groups of helminths (trematodes, cestodes, and nematodes) in the wolf are represented unevenly. The maximum number of helminth species is registered among nematodes – 12 species (Aonchothe caputorii, Eucoleus aerophilus, E. boehmi, Pearsonema plica, Calodium hepaticum, Trichinella nativa, Toxocara canis, Toxaskaris leonina, Uncinaria stenocephala, Crenosoma vulpis, Molineus patens, Dirofilaria repens). We have identified three species of cestodes (Dypilidium caninum, Taenia hydatigena, T. krabbei) and one species of trematodes (Alaria alata). The highest infection rates were registered for A. alata: the extensiveness of invasion (EI) is 92.3%, the intensity of invasion is 149.7 specimens, an abundance index is 138.2 specimens. High rates of wolf invasion by certain nematode species were revealed: E. boehmi (69.2%); P. plica (46.2%); E. aerophilus (38.5%); C. hepaticum (15.4%) and nematode T. nativa (27.9%). More than ten species of helminths of the wolf are of significant epidemiological and epizootological importance for this area.

Foxes (Vulpes vulpes) as sentinels for parasitic zoonoses, Toxoplasma gondii and Trichinella nativa, in the northeastern Canadian Arctic.

Outbreaks of Toxoplasma gondii and Trichinella spp. have been recurring for decades among Inuit of Nunavik, northeastern Canada. Contact with wildlife has been identified as a risk factor for Inuit exposure to T. gondii, but reservoirs have yet to be confirmed based on direct detection of DNA or organism. Similarly, little is known about the occurrence of Trichinella spp. in wildlife species of Nunavik other than walrus (Odobenus rosmarus) and bears (Ursus americanus, Ursus maritimus). Foxes (Vulpes vulpes) were targeted as possible sentinels for T. gondii and Trichinella spp. because of their high trophic position within the Arctic food chain as carnivorous scavengers. A total of 39 red foxes were sampled from four communities in southern and western Nunavik between November 2015 and September 2016. For the first time in wildlife, a novel magnetic capture DNA extraction and real-time PCR technique was used to isolate and detect T. gondii DNA from the heart and brain of foxes. A double separatory funnel digestion method followed by multiplex PCR was used to recover and genotype larvae of Trichinella spp. from tongues of foxes. Seroprevalence based on detection of antibodies to T. gondii was 41% (95% CI: 27–57%) using a commercially available modified agglutination test (MAT). Detection of DNA of T. gondii and larvae of Trichinella nativa (T2) occurred in 44% (95% CI: 28–60%) and 36% (95% CI: 21–51%) of foxes, respectively. Coinfection with both T. nativa and T. gondii occurred among 23% (95%CI: 13–38%) of foxes which can be attributed to co-transmission from prey and scavenged species in their diet. There was only moderate agreement between T. gondii serology and direct detection of T. gondii DNA using the MC-PCR technique (Kappa test statistic: 0.321), suggesting that using both methods in tandem can increase the sensitivity of detection for this parasite. These findings show that foxes are good sentinels for circulation of parasitic zoonoses in terrestrial northern ecosystems since they are highly exposed, show measurable indicators of infection and do not serve as exposure sources for humans.

A survey of helminths of polar bears in the Russian Arctic

The polar bear Ursus maritimus is a circumpolar species classified as vulnerable and included in the IUCN Red List. It is considered to be practically free of helminth parasites with the only species reported being Trichinella spiralis s. l. Samples of feces were collected on Chukotka coast, Wrangel Island and on ice floes in the Kara, Laptev and Chukchi seas in 2013-2015 in different seasons of the year. Coprological diagnostics was carried out using the standard flotation and sedimentation methods. In the samples collected in the snow-free period, a single sample (3.7%) was found to contain eggs of the nematode Toxascaris sp. In three out of 9 samples collected in the winter, eggs of a cestode Diphyllobothrium sp., of unidentified trematodes (presumably Heterophyidae) and of the strongylid nematode Uncinaria stenocephala were found as well as the first stage nematode larvae tentatively identified as Crenosoma sp. Viable Trichinella nativa larvae were recovered from the muscles of a female animal from north of Yakutia.

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

Although trichinellosis is known to cause thrombotic disease, serious thrombotic events are rare and have not been previously associated with Trichinella nativa infection. 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. 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. 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.

Genetic evidence of interspecies introgression of mitochondrial genomes between Trichinella spiralis and Trichinella britovi under natural conditions

Trichinellosis is a zoonotic disease caused by Trichinella muscle larvae (ML) through ingestion of raw or undercooked meat. To date, 12 taxa are recognized in this genus, of which four are circulating in Europe (Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis). T. spiralis and T. britovi circulate in European wildlife and occur simultaneously in the same host species. The possibility of hybrid formation between T. britovi and T. spiralis has hardly been addressed and so far, results of experimental hybridisation attempts between T. britovi and T. spiralis are inconclusive. The aim of the present study was to analyse molecular polymorphisms of single T. spiralis and T. britovi ML from natural infections based on nuclear 5S rDNA intergenic spacer region (5S rDNA-ISR) and mitochondrial cytochrome c oxidase 1 (CO1) gene sequences. Six haplotypes of the 5S rDNA intergenic spacer region (5S rDNA-ISR) and 14 of the cytochrome c oxidase 1 (CO1) gene were demonstrated in 89 individual T. britovi ML from Latvia and Poland. In contrast, only two haplotypes were observed at both 5S rDNA-ISR and CO1 of 57 individual T. spiralis ML from Polish wild boar and red foxes. Moreover, this study demonstrates hybridisation in eight individual ML between T. britovi and T. spiralis under natural conditions in four Polish wild boar and two red foxes, revealed by combining 5S rDNA-ISR and CO1 sequence information of individual Trichinella ML. To our knowledge, this is the first report of interspecies hybridisation between T. spiralis and T. britovi under field conditions.

Polymerase chain reaction – Restriction fragment length polymorphism analysis for the differentiation of Trichinella nativa and Trichinella britovi

Recently, Trichinella nativa was identified in foxes in Germany and Poland, indicating that the geographical distribution of T. nativa is not restricted to areas north of the isotherm −4°C in January.In the European Union, legislation requires that a regular monitoring of the occurrence of Trichinella spp. in indicator animals such as foxes or raccoon dogs be carried out. The Trichinella isolates must also be identified on a species level.The multiplex PCR recommended by the Community Reference Laboratory for Trichinella allows species identification, yet the differentiation of T. nativa and Trichinella britovi, a widespread Trichinella species in the temperate regions of Europe, is unstable.We therefore describe an easy and reliable method for the differentiation of the two species, which can be utilised to monitor a potential spread of T. nativa in Central Europe.

Survival of North American genotypes of Trichinella in frozen pork.

North American genotypes of Trichinella spiralis (T-1), Trichinella nativa (T-2), Trichinella pseudospiralis (T-4), Trichinella murrelli (T-5), and Trichinella T-6 were examined for susceptibility to freezing in pork using time-temperature combinations that have been proven to inactivate T. spiralis. Infections were established in 3-month-old pigs of mixed sex and breed by oral inoculation of 10,000 muscle larvae (ML) (all genotypes, rodent-derived ML), 20,000 ML (T-1, T-4, and T-5; cat-derived ML), or 30,000 ML (T-2 and T-6; cat-derived ML). Pigs were euthanized 60 days postinoculation. Muscles from the tongue, masseter muscles, diaphragm, triceps, hams, neck, rump, and loins were ground, pooled, and mixed to ensure even distribution of larvae. Samples (20 g) containing each Trichinella species, genotype, and source combination were placed in heat-sealable pouches, transferred to a constant temperature refrigerant bath, and maintained according to defined time and temperature combinations. Larvae recovered from cold-treated pork samples were inoculated into mice to determine infectivity. Results indicated that the time-temperature combinations known to render pork safe for T. spiralis are sufficient to inactivate T. nativa and T-6 (the freeze-resistant isolates), T. murrelli (the most common sylvatic species in the United States excluding Alaska), and T. pseudospiralis (a species that lacks a muscle nurse cell). These data close a gap in knowledge about the effectiveness of freezing for inactivating these parasites in pork and should alleviate concern about the safety of frozen pork products from the United States.

A Nonhealing Ulcerative Skin Lesion Associated with Trichinella Nativa Infection in a Cat

Trichinella spp. can infect various domestic and wild species, including companion animals. Infection occurs because of the ingestion of raw meat (e.g., infected prey). In experimental studies, cats have been found to be a very susceptible host to infection by Trichinella spp.; naturally occurring feline infections have also been reported. However, clinically apparent disease seems to be a rare manifestation of this infection in cats. The skin biopsy of an 8-year-old, neutered, male, domestic cat revealed an inflammatory granulation tissue that surrounded a well-preserved cyst that contained a Trichinella sp. larva. Distinct seropositive reaction against Trichinella spp. antigens was demonstrated by enzyme-linked immunosorbent assay and Western blot. Immunohistochemistry, by using serum from the infected cat as the source of antibody, showed strong immunostaining of Trichinella spp. larvae. During a 1-year follow-up, a postexcisional local tissue reaction was observed. This manifested as a firm, poorly circumscribed subcutaneous mass adjacent to the eye, which demonstrated clinical features and histopathologic findings indicative of chronic inflammation associated with granulation tissue and fibrodysplasia. Digestion of the muscle biopsy revealed one Trichinella sp. larva, which was identified by multiplex polymerase chain reaction as Trichinella nativa. To the authors' knowledge, this is the first documented case of trichinellosis in a cat with a nonhealing ulcerative skin lesion as the main clinical manifestation of the infection.

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.

Molecular epidemiology of Trichinella spp. in three Baltic countries: Lithuania, Latvia, and Estonia

Meat of domestic pigs and wild boars has been the significant source of emerged human trichinellosis in Lithuania, Latvia, and Estonia over the past two decades. However, there is very little known on the occurrence of Trichinella spp. in main wildlife reservoirs and its transmission in domestic and sylvatic cycles in these countries. The present study demonstrated considerably higher endemicity of Trichinella spp. in main sylvatic reservoirs (28.9-42% in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in all three countries than previously reported. Molecular identification of Trichinella larvae from more than 500 sylvatic and domestic animals revealed four Trichinella species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, and Trichinella pseudospiralis) sympatric in a relatively small area and several as the first records for the respective countries. The nonencapsulating T. pseudospiralis is found for the first time in the Eastern Europe. Sylvatic T. britovi was found in domestic pigs in Lithuania and Latvia (16 and 57.1%, respectively) and only in these countries, domestic T. spiralis was detected in sylvatic animals in areas where domestic trichinellosis was registered. The study suggests that transmission of Trichinella between domestic and sylvatic cycles in Lithuania and Latvia is favored by improper human behavior, e.g., pig and slaughter waste management.

MOLECULAR CHARACTERIZATION OF TRICHINELLA GENOTYPES BY INTER-SIMPLE SEQUENCE REPEAT POLYMERASE CHAIN REACTION (ISSR-PCR)

A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.

HELMINTHOLOGIC SURVEY OF THE WOLF (CANIS LUPUS) IN ESTONIA, WITH AN EMPHASIS ON ECHINOCOCCUS GRANULOSUS

Carcasses of 26 wolves were collected during the 2000/2001 and 2003/2004 hunting seasons and examined for helminths. Thirteen helminth species were recorded: one trematode (Alaria alata), seven cestodes (Diphyllobothrium latum, Mesocestoides lineatus, Taenia hydatigena, Taenia multiceps, Taenia ovis, Taenia pisiformis, and Echinococcus granulosus), and five nematode species (Uncinaria stenocephala, Toxascaris leonina, Toxocara canis, Trichinella nativa, and Trichinella britovi). The most common species were A. alata and U. stenocephala. Mature Echinococcus granulosus was found and described for the first time in Estonia, and its identity verified using PCR-RFLP analysis. Sequencing a fragment of the mitochondrial DNA NADH dehydrogenase 1 (mtND1) gene showed that the E. granulosus strain from Estonia was identical to strain G10, recently characterized in reindeer and moose in Finland.

Intestinal establishment and reproduction of adult Trichinella spp. in single and mixed species infections in foxes ( Vulpes vulpes)

Intestinal establishment and reproduction of adult Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis were examined as single species or mixed species infections in foxes. This is the first study of intestinal dynamics of Trichinella spp. in a carnivore model and the results suggest that the intestinal phase is relatively short as only very few worms were recovered 10 days post-inoculation (dpi). In mixed species infection with equal doses of T. nativa and T. spiralis, molecular typing demonstrated that 64% of the intestinal worms and 78% of the muscle larvae were T. nativa. Conversely, T. spiralis dominated in the mixed species infections with T. pseudospiralis, constituting 66% of the intestinal worms and 94% of the muscle larvae. Although, the individual recoveries of intestinal worms were only up to 5.6% on day 1, and up to 1.5% on day 4 post-infection, the muscle larvae establishment was comparable to other fox studies. Infectivity, measured as muscle larvae burden did not differ among the four species of Trichinella, which is in contrast to other models with mice, rats, pigs or herbivores. Although statistically significant differences in intestinal worm burdens were found for some days, no distinct species were recovered in consistently higher numbers than the others.

Tolerance to low temperatures of domestic and sylvatic Trichinella spp. in rat muscle tissue.

The present study was designed to investigate the tolerance to low temperatures of 9 Trichinella isolates in rat muscle tissue. Nine groups of 24 rats were infected with encapsulated Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella T6, Trichinella nelsoni, and 3 nonencapsulated Trichinella pseudospiralis strains. Six rats from each of the groups were necropsied at 5, 10, 20, and 40 wk postinfection (wpi). Muscle tissues containing Trichinella larvae were exposed to temperatures of -18, -5, and 5 C for 1 or 4 wk, and afterward the reproductive capacity index (RCI) in mice was determined for the 9 individual Trichinella isolates. Only T. nativa muscle larvae were infective after freezing at a temperature of -18 C. At 5 wpi all encapsulated isolates, except for the tropical species T. nelsoni, remained infective after exposure to a temperature of -5 C for both 1 and 4 wk, whereas nonencapsulated T. pseudospiralis survived only 1 wk of exposure. All Trichinella spp. remained infective after exposure to a temperature of 5 C. Muscle larvae for all investigated species remained infective as long as they persisted in live rats during the experiment. Analysis of variance showed a significant effect of age on the temperature tolerance of encapsulated T. spiralis and nonencapsulated T. pseudospiralis. In addition, significant interaction between age of muscle larvae and length of exposure was found. In general Trichinella muscle larvae of medium age (10 and 20 wpi) tolerated freezing better than early and late stages of infection (5 and 40 wpi). This is the first study to demonstrate such a relationship between age of infection and temperature tolerance of Trichinella spp. muscle larvae.

Molecular identification of natural hybrids between Trichinella nativa and Trichinella T6 provides evidence of gene flow and ongoing genetic divergence

To date, there are no data available on the population genetics of Trichinella due to the lack of genetic markers and the difficulty of working with such small parasites. In the Arctic region of North America and along the Rocky Mountains, there exist two genotypes of Trichinella, Trichinella nativa and Trichinella T6, respectively, which are well differentiated by biochemical and molecular characters. However, both are resistant to freezing, show other common biological characters (e.g. low or no infectivity to rodents and swine) and produce fertile F1 offspring upon interbreeding. To data, these two genotypes have been considered allopatric. In this study, we detected both genotypes in wolves of the same wolf packs in Alaska, suggesting sympatry. A single GTT trinucleotide present in the ITS-2 sequence of T. nativa but not in Trichinella T6 was used as a genetic marker to study gene flow for this character in both a murine infection model and in larvae from naturally-infected Alaskan wolves. Only F1 larvae originating from a cross between T. nativa male and Trichinella T6 female were able to produce F2 offspring. Larvae (F1) originating from a cross between Trichinella T6 male and T. nativa female were not reproductively viable. As expected, all F1 larvae showed a heterozygote pattern for the GTT character upon heteroduplex analysis; however, within the F2 population, the number of observed heterozygotes ( n=52) was substantially higher than expected ( n=39.08), as supported by the F is index, and was not in the Hardy–Weinberg equilibrium. Larvae from two of the 16 Trichinella positive Alaskan wolves, showed the Trichinella T6 pattern or the T. nativa/ Trichinella T6 hybrid pattern. Our data demonstrate that T. nativa and Trichinella T6 live in sympatry at least in Alaskan wolves, where T. nativa occurs more frequently (69%) than Trichinella T6 (31%). One explanation for this phenomenon is that glacial periods may have caused a geographical relocation, colonisation and independent evolution of T. nativa within the Rocky Mountains, resulting in a bifurcation of the freeze-resistant genotype. Additional studies will be required to test this hypothesis.