Sylvatic and domestic Trichinella spp. in wild boars; infectivity, muscle larvae distribution, and antibody response

Thirty-six wild boars were inoculated with Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (USSR), T. pseudospiralis (USA), T. pseudospiralis (AUST), Trichinella murrelli, Trichinella T6, and Trichinella nelsoni. The wild boars were killed at 5 and 10 wk postinoculation (PI), and the number of muscle larvae per g (lpg) of tissue was determined for 18 muscles or muscle groups. Five weeks PI, all Trichinella genotypes had established as muscle larvae, but their infectivity varied widely: T. spiralis established in high numbers (mean = 296 lpg), T. britovi, T. nelsoni, and 1 of the T. pseudospiralis genotypes (AUST) in moderate numbers (mean = 53-74 lpg), whereas the remaining genotypes were poorly infective (mean 2-16 lpg). Because of considerable weight gain of the wild boars, an estimated total larval burden (live weight x lpg) was calculated for each animal. The total larval burden did not change significantly over time for T. spiralis, T. murrelli, T. britovi, T. nelsoni, and T. pseudospiralis (USA and USSR), whereas a significant reduction could be demonstrated for T. nativa, Trichinella T6, and T. pseudospiralis (AUST). Diaphragm and tongue were predilection sites in wild boars, independent of Trichinella genotype and infection level. At low infection levels, a greater percentage of larvae were found in diaphragm and tongue at 10 wk than 5 wk PI. Antibody responses increased rapidly between weeks 3 and 5 PI. For T. spiralis and T. nelsoni, the high antibody level persisted throughout the experimental period, but for T. nativa, T. britovi, T. murrelli, or Trichinella T6, the levels declined. For T. pseudospiralis, the antibody response increased more gradually between weeks 3 to 10 PI. Infection with all genotypes of Trichinella were detected using any of 7 excretory-secretory antigens, which points to the potential use of 1 common antigen for epidemiological studies on Trichinella in wild boars. In conclusion, T. spiralis is highly infective to wild boars, T. britovi, T. nelsoni, T. pseudospiralis (USA), and T. pseudospiralis (USSR) are moderately infective, and T. nativa, T. murrelli, T. pseudospiralis (AUST), and Trichinella T6 are poorly adapted to this host species.     

Trichinella spp.: differential expression of two genes in the muscle larva of encapsulating and nonencapsulating species.

Kuratli, S., Lindh, J. G., Gottstein, B., Smith, D. F., and Connolly, B. 1999. Trichinella spp.: Differential expression of two genes in the muscle larva of encapsulating and nonencapsulating species. Experimental Parasitology 93, 153-159. The expression of the two genes tsmyd-1 and tsJ5 was studied in the muscle stage larva of three different species of Trichinella. T. spiralis and T. britovi are both encapsulating species, while T. pseudospiralis is a nonencapsulating species. Expression of tsJ5 is developmentally regulated in T. spiralis and has been shown in this study to be down-regulated in the T. pseudospiralis muscle larva compared with the other two species. Immunoblot analysis has also revealed that the relative abundance of the protein product of this gene, TSJ5, is lower in T. pseudospiralis muscle larvae. It has previously been shown that expression of tsmyd-1 is not developmentally regulated in T. spiralis (Connolly et al. 1996). In contrast, expression of this gene is slightly increased in the muscle larvae of T. pseudospiralis. Southern analysis of genomic DNA from the three Trichinella species shows that both genes are highly conserved.     

Differences and similarities of nurse cells in cysts of Trichinella spiralis and T. pseudospiralis

The nurse cell in the cyst of Trichinella spiralis comprises at least two kinds of cytoplasm, derived from muscle or satellite cells, as indicated by the pattern of staining using regular dye (haematoxylin and eosin, or toluidine blue), alkaline phosphatase (ALP) expression, acid phosphatase (ACP) expression and immunostaining with an anti-intermediate filament protein (desmin or keratin). Muscle cells undergo basophilic changes following a T. spiralis infection and transform to the nurse cells, accompanied by an increase in ACP activity and the disappearance of desmin. Satellite cells are activated, transformed and joined to the nurse cells but remain eosinophilic. The eosinophilic cytoplasm is accompanied by an increase in desmin and ALP expression but not an increase in ACP activity. Differences in the staining results for ALP or ACP suggest that the two kinds of cytoplasm have different functions. Trichinella pseudospiralis infection results in an increase of ACP activity at a later stage than T. spiralis. There is also a difference in the location pattern of ACP in the cyst of T. spiralis compared with T. pseudospiralis. In T. spiralis, ACP is diffused within the cell, but in T. pseudospiralis, ACP distribution is spotty corresponding to the location of the nucleus. Trichinella pseudospiralis infection is accompanied by a slight increase in ALP activity. Activated satellite cells following a T. pseudospiralis infection exhibit an increase in desmin expression. The present study therefore reveals that nurse cell cytoplasm differs between the two Trichinella species and between the two origins of cytoplasm in the cyst of T. spiralis.     

Trichinella spp.: differential expression of acid phosphatase and myofibrillar proteins in infected muscle cells

Major alterations are induced in muscle cells infected by either Trichinella spiralis or Trichinella pseudospiralis. To investigate the response of muscle to these infections we have analyzed the expression of acid phosphatase (ACP, EC 3.1.3.2), adult skeletal muscle myosin heavy chain, and muscle tropomyosin proteins in infected mouse skeletal muscle cells. Using T. spiralis-infected cells, we provide strong evidence that the tartrate-sensitive ACP of these cells was synthesized by the infected cell and localized in lysosomes. Isoenzyme analysis indicated that the ACP activity was of host muscle cell origin and the specific activity of this ACP was 2.5 times greater than that in associated inflammatory cells. Increased ACP activity was also demonstrated in muscle cells infected by T. pseudospiralis. In synchronized muscle infections, increased ACP activity was detected at 5 days post-muscle infection for both parasites. ACP activity was further increased in infected muscle cells at later times tested. This increased infected cell ACP activity represents the earliest positive enzyme marker yet described indicating expression of the infected cell phenotype. In contrast, myofibrillar proteins were not detected in muscle cells chronically infected by T. spiralis but were detected in muscle cells infected by T. pseudospiralis. Decrease in myofibrillar protein levels was detected by 10 days post-muscle infection by T. spiralis. The data presented demonstrate significant differences and similarities in the phenotypes of muscle cells infected by these two parasites and establish criteria that could facilitate identification of parasite factors that may be involved in these phenomena.     

Biological variation in Trichinella species and genotypes

At present, the genus Trichinella comprises seven species of which five have encapsulated muscle larvae (T. spiralis, T. nativa, T. britovi, T. nelsoni and T. murrelli) and two do not (T. pseudospiralis and T. papuae) plus three genotypes of non-specific status (T6, T8 and T9). The diagnostic characteristics of these species are based on biological, biochemical and genetic criteria. Of biological significance is variation observed among species and isolates in parameters such as infectivity and immunogenicity. Infectivity of Trichinella species or isolates is determined, among other considerations, by the immune status of the host in response to species- or isolate-specific antigens. Common and particular antigens determine the extent of protective responses against homologous or heterologous challenge. The kinetics of isotype, cytokine and inflammatory responses against T. spiralis infections are isolate-dependent. Trichinella spiralis and T. pseudospiralis induce different dose-dependent T-cell polarizations in the early host response, with T. spiralis initially preferentially promoting Th1-type responses before switching to Th2 and T. pseudospiralis driving Th2-type responses from the outset.     

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

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.     

Ribo HRM--detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards

Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates. Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020-JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1). Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus.     

Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs

Groups of pigs were inoculated with genotypes of Trichinella belonging to: Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (from Caucasus), T. pseudospiralis (from USA), Trichinella murrelli, Trichinella sp. (from North America), and Trichinella nelsoni. The pigs were sacrificed between 5 and 40weeks p.i., and the number of muscle larvae per gram (l.p.g.) of tissue was determined as an average of 18 muscles. All Trichinella genotypes were infective for pigs, but both their infectivity and persistence varied: 5weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24-52l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05-5. 00l.p.g.). The total larval burden (live weight of pigxl.p.g.) was constant over time for T. spiralis, T. britovi, and T. nelsoni, but declined significantly (P<0.05) for the other genotypes. Antibody responses could be detected 3-4weeks p.i. by seven different Trichinella ES antigens, but the antibody levels and dynamics differed significantly among the experimental groups. In pigs inoculated with T. spiralis, T. britovi, or T. nelsoni, the antibody level increased rapidly between weeks 3 and 5 p.i. and was stable or increased slightly throughout the experimental period. In pigs inoculated with T. nativa, T. murrelli, or Trichinella (T6) (from North America), a rapid increase was detected between weeks 3 and 5 p.i., but for these genotypes a reduction in the antibody levels was seen thereafter. In the pigs inoculated with T. pseudospiralis, the antibody level increased more gradually over a period from week 3 p. i. to weeks 15-20 p.i., and decreased thereafter. In general, all species of Trichinella were detected by any of the seven ES antigens, which points to the potential use of one common antigen for surveillance and epidemiological studies on both domestic and sylvatic Trichinella in pigs. Homologous ES antigens were slightly more sensitive in detecting antibodies to the corresponding Trichinella species.     

Oxidative,heat and anthelminthic stress responses in four species of Trichinella: comparative study

The aim of this study was to compare levels of stress proteins in four Trichinella species when exposed to different stressors. Heat shock protein (HSP) 60, 70 and 90 responses were evaluated in infective larvae (L(1)) of four classic Trichinella species following exposure to oxidative, anthelminthic and thermal stress. Larvae of T. nativa, T nelsoni, T. pseudospiralis and T. spiralis were exposed to peroxide shock (0.2%, 1%, or 2% H(2)O(2)for 2h), high temperatures (40 degrees C or 45 degrees C for 2h), or 0.1 microg/ml of the benzimidazole anthelminthics: mebendazole (MBZ), albendazole (ALB) or thiabendazole (TBZ) for 4h. Following exposures, the L(1) were tested for induced morphological changes. Those observed were: (i) no change (in all species exposed to 40 degrees C) (ii) aberrant forms (in all species exposed to anthelminthics, in T. nativa, T. nelsoni and T. spiralis exposed to 45 degrees C, and in T. spiralis and T. nelsoni exposed to 0.2% H(2)O(2)) and (iii) severe degradation or death (in T. nativa and T. pseudospiralis exposed to 0.2% H(2)O(2), and in all species at 1% and 2% H(2)O(2)). In Western blot analyses, L(1) proteins were probed with monoclonal antibodies (mAbs) specific for the three HSPs. Greater changes in HSP levels occurred following H(2)O(2) exposure than with other stresses in all Trichinella species, while accumulation of a 50 kDa HSP was only observed in T. spiralis and T. pseudospiralis. Anthelminthic stress only caused decreased HSP levels in T. nativa. Thermal stress caused no significant changes in the HSP response of any species. It is suggested that other stress proteins (e.g., glucose-regulated proteins) may be involved in adaptation to thermal stress.     

Identification of Trichinella isolates by polymerase chain reaction--restriction fragment length polymorphism of the mitochondrial cytochrome c-oxidase subunit I gene.

We developed a polymerase chain reaction based approach using restriction fragment length polymorphisms of the mitochondrial cytochrome c-oxidase subunit I to identify nine genotypes (Trichinella spiralis, Trichinella britovi-European strains, Trichinella britovi-Japanese strains, Trichinella nativa, Trichinella nelsoni, Trichinella T5, Trichinella T6, Trichinella T8 and Trichinella pseudospiralis) in the genus Trichinella. Partial mitochondrial cytochrome c-oxidase subunit I genes of nine genotypes were amplified by polymerase chain reaction, sequenced, and digested with three restriction endonucleases (Mse I, Alu I and Bsp1248 I). This polymerase chain reaction based restriction fragment length polymorphism method allowed the identification of Trichinella genotypes. Trichinella spiralis, Trichinella britovi-Japanese strains, Trichinella nelsoni, T5 and Trichinella pseudospiralis were distinguishable by digestion with Mse I. Trichinella britovi-European strains and Trichinella T8 were distinguishable by digestion using Alu I, and Trichinella nativa and Trichinella T6 were distinguishable by double-digestion with Mse I and Bsp1286 I. The results obtained with this polymerase chain reaction based restriction fragment length polymorphism assay confirmed those previously reported by others and support the separation of the Japanese isolates as a new genotype, namely Trichinella T9.     

Muscle fiber selectivity of Trichinella spiralis and Trichinella pseudospiralis (1983)

The biceps, semimembranosus, biceps femoris, and soleus muscles of female Rockland Wistar mice infected with either 1,000 Trichinella spiralis or 1,000 Trichinella pseudospiralis larvae were removed on days 12, 14, 16, and 18 post-infection (PI), sectioned and stained histochemically for their myosin ATPase activity. Light microscopic examination of the sections revealed that larvae of T. spiralis invade only the slow twitch muscle fibers, and those of T. pseudospiralis invade both the fast twitch and the slow twitch fibers. In sections obtained from mice infected with either parasite and killed on days 16 and 18 PI, identification of the majority of the infected fibers as fast twitch or slow twitch was not possible due to pathological modification of infected fibers.     

Freeze-resistant Trichinella (Trichinella nativa) Established on the Scandinavian Peninsula

Until the 1970’s, Trichinella spiralis (Owen 1835) was considered the only species within the genus Trichinella. Then T. pseudospiralis (Garkavi 1972) was classified as a separate species on the basis of morphological and biological features. The remaining morphologically homogenous “T. spiralis-group” has been split into 4 different species (or subspecies) on the basis of their biological and biochemical characteristics; T. nativa (Britov & Boev 1972), T. nelsoni (Britov & Boev 1972), T. spiralis sensu stricto and T. britovi (Pozio et al. 1992).     

Allozymic and biological characters of Trichinella pseudospiralis isolates from free-ranging animals.

To evaluate biological and biochemical variability in nonencapsulated Trichinella isolates, biological and allozymic studies were conducted on isolates of Trichinella collected from a raptoral bird (Aquila rapax) and a fox (Vulpes corsac) in Kazakhstan and from a dasyurid marsupial (Dasyurus maculatus) on the island of Tasmania, Australia. Allozyme profiles of bird and marsupial isolates showed close similarity with the type isolate of Trichinella pseudospiralis. The avian and fox isolates successfully interbred with the type T. pseudospiralis isolate, but they failed to interbreed with 3 encapsulating species, Trichinella spiralis, Trichinella nativa, and Trichinella britovi. The reproductive index assessed in 4 inbred and 1 outbred strains of mice was lower for the avian isolate than for the marsupial and the type T. pseudospiralis isolates (P < 0.001).     

Alterations in the longevity and fecundity of adult Trichinella pseudospiralis related to method of isolation of infective larvae

The infectivity of Trichinella pseudospiralis infective larvae was reduced significantly following exposure to low pH or a combination of 1% pepsin at low pH compared to that for larvae isolated in phosphate-buffered saline (PBS) at pH 7.0. Reduction of host gastric pH by administration to mice of sodium bicarbonate solution in PBS was accompanied by an increase in the infectivity of larvae isolated in 1% pepsin/HCl (P/HCl) compared to that for worms inoculated into hosts given PBS alone. Fewer adult worms developing from larvae isolated in P/HCl became established in the host small bowel than was seen with larvae isolated in PBS; moreover, the fecundity in vitro of adult worms developing from P/HCl-isolated larvae was reduced below that for adults developing from larvae isolated from host muscle in PBS. More adult worms were recovered following infection of immune hosts with PBS-isolated larvae than were recovered from immune mice challenged with larvae isolated in P/HCl. Similar findings were observed in mice immunized by infection with Trichinella spiralis and challenged with T. pseudospiralis larvae isolated in either P/HCl or PBS. Immunization of mice with T. pseudospiralis larvae isolated by either method and challenged with larvae of T. spiralis resulted in recovery of similar percentages of the challenge inoculum.     

Stimulated chemotactic response in neutrophils from Trichinella pseudospiralis-infected mice and the neutrophilotactic potential of Trichinella extracts.

During infection with Trichinella pseudospiralis a strong neutrophil response is evident in the peripheral circulation of the mouse. This study compared the chemotactic response of neutrophils from uninfected, T. pseudospiralis-infected and Trichinella spiralis-infected mice to extracts from adult worms, newborn larvae and muscle-stage larvae of both species of parasite. The chemotactic response of neutrophils from T. pseudospiralis-infected mice to Zymosan-activated mouse serum (ZAMS) was significantly greater than that seen with neutrophils from either uninfected or T. spiralis-infected mice. Unstimulated chemotactic response of neutrophils from these three groups of animals to medium alone was similar. The chemotactic response of neutrophils from the three groups of animals was unaffected by either the concentration or source of serum. The chemotactic response of neutrophils from T. pseudospiralis-infected mice was significantly greater than that observed with cells from uninfected or T. spiralis-infected mice. Among parasite extracts, those from newborn larvae displayed the strongest chemotactic potential for neutrophils. Extracts from muscle larvae of T. spiralis and T. pseudospiralis and extracts of T. spiralis adult worms showed the weakest attraction for neutrophils. Extracts from adult T. pseudospiralis and from newborn larvae of both species elevated the chemotactic response of uninfected mouse neutrophils to a significantly greater level than that seen with ZAMS alone, while a significant reduction in this response was evident only when ZAMS was presented to neutrophils with 500 micrograms of extract from muscle larvae of T. pseudospiralis or T. spiralis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella spiralis infections of inbred mice: genetics of the host response following infection with different Trichinella isolates

The immune response of inbred strains of mice was studied following infection with isolates of Trichinella from a pig (P1), an arctic fox (AF1), and T. spiralis var. pseudospiralis (TP). Strains of mice previously characterized as highly resistant to a separate pig isolate of T. spiralis responded to the P1 and AF1 isolates by expelling over 80% of the worms by day 10 postinfection (PI), and by suppressing the in vitro release of newborn larvae by female worms. However, the response induced by AF1 worms was expressed more quickly when compared to responses induced by the P1 and TP isolates. The host response to TP was less as recovery was always higher at day 10 PI and antifecundity effects were not induced in TP worms even in highly resistant strains of mice. Strains of mice previously characterized as susceptible to T. spiralis infection were slow to develop resistance when compared to the resistant mouse strains, but even among the susceptible strains, infection with AF1 induced a more rapid response. The mouse strains used in these experiments allowed us to assess the role of the major histocompatibility complex (MHC) and/or non-MHC genes in influencing the responses observed. As previously reported for a pig isolate of T. spiralis, both MHC and non-MHC genes influenced the rate at which worms were expelled from the gut and the host response that limits the fecundity of adult female worms.     

Infection of the Chinese hamster with Trichinella pseudospiralis

A mean of 2,862 muscle larvae was recovered on day 45 postinfection (PI) from the total body musculature of Chinese hamsters infected with 498 Trichinella pseudospiralis. Infection of the Chinese hamster with 494 Trichinella spiralis resulted in recovery of a mean of 225 muscle larvae on day 45 PI. The reproductive capacity index for T. pseudospiralis was 5.74, whereas that for T. spiralis was 0.46 in this host species.     

Genetic diversity within the genus Trichinella as shown by cleavage fragment length polymorphism analysis

The genetic diversity within the genus Trichinella was studied using cleavage fragment length polymorphism (CFLP) analysis. The CFLP method generates specific fingerprints based on single nucleotide mutations. By this method the amplified intergenic regions of the 5S rRNA genes of the eight different genotypes of Trichinella were analysed. The CFLP pattern of T. spiralis was completely different compared with the sylvatic species T. britovi, T. nativa, T. nelsoni, and the genotypes Trichinella T5, Trichinella T6 and Trichinella T8. The T. pseudospiralis intergenic region can be differentiated by size from the other species of Trichinella.