PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.     

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).     

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

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

Trichinella spiralis infections of inbred mice: immunologically specific responses induced by different Trichinella isolates

The immune response of inbred mice was studied following infection with Trichinella spiralis var. pseudospiralis (TP) or with isolates of T. spiralis derived from a pig or from an arctic fox. Animals given a primary infection with 1 isolate of Trichinella and challenged 21 days later with the same or different isolates responded more quickly by expelling worms from the homologous challenge. In addition, although mesenteric lymph node cells from mice infected with each isolate of Trichinella would proliferate in vitro when cultured with antigen derived from each of the others, the strongest proliferation response always occurred when cells were cultured in the presence of antigen prepared from the specific isolate used to infect the mouse from which the cells were derived. In addition, it was possible to prepare monoclonal antibodies that recognized an antigen expressed by TP which was not shared by T. spiralis isolates and vice versa. Collectively, these data support the conclusion that the differences observed in the kinetics of immune responsiveness to different Trichinella isolates are referable, at least in part, to differences among the isolates in the expression of functionally relevant antigens.     

Molecular investigation of African isolates of Trichinella reveals genetic polymorphism in Trichinella nelsoni

Molecular genetic studies were carried out on three isolates of Trichinella nelsoni (from Kenya, Tanzania and South Africa) and three isolates of Trichinella T8 (from South Africa and Namibia) from sylvatic carnivores and from a sylvatic swine. A probe (pT7.3) specific for T. nelsoni was obtained by screening a pUC18 genomic library. The pT7.3 sequence was 346 bp in length with an AT content of 70%. The sequence is present approximately 200 times per haploid genome. Southern blot analysis of Hind III digested DNAs of the three isolates of T. nelsoni revealed that the hybridisation patterns of the isolates from Kenya and Tanzania were identical and that they differed from that of the isolate from South Africa, indicating the presence of polymorphism in this species. A pUC18 genomic library of Trichinella T8 was also screened, and one clone (pT8.3) was found to be specific for homologous DNA by dot blot, but Southern blot analysis of DNA samples from eight genotypes showed different hybridisation signals for both Trichinella T8 and Trichinella britovi DNAs. No differences in the nucleotide sequences of the expansion segment V were observed for the T. nelsoni isolates. However, they differed from those of Trichinella T8. The presence of Trichinella T8 in Africa south of the Sahara and its genetic relationship with T. britovi remain unclear and warrant detailed investigations.     

Molecular identification of Korean Trichinella isolates

Muscle larvae of Trichinella isolates from two outbreaks in Korea were analyzed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiple-PCR. All of the muscle larvae showed a band similar to that of T. spiralis larvae of the reference strain. The two Korean Trichinella isolates (isolate code ISS623 and ISS1078) might be classifiable to Trichinella spiralis.     

Comparison of IgG3 responses to carbohydrates following mouse infection or immunization with six species of Trichinella

The IgG3 antibody responses to carbohydrate epitopes were compared in BALB/c mice infected or immunized with six species of Trichinella: T. spiralis (T1), T. nativa (T2), T. britovi (T3), T6, T. nelsoni (T7), and T8. The dynamics of IgG3 responses and antigen recognition following infection or immunization were measured by ELISA and Western blot respectively, using glycosylated and deglycosylated larval crude extracts (LCE) prepared from homologous isolates. A high degree of protein glycosylation was found in all species and with similar profiles. Deglycosylation was completely achieved only in LCE from T1 and T6 isolates. The dynamics of IgG3 responses following infection or immunization significantly differed whereas the antigen recognition profiles appeared similar. Variations in the levels and antigen recognition patterns of IgG3 among the different species were apparent. The highest IgG3 levels were recorded in infections by the T8 isolate and the lowest in infections by the T6 isolate, whereas for immunization the highest IgG3 response was induced by T7 and the lowest by T8. Following antigen deglycosylation, the IgG3 responses were significantly reduced or abrogated and the recognition patterns markedly modified or suppressed in the different species of Trichinella.     

Conservation of diagnostic antigen epitopes among biologically diverse isolates of Trichinella spiralis

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.--Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis--and compared by electrophoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates.     

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

An isolate of Trichinella obtained from a wild boar in Yugoslavia did not form cysts in the musculature of its natural host. Subsequent inoculation into experimental hosts demonstrated that some larvae became encysted only after extended time periods, whereas others remained unencapsulated. Histological staining of larvae in the musculature demonstrated no deposition of collagen typically seen for Trichinella spiralis spiralis, Trichinella spiralis nativa, or Trichinella spiralis nelsoni. The Yugoslavian isolate, given the name of Zagreb isolate after the University where it was first studied, had low infectivity for pigs and mice. Isozyme analysis demonstrated greater homology with T. s. nelsoni than with other subspecies of Trichinella. Restriction fragment length polymorphisms and dot blot analyses further demonstrated the distinctive nature of this isolate. These results suggest that lack of cyst formation might be characteristic of isolates other than those designated Trichinella pseudospiralis and that this character might be important in the classification of Trichinella.     

Trichinella britovi etiological agent of sylvatic trichinellosis in the Republic of Guinea (West Africa) and a re-evaluation of geographical distribution for encapsulated species in Africa

In West Africa, Trichinella infection was documented in humans and animals from Senegal in the 1960s, and the biological characters of one isolate showed a lower infectivity to domestic pigs and rodents when compared with that of a Trichinella spiralis pig isolate from Europe. To identify the Trichinella species present in West Africa, a survey was conducted in a total of 160 wild animals in the Republic of Guinea. Three Viverridae, one true civet (Viverra civetta) and two African palm civets (Nandinia binotata) from the Fouta Djallon Massif, Pilimini Subprefecture, were found positive by artificial digestion of muscle samples. Trichinella larvae from these three viverrids were identified as Trichinella britovi and no difference was detected in three examined sequences from these African isolates and the reference strain of T. britovi from Europe, indicating common ancestry, an historically continuous geographic distribution, and recent isolation for African and European populations. The detection of T. britovi in West Africa modifies our knowledge about the distribution of encapsulated species of Trichinella in Africa. Thus, Trichinella nelsoni is now considered to have a distribution limited to the Eastern part of the Afrotropical region from Kenya to South Africa. This provides a plausible explanation for the presence of Trichinella T8 in Namibia and South Africa, and further suggests that T. britovi could be the Trichinella species circulating among wild animals of Northern Africa.     

Trichinella pseudospiralis populations of the Palearctic region and their relationship with populations of the Nearctic and Australian regions

Since few non-encapsulated isolates of Trichinella have been studied to date, their level of differentiation from encapsulated species and the taxonomic value of the observed polymorphisms remain to be determined. To this end, biological, biochemical and molecular data from 11 isolates of Trichinella pseudospiralis and one isolate of Trichinella papuae were examined using the broad group of encapsulated species and genotypes for comparison. Single-worm cross-breeding experiments and reproductivity capacity indices revealed F1 progeny only among T. pseudospiralis isolates from different zoogeographical regions, whereas no F1 were produced when T. pseudospiralis was crossed with T. papuae. Furthermore, unlike T. pseudospiralis, T. papuae failed to infect chickens. Comparative analysis of 12 allozymes revealed a single difference between Nearctic and Australian isolates of T. pseudospiralis, but substantial differences when compared with T. papuae (i.e. two unique and six diagnostic markers). Molecular studies involving mitochondrial-derived genes encoding cytochrome oxidase I and the large subunit ribosomal DNA indicated a high level of sequence similarity among T. pseudospiralis isolates; however, a concomitantly high level of variation was observed in expansion segment five of the genomic large subunit ribosomal DNAs among T. pseudospiralis isolates and between this species and T. papuae. Collectively, these results demonstrate high uniformity among isolates of T. pseudospiralis from Eurasia and polymorphism among isolates of T. pseudospiralis belonging to different zoogeographical regions; the results corroborate the classification of T. papuae as a differentiated species.     

Molecular characterization of sylvatic isolates of Trichinella spiralis

Genetic relationships of 20 Trichinella isolates from Indiana wildlife were assessed and compared to Trichinella isolated from an infected swine herd. Trichinella larvae were isolated from coyotes, mink, raccoons, and red foxes. The larvae were maintained and amplified in white mice (ICR) and wild mice (Peromyscus leucopus). Differences in phenotypic characters of sylvatic isolates in the 2 laboratory hosts included an approximately 10-30-fold increase in parasite fecundity in wild mice. DNA for each isolate was extracted from Trichinella larvae and analyzed by dot-blot hybridization using a repetitive DNA probe pBP2 that recognizes DNA sequences specific for swine Trichinella. The probe hybridized only to Trichinella from swine and a single coyote isolate. Restriction endonucleases were used to digest DNA and the resulting fragments were separated by gel electrophoresis. Based on the presence of repetitive DNA sequences in the Trichinella genome, distinctive banding patterns were seen among the isolates. Trichinella isolated from swine had a pattern distinct from all sylvatic isolates except 1 from a coyote. Because this coyote was from the same general locality as the swine Trichinella outbreak, it was concluded that the isolate represents transmission of swine trichinellosis to the wildlife population. Further analysis using the enzyme Cla I identified unique banding patterns for wild isolates, suggesting that the sylvatic group is a genetically heterogeneous complex.     

The use of the polymerase chain reaction to identify porcine isolates of Trichinella.

A method was developed to identify domestic isolates of Trichinella using the polymerase chain reaction. Oligonucleotide primers, based on the repetitive DNA sequence (pPRA) from the P1 isolate of Trichinella, were used to amplify genomic DNA from 13 domestic isolates and tested against sylvatic isolates of Trichinella. Pattern differences were observed among domestic isolates, indicating divergence of this repetitive sequence. The primers were specific for domestic Trichinella as no amplification was detected for sylvatic isolates or Trichinella pseudospiralis. It was possible to identify an isolate from a single larva following digestion or in situ in muscle tissue.     

Allozyme analysis of Trichinella isolates from various host species and geographical regions.

Allozyme analysis was carried out on 152 Trichinella isolates from synanthropic and wild animals and from humans; the isolates were collected from 5 continents. The analysis, involving 27 enzymes, revealed the presence of 8 distinct gene pools, termed T1-T8. Four of the genetic groups represent the 4 previously proposed species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella nelsoni (T7), and Trichinella pseudospiralis (T4). The other 4, T3, T5, T6, and T8 are distinct from previously described species. The absence of allozymic hybrid patterns among even sympatric groups indicates a lack of gene flow among the groups. Principal component analysis and the unweighted pair group method of analysis were used to assemble allozyme patterns of the 152 isolates into discrete groups and to show their relative relationships. Both analyses indicated the presence of 8 primary clusters that correlated with the gene pools revealed by direct allozyme profile analysis. The absence of evidence of gene flow among the gene pools and the high level of allozymic differentiation between the cluster groups support the concept that the genus Trichinella is composed of several sibling species.     

Identification of two isolates of Trichinella recovered from humans in France

Two Trichinella isolates from humans in France were characterized using reproductive capacity indices and a combination of molecular methods. The isolate TRLL hybridized with the pig type-specific probe pPra and had pig type restriction profiles and rDNA patterns. It was therefore identified as a domestic or pig type isolate. The isolate CTRD-85 had similarities and differences in restriction profiles and rDNA patterns with both AF1 and Trichinella nelsoni and was identified as a sylvatic type. Pattern comparisons also show that T. nelsoni is similar to variants of the North American sylvatic type.     

Biological characterization of Trichinella isolates from various host species and geographical regions.

Forty isolates of Trichinella collected from 5 continents were compared for 7 biological characters: newborn larvae produced per female worm cultured in vitro at the seventh, eighth, and ninth day postinfection, host muscle nurse cell development time, reproductive capacity index in rats and chickens, and resistance of muscle larvae to freezing. The isolates also were compared by analyses of an environmental character of the location from which they were isolated: the isotherms for January and July. By factorial analysis of correspondence of the biological and environmental data, the 40 isolates were grouped into 8 gene pools (T1-T8). The environmental temperature-related distribution was more evident for the sylvatic isolates (T2, T3, T5, T6, T7, T8), than for T1, which was isolated from domestic pigs, and for T4, a bird-adapted, nonencapsulating genetic type. The 8 biological groups correlated closely with the 8 gene pools previously identified on the basis of allozyme analysis. These results support the concept that the genus Trichinella is composed of at least 5 distinct gene pools or sibling species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella sp. (T3), Trichinella pseudospiralis (T4), and Trichinella nelsoni (T7), and 3 other groups of uncertain taxonomic status (i.e., T5, T6, and T8).     

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.     

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.     

Trichinella murrelli n. sp: etiological agent of sylvatic trichinellosis in temperate areas of North America

Trichinella T5, collected from sylvatic carnivores in North America, was identified previously as a different phenotype of Trichinella, with an uncertain taxonomic level due to the availability of only 2 isolates. Cross-breeding experiments carried out with single female and male larvae of 2 strains of Trichinella T5, with single female and male larvae of 2 strains of Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis, Trichinella nelsoni, and Trichinella T6, showed a reproductive isolation of Trichinella T5. Viable offspring were obtained only when a female of Trichinella T5 was crossed with a male of T. britovi, but not vice versa. Furthermore, the analysis of biological, biochemical, and molecular data of 32 isolates collected from sylvatic animals in the Nearctic region and identified as Trichinella T5 permitted its reassessment at the species level. Trichinella murrelli n. sp. is characterized by the following: distribution in temperate areas of the Nearctic region; newborn larvae production in vitro of 29-36/72 hr; nurse cell development time between 24 and 70 days postinfection; reproductive capacity index in Swiss mice 1.2-9.5, in wild mice 29.5-159.8, in rats 0.7-2.4, and in pigs 0.03-0.0004; no resistance to freezing; ribosomal DNA fragments of 7.2 kb and/or 11.4 kb, plus 2.2 kb and 1.8 kb present after Dra I digested DNA when probed with total T. spiralis RNA; a specific amplicon of 179 bp after polymerase chain reaction (PCR) amplification with the primer set SB147G; a specific fragment of 1,600 bp after PCR amplification with the primer set Ts43CA and Hhb I digestion; long incubation period; and moderate to severe pathogenicity for humans. The new species is most similar to T. britovi, though it differs from T. britovi in the pattern of 2 allozymes, in the patterns of major ribosomal DNA and PCR-restriction fragment length polymorphism fragments, and in geographical distribution.     

Genetic differentiation of the Trichinella genus with isoenzymatic analysis

Twenty-six Trichinella isolates have been examined by the isoenzyme typing of ten enzyme systems (LDH, ME, 6PGDH, G6PDH, GOT, AK, PGM, ACON, MPI, GPI). Four different zymodemes were obtained. All the examined isolates have shown an electrophoretic behaviour like one or other of four reference strains. The isolates from Italy and Yugoslavia have an electrophoretic mobility like T. nelsoni reference strain. The isolates from France, Holland, Great Britain, Poland and USA have an electrophoretic mobility like T. spiralis reference strain. For T. nativa and T. pseudospiralis we have tested only the reference strains. These results support the validity of the taxonomy of Trichinella genus in four good species.