Trichinella Genotypes Research Articles

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.

Host diversity and biological characteristics of the Trichinella genotypes and their effect on transmission

The host spectra and biological diversity of the Trichinella genotypes are reviewed. While all genotypes appear to reproduce equally well in carnivore hosts, their infectivity and persistence in omnivores and herbivores show remarkable differences. Most of the genotypes found in wildlife have low infectivity for pigs and some persist only for a few weeks; in herbivores this tendency is even more profound, but malnourished, environmentally stressed, or otherwise immuno-suppressed hosts are likely to be more susceptible to Trichinella genotypes that would otherwise cause no, or only low level infection in that particular host species. In the domestic habitat (e.g. domestic pig farms), Trichinella spiralis is found almost exclusively, but in the sylvatic habitat the other Trichinella genotypes have found individual ecological niches. Thus, when environmental stress is limited in the domestic habitat, the high reproductive capacity of T. spiralis has a selective advantage, but in nature, the tolerance of other (sylvatic) genotypes to high and low temperatures and decomposition of host tissue might be more important. Parasite distribution according to muscle appear to be independent of the genotype of Trichinella and predilection sites are primarily determined by host species and secondarily by the age and level of infection. The biological diversity of the Trichinella genotypes should definitely be considered when planning experimental studies, as the uniform high infectivity of all genotypes in carnivores probably make them more suited for comparative studies than rodents.

Factors affecting the flow among domestic, synanthropic and sylvatic cycles of Trichinella

Nematodes of the genus Trichinella are maintained in nature by sylvatic or domestic cycles. The sylvatic cycle is widespread on all continents, from frigid to torrid zones, and it is maintained by cannibalism and scavenging behavior of carnivores. Trichinella is primarily a parasite of carnivorous mammals, although one non-encapsulated species, Trichinellapseudospiralis, has also been detected in birds. The anaerobic metabolism of larvae in nurse cells allows their survival in extremely decayed meat. Encapsulated larvae in the decomposing carcass function similarly to the species-dispersing population of eggs or larvae of other nematodes, suggesting that the natural cycle of Trichinella includes a free-living stage when the parasite is no longer protected by the homeothermy of the host. Consequently, environmental temperature and humidity play an important role in the transmission of Trichinella among wildlife. Of the 10 recognized genotypes of Trichinella, only Trichinellaspiralis is transmitted and maintained in a domestic cycle, although it can be present also in wildlife. All other genotypes (Trichinellanativa, Trichinellabritovi, T.pseudospiralis, Trichinellamurrelli, Trichinellanelsoni and Trichinellapapuae, Trichinella T6, T8, and T9) are transmitted and maintained only in a sylvatic cycle. This generalization does not preclude sylvatic species of Trichinella from invading the domestic habitat, and T.spiralis may return to this habitat when humans fail in the management of wildlife and domestic animals. However, the presence of sylvatic genotypes of Trichinella in the domestic habitat represents a “dead-end” for the sylvatic cycle. Synanthropic animals (rats, foxes, mustelides, cats, dogs, etc.) contribute to the flow of sylvatic Trichinella genotypes from wildlife to domestic animals and of T.spiralis from domestic to sylvatic animals. Furthermore, human behavior not only influences the transmission patterns of Trichinella genotypes in the domestic habitat, but also it can contribute to the transmission and spread of this infection among wildlife, for example by improper hunting practices.

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 40 weeks 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: 5 weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427 l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24–52 l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05–5.00 l.p.g.). The total larval burden (live weight of pig×l.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 3–4 weeks 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.

A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella

We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognised genotypes of Trichinella. Partial DNA sequence data were generated from internal transcribed spacers ITS1 and ITS2, and from the expansion segment V region of the ribosomal DNA repeat from five species of Trichinella and two additional genotypes, designated T5 and T6. Five different PCR primer sets were identified which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern for each species and genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite genotype consist of no more than two well-defined DNA fragments, except isolates of T. pseudospiralis which generate multiple, closely migrating bands. The expansion segment V-derived primer set contributes at least one fragment to each genotypic pattern and, therefore, functions both as a means for differentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were verified using multiple geographical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae using a nested, multiplex PCR, whereby the entire internal transcribed spacer region as well as the gap region of the expansion segment V of the large subunit ribosomal DNA are amplified concurrently in a first-round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis.

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.

Differentiation of Trichinella Genotypes by Polymerase Chain Reaction Using Sequence-Specific Primers

A method was developed to identify species and genotypes within the genus Trichinella using polymerase chain reaction (PCR) and specific primers. Enzymatic amplification of 2 partially conserved and repetitive genomic DNA sequences that have been shown to be variable in length within the different Trichinella genotypes form the basis of this test. Within these regions of the genome, 4 sets of primers were evaluated from which 2 were chosen for their ability to differentiate among the genotypes under stringent primer annealing conditions while maintaining high yields of amplification product. Differences in the size of PCR products from multiple isolates of each genotype indicate sufficient variation to identify 7 of the 8 parasite groups within this genus. One primer set can differentiate among some genotypes working from a single larva. Identification of Trichinella genotypes will assist in distinguishing between sylvatic and synanthropic life cycles. Such information will be critical in tracing sources of trichinellosis by easily and unambiguously identifying likely host reservoirs and will provide valuable information for instituting methods of control.