Trichinosis continues to be a serious risk for man in some parts of the world. In Korea, human trichinosis outbreak was observed in 1997 at the first (Sohn et al., 2000). The genus Trichinella has a broad geographical background and host range, and different isolates can often display distinct biological characteristics. Variability within the genus Trichinella has been reported in terms of morphology, infectivity, virulence, antigenicity, and enzyme polymorphism. During the last years, diversity has been analysed using parasitological, biological, morphological, immunological and biochemical (allozyme electrophoretic) parameters (Flockhart et al., 1982; Pozio, 1987; Bolas-Fernandez and Wakelin, 1990).

DNA technology has also provided useful approaches to reveal genetic difference within the genus. Polymerase chain reaction (PCR)-based methods are popular technique recently (Chambers et al., 1986; Soule et al., 1993; Minchella et al., 1994; Zarlenga et al., 1999). It has been reported that PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) analysis of the rDNA repeat can readily distinguish a species or strain from others (Boyd et al., 1989; Zarlenga and Dame, 1992; Gasser and Monti, 1997; Nagano et al., 1999). The rDNA internal transcribed spacer (ITS) gene family consists of regions of highly conserved gene sequences that is flanked by transcribed and non-transcribed spacer regions, which may contain variations that can be useful for identifying the relatedness of strains. The ITS1 (internal transcribed spacer 1) PCR product used for a genetic marker to show differences among isolates studied here spans the ITS1 region of the rDNA repeat unit and includes most of the 5.8S gene and a portion of 18S gene (Bowles et al., 1994). Here we have used the PCR-RFLP analysis of ITS1 rDNA and examined the patterns of four isolates of Trichinella.

The PCR products of the ITS1 region of all Trichinella isolates consisted of a fragment of approximately 910 bp and an additional band (525 bp) in the Japanese unknown isolate (Fig. 1A). It was confirmed that the anticipated ITS1 product is indeed a 910 bp fragment by Southern blotting analysis with 5.8s rDNA probe that is included in ITS1 sequences (Fig. 1B). The PCR products were digested with Alu I, Cfo I, Msp I, Rsa I, and Taq I. The RFLP pattern of four isolates digested using Alu I and Msp I were all the same at 910 bp uncut while digestion with Cfo I and Taq I produced the same two bands, 653 bp and 210 bp in every isolate respectively (Fig. 2). But the pattern digested with one of 4 base recognizing enzymes, Rsa I, distinctly showed the different patterns in ITS1 of four isolates. The Shanghai isolate showed a 308 bp band, the Thai isolate had two bands at 392 bp and 336 bp, the Japanese unknown isolate had 372 bp and 308 bp band, while the Korean isolate had bands of 385 bp and 313 bp (Fig. 3).

Estimated genetic convergence was analysed using RESTSITE v.1.2 (Table 1). Genetic divergence between the Korean isolate and Shanghai isolate was 0.027218. It was revealed genetic divergence between the Korean isolate and the Japanese unknown isolate and between the Korean isolate and the Thai isolate was 0.03488 and between the Shanghai isolate and the Japanese unknown isolate was 0.008005. The phyllogenic dendrogram was drawn using UPGMA v.2.0 as shown in Fig. 4.

In this study, we have shown the genetic difference of four isolates of Trichinella using PCR-RFLP analysis. The genus Trichinella has been tried to classify to species. Thus, nameless taxa (T5, T6 and T8) have been compared with already known species using various molecular and biochemical methods including RFLP (Wu et al., 1999), RAPD (random amplified polymorphic DNA; Bandi et al., 1993), SSCP (single-stranded conformational polymorphism; Gasser and Monti, 1997) and isoenzyme assay, etc. (La Rosa et al., 1982). These methods were also applied to the classification of many subspecies and isolates obtained from geographically distinct areas or different hosts. Dame et al. (1987) grouped 18 isolates of the T. spiralis from various origin to subspecies, T. spiralis spiralis and T. spiralis spp. by probe hybridization to restriction fragments of genomic DNA. If T. spiralis isolates in the present study were compared with appropriate reference subspecies, they might have also been separated into the subspecies groups as known.

It is not surprising as heterogeneity among repeat units of rDNA is characteristic of eukaryotic DNA. Bowles et al. (1994) genetically characterized several strains of Echinococcus granulosus through PCR-RFLP analysis of the mitochondrial DNA as well as the nuclear ITS1 region and reported that the ITS1 region of rDNA repeat can distinguish the cervid strain from other strains of E. granulosus. For the species T. spiralis, it is apparent that the internal repeats of the IGS (intergenic spacer) and ITS region vary between isolates and are thus useful in distinguishing from others (Boyd et al., 1989).

Here we tried to characterize the molecular genetic differences on the nuclear ITS1 region between the newly found Korean isolate and other isolates maintained experimentally. The results showed that the ITS1 PCR product amplified with ITS1 specific primers was identical in four isolates and Japanese unknown isolate had an additional band. Southern blot analysis confirmed that the band in all four isolates is the appropriate ITS1 product. And it's restriction enzyme fragments were different from each other when only one of the restriction enzymes, Rsa I, was reacted. It would appear that small genetic changes had occurred during the courses of evolution. However, it is not known at the present whether these differences are of any significance.

The estimated genetic divergence between the isolate obtained from a Korean patient and the Thai or the Japanese unknown isolate was comparatively higher than between the others. The result indicates that the Shanghai and Japanese unknown isolates are significantly genetically closer due to their proximity on the phyllogenic dendrogram. Thus the Shanghai isolate may be genetically more closer to the Japanese unknown isolate than others. It was thought that geographic location may contribute to the diversity in development of each isolate. Dame et al. (1987) had determined that genetic differences do exist between sylvatic isolates and domestic isolates. In this vain, it is important to note that the origin of the Shanghai isolate is domestic (from the domestic pig) while the other isolates are known to be from wild hosts (the Korean strain originating from ingestion of a wild badger). However, there appears to be no significant relations with differing origins in our study. According to the dendrogram analysed, the Thai isolate may have evolved earlier than the others. This may be due to the geographic isolation of that isolate. In addition, it is suggested that Korean isolate is genetically distinct from the other three isolates presumed T. spiralis (Shanghai, Thai and Japanese unknown isolate) from foreign countries and that three isolates also differ from one another.

In conclusion, it can be stated that the four isolates of the Trichinella genus display genetic difference in their respective ITS1 regions of rDNA. However the significance of these observations can only be determined when the rDNA of the various Trichinella isolates is sequenced. In turn, it will be possible to determine if the genetic differences found during RFLP analysis are due to differences in genetic development in the whole range of the gene or just due to point mutations. Further PCR-based molecular studies of other genes and sequences must be performed for proper molecular characterization and identification of subspecies of T. spiralis.