Molecular identification of Korean Trichinella isolates

Abstract

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.

Trichinosis is one of the most wide spread helminthic zoonoses. In Korea, its presence was first confirmed in 1998 as encysted larvae in the biopsied muscle of patient, who ate the raw flesh of a badger (Sohn et al., 2000). Another a small outbreak originating from a wild boar took placed in a mountainous area of Kangwon-do, Korea (Park et al., 2001). In this study, we performed a molecular biological study to identify the two Korean Trichinella isolates from two outbreaks at the species level.

Parasites were isolated and maintained in ICR mice. The mouse carcasses, infected with two Korean isolates, were sent to the Trichinella Reference Center at the Laboratory of Parasitology of the Instituto Superiore di Sanita in Rome, Italy. Muscle larvae (ML) were collected after artificially digesting the mouse carcass (Pozio, 1987) and washed three times in distilled water. After washing, individual ML were stored in 5 µl of H₂O at -30℃ until used.

For the identification of the ML of the first outbreak (isolate code ISS623), a single ML was placed in 14 ml of Tris-HCl (pH 7.6), overlaid with mineral oil, heated at 90℃ for 10 min, treated with 100 mg/ml of proteinase K at 55℃ for 3 hr, and heated again at 90℃ for 10 min. Polymerase chain reaction (PCR) was carried out using SB2 primers, which are specific for Trichinella spiralis (forward 5'-CTCCACTTACGCAATGCACG-3' and reverse 5'-ACACCAAACGGCAACTGCTA-3') (Wu et al., 1997) and by PCR-RFLP with the primer set Ts43CA (forward 5'-ATGCGAAATATACATTTTTCTTA-3' and reverse 5'-TTAGCTGTATGGGCAAGG-3'), and this was followed by RsaI restriction for T. nativa and T. britovi, in accordance with the protocol of Wu et al. (1999). Muscle larvae from the reference strains of T. spiralis (code ISS3), T. nativa (code ISS10), and T. britovi (code ISS2) were used as controls (La Rosa et al., 1992).

To identify ML originating from the second outbreak (isolate code ISS1078), a 0.1 µl solution of 0.1 M Tris-HCL (pH 7.6) and 1.9 µl of H₂O was added to the larva, overlaid with mineral oil, and heated at 90℃ for 10 min. Then, 0.4 µl of proteinase K and 2.6 µl of H₂O were added to the larva at 48℃ for 3 hr and at 90℃ for 10 min. PCR was performed using 4 µl of a single larva preparation, 0.1 µl Taq DNA polymerase (Takara, Otsu, Shiga, Japan), 5 µl 10X PCR buffer, 4 µl dNTPs, 0.7 µl of primers (I-V primer sets) (Zarlenga et al, 1999), 0.5 µl DMSO, and H₂O up to 50 µl. Amplifications consisted of 35 cycles of: denaturation at 94℃ for 30 sec, annealing at 62℃ for 20 sec and at 58℃ for 30 sec, and elongation at 72℃ for 1 min. The MLs of reference strains were used as controls. The ISS numbers of the isolates refers to the code of the Trichinella at the Reference Center (Pozio et al., 1989).

All of the ML from the mice infected with two Korean Trichinella isolates showed a band similar to that of T. spiralis larvae of the reference strain (Fig. 1).

Fig. 1.

Electrophoretic patterns after multiplex-polymerase chain reaction amplification of Trichinella larvae originating from a badger and from a wild boar in Korea. Lane M, 100 bp ladder; lanes 1-3, larvae from the badger (isolate code ISS623); lanes 4-6, larvae from the wild boar (isolate code ISS1078); lane 7, Trichinella spiralis reference larva (code ISS3); lane 8, T. nativa reference larva (code ISS10); lane 9, T. britovi reference larva (code ISS2); lane 10, T. pseudospiralis reference larva (code ISS13).

Trichinosis is generally diagnosed by detecting larvae in biopsied muscle and/or by the detection of antibody in the serological test without species differentiation. Recently, rapid and sensitive genotyping tools for Trichinella have been developed, and studies on the differentiation of genotypes and species of Trichinella have been performed successfully by several investigators (Gasser et al., 1998; Appleyard et al., 1999; Nagano et al., 1999; Wu et al., 1999; Zarlenga et al., 1999). Gasser et al. (1998) identified 7 isolates from mainland China by PCR-based SSCP. Wu et al. (1999) applied PCR-RFLP analysis to identify 5 species, i.e. T. spiralis, T. nativa, T. britovi, T. pseudospiralis and T. nelsoni, and 3 phenotypes of uncertain taxonomic status (Trichinella T5, T6 and T8).

Of the 7 isolates from the mainland China, 5 were identified as T. spiralis and the results upon the other two were identical with those of T. nativa and Trichinella T6 (Gasser et al., 1998). The Japanese isolates from wild animals were identified as T. britovi by random amplified polymorphic DNA (Pozio et al., 1996). In the present study, PCR-RFLP and multiple-PCR found that both Korean isolates (ISS623 and ISS1078) showed the same molecular pattern as that of a T. spiralis reference strain.

According to the new taxonomic scheme, nematodes belonging to the genus Trichinella are divided into 7 valid species, T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. nelsoni, T. murrelli and T. papuae (Murrell and Pozio, 2000). Among them, T. spiralis has the widest geographical distribution and the largest range of host species. The majority of human infections are caused by this species (Capo and Despommier, 1996; Murrell and Pozio, 2000). However, since the various genotypes or species of Trichinella are distributed worldwide including Asia, Trichinella isolates should be examined in detail. In this study, we have identified two Korean Trichinella isolates infecting human as T. spiralis.