Metacercariae of C. sinensis were collected after the artificial digestion of the muscle of the freshwater fish, Psudorasbora parva in Kimhae, Korea (A), Guangxi (B) and Shenyang (C), China. The adult worms were obtained from the rat (Sprague-Dawley, 4 to 6-weeksold male) liver one month post-infection with metacercariae (A and B) or from the rabbit liver five months post-infection (C). The worms collected were then stored at -70℃ until assayed. The frozen worms were lyophilized and lysed with a lysis buffer containing 1% SDS, proteinase K (500 µg/ml), and RNase at 37℃for 2-3 hr. The DNA was extracted in phenol/chloroform and precipitated in ethanol as reported by Sambrook and Russell (2001). The PCR primers previously designed for amplification of target DNA region were used (18S, Barker and Blair, 1996; ITS1, Bowles et al., 1993; ITS2, Bowles et al., 1995; cox1, Bowles et al., 1992). Each PCR was carried out in a volume of 50 µl, as follows: 10-100 ng of the extracted genomic DNA as template, 200 µM of each dNTP, 10 pmoles of each primer and 0.5 unit of Ex Taq enzyme (TAKARA Shuzo Co., Japan). PCR amplification consisted of 40 cycles of 20 second denaturation at 95℃ 30 second annealing at 50℃ 30 second extension at 72℃ followed by a final extension at 72℃for 6 minutes. The amplified PCR products were extracted using QIAEX II Gel extraction Kit (QIAGEN Co., Germany) and ligated into a T cloning vector (pT7Blue Perfectly Blunt Cloning Kit, Novagen Co., USA). Transformation was carried by E. coli NovaBlue competent cells provided in T cloning kit. Positive recombinant clones were picked and grown overnight in 2 ml of LB broth (in the presence of 50 µg/ml ampicillin) at 37℃ and the positive plasmid DNAs were purified using a QIAprep spin plasmid kit (QIAGEN Co.). The recombinant plasmids were selected by blue/white screening using isopropyl-β-thiogalactoside and 5-bromo-4 chloro-3-indolyl-β-D-galactoside. Plasmids DNAs of white colonies were digested with BamHI and HindIII restriction enzymes at 37℃ run in 1% agarose gels and stained with ethidium bromide. DNA sequencing was performed by the dideoxy chain termination method (Sanger et al, 1977) using a Sequenase kit (ABI Prism Dye Terminator Cycle Sequencing Core Kit, Perkin Elmer) and an automated DNA sequencer (Applied Biosystems model 373A, Perkin Elmer) according to manufacture's instructions. Sequencing primers included the universal primers T3 and T7 for both directions. At least two clones were sequenced per isolate with additional clones being sequenced as necessary to resolve ambiguous sites. The aligned sequence was done using the CLUSTAL W program (European Bioinformatics Institute, http://www.ebi.ac.uk/clustalw/) among three isolates. Alignment gaps were treated as missing data (Higgins et al., 1992). For sequence analysis, we used BLAST in National Center for Biotechnology Information, NIH, Bethesda, USA. We also calculated the fractional GC content of nucleic acid sequences using an EMBOSS GEECEE program in Sanger Institute, Cambridge, U.K. (http://analysis.molbiol.ox.ac.uk/pise_html/geecee.html).

As results of sequencing, the length for each product of isolates was 1,030-1,031 bp for 18S; 762-776 bp for the ITS1; 451 bp for ITS2 and 393-395 bp for cox1 (Table 1). A G+C content was 51% (18S), 54% (ITS1), 52% (ITS2), and ranged from 41% to 42% (cox1) (Table 1). Sequences were deposited in GenBank by the accession numbers listed in Table 1. The nucleotide substitution differences among the isolates were 1-3 bp for the 18S, 1 bp for ITS1 and 3 bp for cox1. For the ITS2, no variation was detected among and within isolates from Korea and China. The nucleotide gap (insertion, deletion) differences among the isolates were 1 bp for the 18S, 8-22 bp for the ITS1 and 2 bp for cox1. For the ITS2, no gap was detected among isolates from Korea and China (data of multiple sequences alignment not shown). The ITS1 sequences showed much higher gap differences at nucleotide composition, 8-22 bp insertion and deletion at the same position among three isolates (Table 2).

According to the above results, it was found that Korean and Chinese isolates of C. sinensis are remarkably similar at the DNA sequential level for ribosomal DNA and mitochondrial DNA. A few intraspecific variations in C. sinensis were found in the rDNA repeat (18S, ITS1 and ITS2) and cox1, and a few base insertion and deletions were detected in the ITS1 sequences. Extensive intraspecific sequence variations were not found between the Korean and two Chinese isolates. There were other comparable data on the intraspecific variations of ITS2 and cox1 sequence of parasitic trematodes in human. The nucleotide sequence of ITS 2 and cox1 of Opisthorchis viverrini in northeast Thailand showed intraspecific variation, that has been classified into 5 patterns, but no areaspecific pattern was observed. Nucleotide sequences in a region of the ITS2 from different areas were identical (Ando et al., 2001). Those data compared very well to the present results suggested that the geographic differences are not so significant for some DNA markers. The average sequence divergence among the Echinostoma species range from 2.2% in the ITS rDNA to about 8% for the cox1 (Morgan and Blair, 1988). DNA sequence similarities observed between the Korean and Chinese isolates of C. sinensis are indicative of a common ancestry. There is negligible intraspecific variation among one Korean and two Chinese isolates of C. sinensis.