Sequence analysis of the first internal transcribed spacer of rDNA supports the existence of the intermediate Fasciola between F-hepatica and F-gigantica in mainland China

In the present study, a polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) approach combined with DNA sequencing was used to characterise samples of Fasciola spp. from different host species and geographical locations in mainland China. The first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by PCR from individual Fasciola and analysed by SSCP. SSCP analyses displayed three different banding profiles that allowed the identification of all Fasciola samples examined into three groups: Fasciola hepatica, F. gigantica and the intermediate Fasciola. Then, the ITS-1 rDNA was sequenced from representative Fasciola samples, and analysis of the complete ITS-1 sequences supported the identification of all Fasciola samples by SSCP approach. The length of the ITS-1 sequences was 422 bp for all Fasciola samples sequenced. Although there was no variation in length or composition of the ITS-1 sequences among multiple specimens within each of the taxa, F. hepatica and F. gigantica differed by 1.2% in their ITS-1 sequences, whereas the intermediate Fasciola was unique, in which two different ITS-1 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is identical to that of F. gigantica. This study demonstrated that PCR-SSCP analysis of the ITS-1 rDNA followed by selective sequencing provides a reliable approach for the accurate identification of Fasciola spp., and also supports the existence of the intermediate Fasciola between F. hepatica and F. gigantica in mainland China.