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

Abstract

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.

Genetic characterisation of Fasciola samples from different host species and geographical localities revealed the existence of F. hepatica and F. gigantica in Niger

“…Previous studies have characterized Fasciola from different countries using genetic approaches (Itagaki and Tsutsumi 1998;Agatsuma et al 2000;Huang et al 2004;Itagaki et al 2005a, b;Alasaad et al 2007;Lin et al 2007;Vara-Del Río et al 2007); however, before the present study, there had been no any reports characterizing Fasciola from Niger using genetic approaches. Therefore, the objective of the present study was to characterize Fasciola samples from Niger from different host animals and geographical localities by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA (rDNA) because these sequences have been shown to provide specific markers for the delineation of F. hepatica, F. gigantica, and the "intermediate Fasciola" (Itagaki and Tsutsumi 1998;Agatsuma et al 2000;Huang et al 2004;Itagaki et al 2005a, b;Lin et al 2007).…”

Characterization of Fasciola samples from different host species and geographical localities in Spain by sequences of internal transcribed spacers of rDNA

“…The 5′ and 3′ ends of the Fasciola ITS-1, 5.8S and ITS-2 sequences were determined by comparison with previously published Fasciola ITS-1, 5.8S and ITS-2 sequences (Itagaki and Tsutsumi 1998;Agatsuma et al 2000;Huang et al 2004;Itagaki et al 2005a, b;Lin et al 2007; also see GenBank™ accession numbers AJ557567, AJ557569, AJ557570, AJ557571, AJ628430, AJ628431, and AJ628043). The sequences were aligned and compared among themselves and with those of F. hepatica, F. gigantica and the "intermediate Fasciola".…”

“…Several species have been described within the genus Fasciola, but only two species, Fasciola hepatica and Fasciola gigantica, are commonly recognized as taxonomically valid occurring in animals and humans (Yamaguti 1958;Mas-Coma et al 2005), with F. hepatica mainly occurring in temperate areas, F. gigantica in tropical zones, and both overlapping in subtropical areas (Krämer and Schnieder 1998;Mas-Coma et al 2005). In addition to F. hepatica and F. gigantica, several recent studies using the first and/or second internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA (rDNA) as genetic markers have identified a so-called "intermediate Fasciola" between F. hepatica and F. gigantica from Japan, Korea, and China (Itagaki and Tsutsumi 1998;Agatsuma et al 2000;Huang et al 2004;Itagaki et al 2005a, b;Lin et al 2007). …”

Species discrimination and phylogenetic inference of 17 Chinese Leishmania isolates based on internal transcribed spacer 1 (ITS1) sequences

“…Sequence data of the ribosomal RNA (rRNA) gene, in particular the two highly variable internal transcribed spacer regions (ITS1 and ITS2), have been successfully used to resolve taxonomic questions and to determine phylogenetic affinities among closely related Leishmania species (e.g., Dávila and Memen 2000;El Tai et al 2001;Kuhls et al 2005;) and other parasites (e.g., Chen et al 2007;Lin et al 2007;Al-Kandari and Al-Bustan 2010). Thus, the starting point of our work is to obtain a better understanding of the status of different isolates from different epidemic areas in China by DNAbased taxonomy.…”