Studies On Polymorphism In The Ribosomal And Mitochondrial DNA Fragments Of Fasciola From Mainland China

The objectives of the present study were to examine polymorphisms in the ribosomal and mitochondrial DNA (rDNA and mtDNA) of Fasciola from 76 specimens from different host species from provinces of Guangxi, Sichuan, Guizhou, Jiangsu, Gansu, and Heilongjiang in China, with Fasciola from France for comparative purposes, as well as to elucidate level of genetic variation in Fasciola from mainland China. The sequences of the representative samples were compared with that available in GenBank?Based on the published sequences of the second internal transcribed spacers (ITS-2) ribosomal DNA (rDNA) of F. gigantica and F. hepatica from mainland China, two species-specific reverse primers were designed in the ITS-2 sequence positions of 270-290bp with a mismatch at sequence position 271. Together with the conserved forward primer which was located within the 5'end of the ITS-2 sequence, these two specific primers were used to establish specific PCR assays for the identification of Fasciola specimens after optimizing amplification conditions. The specificity and sensitivity of these assays were evaluated. Using these assays, 76 specimens of Fasciola were identified as F. hepatica, F. gigantica or the "intermediate" type between the two species. Sequencing results of the amplified specific partial ITS-2 fragments confirmed the validity of the specific PCR assays. The specificity test indicated that there was no amplification from genomic DNAs of other parasites commonly found in ruminants, such as Paramphistomum spp., Eurytrema pancreaticum, Schistosoma japonicum and Neoascaris vitulorum. The specific PCR assays could detect DNA concentration of 110pg for F. hepatica and 350pg for F. gigantica.A fragment of the first internal transcribed spacer(ITS-1) was amplified from 36 specimens representing three groups of Fasciola, which were identified using specific PCR assays. The amplicons were then subjected to analyses by single-strand conformation polymorphism(SSCP), and the results revealed that these specimens of Fasciola could be classified into three groups, F. hepatica, F. gigantica and the "intermediate" Fasciola,consistent with results of specific amplifications using the ITS-2 as genetic makers. Representative specimens were sequenced, and the results revealed that the ITS-1 was 422bp in length for all the three groups of Fasciola. F. hepatica differed from F. gigantica at 5 variation positions in the ITS-1, and the "intermediate" type had both nucleotides of F. hepatica and F. gigantica at these variation positions. The findings were consistent with that of the specific PCR assays, indicating that ITS-1 sequence could be used as genetic marker to study DNA polymorphism in Fasciola.In order to assess level of polymorphisms in mtDNA among specimens of Fasciola from mainland China, a portion(approximately 450bp in length) of the mitochondrial cytochrome c oxidase subunit 1 gene(pcoxl) and a portion(approximately 200bp in length) of the dehydrogenase subunit 1 gene(pnad1) of Fasciola from 76 specimens was amplified separately by PCR and analysed by SSCP. Results revealed polymorphisms in pcoxl and pnadl SSCP profiles among samples from the same or different geographical locations. 18 and 11 representative specimens were sequenced for pcoxl and pnadl sequence, respectively. Sequence analyses revealed that interspecific nucleotide difference in pnadl and pcoxl was significantly higher than intraspecific variation among specimens of Fasciola. Phenograms depicting genetic differences in pnadl and pcoxl were similar, and the same grouping of specimens was achieved using pnadl and pcoxl sequences. With the exception of the specimens from Nanjing (FhNJG4) and Gansu (FhGSG3), the pnadl and pcoxl sequences of specimens representing the "intermediate" type(identified by specific PCR assays) were more similar to that of F. gigantica than they were to that of F. hepatic. These findings were consistent with that of studies in Korea and Japan, suggesting that F. gigantica could be the maternal ancestor of the "intermediate" ty...