Research On Molecular Phylogeny Of Orientobilharzia Turkestanicum

Adult worms of Orientobilharzia turkestanicum live in the portal veins or intestinal veins of cattle, sheep and other animals and cause orientobilharziasis, which have an important impact on livestock industry and have a large distribution in Asia and several areas of Europe. In recent years, researches are mainly focused on the morphology, life cycle of this pathogen, and epidemiology, diagnosis, treatment, prevention of orientobilharziasis. However, little has been done for the classification and molecular phylogeny of O. turkestanicum. Ribosomal DNA (rDNA) was generally existed in the living nature, it has numerous characters for using as the DNA molecular maker, and for example it has a lot of multicopy and different revolutionary rates in different regions; it is suitable for re-constructing the deep phylogenic trees due to its revolutionary rate is slow in the coding region of the rDNA; it is easy to contrive the universal primers, align and amplify, in hence, it is widely used as the molecular maker in the phylogenetic analysis. The ribosomal DNA contains non-transcribed spacer (NTS), internal transcribed spacer region (ITS), three ribosomal gene coding regions (18S, 5.8S and 28S), which be applied in molecular phylogeny. Since mitochondrial DNA (mtDNA) is maternal inheritance, seldom recombination between them, and one of the mitochondrial genomes may represent the whole variation, they can be used to determine the molecular phylogeny. The ITS, 28S rDNA-LSU(28S ribosomal DNA large-subunit sequences)in the ribosomal DNA and the cox1 (cytochrome c oxidase subunit 1 gene) and nad1 (nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene) genes were amplified by PCR and cloned into pMD18-T easy vector and then sequenced and analyzed, and compared with other sequences of schistosome, counstucted phylogeny tree then determined the molecular phylogeny.O. turkestanicum was isolated from the portal veins or intestinal veins of cattle, sheep, cashmere goat and goat, and identified by morphology. Genomic DNA was extracted from parasites isolated from individual host by SDS-proteinase K method. The ITS, 28S rDNA-LSU, cox1 and nad1 genes were amplified by PCR and cloned into pMD18-T easy vector and then sequenced and compared with by Chromas and DNAStar software, and the RNA secondary structure of 28S-LSU were analyzed by DNAMAN software. The sequences were compared with other schistosomes published in GenBankTM using the Clustal X1.83 software and the phylogenic trees were generated using the MEGA software. Phylogenetic relationships between them were reconstructed using the neighbor-joining method (NJ) and the maximum parsimony method (MP). Sequencing results showed that ITS, 28S rDNA-LSU, cox1 and nad1 were 874 bp, 1 304 bp, 1 125 bp and 518 bp, respectively. The sequences from different definitive hosts exist nucleotide variations to some extent and the RNA secondary structure of 28S rDNA-LSU from caprine are identical or resemble, which have large variation compared with that of bovine. The phylogenetic tree revealed that O. turkestanicum is placed within the African schistosomes, and O. turkestanicum should be considered a sister species of Schistosoma spp.