Phylogenetic Study Of Dicrocoelium Dendriticum In Sheep And Goats In Shaanxi

Dicrocoelium dendriticum, a commom parasite, inhabits the bile ducts and gall bladderof ruminants (e.g. goats, sheep cattle, camle). In the present study, three partial mitochondrialDNA (mtDNA), the internal transcribed spacer (ITS) and intergenic spacer (IGS) of nuclearribosomal DNA (rDNA) of D. dendriticum were sequenced and characterized. Based on thesesequences, phylogenetic relationships were re-constructed using molecular biology softwares.The ITS rDNA of25D. dendriticum isolates from Shaanxi province were amplified andsequenced. The total length of ITS rDNA was1144bp, with749bp for ITS1,161bp for5.8Sand234bp for ITS2rDNA. Intra-specific sequence variations were0~0.5%for ITS1and0~1.3%for ITS rDNA, Since very small variation in5.8S rDNA sequences and limitinformation in GenBankTMfor ITS1of related species, only the inter-specific variationsamong species in genus Dicrocoelium in ITS-2rDNA were examined, with sequencedifference of3.4%~12.3%. Phylogenetic analysis based on sequences of ITS2rDNA showedthat all parasites in the present study were grouped with reference D. dendriticum isolatesfrom sheep and goats, and D. dendriticum isolates from cattle and Japanese serow wereclustered in a sister clade. However, the phylogenetic tree could not reveal geographicallygenetic relationships of D. dendriticum isolates in different origins and hosts.The intergenetic spacer (IGS) rDNA of16D. dendriticum isolates from Shaanxi andGansu provinces were sequenced, and the intra-species variation, direct and inverted repeats,and phylogenetic relationship were also studied. The length of IGS rDNA was3029~3012bp,with sequence difference of0.1%~1.3%, and4direct and4inverted repeats were found. Thephylogenetic analyses showed that the IGS was not a suitable molecular maker for thephylogenetic study of D.dendriticum, and it can not differentiate the isolates from differentregions and hosts.Based on the partial mitochondrial DNA (mtDNA) cox1, nad1and cytb (pcox1, pnad1and pcytb), the genetic variations and phylogentic relationships were examined. The lengthsof pcox1、pnad1and pcytb were approximately400bp,500bp and400bp, respectively. Theintra-species variation of isolates from different geographical regions in Shaanxi provincewere0~0.52%for pcox1,0~0.73%for pnad1and0~0.58%for pcytb. Phylogenetic study based on the combined sequences pcox1, pnad1and pcytb showed that all samples in thisstudy were clustered together in same clade of Paragonimus westermani. But thephylogenetic trees could not reveal geographically genetic relationships of D. dendriticumisolates in this province.The present study examined the genetic variations and population structure of D.dendriticum based on the three mt DNA, ITS and IGS rDNA. The results were able to reflectthe genetic variability and population structure of D. dendriticum, and play an important rolein prevention and control of Dicrocoeliosis in different regions of China.