Development Of PCR-RFLP For Taxonomy And Sequencing Of Some Mark Genes Of Bovine And Ovine Piroplasms

Piroplasm is a tick-born parasitic protozoa which differentiated into the genera Theileria and Babesia, containing number of species which infect wild and ruminants in the tropical and subtropical regions of the world. B. bovis, B. sp, B. ovata, B. major, B. bigemina and B. divergens are suspected to cause bovine babesiosis. Also, T. annulata, T. sergenti, T. sinensis, T. parva and T. mutants are suspected to cause bovine theileriosis in the world. The disease is usually characterized by hemogolobinuria, hemolytic fever, and even death in some acute cases. And also take the great the great economic losses in the animal husbandry.In this study, we explored the DNA bar-coding gene of the piroplasm, such as the Co I and the cyt b gene and analysis the phylogenetic relationship of the Theileria and Babeisia. The result in our study were presented as follow:1: Extract 18S rRNA gene sequence of the piroplasm from the NCBI's nucleotide sequence database, then kept in FASTA format for the follow multiple sequences alignment. Compiled sequences were aligned using CLUSTAL_ X for complete alignment, default parameters (gap opening penalty 15.00,gap extension penalty 6.66, deplay divergent sequence 30%, DNA transition weight 0.5). Each aligened sequence was balanced to an appropriately 1.7 Kb fragment .Also the universal primer set A and set B described above bounded two conserved regions. Then choosed the different enzyme to digest the amplification products of the 18S rRNA gene. This method can detect and differentation the Babeisia and the Theileria species and also can be used for the diagnose and perform epidemiologic survey.2: Extracted the piroplasm genomic DNA and then amplification the Co I and cyt b gene by the PCR method. Then construct the phylogenetic based on the Co I and cyt b gene sequence and analysis the homology among Babeisia and Theileria. The result shows: The homology of the Co I between the piroplasm species was 98.9%—38.1%, the homology between the Theileria species was 98.6% and 38.1%,Babeisia was 98.9%—74.1%,the divergence of Theileria is higher than the Babeisia; The homology of the cyt b between the piroplasm species was 99.4%—37.2%, the homology between the Theileria species was 99.4%—52.5%,Babeisia was 97.9%—83.8%,the divergence of Theileria is higher than the Babeisia. The phylogenetic trees showed that the piroplasm and Plasmodium falciparum were placed onto one branch, and relationship between piroplasm and the other protozoa is a little far. The result illustrated here showing coincidence with the tradition classification based on the 18S rRNA gene.