Development Of Novel Diagnostic Method And Biological Characterization Study Of Unidentified Theileria Sp.

Bovine theileriosis is caused by Theileria, a tick-borne, intraerythrocytic protozoan parasite, which of worldwide economic and veterinary importance in ruminants. Variety of bovine Theileria have been reported in China and abroad, and the taxonomic status of some Theileria spp. are still controversial, especially Theileria sergenti, T. buffeli and T. orientali, the nomenclature of these parasite is usually based upon their geographic origin or the host. With the rapid development of biological technology, various diagnostic techniques for Theileria detection has been reported, however, each diagnostic method has its advantages and disadvantages, more sensitive, rapid and simple diagnostic methods for the diagnosis are urgently needed.In this study, diagnostic methods for Theileria spp. were established and evaluated. The 18S rRNA gene and ITS (ITS1-5.8S rRNA-ITS2) gene of Theileria isolated from different area in China were cloned and sequenced, the obtained sequences were aligned, and the phylogenetic tree was constructed by MEGA software, from the phylogenetic analysis, an unidentified Theileria spp. was considered as a novel Theileria sp. The animal experiment and tick-transmited experiment for the nevel Theileria sp. were performed. The details are following:1. Serological survey of T. sergenti228 water buffalo (or cattle) collected from China and 230 buffalo blood samples collected from South Africa were tested by an indirect ELISA, which was established for detection of T. sergenti antibodies based on p23 protein, the positive level of T. sergenti in China is 46.9%(107/228), and in South Africa is 51.3% (118/230), the positive level in South Africa is a little higher than it in China, but the difference is not significant. The antibody level of two cattle which were T. sergenti positive tested by PCR were monitored by the ELISA, the result showed that antibody levels were hold in a high level in about two years.2. Development of LAMP method for Piroplasma detectionA group of primers were designed according to the 18S rRNA gene of Piroplasma, and the LAMP was eatablished. The sensitivity of the LAMP is 0.000008%, and the method could be used in T. sergenti, Theileria sp., T. annulata, T. orientali, Babesia oritalis and B. bigemina detection, Mycoplasma wenyonii, Anaplasma marginale and Toxoplasma gondii could not amplified by the LAMP, suggested the method is specific for Piroplasma. This LAMP is suit for Piroplasma detection in clinically to obtain the prevalence status and character of the piroplasmosis.3. Development and evaluation of LAMP and PCR methods for T. sergenti detectionThe main prevalence of Theileria in China is T. sergenti, a LAMP method was developed for detecting T. sergenti. Primers targeting the p33 gene of T. sergenti were designed, LAMP and PCR method for T. sergenti detection were established, respectively. The specificity assay showed the two methods were specific for T. sergenti, and the sensitivity of LAMP and PCR was 0.000002% and 0.00002%. A total of 313 filed samples were subjected to LAMP and PCR,154 field samples from water buffalo and 159 field samples from cattle were analyzed using the LAMP method. The positive level of PCR is a little lower than LAMP, but the difference is not significant.4. Phylogenetic analysis of Theileria sppThe ITS gene and 18S rRNA gene of Piroplasma isolated from different area were subjected to amplification, cloning and sequencing. The phylogenetic tree showed there were 6 Piroplasma species prevalence in China:Theileria sergenti/buffeli/orientalis, T. annulata, T. sinensis, an unidentified Theileria sp., B. orientalis and B. bigemina. The gene sequence analysis showed that the～2400 bp and～1200 bp products represented T. sergenti/buffeli/orientalis group and T. sinensis, the～1500 bp product represented the unidentified Theileria sp., the～1000 bp product was representative of T. annulata, the～900 bp product was representative of B. bigemina or B. orientalis. These results suggesting the Theileria and Babesia species in China could be distinguished by the PCR product size of the ITS gene.5. Study on biological characterization of Theileria sp.A PCR method targeting ITS gene for the Theileria sp. detection was developed. Animal experiment and tick transmission were performed, the results showed it has not detected the Theileria sp. in cattle, water buffalo was tested positive after infection. The hematologic study showed the parasite could change the hematologic index of it's hosts. Hyalomma asiaticum asiaticum and Rhipicephalus haemaphysaloides could not transmit the Theileria sp..In summary, the diagnostic method provides a powerful tool to monitor the prevalence and clinical characteristics of Piroplasma in China. The phylogenetic analysis of Theileria spp provides a scientific basis of taxonomic studies in Theileria spp. The biological characteristics study of the novel Theileria sp. has riched the parasite research resources.