Molecular Phylogenetic Studies On Theileria Sergenti In Hubei Province And Establishment Of Molecular Indirect ELISA Methods For Diagnosing Theileriosis

T.sergenti is an tick-borne hemoprotozoan belonging to the phylum Apicomplexa, Theileriidae,Theileria,which parasitizes in macrophagocyte,lymphocyte and erythrocyte of cattle or bufflo.It is the cause of theileriosis,some main clinical symptoms characterised by persistent ardent fever,anaemia,emaciation and lymphadenectasis of body surface.The disease happens in many provinces in China and causes enormously economical losses to animal husbandry.In this research,the sequences of p33 gene and p23 gene from 5 geographic Theileria strains which were isolated from different areas in Hubei province were sequenced,and the molecular taxonomys to the five isolates of T.sergenti by p33 gene sequence were investigated.Moreover,the entire ORF of the p33 and p23 gene were expressed in prokaryotic cells.The molecular indirect ELISAs were developed by using the two fusion-proteins as diagnostic antigen.(1) The molecular taxonomy study of T.sergentiThe p33 genes of five geographic Theileria strains were amplified by PCR using the specific primers of p33 gene.The result of sequence indicated that the complete open read frame(ORF) was 852bp or 855bp,which encoded 283 amino acids(AA) or 284 amino acids(AA).The complete sequences from the genes and amino acids of p33 were compared with other twenty relative sequences available on GenBank.The datas were analyzed and a phylogenetic tree was established.The result displayed that the hereditary distance of the Wuchang -1 stock was close to that of a unclassed Theileria sp.from Nha_Trang in Vietnam,Which were alone branch of the phylogenetic tree,and far from any other species.Meanwhile,other features were considered,for instances,result of phylogenetic tree by 18SrRNA and pathogenic feature of the stock et al.So it was initially suggested that the stock was probably a new species.But other four stocks resided on small one branch and were close to that of Theileria buffeli from Marula(Tpye A,MPSP-3 in Japan.So the four stock could be classified into the group of MPSP-3 or subgroup.(2) Prokaryotic expression of the p33 and p23 gene of T.sergentiThe entired ORF of the p33 and p23 gene of T.sergenti were cloned into the vectors of pGEX-KG vector and pET-28a one after another,and the recombinant plasmids pGEX-KG KG- p33,pGEX-KG-p23,pET-28a-p33 and pET-28a-p23 were then constructed.The plasmids were introduced into E.coli-BL_21-CodonPlus.After inducement by IPTG,stable expression products were all obtained.Every expressed product could specifically bind to the antibody against T.sergenti by Western-blot.(3) Development of p33-ELISA and p23-ELISA for the diagnosis of T.sergentiAt first,the diagnostic methods of p33-ELISA and p23-ELISA for T.sergenti were developed based on the fusion protein of GST-p33 or GST-p23.But,it was found that the tag of GST in the two ELISAs coule not distinguish bovine Theileria spp.and bovine Schistosoma japonicum.So the two methods of ELISAs for T.sergenti were developed by another two fusion proteins,His-p33 and His-p23.The rusults indicated that the percents of negative coincidence,positive coincidence and total coincidence were all 100%between p33-ELISA and p23-ELISA.After detected tests of positive sera of bovine babesia,the results were all negative:the rusults of blocking test from His-p33 and His-p23 suggested that it could be completely blocked when serum dilutions of T.sergenti was 1.100:the maximal serum dilutions of T.sergenti exceeded 1:6400;the variation coefficient intra-plates and inter-plates were both less than 10%;store times of p33-ELISA and p23-ELISA products in 37℃,25℃were both up to eight days,it was equivalent to eight months in -20℃.All of the results revealed that the two methods had both good specificity,good sensitivity,good stability and longer shelf life of products in common temperature and the blocking effects from the two antigens of 6His-p33 and 6His-p23.(4) The seroepidemiologieal survey of bovine T.sergenti in Hubei provinceThe 178 serum samples from eight areas in Hubei province were tested by the methods of p33-ELISA and p23-ELISA,the total of positive percent was 27.53 which was higher than speciesspecific PCR of T.sergenti(21.91%).The percent of negative coincidence,positive coincidence and total coincidence was 87.05%,82.05%and 85.96%one by one between ELISA and PCR.In this research,the molecular taxonomy study of T.sergenti from five areas in Hubei province were carried out by p33 gene,the rusult based on molecular biological grounds will provid other evidences for the classification of T.sergenti.Otherwise,the eukaryotic expression of p33 and p23 gend had been studied,and diagnostic methods of p33-ELISA and p23-ELISA of T.sergenti had been established by use of 6His-p33 and 6His-p23. Undoubtedly,all of these results indicate that p33 and p23 has both a bright prospect in study of clinical diagnostic methods of T.sergenti,seroepidemiological survey as well as of prevention and control of theileriosis.