A Proof For Taxonomic Status Of Theileria Equi And The PCR, ELISA Diagnostic Method Established For Equine Theileriosis

Theileria equi and Babesia caballi ware included in equine piroplasmosis. The pathogenic parasites form in equine erythrocytes. They are an important tick-borne blood protozoan disease. The clinical signs and the pathogenic forms were described. The frist sign was fever, inappeteence, cessation, dyspnea, weakness, listlessness and swelling of the superficial lymph nodes, occasional reports of deaths. The epidemic period is from late March to July with April-May being the peak months. Endemic regions including Europe, Africa, the Americas, Asia and most parts of Australia, with some regional and seasonal, more distributed in tropical, subtropical and temperate regions as well as related reports. Equine piroplasmosis ware reported in northeast China, northwest, north and other regions. So far, the equine piroplasmosis on the serious harms the industry, and has established a series of diagnostic techniques.There exsited a dispute on the taxonomic status of Theileria equi (T. equi). To elucidate the taxonomic features of equine piroplasmid in China, We designed primers in hypervariable region within 18S rRNA gene sequence of equine piroplasmid, and obtained a fragment with the length of 913 bp for T. equi and 451bp for Babesia caballi (B. caballi) by PCR. Subsequently the PCR products were ligated into pMD18-T vector and sequenced. The phylogenetic status of equine piroplasmid in China have been established with 18S rRNA gene sequences of others equine piroplasmid available in GenBank. The result of phylogenetic analysis indicated that T. equi clustered into the same branch with others theileria spp, which showed that T. equi should appropriately belong to Theileriidae. Furthermore, 18S rRNA gene sequences of B. caballi are highly conserved across different geographical strains. The present study provided the molecular data for establishing the molecular diagnosis tool that used for investigating the epidemiology of equine piroplasmosis. The 18S rRNA gene recently discovered was shown to be species-specific. A pair of primers was designed to specifically amplified a 452bp fragment. The PCR result of specificity assay showed that one references B.caballi could be detected by the PCR test,But no amplification was observed when other five bacterial species isolated from T. equi, B. bigimina, B. motasi, B. ovata, B. major tissue were detected. And the sensitivity result showed that the minimum dose of B. caballi that could be detected by PCR assay was10^-10,45 clinical sample,from horses farm in China,doubtedly infected with B. caballi were tested by PCR. The results showed that 12 positive samples could be detected by the PCR assay. At the same time,microscope were also used for the clinical samples. The test revealed that the sensitivity of the PCR assay was higher than that the microscope test. To establish a PCR detection method of Theileria equi. The 18S rRNA gene recently discovered was shown to be species-specific. Apair of primers was designed to specifically amplify a 531bp fragment. And the T. equi, T. equi and B. caballi mixture, B. caballi, T. uilenbergi, T. sinensis, T. annulata, T. ovis, T. luwenshuni, T. sergenti are tested by PCR. While T. equi genome templates were amplified with different concentrations diluted in order to determine the sensitivity of the experiment. This PCR method and of microscopic method examination of 45 equine blood samples. The PCR result of specificity assay showed that one references T. equi and mixed with B. caballi could be detected by the PCR test, But no amplification was observed when other 7 bacterial species B. caballi, T. uilenbergi, T. sinensis, T. annulata, T. ovis, T. luwenshuni, T. sergenti tissue were detected. And the sensitivity result showed that the minimum dose of T.equi that could be detected by PCR assay was10^-13 , 45 clinical sample, from horses farm in China, doubtedly infected with T.equi were tested by PCR. Relevance ratio of T.equi was 40.0% (18/45) by PCR and microscopic examination found that only 8.89% (4/45). Between the coincidence rate was 100%. This research established T.equi PCR detection method is definitely a good detection method. To determine the taxonomic status of Theileria equi(T. equi). According to Hsp70 gene sequence (AB248743.1) of Babesia equi (B.equi) in GenBank, the primers TeHsp70F, TeHsp70R ware designed. And obtained a fragment with the length of 1920 bp by PCR. The gene sequence of T. equi was compared with others 23 amino acids sequences. The fragment encoded 639 amino acids, 208 hydrophobic amino acids and 167 polar amino acid. Identity analysis, T. equi isolates in Zhaoyuan has closest relationship with B.equi (BAF02625.1), T. parva (XP764717.1) and T. annulata (AAA30130.1), while T. equi has distantly related with B. caballi (BAF02619.1), which infect equine. It was correct taxonomic status for T. equi to Theileria species. And the taxonomic status was error before, for T. equi to Babesia species. Meanwhile, the present study provided the data for establishing the molecular diagnosis and serological diagnosis tool that used for investigating the epidemiology of equine piroplasmosis.