Development And Application Of PCR Assay For Diagnosing Theileria Sp Infection

Ovine and caprine theileriosis is one of blood protozoal disease, caused by Theileria spp, It multiplies in small ruminants intererthrocytes. The disease is of grate disservice, it can cause the mass mortality death of lambs and the sheep initiated from other places, make the growth and development of goats and sheep with chronicity invasion to be tardy, production of meat and wool volume dropped significantly, it finally result in the sheep industry weighty economic losses. To date, the diagncsis of Theileria sp. (China) infection depends on the detection of merozoites in Giemsa-stained blood smears, indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) et al. But the specificity, sensitivity and accurate were lower in these methods just mentioned. It is necessary to establish a kind of molecular bioogy diagnostic method which is fast, sensitive and specific to carry out early diagnosis and prevention for the Theileriosis.In this study a pair of suitable PCR primers were designed according to gene sequence of Theileria species which were reported in GenBank utilizing Oligo6.0 and Primer Premier 5.0 software, the amplification fragment were 671bp, and cloned into pGEM-T Easy vehicle, and the positive clonal recobinant plasmid was screened and the nucleotide sequence analysis and homology comparison were undertake after the vehicle was identified by restriction enzyme,PCR. The results suggest that the homology of Theileria species Jilin isolated strain 18S rRNA is 100% and 96.0% respectively compare with gene order of Theileria sp China 1 (AY492086) and Theileria sp China 2 (AY262121) ,97.0% compare with Theileria ovis ( AY260172 ) which was reported abroad, 96.4% with T. lestoquardi (AF081135) and the homology is 99. 5% when compareing the superficial protein gene of Theileria sp Jilin isolated strain with that of Theileria sp-China 1 SP China 1. Bases on above study, we established two kind of conventional routine PCR diagnostic method which can detect Theileria sp 18S rRNA gene and superficical protein gene end a semi-nested PCR to detect Theileria sp. The three kinds of PCR method were compared by detecting 51 blood samples parallelly. The positive detection rate of Semi-nested PCR is better than other two methods as the results shown.Semi-nested PCR which was estimated in this suudy can detect as few as 0.016fg Theileria sp genomic DNA, which is more sensitive than the conventional PCR methods at 100 times. Semi-nested PCR which was established in this study is sensitive, specific, high stability, so it can be used as the method for early diagnosis and epidemic situation detection of Theileria sp.130 blood samples from 3 farms in Hunchun area Jilin province were detected using the semi-nested PCR. The results were compared with those of dyeing blood smear tests under microscope. 35.38% were positive for Theileria sp by semi-nested PCR, whereas only 15. 38% were positive under microscope.