Establish Of Sheep Taylor Insects Merozoite Protein Analysis And ELISA Diagnostic Methods

There was clear difference in pathogenic ability of Theileria sp. isolated from sheep in different districts in China. Merozoite proteins of four strains of Thelleria sp. (Gannan strain, Longde strain, Zhangjiachuan strain and Madang strain) were analyzed with SDS ?PAGE and Western blotting in order to ascertain whether there are differences in protein polypeptides and characteristics of antigen of Theileria sp. isolated from sheep in different districts. SDS ?PAGE indicated that 16 bands were shown in the lane of Thelleria sp. (Gannan strain), including 6 wide bands: 102, 71, 41, 33, 29 and l4kD; 12 bands of Theileria sp. (Longde strain), including 6 wide bands: 102, 71, 41, 31, 29 and I4kD; 14 bands of Theileria sp. (Zhangjiachuan strain), including 7 wide bands: 102, 38, 33, 22, 17, 16 and I4kD; 12 bands of Theileria sp. (Madang strain), including 7 wide bands: 102, 38, 37, 27, 17, 16 and l4kD. There were differences in the number, content and molecular weight of protein polypeptides. The lanes of electrophoresis of four strains of The ileria sp. were analyzed. The results indicated that Gannan strain and Longde strain of Theileria sp. had a very close relationship, that Gannan strain and Zhangjiachuan strain had a close relationship, and that Zhangjiachuan strain and Madang strain had a very close relationship. When the proteins were transferred to PVDF membrane and probed with positive sheep sera against those four strains of Theileria sp,The positive sheep sera against those four strains of Theileria sp. reacted all with the protein polypeptides of Gannan strain and Zhangjiachuan strain in 38kD. The protein polypeptides of Longde strain in 38kD and Madang strain in 35kD were found in the experiment of Western blotting. An indirect enzyme-linked imrnunosorbent assay (ELISA) was first reported for successfully detecting specific antibody to Theileria sp.. The merozoite antigen was used to coat ELISA plates. The optimal condition (concentration of antigen, serum and conjugate) of the ELISA was evaluated. Furthermore, the coating time, Tween-20 concentration, dilution and wash were evaluated. The cut-off value was determined by the mean of negative control plus three standard deviation. The positive coincident rate of the ELISA was 100% according to the results examining 46 positive sera and the negative coincident rate of the ELISA was 98.0% according to the results examining 48 negative sera. The antibody level of sheep experimentally and naturally infected with Theileria sp. was examined by the ELISA. The antibody to Thelleria sp. could be detected by the ELISA in two sheep experimentally infected at 5 days post-infection. The peak antibody levels (1:3200) reacted between 30 and 40 days post-infection and then gradually fell to I :400-1:800. The antibody could be detected at 270 days post- infection. The antibody levels reached two peaks in two naturally infected sheep. The first peak was reached between March and July and the other was reached between September and October. The antibody to Theileria sp. was detected in samples obtained from four counties of Gansu province (including Madang County, Lintaxi County, Zhangjiachuan County and Tianzhu County) by the ELISA. The positive-rates was respectively 5.7%, 93.80/0, 82.5% and 80.0%.