| Theileria is a tick-borne hematic protozoosis, which parasitized on bovine leukomonocyte cells and red blood cells, leading to the main symptoms of fever, anemia, jaundice and surperficial lymph node enlargement. There are three main important Theilerial species,namely Theileria annulata, Theileria orientalis and Theileria sinensis in China. Theileriosis is prevalent seasonaly and regionaly, related with the activity of ticks, which makes it difficult to wipe out. Sometimes severe infection may even cause death, bringing about huge economic loss and potential threats for breeding. In the present study, three sets of special probes and primers were respectively designed for Theileria annulata, Theileria sinensis and Theileria orientalis, with target gene of the ribosome transcribed spacer of ribosomal RNA(internal transcribed spacer, ITS). Then real-time fluorescent PCR methods were established to distinguish and identify three Theilerial disease, and then had an investigation on 150 filed blood DNA samples from some regions in China. The prevalence and sensitivity of samples detection were evaluated compared to conventional PCR. The main research contents were as follows:1. ITS gene sequences of 18 strains of Theileria and 5 strains of Babesia were sequence aligned and homology analyzed. The results showed that each geographic strains of T.annulata, T. sinensis, T.orientalis were high conservative, while intraspecific idendities between local strains were successively 91.8%~94.2%, 99.2%~97.9%, 99.1%~59.2%. The similarities of Theileria annulata between T.sinensis and T.orientalis were 17%~35.6%, 16.1%~35.7%, and the similarity between T.sinensis and T.orientalis was 40.2%~69.9%. The variability between Babesia and Theileria was high, with identity only 15.2%~40.8%. All these features of ITS gene for Theileria made it a good target gene. Specific primers and probes were designed for T.annulata, T.sinensis, T.orientalis from interspecific variation regions and intraspecific conserved regions. To construct plasmid standards for sensitivity analysis, normal PCR primers were designed of three Bovine Theileria ITS gene, and general PCR methods published were adopted.2. Three TaqMan real-time PCR methods respectively of T.annulata, T.sinensis and T.orientalis were established. The reactivity was analysised by optimized reaction conditions and system of real-time PCR, which showed that reaction type in an s-shaped curve had a good reactivity, amplification efficiency and linear dependence. Three methods presented good specificity, which showed that there was no cross reaction between three Theilerial species, and no cross reaction among neighborhood piroplasma including B.major, B.bovis, B.bigemina, B.ovata and B.sp. Kashi. Minimum concentrations for standard plasmid detected were 100, 101, 101copies/μL, which were respectively 100, 1000, 1000 times as the sensitivity of conventional PCR. Ct values of coefficient of variation for experiment in the group were both less than 5%, meanwhile Ct values between the experimental groups were both less than 10% with good repeatability.3. 150 blood samples were tested from three areas Xinjiang, Gansu and Inner Mongolia. The results showed that the positive rates of T.annulata were 18% and 8.66% respectively by real-time PCR and by conventional PCR of T.annulata. For T.sinensis detecting, the positive rates were 22.6% and 10% respectively by real-time PCR of T.sinensis and by conventional PCR. The positive rates of T.orientalis were 43.7% and 18% respectively by real-time PCR and by conventional PCR of T.orientalis. To analyze the detection results furthermore, positive samples detecd by common PCR were included by the detection of real-time PCR, while positive samples detecd by real-time PCR were not detected by common PCR. The investigation confirmed that TaqMan real-time PCR methods had high reliability and sensitivity. |
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