| Neosporosis is a protozoosis, which was caused by parasitic Neospora caninum in cells of dogs, cattle, sheep and other animals. It can cause abortion, stillbirth and weak fetus for pregnant animals, to cause huge economic losses for livestock farming. Currently, there is no effective drug and vaccines to control Neosporosis, timely detection, diagnosis, prevention and out of livestock are the ways to control it. Therefore, isolation of local strain, in vitro cultivation, R&D and application to sensitive and quick kit have great significance for early diagnosis, epidemiological studies, treatment, prevention and control.(I) This experiment inoculated N. caninum tachyzoites for support cells by Vero cells with adherent culture to establish the culture system in vitro. The N. caninum tachyzoites were put on the microscope to be observed parasitic condition and proliferation at intervals of24hours. Simultaneously look for the optimal experiment conditions in subculturing, reserving and anabiosis. The result showed that:we can get the vigorous growth of Vero cells in logarithmic growth phase, when cells were cultured in DMEM at37℃in humid air with5%CO2. On this basis, has successfully established the local insect strains Nc-1of N.caninum tachyzoites in vitro culture. The experiment showed that, N.caninum Xinjiang strain Nc-1tachyzoites can be stably increased in Vero cells, and the optimum medium was RPMI-1640(including100μg/ml penicillin-streptomycin and5%fetal bovine serum). At37℃in humid air with5%CO2was the best culture condition The best time of strains of recovery and save is six months post freezing. This experimental took N. caninum of local strain was cultivated on cells. It provided rich pathogenic parasites and laid the groundwork for subsequent in-depth study of Neosporasis.(Ⅱ) In this experiment, using the purified recombinant protein NcSRS2t coating antigen to assembled four batches rELISA kit. Series of tests were conducted respectively in the specificity, sensitivity, stability, reproducibility, stored period and sample test (N=1613) of the kit, and clinical trials were also carried out. The results show that, in sensitivity test, still tested antibodies under the condition of kit with positive serum1:1600dilution; intra-assay and inter-assay were tested repeatedly and the variation coefficients were less than10%. The coincidence of test results between self-assembled kit and IDEXX commodity kit was97.5%; what of test results with Bio-X commodity kit was98.0%. Tested212Neospora caninum antibody positive serum samples by assembled kit to test bovine serum samples (N-1613), the positive rate is13.1%, the results were also verified by commodity kit. The above results showed that the sensibility of self-assembled kit was higher, specificity and repeatability were better and can be used to clinical test of disease.(Ⅲ) This experiment tested Neospora caninum antibody to543cashmere goats’serum of Xinjiang Hejing County by applying assembled Neospora caninumNcSRS2t-rELISA kit. The results showed that, the average antibody positive rate of Neospora caninum in cashmere goats was13.8%. In33goats flock samples, the infection rate of51.5%flock was10%-20%. The average antibody positive rate of Neospora caninum samples with abortion was higher than random samples but it needs to further investigation and research for whether the neosporosis is major reason of causing abortion of cashmere goats in our region. |
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