| Chlamydia abortus( C. abortus), which is an obligate intracellular gram-negative bacterium, is the causative agent of ovine enzootic abortion( OEA). It also can cause pigs, cattle and other vertebrates abortion in late pregnancy. The clinical features will be showed such as the genital tract mucosa injury,the female animals abortion, stillbirth, weak fetus and the male animals orchitis, acrobystitis, urethritis if Chlamydia abortus exists in animals’ reproductive tract mucosa surface epithelial cells. C. abortus is also a serious zoonotic pathogen and it can infect pregnant women occasionally, cause acute infections or abortion. So it is very important to research on it’s prevention and control measures for public health and livestock economy.In this study, a sheep strain of Chlamydia in Sunan County of Gansu Province was isolated by inoculating the sample into the SPF chick embryo yolk sac. Primers of PCR amplfication test coupled with RFLP analysis have been designed according to the published sequence of helicase gene clone 8gene on the GenBank, The PCR products were digested with the Alu I enzyme and a 479 bp band was cut into two small fragments, which has the same feature with the standard C. abortus S26/3 strain, can be identified as C. abortus.In this study, HeLa cells, BHK cells, L929 cells and McCoy cells were used to inoculate with the isolated ovine C. abortus, respectively. The infection rates of different cells were observed by Giemsa staining. The most sensitive cell to C. abortus is HeLa cell according to the results. So an expanded cultivation experiment of C. abortus was done in HeLa cells and the titer of collected C. abortus is1.2735 x 105 IFU/mL. The elementary body(EB), 0.2 to 0.5 μm in diameter, a spherical or oval, small and dense granular structure and the reticulate body(RB), 0.6 μm in diameter, large and loose structure can also be observed by using electron microscope.In this study an inactivated cell vaccine of Ovine C. abortus has been done, which were mainly carraied out on the aspect of inactivated agent and adjuvant. Balb/c mice were used finally as the experimental animal, and the inactivated vaccine was prepared with formaldehyde inactivated and206+CpG as adjuvant. The serum antibody IHA titers of the immunized mice was 1: 65536 and the subtype of the serum antibody was IgG1. The light chain of the serum antibody was mainly kappa chain.The content of IFN-γ in serum and lymphocyte culture supernatant were both higher than that in normal saline group according the results. The thickness of the foot pad of the immunization group was significantly higher than that of the normal saline group in DTH test and the lymphocyte proliferation of C. abortus stimulation was significantly greater than that of Con A negative control in the lymphocyte proliferation test. The number of IFU formed in the immune group cells, which were incubated by the C.abortus in serum for an hour, was much less than that in the normal saline group in the neutralization test. The spleen weight of the mice in the normal saline group was significantly higher than that in the spleen of the mice in the immune group after challenge and the DNA amplification of the immune group was negative. It was indicated that the immune group can effectively resist the invasion of C.abortus. The results of these evaluation measures showed that the body was induced to produce specificimmune responses, including Th1 cellular immune response and Th2 humoral immune response after vaccination.In summary, in this study, the ovine C. abortus was isolated, identified and culture in HeLa cells successfully. In addition, the inactivated C.abortus with 206+CpG adjuvant has a good immune effect,which providing a new method for the development of inactivated cell culture vaccine of ovine C.abortus. |
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