牛种布鲁氏菌2308紫外诱变株毒力检测及其对昆明系小鼠的免疫效果研究

The virulence detection of B.abortus 2308 mutation strain and the immunize effect on Kunming Mice

为了检测牛种布鲁氏菌2308紫外诱变株毒力和免疫效果,本实验以S19疫苗株为对照,用牛种布鲁氏菌基因缺失株紫外线照射组(2308△rwbk A、2308△romp25和2308△rery)和非照射组(2308△wbk A、2308△omp25和2308△ery)分别侵染小鼠巨噬细胞RAW264.7,对小鼠巨噬细胞内的细菌进行计数评价毒力致弱情况;用牛种布鲁氏菌基因缺失株紫外照射组、非照射组以及S19疫苗株分别以1×107CFU/0.2 m L免疫4-6周龄昆明系小鼠,通过ELISA检测小鼠血清中布鲁氏菌Ig G抗体和IFN-γ细胞因子,同时分离脾脏,CFU计数布鲁氏菌胞内菌数量;用2308强毒株以3×108CFU/0.2 m L腹腔感染各组免疫10周后小鼠,脾脏CFU评价各受试组小鼠的免疫效果。结果表明:在细胞水平,紫外线照射后的布鲁氏菌基因突变株其CFU较紫外线非照射组有不同程度的下降,统计学分析无显著性差异(P>0.05),但均高于S19胞内菌CFU;与S19疫苗株相比,2308△rwbk A和2308△rery紫外线照射组与其对应的2株紫外线非照射组在不同检测阶段,其Ig G和IFN-γ含量水平相当,但2308△romp25紫外线照射菌株组在第6、8周,其Ig G和IFN-γ含量均高于2308△omp25紫外线非照射菌株组和S19免疫组,统计学分析有显著性差异(0.01<P<0.05);攻毒实验表明:经过紫外照射的romp25缺失株产生的保护力高于其它未经过紫外照射的缺失株,但低于S19疫苗株免疫效果。结论:紫外诱变株romp25毒力进一步降低而且能够产生一定的保护效果。

To detect the virulence and the immune effects of Brucella abortus 2308 mutant strains irradiated with the ultraviolet（UV） rays. RAW 264.7 mouse macrophages were respectively infected with B.abortus 2308 UV-irradiated mutants（2308△rwbk A, 2308△romp25 and 2308△rery）, and UV-free mutants（2308△wbk A, 2308△omp25 and 2308△ery）. The S19 vaccine was the positive control. Bacteria in macrophages were counted by colony formation unit（CFU） for the virulence evaluation of different mutant strains. The 4-6 week Kunming mice were immunized with 1×10^7CFUs/0.2 mL UV-irradiated mutants and UV-free mutants. The concentration of Brucella-specific Ig G antibody and IFN-γ cytokine was detected by ELISA kits in post-immunization（pi） 2, 4, 6, 8 and 10 weeks. Meanwhile, the intracellular bacteria were counted by splenic CFUs. Then the immunized mice were intraperitoneally injected with 3×10^8CFUs /0.2 mL B.abortus 2308 conference strain. The results showed that the number of splenic CFU of brucella UV-irradiated mutants was less than that of UV-free mutants, but still higher than S19 vaccine group. There was not statistically significance（P〉0.05） between the groups of UV-free mutants and UV-irradiated mutants.） Compared with S19 vaccine group, the titers of Ig G and IFN-γ immunized with 2308△rwbk A, 2308△rery, and these two UV-irradiated mutants was similar in different pi stages. The Ig G and IFN-γ level that induced by 2308△romp25was higher than its UV-free mutants, and also higher than S19 vaccine group, especially in pi 6-8 weeks, which was statistically significant（P〈0.05）. The results of animal experiment showed that the protection induced by mutant 2308△romp25was higher than the other two UV-irradiated mutants（2308△rwbk A and 2308△rery）, but lower than S19 vaccine group. The mutant 2308△romp25 showed less virulence and more protective effect, which is helpful in the development of marked brucella vaccine in the future.