Experimental Study On Prevention Of Tuberculosis By Mycobacterium Tuberculosis H37Ra In Mice

Objectives: 1. To preliminarily evaluate the virulence based on the growth of M. tuberculosis H37Ra in Balb/c mice. 2. To study the activation capacity of M. tuberculosis H37Ra in macrophages. 3. To evaluate the immunogenicity and the protective efficacy against a virulent M. tuberculosis strain H37Rv after immunization with Mycobacterium tuberculosis H37Ra in mice.Methods: 1. BALB/c mice were injected with H37Ra, BCG and H37Rv respectively,the bacteria growth rate in the mice,the weight indexes and the pathological changes of lung were determined. 2. Macrophages were infected with H37Ra and BCG,cytokine expression was measured by RT-PCR at 6 and 24h post-infection. 3. BALB/c mice were divided into H37Ra, BCG and NS group randomly, and intracutaneously injected with H37Ra, BCG and NS respectively. After 6 weeks of immunization, the delayed type hypersensitivity reaction was observed. 4. At 8 weeks after immunization, some mice were sacrificed, the percentage of CD3+T, CD4+T and CD8+T lymphocyte in spleen was detected by flow cytometry, and the ratio of CD4+T/CD8+T was also determined. The spleen lymphocyte of mice was cultured with PPD in vitro, the proliferation of splenocyte was detected by MTT assay. The product of IFN-γin the supernatants of culture was determined by ELISA. 5. After 8 weeks of immunization, BALB/c mice were challenged via intraperitoneal route with MTB H37Rv strain. Four weeks after challenge, mice were sacrificed, the lung,liver and spleen tissues were harvested to quantitate the viable mycobacteria respectively. 6. And the organ weight indexes were determined. The lung tissue was also harvested to have the histopathological examination.Results: 1. H37Ra grew significantly lower than H37Rv in mice (P<0.05),but slightly faster than BCG; and the weight indexes of lung were lower than that of H37Rv group (P<0.05); Histopathology of the lungs showed that the lung structure of the H37Ra group changed slightly as BCG group, but the pathological changes of H37Rv were extensive and severity. 2. Macrophages infected with H37Ra could expressed more IL-12 and TNF-α. 3. The delayed-type hypersensitivity reaction could be induced in mice after immunization with H37Ra, 24h after injection was the most optimal observing time. 4. The percentage of CD3+T-lymphocyte in mice of H37Ra vaccination was significantly higher than that of the NS control subjects (41.63±1.68)% vs (38.44±1.87)% (P<0.05). Compared with the BCG group, the percentage of CD3+T,CD4+T and CD8+T- lymphocyte in mice of H37Ra vaccination were no significant difference (P>0.05);the proliferation of antigen-specific lymphocyte, the product of IFN-γin the H37Ra group were significantly higher than those of the NS group (P<0.05). 5. The spleen,liver and lung of mice were isolated, dissociated and suitable dilutions of the homogenates were plated on Lowenstein-Jensen culture. The numbers of CFU were counted 21 days after incubation. Compared with the NS group, the mean numbers of viable bacteria in the lungs,spleens,livers of the H37Ra group reduced 0.954,1.228 and 0.883log10CFU respectively (P<0.05). The viable bacteria in the lungs,spleens,livers were no difference between the H37Ra and BCG groups. 6. Compared with the NS group, weight indexes of lung, spleen, kidney and liver of the BCG and H37Ra groups were significantly decreased (P<0.05), while there was no significant difference between the H37Ra and BCG groups. Histopathology of the lungs showed: there were a little of hemorrhagic and effusion in alveolar space of the H37Ra group, and alveolar septa was thickened along with the massive infiltration of macrophages, some granulomas were appeared. The structure of alveoli of the BCG group was integrity, some macrophages were infiltrated, alveoli wall was a little thickened. The pathological changes of the NS subjects were extensive and severity. There was much serous and hemorrhagic effusion in alveolar space. Most of alveoli wall was thickened. The structure of alveoli was partly destroyed. Conclusions: 1. The virulence of M. tuberculosis H37Ra significantly lower than H37Rv, but slightly strong than BCG. It's feasible as a vaccine candidate. 2. M. tuberculosis H37Ra could activate macrophages, produce more helpful cytokines to control MTB. 3. M. tuberculosis H37Ra could induce specific immune responses in mice and protective efficacy in mice, and the effect of H37Ra was similar to BCG.