Study On Mouse Model Of Tree Shrew Tuberculosis And Drug - Resistant Tuberculosis

objective To establish Mycobacterium tuberculosis infection model in tree shrew and study the pathogenic mechanisms of tuberculosis.Method Tree shrews were intravenously infected of2.5×106CFU or2.5×103CFU Mycobacterium tuberculosis H37Rv, then temperature, weight, vitality, blood routine, erythrocyte sedimentation rate, tissue homogenate of bacterial culture, pathological changes in tissues and protein mass spectrometry analysis were analyzed at week5,8and11post-infection.Results Low dose group:body temperature, body weight and vitality didn’t present significnat change. No obvious gross pathology changes were observed.The MTB-related histopathologic changes didn’t found in low dose group.High dose group:body temperature didn’t present obvious change.One tree shrew in high dose became seriously ill and died within5weeks, and lost51.52gram. Another lost40.74gram and vitality decreased apparently at8weeks post-infection. The ratio of lympHocyte of two groups was increased in blood count. Erythrocyte sedimentation rate<5mm/h. Mycobacterium tuberculosis growth could be found in many tissues with tissue homogenate bacterial culture.Afer acid-fast staining, acid bacillus were be visible in these tissues. Many nodules could be found at different tissues of the spontaneous death animal and kidney had gray lesions. Skin ulcer was observed in injection sites.Pleural effusion and subcutaneous abscesses of head were discovered.Nodular lesions were found at lung tissue. Assayed pathology shows Lung cells dramatically decreased, and the presence of necrosis in the central portions of nodules, around which lots of inflammatory cells infiltrated. Skin ulcer was observed in injection sites, in which derma and subcutaneous tissues were necrosis and inflammatory cells infiltrated.Chinese shrew with poor mental state have lesions in the cerebellum. Differential proteins that were found in protein mass spectrometry analysis. Such as regulation of actin cytoskeleton, pHagosome, calcium signaling pathway, Jak-STAT signaling pathway, antigen processing and presentation, CD45/MAPK signaling pathway induced autopHagy pathway, all of them maybe connected with mycobacterium tuberculosis infection. Jak-STAT signaling pathway linked with tuberculosis infection which were proved in human and other animals tuberculosis infection. Regulation of actin cytoskeleton may play important role in tuberculosis infection. With STAT protein as reference,10random altered proteins validated at transcriptional level with STAT as reference. The results showed transcriptional express change of8in10altered proteins keeped consistent with protein expression profiling.Conclusions Tree shrew was susceptible to tuberculosis.Histopathology after infection had its character.The high dose group:lung and spleen had TB granulomas, with caseous necrosis in the center and a large number of inflammatory cells outside the center. Focal necrosis with inflammatory cells was distributed in the adrenal cortex and adrenal capsula, and in the liver and kidney, there was focal inflammatory cell infiltration.Skin ulcer,tuberculosis pleural effusion and cerebellum necrosis were observed.Infective dose and individual difference caused different results, no obvious clinical symptoms in mycobacterium tuberculosis infected tree shrew,. It had been proved that chinese shrew was susceptible to tuberculosis with its different character.Cytoskeleton regulated protein may play important role in tuberculosis infection. Tree shrews could be tuberculosis transmission intermediate host and new tuberculosis animal model for nosogenesis, pathology and immunity research. Objective To study the in vivo virulence of rifampin-resistant Mycobacterium tuberculosis in mice and screen out standard clinic strains used for drug-resistant Mycobacterium tuberculosis animal model.Method The strains were collected from TB patients in several provinces of China. Drug susceptibility test with first-line and second-line anti-tuberculosis drugs and drug-resistant gene mutant analysis were performed for98clinic isolations. After gene sequencing, the strains with explicit durg resistant pHenotype and mutation were selected for in vivo virulence experiment in mice.Result Among the40strains with explicit drug resistant patterns,35strains were demonstrated stronger virulence compared with H37Rv, since the half lethal time of35strains were shorter than that of H37Rv’s in the virulence experiment in mice.18in35strains were rifampin and isoniazid-resistant.5strains were rifampin-resistant,7strains were isoniazid-resistant,5strains were isoniazid and rifampin sensitive. Standard strain with moderate virulence and explicite genotype was screened out for each drug resistant pHenotype. Isoniazid-resistant and rifampin-resistant gene sequencing revealed that katG315genetic mutations and rpoB531,526genetic mutations were common in these strains and the virulence wasn’t declined compared with its clinic drug sensitive strains.25strains in35were Beijing strains.Conclusion As for rifampin-resistant combined with isoniazid-resistant strains, one with LT50≤7days was selected as its standard strong virulence strain, and for rifampin-resistant or isoniazid-resistant strains one with LT50≤7days was selected as its standard strong virulence strain respectively. Each standard strain can be used as candidate standard strain to infect guinea pig or other animal model. Isoniazid-resistant strains with katG315genetic mutations and rifampin-resistant strains with rpoB531,526genetic mutations were common and the virulence wasn’t declined compared with their clinic drug sensitive strains and most of them were Beijing strains. It is implicated that the virulence of rifampin-resistant strains with these genetic mutations were still with strong virulence and might be threaten to the health of population.