Research On Epidemiology Of Bovine Tuberculosis And Transcriptional Profile Of Dendritic Cells Infected By Virulent M. Bovis

Bovine tuberculosis is a chronic consumptive zoonosis casued by Mycoabcterium tuberculosis complex,and is considered as B type infectious disease by OIE.Bovine tuberculosis is referred as one of List 2 disease of cows,causing great economic loss in cow industry and a continuing threat to public health.Currently,more than 50 million cattle were affected by bovine tuberculosis,and caused 3 billion US dollars of economic loss annually.It is reported that 15%tuberculosis cases in the world due to drinking milk from sick cows.Therefore,the experts in World Health Organization(WHO) committee said that tuberculosis can not be controlled unless bovine tuberculosis is eradicated.In 1950s,the positive rate of bovine tuberculosis is up to 80%in China.Along with the rapid development of dairy industry,bovine tuberculosis is becoming more and more a serious threat to Chinese dairy industry and public health.Thus,control of bovine tuberculosis attracts much more attention nowadays.However,lack of the epidemic data and effective vaccine of bovine tuberculosis posed the greatest obstacle to control bovine tuberculosis in China.Furthermore,the knowledge of immunity to tuberculosis is essential to develop new vaccines and drugs to eradicate tuberculosis.Dendritic cells are potent professional antigen presenting cells.Therefore,this work was aimed to investigate the molecular epidemiology of bovine tuberculosis and the interaction between Mycobacterium bovis and dendritic cells.The main results were summarized as follows:1.National serological survey of bovine tuberculosis1344 bovine sera from national serum bank representing 30 provinces of China were used to make serological epidemiological investigation.The sera were collected by stratified random sampling and subjected to antibody detection to specific antigens MPB70/MPB83/Esat-6/CFP 10 of M.bovis.As a result,22 sera were found to be positive, and the overall positive rate was 1.6%.19 of 30 provinces did not possess positive sera, but 11 did.The positive rates ranged from 1.1%to 20%among provinces.Moreover,782 dairy cow sera from 16 local cow herds were collected,and subjected to antibody test with ELISA.59 sera were found to be positive,and the overall positive rate is 7.54%.13 of 16 herds were found positive samples,and the positive rate ranged from 0.87%to 41.18%.2.Mycobacterial molecular epidemiology investigationNasal swab collected from antibody positive cows were subjected to culture,and 17 isolates were obtained.By using biochemical and PCR identification,1 of 17 isolates was non-tuberculous mycobacterium,3 were M.bovis and 13 were M.tuberculosis. Furthermore,13 M.tb isolates were identified to be Beijing genotype by spoligotyping and share a 2223-2517-3533 MIRU pattern.Meanwhile,the investigation of clinical isolates from local human TB patients revealed there is no M.bovis in 105 clinical isolates.Nevertheless,the same genotype(2223-2517-3533,Beijing genotype) were found to be a main cluster of human infection,which there was an molecular epidemiological link between bovine and human tuberculosis.3.The interaction between murine dendritic cells and Mycobacterium bovisMurine bone marrow derived immature dendritic cells were respectively infected by virulent Mycobacterium bovis and avirulent Mycobacterium bovis BCG.at an MOI of 10. The total RNA of dendritic cells was extracted at 16h post-infection,and subjected to murine whole genome microarray to screen the gene transcription difference.36 transcripts were found to have different transcription amount.Among them,27 transcripts were up-regulated in virulent M.bovis infected dendritic cells,including genes coding prostaglandin synthesis enzyme(ptgs2,ptges),cytokines(il-1,il-12,tnf) and chemokines (ccl4,cxcl1),while other 9 transcripts were down-regulated in virulent M.bovis infected dendritic cells,including dffa,ccr2,cnr2 and so on.By verification using Realtime PCR, the up- or down-regulated of ptgs2,il-12a,cxcl1,tnf and dffa were found to be consistent with microarray detection.The protein level changes of IL-12 and IL-1βwere also tested with commercial ELISA kits.Virulent M.bovis infected dendritic cells secreted 1.6-fold and 1.9-fold more IL-12 and IL-1βthan those of avirulent M.bovis BCG infected dendritic cells.These data suggest that murine dendritic cells infected by virulent M. bovis manipulate immune system and help virulent M.bovis survive,spread and activate immune system by increasing synthesis of prostaglandin,cytokines and chemokines and decrease some other genes.