【作者】 晁彦杰；

【导师】 郭爱珍；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 牛结核病是由分枝杆菌引起的包括人和家畜以及野生动物发病的一种慢性消耗性人兽共患传染病,被国际动物卫生组织(OIE)定为B类动物传染病。奶牛结核病,与布鲁氏菌病并称奶牛的“两病”,历史是严重威胁奶业健康发展的重大传染病。目前全球有约5000万头以上的牛感染了牛结核,每年造成大约30亿美元的经济损失。另据有关资料报道,世界上结核病人中约有15%是通过饮用了结核病牛的奶而生病的。因此,早在1960年世界卫生组织专家委员会第七次会议中便指出:“在那些还存在牛结核病问题的国家中,人类始终受到它的威胁,除非扑灭牛结核病,否则人类结核病的控制是不会成功的”。有数据显示,解放初期,我国奶牛结核阳性率高达80%。随着近年来我国奶业的迅速发展,牛结核病的对奶业健康发展具有越来越严重的威胁,奶牛结核病的防控也愈来愈受到重视。但是我国牛结核病的流行状况却十分不清楚,背景不明使我国对牛结核病的防控遇到的最大障碍之一。另外,牛结核病因缺乏疫苗而无法对易感牛群进行免疫预防,了解结核病的免疫特点是开发新是开发新疫苗、新药物来防治结核病的基础性工作。树突状细胞是体内专职的免疫提呈细胞。因此,本研究从牛结核病的分子流行病学和牛分枝杆菌与树突状细胞的相互作用等角度进行了初步探讨,主要获得以下结果:1.奶牛牛结核病血清学调查严格按照分层随机采样方法,从全国30个省、市、自治区采集到1344份奶牛血清,使用牛结核病特异性抗体ELISA检测试剂盒检测牛结核病,结果发现22份阳性血清,总体阳性率为1.6%。30个省中有19个省未检出阳性血清,有11个省发现有阳性结果,各省份的阳性率从1.1%至20%不等。另外对某省进行16个奶牛场进行高密度采集,对782份血清进行ELISA检测,结果共检测出59份阳性血清,总体阳性率为7.54%。16个牛场中,有3个奶牛场未检出阳性,其余13家奶牛场均有阳性结果。牛场间阳性率从0.87-41.18%不等,说明牛结核病在牛场间呈散在分布。2.分枝杆菌分子流行病学研究采集结核病抗体阳性奶牛的鼻腔分泌物进行分枝杆菌培养,结果共培养出17个阳性培养物。使用生化方法和PCR鉴定,17株阳性培养物,有1株为非结核分枝杆菌,3株牛分枝杆菌和13株结核分枝杆菌。使用Spoligotyping和MIRU分子分型方法,发现13株结核分枝杆菌基因型为北京基因型,MIRU pattern为2223-2517-3533。同时对当地结核病防治院所的105株结核分枝杆菌临床分离株进行的分子分型未发现人中有牛分枝杆菌感染,且MIRU pattern为2223-2517-3533的北京基因型菌株为人中感染的主要菌株之一,说明奶牛感染的结核分枝杆菌与人结核病之间具有分子流行病学联系。3.牛分枝杆菌与小鼠树突状细胞相互作用研究分别用毒力牛分枝杆菌和卡介苗感染小鼠未成熟树突状细胞,感染比(MOI)为10,感染后16小时采用小鼠全基因组表达谱芯片检测感染的树突状细胞差异基因的表达。结果共发现差异基因36个,基中上调基因27个,主要包括ptgs2、ptges等前列腺素合成酶基因、il-1、i1-12、tnf等细胞因子基因和ccl4、cxcl1等趋化因子基因;下调基因9个,主要包括dffa、ccr2和cnr2等。利用实时定量RT-PCR证实进一步证实基因芯片的检测结果,发现ptgs2、il-12a、cxcl1、tnf,dffa等基因的差异表达结果与芯片结果基本相符。用ELISA检测试剂盒检测感染树突状细胞分泌的细胞因子水平的变化,发现感染毒力牛分枝杆菌的树突状细胞分泌IL-12和IL-1β细胞因子的量分别是感染无毒卡介苗的树突状细胞的1.6倍和1.9倍,t检验表明两组数据间差异显著。以上结果表明,和感染卡介苗相比,感染牛分枝杆菌毒力株能促进树突状细胞合成大量前列腺素、细胞因子和趋化因子,同时下调另一些相关基因。这些基因表达谱的差异可能通过调控机体免疫系统功能,使毒力牛分枝杆菌得以在体内存活、散播、并激活特定的免疫反应。更多还原

【Abstract】 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.更多还原