中国羊种布氏菌遗传与分子流行病学特征研究

Brucellosis is a zoonotic disease of major public health, animal welfare, and economic significance worldwide. In humans, infection with Brucella can lead to a chronic debilitating infection; in domesticated animals, the main symptom is reproductive failure. Brucellosis remains a major problem in China, especially in the northern China, where people are economically dependent on ruminant livestock. The epidemiological characteristics of humans brucellosis in China are as follows: during 1960s, humans brucellosis was very serious. During 1970s and 1980s, humans brucellosis was relatively low. Until 1995, the incidence of humans brucellosis increased. Approximately 30,000 human cases are reported annually over the past 5 years. Over the last 20 years, the geographic distribution of brucellosis in China had been changing from pasturing areas to regions of with reduced agricultural interests (or alternatively more industrial concentrations); in these areas the infection rates, reported incidence, and number of outbreaks of brucellosis have increased markedly. In China, B.melitensis was the predominant strain associated with humans brucellosis outbreaks. Based on A or M antigen variation, B.melitensis included three biotypes (biovar 1, 2 and 3), which of virulence seemed to be different. B. melitensis biovar 1 strains were predominant before 1990s. However B. melitensis biovar 3 stains were predominant for the entire country since 1995. Species identification and subtyping of Brucella isolates is very important for epidemiologic surveillance and investigation of outbreaks in Brucella-endemic regions. High-resolution genetic subtyping tools that provide important information about disease transmission patterns and the molecular epidemiology of Brucella have been difficult to develop and implement due to the genetically monomorphic nature of Brucella species. Since the genome sequencing of four strains, B. abortus 2308, B. abortus 9-941, B. suis 1330, and B. melitensis 16M, multilocus sequence typing(MLST) and multiple-locus variable-number tandem-repeats analysis(MLVA) have been developed. Recent studies have confirmed that MLST has been applied widely to microbial typing and epidemiological studies at both local and global levels as well as generating data that is ideal for studies of population structure and phylogenetic relationships. Tools such as VNTR based typing, indexing variation at more rapidly evolving markers, are likely to be far more informative in their ability to distinguish geographic origin and resolve genetic relationships. The comparative analysis of genome sequences can provide an important resource for further investigations into virulence differences and genetic variation of strains. Genetic and molecular epidemiological characterization of predominant strains isolated from the different epidemic regions and years can display epidemic patterns and provide strategies for new diagnosis technology and vaccine of Brucellosis.

布氏菌病作为一种人畜共患传染病,已成为我国重要的公共卫生问题之一。人患此病后，发生多器官受损，许多转为难以治愈的慢性病人，致残致贫，严重降低生存质量；动物患病引起流产不孕，影响畜牧业经济发展。人畜间疫情近20年强势走高，疫情从牧区半牧区向全国农区和城市蔓延。不同阶段的布氏菌存在菌种（型）变迁，但羊种菌一直都是我国的流行优势菌。根据其表面抗原A和M的成分和比例的差异分为3个生物型，各个型间毒力有所差异, 一般羊3型菌毒力最强。60-80年代主要以羊1型为主；近20年羊种3型菌比例明显增加，高达80%以上，与当前愈演愈烈的疫情形势相吻合。根据目前中国布病流行特征变化，利用多种基因分型方法系统地对不同年代、不同疫区羊种流行菌株进行遗传多态性分析；利用基因组测序技术对代表菌株进行毒力及差异基因研究，全面掌握羊种菌遗传变异特征，诠释布病疫情流行规律,为新诊断技术和新型布氏菌疫苗的研究提供新线索。

本研究利用多种基因分型方法系统地对不同年代、不同疫区羊种流行菌株进行遗传多态性分析；利用基因组测序技术对代表菌株进行毒力及差异基因研究，全面掌握羊种菌遗传变异特征，诠释布病疫情流行规律，为新诊断技术和新型布氏菌疫苗的研究提供新线索。建立了300株流行羊种菌菌株信息库和核酸库。通过rpoB基因多态性分析发现：基因2型菌株共264株（88%）、基因1型菌株3株、基因1型变种3株、基因2型变种15株、基因2型亚变种15株。本研究提出：rpoB基因多态性特征与文献报道不一致，中国羊种菌和国外羊种菌可能来自不同的进化路线。羊种菌rpoB SNP基因分型尽管与传统的生物型鉴定不吻合，不能作为PCR-RFLP方法的基础，但也可以作为一种新的基因分型方法对羊种菌种内微进化提供新的工具。首次建立了我国布鲁氏菌MLST和MLVA分型数据库。除了ST8型为优势基因型外，又发现了新的ST34和ST35型。绝大多数panel 1基因型是东地中海42型，为我国的主要流行基因型。MLST和MLVA分型数据库为病原监测和流行病溯源提供信息平台。首次建立了布鲁氏菌最小核基因组分型方法，可分为6个MCG Group，MCG Group 2 和MCG Group 3分别为羊种和牛种布鲁氏菌。找到了鉴别羊种和牛种布鲁氏菌的靶标和SNP位点，第一个靶基因ORF为BOV 0551，第二个靶基因ORF为BOV_A0392。可用于羊种和牛种布鲁氏菌的快速识别和鉴定，并对布病流行病学研究提供技术支撑。通过比较基因组学研究发现了菌株特异的毒力基因，羊种菌特异毒力基因：Y3BB15gene103(Y3BB15) \Y3BB20gene889(Y3BB20) \Y1BB17gene1730(Y1BB17) \Y1BB9gene741(Y1BB9) \Y3BB15gene3248(Y3BB15)(Y3BB5)。国外羊种菌有，国内菌株没有的毒力基因：Y1BB21gene965(Y1BB21)、Y1BB21gene869(Y1BB21)、Y1BB21gene2174。国外羊种菌株没有，国内菌株有的毒力基因：Y1BB10gene2867(Y1BB10)、Y1BB10gene2866(Y1BB10)、Y1BB10gene2946(Y1BB10)，这些研究结果为下一步研究布鲁氏菌毒力进化和疫苗靶点筛选提供关键数据。