犬种布鲁氏菌的遗传多样性分析

Genetic diversity of Brucella canis

摘要:

【背景】犬种布鲁氏菌是犬种布病的病原菌，主要导致犬流产和繁殖障碍。虽然犬种布鲁氏菌感染人群的病例极为少见，但是犬种布鲁氏菌对人的安全风险仍存在争议。目前，我国犬种布病的流行病学特征及犬种布鲁氏菌的遗传多样性的研究相对缺乏。开展犬种布病的流行特征及遗传多样性调查对加强犬种布病的监测防控具有重要意义。【目的】对犬种布病的流行病学特征和犬种布鲁氏菌的遗传多态性进行调查，为犬种布病的防控提供参考。【方法】采用常规鉴定方法和BCSS-PCR对63株试验菌株进行鉴定。采用HGDI (Hunter and Gaston diversity index)多态性指数调查犬种布鲁氏菌的遗传多态性，用MLVA方法基于BioNumerics 5.0软件对菌株进行聚类分析，揭示犬种布病的流行病学特点。此外，基于MLVA-11采用goeBURST软件构建犬种布鲁氏菌的最小生成树(Minimum spanning tree，MST)，阐述我国犬种布鲁氏菌的地理起源特征。【结果】常规鉴定方法和BCSS-PCR扩增结果显示63株试验菌株全部为犬种布鲁氏菌。BCSS-PCR与常规鉴定方法的符合率为100%，BCSS-PCR的分析敏感性为10?3 (即50 pg/μL犬种布鲁氏菌DNA)。我国犬种布鲁氏菌具有较高的遗传多样性，基于HGDI分析表明Panel 2B的5个位点具有较高的变异度，等位基因型由高到底依次为bruce09(11)> bruce07(8)>bruce16(7)>bruce04(6)>bruce30(5)。MLVA聚类分析表明北京地区出现了3次较小规模的犬种布病暴发流行，其余地区均为零星散发。我国犬种布鲁氏菌可分为5个地理集群，以MLVA-11基因26型克隆群为主导种群，该种群与来自美国、希腊、加拿大、法国、罗马尼亚和韩国等国家的菌株具有共同的地理起源，其余4个种群为中国特有。【结论】我国犬种布鲁氏菌呈现高度的遗传多样性并有广泛的地理来源，表现为输入性和中国特有血统共存的起源进化特征。

Abstract:

[Background] Brucella canis is a pathogenic bacterium of canine brucellosis and mainly leads dogs to abortus and reproductive disorders. B. canis is a rare source of infection for human brucellosis, but safety risk of human posed by B. canis remains controversial. At present, the epidemiology characteristics and genetic diversity of B. canis research in China were lacking relatively. It is significant to investigate epidemiology characteristics and genetic diversity of canine brucellosis for strengthening surveillance and control of canine brucellosis. [Objective] Epidemiology characteristics and genetic diversity of B. canis strains were investigated in order to provide a reference for control and prevention of canine brucellosis. [Methods] Both conventional bio-typing methods and BCSS-PCR were applied for species identified of 63 B. canis strains. The genetic diversity of B. canis strains were investigated by the means of HGDI. MLVA method was used for cluster analysis of B. canis to reveal the epidemiological characteristics of canine with BioNumerics 5.0 software. goeBURST software was applied to construct minimum spanning tree of B. canis strains for elucidate characteristics of geographic origin of B. canis strains in China. [Results] Both conventional bio-typing methods and BCSS-PCR were used and 63 tested strains were all B. canis, and 100% identification coincidence rate was found between conventional bio-typing method and BCSS?PCR assay. The analytical sensitivity of BCSS-PCR at DNA sample was 10?3 (50 pg/μL DNA of detecting B. canis). The higher genetic diversity was observed in B. canis strains in China. The results of genetic diversity investigated have showed that five loci in Panel 2B presented higher variability, the orders of allele genotypes from high to low were bruce09 (11), bruce07 (8), bruce16 (7), bruce04 (6), bruce30 (5). MLVA cluster analysis showed that there were three small-scale outbreaks of canine brucellosis in Beijing and sporadic in other areas. B. canis strains in this study were divided into five geographic groups (I?V), MLVA-11 genotype 26 clone group was predominated population. The strains of this clone group had common geographic origin with the strains from countries such as the United States, Greece, Canada, France, Romania and Korea. The others four geographic groups were unique lineages of B. canis in China. [Conclusion] There were higher genetic diversity and extensive of geographic origin of B. canis strains observed in China, and the results exhibited characteristics of origin and evolution of co-existing of imported and native specific lineage.