一株鹿源牛种布鲁菌的鉴定及全基因组分析

doi:10.11843/j.issn.0366-6964.2019.08.016

摘要/Abstract

摘要： 本研究旨在对一株鹿源牛种布鲁菌（Brucella abortus） BJ1进行全面鉴定，为研究鹿的布鲁菌病提供参考。将B.abortus BJ1培养后，挑取单菌落分别接种到含硫堇或碱性品红的TSA培养基，观察其生长状态；将接种有B.abortus BJ1的TSA平板分别置于普通生化培养箱和CO₂培养箱37℃培养72 h，观察其对CO₂的依赖性；通过醋酸铅培养基测定B.abortus BJ1生长过程中是否产生H₂S；通过结晶紫染色和热凝集试验测定B.abortus BJ1的表型；通过平板凝集试验测定B.abortus BJ1单因子血清反应性；采用AMOS-PCR鉴定细菌种属；为测定其毒力，以1×10⁹CFU感染豚鼠，14 d后测定豚鼠每克脾组织的含菌量。使用二代测序方法测其全基因组序列并对其进行分析和注释，将结果与B.abortus 2308比对，并通过Mauve软件进行系统进化分析。结果显示：B.abortus BJ1不依赖CO₂，产H₂S，可以在含硫堇和碱性品红的培养基上生长；表型为光滑型，与M因子血清发生凝集；豚鼠脾含菌量为1.17×10⁶～2.05×10⁶CFU·g^-1；基因组大小为3 270 584 bp，在基因编码区内与B.abortus 2308株相比有46处Indel差异，进化树分析显示与B.abortus 8416有较高的同源性。通过全面鉴定，确定B.abortus BJ1为牛种9型，为更深入地了解我国布病发生情况和鹿的布病防控提供参考。

关键词: 布鲁菌, 生化鉴定, 全基因组测序

Abstract: This study aimed to comprehensively identify a strain of Brucella (B. abortus), tentatively designated BJ1, to provide a reference strain for the research of deer brucellosis. B. abortus BJ1 was diluted and cultivated on TSA plate, and single colony was inoculated into thiopurine and basic fuchsin medium for dye sensitivity test; The TSA plates inoculated with B. abortus BJ1 were placed in a common biochemical incubator and CO₂ incubator for 72 h at 37℃ to observe its dependence on CO₂; B. abortus BJ1 was inoculated into lead acetate medium for H₂S release test; Bacterial S/R type was identified by crystal violet staining and thermal agglutination test; mono-specific serum reactivity was determined by plate agglutination test; bacterial species were identified by AMOS-PCR. Guinea pigs were infected with 1×10⁹ CFU of BJ1 for 14 days to determine the virulence of BJ1 by measuring the number of B. abortus in one gram of spleen of the infected guinea pig. The entire genome was sequenced by next-generation sequencing approach, and phylogenetic analysis was performed using Mauve software to compare with B. abortus 2308. Results were as follows:B. abortus BJ1 was demonstrated to grow independent of CO₂, and produced H₂S. BJ1 was able to grow on medium containing thiopurine and basic fuchsin, and it was confirmed to be smooth phenotype with capability of agglutination with M factor serum. A number of 1.17×10⁶- 2.05×10⁶ CFU of the strain could be obtained from one gram of spleen 14 days post infection. The genome size of BJ1 was 3 270 584 bp, and a total of 46 Indel difference were found in the coding region of BJ1 genome, compared to the B. abortus 2308 strain. Phylogenetic analysis showed that BJ1 exhibited high homology with B. abortus 8416. B. abortus BJ1 was identified to be a bovine species type 9 and enriched the Brucella species. Our findings provide a reference for in-depth understanding of the epidemiology of brucellosis in wild animals of China.

Key words: Brucella, biochemical identification, whole genome sequencing

中图分类号:

S852.614

牛凯, 程汝佳, 许冠龙, 冯宇, 孙石静, 范学政, 彭小薇, 朱良全, 秦玉明, 丁家波, 常维山, 蒋卉. 一株鹿源牛种布鲁菌的鉴定及全基因组分析[J]. 畜牧兽医学报, 2019, 50(8): 1666-1675.

NIU Kai, CHENG Rujia, XU Guanlong, FENG Yu, SUN Shijing, FAN Xuezheng, PENG Xiaowei, ZHU Liangquan, QIN Yuming, DING Jiabo, CHANG Weishan, JIANG Hui. Identification and Complete Genomic Analysis of a Brucella Strain Isolated from Deer[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1666-1675.

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