呼伦贝尔地区牛体表寄生蜱中羊种布鲁氏菌的分离与鉴定

doi:10.3864/j.issn.0578-1752.2022.02.015

摘要/Abstract

摘要：

【目的】阐明牛体表寄生草原革蜱与牛羊布鲁氏菌病的相关性,为内蒙古地区布鲁氏菌溯源调查及防控提供基础数据。【方法】通过传统形态学及分子生物学相结合的方法,对呼伦贝尔地区采集的191只蜱虫进行种型鉴定;以牛体表采集的寄生蜱为材料,应用布鲁氏菌选择性培养基从蜱体内分离可疑细菌;将可疑细菌进行分离培养,菌落的形态学观察,以及革兰氏染色和柯氏染色镜检初步鉴定可疑细菌的种属;经PCR方法扩增布鲁氏菌的Bcsp31和Omp22基因序列,并应用MEGA 7.0软件构建系统遗传进化树;运用AMOS-PCR、单项特异性血清凝集试验、染料抑菌和噬菌体裂解试验对分离的可疑菌进行分型鉴定。【结果】该地区优势蜱种被鉴定为草原革蜱,并成功在其体内分离到4株可疑细菌;经革兰氏染色镜检发现该菌属于革兰氏阴性短杆菌,柯氏染色后菌体呈红色短杆菌,并通过PCR方法成功克隆出布鲁氏菌的Bcsp31和Omp22基因序列,经过基因序列测序及BLAST比对分析,发现与布鲁氏菌参考序列相似度均在99.0%以上。应用MEGA 7.0软件成功构建遗传进化树,分离菌株序列与布鲁氏菌属的已知序列聚类在同一分支;运用AMOS-PCR和布鲁氏菌常规分型鉴定试验,确定4株分离菌均属于羊种布鲁氏菌。【结论】成功在呼伦贝尔地区牛体表采集的草原革蜱体内分离到4株羊种布鲁氏菌,表明草原革蜱作为吸血节肢动物,可能在不同宿主之间的布病传播过程中发挥重要作用。本论文为内蒙古地区布鲁氏菌溯源调查和防控提供基础数据。

关键词: 布鲁氏菌, 草原革蜱, 分子生物学, Bcsp31, Omp22

Abstract:

【Objective】 The aim of this study was to elucidate the relationship between Dermacentor nuttalli and brucellosis in cattle and sheep. 【Method】 The species of ticks collected in Hulunbuir were identified by traditional morphological and molecular biology methods; the ticks collected from the cattle surface were used as materials, and the suspicious bacteria were isolated from their bodies. The isolated bacterial species were determined by methods, such as bacterial isolation and identification, morphological observation, serological detection and molecular biology. 【Result】 The dominant tick species were identified as D. nuttalli in this area, and four strains of suspicious bacteria were successfully isolated in their bodies. The isolated bacteria were gram-negative Brevibacterium by Gram staining, and the bcsp31 and omp22 gene sequences of Brucella were successfully cloned by PCR method. After gene sequencing and BLAST analysis, it was found that the similarity with the Brucella reference sequence was above 99.0%. The phylogenetic tree was successfully constructed by using Mega 7.0 software. The isolated strains were clustered in the same branch with the known sequences of Brucella. Using AMOS-PCR and Brucella typing test, four isolated strains were identified as Brucella melitensis. 【Conclusion】 In this experiment, four strains of B. melitensis were isolated from D. nuttalli collected from cattle in Hulunbuir area, which indicated that as a blood sucking arthropod, D. nuttalli might play an important role in the transmission of brucellosis among different hosts. This paper provided basic data for the investigation and control of Brucella in Inner Mongolia.

Key words: Dermacentor nuttalli, molecular biology, Brucella, Bcsp31, Omp22

黄天鹏,郭旭,孙长云,陈经雕,朝木丽格,武婕,格日勒图. 呼伦贝尔地区牛体表寄生蜱中羊种布鲁氏菌的分离与鉴定[J]. 中国农业科学, 2022, 55(2): 415-424.

HUANG TianPeng,GUO Xu,SUN ChangYun,CHEN JingDiao,CHAOMULIGE ,WU Jie,GERILETU . Isolation and Identification of Brucella melitensis from Ticks on Cattle Surface in Hulunbuir Area[J]. Scientia Agricultura Sinica, 2022, 55(2): 415-424.

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扩增片段 Amplified fragments	引物名称 Primer name	引物序列 Primer sequence (5'-3')	片段大小 Fragment size (bp)
16S rRNA	16Sf	TGACTATACRAAGGTATTG,（R=A或G）	346
16S rRNA	16Sr	GGACGGACTCGTAGCCTG
CO І	LCO1490	GGTCAACAAATCATAAAGATATTGG	658
CO І	HCO2198	TAAACTTCAGGGTGACCAAAAAATCA

扩增片段 Amplified fragments	引物名称 Primer name	引物序列 Primer sequence (5'-3')	片段大小 Fragment size (bp)
Bcsp31	B₄F	TGGCTCGGTTGCCAATATCAA	223
Bcsp31	B₅R	CGCGCTTGCCTTTCAGGTCTG	223
Omp22	Omp22F	TGATGGGAGGGACCGACTA	498
Omp22	Omp22R	TGGTTCTTCAGGTTGTTACGC	498

引物名称 Primer name	引物序列 Primer sequence (5'-3')	片段大小 Fragment size (bp)
A	GAC GAA CGG AAT TTT TCC AAT CCC	498
M	AAA TCG CGT CCTTGC TGG TCT GA	731
O	GCG CGG TTT TCT GAA GGT TCAGG	961
S	GCG CGG TTT TCT GAA GGT TCAGG	285
IS711	TGC CGA TCA CTT AAG GGC CTT CAT

试验与结果 Experiment and result		B. melitensis NMT5	B. melitensis NMT6	B. melitensis NMT7	B. melitensis NMT8	参考标准株 International reference strains
试验与结果 Experiment and result		B. melitensis NMT5	B. melitensis NMT6	B. melitensis NMT7	B. melitensis NMT8	16 M	1330S	544A
宿主Host		牛Cattle	牛Cattle	牛Cattle	牛Cattle
染料抑菌 Dye inhibition	硫瑾 Thionin	+	+	+	+	+	+	+
染料抑菌 Dye inhibition	碱性品红 Basic fuchsin	+	+	+	+	+	+	+
噬菌体裂解 Lysis by phage at RTD	Tb RTD 10⁴Tb RTD BK₂	- - +	- - +	- - +	- - +	- - +	- - +	+ + +
特异性血清 Agglutination on reaction with specific serum	A M R	+ - -	+ + -	+ + -	+ - -	- + -	+ - -	+ - -
CO₂需求 CO₂ requirement		-	-	-	-	-	-	-
产H₂S H₂S production		-	-	-	-	+	+	+
结果 Result	种型 Species biotype	羊种2型布鲁氏菌 Brucella melitensis 2	羊种3型布鲁氏菌 Brucella melitensis 3	羊种3型布鲁氏菌 Brucella melitensis 3	羊种2型布鲁氏菌 Brucella melitensis 2	羊种1型布鲁氏菌 Brucella melitensis 1	猪种1型布鲁氏菌 Brucella suis 1	牛种1型布鲁氏菌 Brucella abortus 1