布鲁氏菌中国分离株遗传多态性研究
摘要
布鲁氏菌病是由布鲁氏菌侵入机体引起的一类传染－变态反应性的全球性分布的人畜共患病，在许多发展中国家流行并造成很大的经济损失。依据对宿主偏好性以及培养和生物学特性的不同，布鲁氏菌分为6个经典的种，其中羊种、牛种、猪种、犬种4个种能够使人致病。布鲁氏菌基因的高度保守性使得建立种型之间的遗传关系极具挑战性。了解布鲁氏菌基因水平的差异，有助于了解布鲁氏菌的进化与毒力特征。目前布鲁氏菌基因分型方法中以多位点序列分型(MLST)和多位点VNTR (MLVA)法重复性好、分辨率高，但是由于布鲁氏菌的变异，在实际工作中已遇到问题。寻找一种更好的分型方法或者是多种方法的联合运用，可能会更好地解决布鲁氏菌的分种分型问题。
通过十株已测序的布鲁氏菌全基因组的比较分析和布鲁氏菌全基因组芯片杂交的结果，共鉴定出53个差异基因区段（DFR），其中位于I号染色体上的DFR有26个，II号染色体上的DFR为27个，II号染色体的DFR密度远远高于I号染色体。探讨这些差异区段在19株标准菌株和129株我国不同种型布鲁氏菌分离株的分布特征表明，差异区段可能是进化过程中获得或缺失的，与菌株的遗传关系没有必然的联系，为研究不同疫源地或不同年代的布鲁氏菌菌株基因水平差异提供了依据。
为了提高传统的MLST方法（CMLST）的分型分辨率，我们在本研究中探讨通过加长基因的PCR扩增片段来用于MLST分型研究，即为改良的MLST方法（EMLST）。对129株我国布鲁氏菌分离株进行了CMST和EMLST的分析，用CMLST方法定义了23个ST型，而用EMLST方法则定义了35个ST型，多种方法分析表明，EMLST较CMLST具有更高的分型分辨率。和已报道的国外布鲁氏菌菌株的CMLST结果比较分析发现，我国布鲁氏菌分离株具有自己的基因型构成特点。基于看家基因的改良的MLST方法适合于亲缘关系较远的布鲁氏菌菌株的分型。
传统的MLVA方法采用PCR扩增并电泳区分的方法，但是存在不稳定及费时费力的缺点，因此本研究采用测序的方法对120株分离株的15个多态性较高的MLVA位点进行分析，共分为87个型。基于VNTR位点的改良的MLVA方法应用于我国布鲁氏菌分离株的结果表明，该方法适合于亲缘关系较近的布鲁氏菌菌株的分型。对120株CMLST、EMLST以及MLVA数据综合比较分析表明，EMLST较CMLST具有更多的分支，有着更高的分辨率，而MLVA则是三种分型方法中分辨率最高的方法。
Bacteria of the genus Brucella are the etiological agents of brucellosis, which is azoonotic disease endemic in many areas of the world. This disease remains endemicin some countries, especially in developing ones and it is still a substantial economicburden for many areas of the world. The genus Brucella is divided into six classicalspecies: B.abortus (bovine), B.melitensis (caprine and ovine), B. ovis (ovine), B.canis(canine), B. suis (porcine), and B.neotomae (only seen in the desert wood rat) on thebasis of host specificity, antigenic differences and biochemical characteristics. Fourout of six species are pathogenic for humans, i.e. B. abortus, B. melitensis, B. suis andrarely B. canis. B. melitensis and B. abortus are highly pathogenic and is a frequentcause of human brucellosis. The conservation of this genus makes it difficult toestablish the true relationships between some classical Brucella species and biovars.
Brucella has several host and widely distributed foci. Different location ofBrucella strains lead differences in biochemical features and pathogenicity. Alsodifferent species of Brucella have diversity in host range and virulence. Gene obtainedand deletion which relevant with adaption to the environment, host specificity andvirulence are an important mechanism of bacterial evolution. Understanding theevolution and virulence characteristics of Brucella depends on the knowlege ofgenetic differences of Brucella. In present, the typing methods of Brucella arephenotyping and genotyping. A variety of methods exist in the genotyping, and typingmethods have advantages and disadvantages, especially the typing results aredifference but have some contacts. In particular, multi-locus sequence typing (MLST)and multi locus VNTR analysis (MLVA) method possess repeatability and highresolution. However, due to the variation of Brucella, some atypical strains cannot betyped in the actual work. A better typing methods, or associated with a variety of methods may be better to solve the typing problem in Brucella.
The comparative analysis of ten Brucella whole genome and Brucellawhole-genome microarray hybridization results identified a total of53DFR, and26in chromosome I, moreover the number of gene differences were102.27DFR werein chromosome II, including a total of194genes, and the DFR density inchromosome II was higher than I. The cluster analysis of53DFR in19standardstrains suggested that all abortus and melitensis, suis biovar4/5were clustered intoone branch, and suis biovar1/2/3, canis, neotomae and ovis were another.129Brucella isolates were also analyzed and it consisted two branches, one was abortusand melitensis isolates, another for suis and canis. DFR may be the acquisition ormissing in evolutionary process and there is no relationship with strains hereditary.18abortus isolates in the1970s and14canis isolates in the1980s were Clusteringanalyzed. It indicates that the propagation of abortus Brucella isolates is only inwestern area in the1970s,basically among several adjacent several provinces.However, in the1980s, the propagation of canis Brucella isolates limites in China'seastern region. The first canis Brucella strain may originate from Xinjiang provinceand then spread in Jiangshu and Zhejiang Provinces.
The product length amplified by traditional MLST(CMLST) primers aregenerally400to500bp, and the valid sequences are between350to450bp afterremoving low quality sequence on both ends. Therefore, the available sequenceinformation by the conventional MLST is greatly limited. With the development ofsequencing instrument and technology, the length per sequencing reaction was greatlyimproved. In this study, all sequences sequenced by the3730sequencer werestatistical analyzed and it showed that at least800bp reading length with high qualitycould be generated. To improve the resolution of MLST for Brucella, and also extendthe application of MLST in other pathogenic bacteria, in the present study, we testedthe feasibility of improving resolution of MLST by increase of the sequencing length,defined as extended MLST (EMLST). To compare with the CMLST method,the nucleic acid length of all loci for EMLST were increased from128bp to301bpwith the proportion increasing about26.12%to71.33%.
129Chinese Brucella isolates were analyzed with CMST and EMLST,23STswere defined by CMLST method, and35STs by EMLST. Except for aroA,gyrB andInt-hyp,alleles identified of six loci were at least increased one allele, such as thatcobQ increased five alleles and omp25increased2alleles. Alleles identified of eachloci dependent on polymorphic sites. polymorphic sites of six loci of nine genes wereincreased by increasing amplified product length.All data of CMLST and EMLSTmethod were analyzed by splittree, eBURST and BioNumerics software. It indicatesthat some of the strains that could be differentiated by CMLST were clearlydifferentiated by EMLST and the resolution of the EMLST is greatly improved.EMLST method can differentiate some canis strains from suis strains.EMLST has ahigher genotyping resolution than CMLST.
27STs were defined in foreign Brucella strains by MLST analysis, and15STswere firstly defined in129Brucella of China which had23STs defined.8STs firstlydefined for abortus Brucella strains were ST8,ST28-33and ST42. ST33,ST34andST35were firstly defined for melitensis isolations. There were4STs and3STs firstlydefined respectively for suis and canis Brucella strains, that is,ST36-37and ST40-41for suis isolations； ST38,ST39and ST40for canis strains.
PCR amplification and electrophoresis were used to distinguish in traditionalMLVA method, but an instability and time-consuming drawback. However,15highlypolymorphic MLVA sites in120isolates were analyzed by this method in our study,and obtained87types. There were13Brucella strains defined the same type81andtype6was consist of5Brucella isolations.The cluster analysis showed that abortusgenotypes were clustered into one branch, suis and canis for one, melitensis were theother one. We analyzed the evolutionary relationships or phylogenetic relationships of all genotypes for all strains by using maximum parsimony method. There is ahypothesis that canis strains may be evolved by the suis strains and abortus strainsmay be due to the melitensis strains.
The CMLST, EMLST and MLVA data of120isolates showed that EMLSTcompared CMLST has more branches and a higher resolution. MLVA possessed thehighest resolution in the three methods, and it can nearly type each strains. However,all three methods can not differentiated biovars of Brucella strains.
通过十株已测序的布鲁氏菌全基因组的比较分析和布鲁氏菌全基因组芯片杂交的结果，共鉴定出53个差异基因区段（DFR），其中位于I号染色体上的DFR有26个，II号染色体上的DFR为27个，II号染色体的DFR密度远远高于I号染色体。探讨这些差异区段在19株标准菌株和129株我国不同种型布鲁氏菌分离株的分布特征表明，差异区段可能是进化过程中获得或缺失的，与菌株的遗传关系没有必然的联系，为研究不同疫源地或不同年代的布鲁氏菌菌株基因水平差异提供了依据。
为了提高传统的MLST方法（CMLST）的分型分辨率，我们在本研究中探讨通过加长基因的PCR扩增片段来用于MLST分型研究，即为改良的MLST方法（EMLST）。对129株我国布鲁氏菌分离株进行了CMST和EMLST的分析，用CMLST方法定义了23个ST型，而用EMLST方法则定义了35个ST型，多种方法分析表明，EMLST较CMLST具有更高的分型分辨率。和已报道的国外布鲁氏菌菌株的CMLST结果比较分析发现，我国布鲁氏菌分离株具有自己的基因型构成特点。基于看家基因的改良的MLST方法适合于亲缘关系较远的布鲁氏菌菌株的分型。
传统的MLVA方法采用PCR扩增并电泳区分的方法，但是存在不稳定及费时费力的缺点，因此本研究采用测序的方法对120株分离株的15个多态性较高的MLVA位点进行分析，共分为87个型。基于VNTR位点的改良的MLVA方法应用于我国布鲁氏菌分离株的结果表明，该方法适合于亲缘关系较近的布鲁氏菌菌株的分型。对120株CMLST、EMLST以及MLVA数据综合比较分析表明，EMLST较CMLST具有更多的分支，有着更高的分辨率，而MLVA则是三种分型方法中分辨率最高的方法。
Bacteria of the genus Brucella are the etiological agents of brucellosis, which is azoonotic disease endemic in many areas of the world. This disease remains endemicin some countries, especially in developing ones and it is still a substantial economicburden for many areas of the world. The genus Brucella is divided into six classicalspecies: B.abortus (bovine), B.melitensis (caprine and ovine), B. ovis (ovine), B.canis(canine), B. suis (porcine), and B.neotomae (only seen in the desert wood rat) on thebasis of host specificity, antigenic differences and biochemical characteristics. Fourout of six species are pathogenic for humans, i.e. B. abortus, B. melitensis, B. suis andrarely B. canis. B. melitensis and B. abortus are highly pathogenic and is a frequentcause of human brucellosis. The conservation of this genus makes it difficult toestablish the true relationships between some classical Brucella species and biovars.
Brucella has several host and widely distributed foci. Different location ofBrucella strains lead differences in biochemical features and pathogenicity. Alsodifferent species of Brucella have diversity in host range and virulence. Gene obtainedand deletion which relevant with adaption to the environment, host specificity andvirulence are an important mechanism of bacterial evolution. Understanding theevolution and virulence characteristics of Brucella depends on the knowlege ofgenetic differences of Brucella. In present, the typing methods of Brucella arephenotyping and genotyping. A variety of methods exist in the genotyping, and typingmethods have advantages and disadvantages, especially the typing results aredifference but have some contacts. In particular, multi-locus sequence typing (MLST)and multi locus VNTR analysis (MLVA) method possess repeatability and highresolution. However, due to the variation of Brucella, some atypical strains cannot betyped in the actual work. A better typing methods, or associated with a variety of methods may be better to solve the typing problem in Brucella.
The comparative analysis of ten Brucella whole genome and Brucellawhole-genome microarray hybridization results identified a total of53DFR, and26in chromosome I, moreover the number of gene differences were102.27DFR werein chromosome II, including a total of194genes, and the DFR density inchromosome II was higher than I. The cluster analysis of53DFR in19standardstrains suggested that all abortus and melitensis, suis biovar4/5were clustered intoone branch, and suis biovar1/2/3, canis, neotomae and ovis were another.129Brucella isolates were also analyzed and it consisted two branches, one was abortusand melitensis isolates, another for suis and canis. DFR may be the acquisition ormissing in evolutionary process and there is no relationship with strains hereditary.18abortus isolates in the1970s and14canis isolates in the1980s were Clusteringanalyzed. It indicates that the propagation of abortus Brucella isolates is only inwestern area in the1970s,basically among several adjacent several provinces.However, in the1980s, the propagation of canis Brucella isolates limites in China'seastern region. The first canis Brucella strain may originate from Xinjiang provinceand then spread in Jiangshu and Zhejiang Provinces.
The product length amplified by traditional MLST(CMLST) primers aregenerally400to500bp, and the valid sequences are between350to450bp afterremoving low quality sequence on both ends. Therefore, the available sequenceinformation by the conventional MLST is greatly limited. With the development ofsequencing instrument and technology, the length per sequencing reaction was greatlyimproved. In this study, all sequences sequenced by the3730sequencer werestatistical analyzed and it showed that at least800bp reading length with high qualitycould be generated. To improve the resolution of MLST for Brucella, and also extendthe application of MLST in other pathogenic bacteria, in the present study, we testedthe feasibility of improving resolution of MLST by increase of the sequencing length,defined as extended MLST (EMLST). To compare with the CMLST method,the nucleic acid length of all loci for EMLST were increased from128bp to301bpwith the proportion increasing about26.12%to71.33%.
129Chinese Brucella isolates were analyzed with CMST and EMLST,23STswere defined by CMLST method, and35STs by EMLST. Except for aroA,gyrB andInt-hyp,alleles identified of six loci were at least increased one allele, such as thatcobQ increased five alleles and omp25increased2alleles. Alleles identified of eachloci dependent on polymorphic sites. polymorphic sites of six loci of nine genes wereincreased by increasing amplified product length.All data of CMLST and EMLSTmethod were analyzed by splittree, eBURST and BioNumerics software. It indicatesthat some of the strains that could be differentiated by CMLST were clearlydifferentiated by EMLST and the resolution of the EMLST is greatly improved.EMLST method can differentiate some canis strains from suis strains.EMLST has ahigher genotyping resolution than CMLST.
27STs were defined in foreign Brucella strains by MLST analysis, and15STswere firstly defined in129Brucella of China which had23STs defined.8STs firstlydefined for abortus Brucella strains were ST8,ST28-33and ST42. ST33,ST34andST35were firstly defined for melitensis isolations. There were4STs and3STs firstlydefined respectively for suis and canis Brucella strains, that is,ST36-37and ST40-41for suis isolations； ST38,ST39and ST40for canis strains.
PCR amplification and electrophoresis were used to distinguish in traditionalMLVA method, but an instability and time-consuming drawback. However,15highlypolymorphic MLVA sites in120isolates were analyzed by this method in our study,and obtained87types. There were13Brucella strains defined the same type81andtype6was consist of5Brucella isolations.The cluster analysis showed that abortusgenotypes were clustered into one branch, suis and canis for one, melitensis were theother one. We analyzed the evolutionary relationships or phylogenetic relationships of all genotypes for all strains by using maximum parsimony method. There is ahypothesis that canis strains may be evolved by the suis strains and abortus strainsmay be due to the melitensis strains.
The CMLST, EMLST and MLVA data of120isolates showed that EMLSTcompared CMLST has more branches and a higher resolution. MLVA possessed thehighest resolution in the three methods, and it can nearly type each strains. However,all three methods can not differentiated biovars of Brucella strains.
引文
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