E-Kobon Teerasak, Leeanan Ratiporn, Pannoi Saengtian, Anuntasomboon Pornchai, Thongkamkoon Pacharee, Thamchaipenet Arinthip
Department of Genetics, Faculty of Science, Kasetsart University, 50, Ngam Wong Wan Rd, Lad Yao, Chatuchak, Bangkok, 10900, Thailand.
Bioinformatics and Systems Biology Unit, Computational Biomodelling Laboratory for Agricultural Science and Technology (CBLAST), Faculty of Science, Kasetsart University, 50 Ngam Wong Wan Rd, Lat Yao, Chatuchak, Bangkok, 10900, Thailand.
BMC Vet Res. 2017 Aug 16;13(1):243. doi: 10.1186/s12917-017-1157-6.
Pasteurella multocida is a Gram-negative bacterium that causes economically significant infections of a broad range of animal species. Pneumonic and septicaemic pasteurellosis caused by this bacterium remain important problems in pigs, cattle, and water buffaloes in Thailand. The aim of this study was to characterise the virulence-associated gene profiles and to develop an OmpA molecular typing scheme for classifying 191 bovine and porcine isolates of P. multocida collected between 1989 and 2012 in Thailand using polymerase chain reactions (PCRs), nucleotide sequencing, and sequence and structural bioinformatics analyses.
PCR screening successfully characterised the profiles of 25 virulence-associated genes in all isolates. The gene profiles separated these isolates into bovine and porcine clusters based on eight genes (hgbB, hsf1, tadD, nanH, pfhA, plpE, pmHAS, and tbpA). Phylogenetic analyses of the nucleotide and protein sequences corresponding to the ompA gene, which encodes a major outer membrane surface protein, showed two major bovine and porcine clusters. Structural prediction and analysis of the dN/dS ratio revealed four hypervariable extracellular loops of the OmpA transmembrane domains. These four loops were used to develop an OmpA typing scheme. This scheme classified 186 isolates into five major loop sequence types (LST8, LST12, LST15, LST18, and LST19), consistent with the phylogenetic results. The loop regions of the bovine isolates were predicted to be more antigenic than those of the porcine isolates. Thus, molecular evolution of the OmpA proteins could be used to classify P. multocida isolates into different capsular types, host types, and, possibly, pathogenicity levels.
Together with the virulence-associated gene profiles, the typing reported in this work provides a better understanding of P. multocida virulence. Effective monitoring and potential strain-specific subunit vaccines could be developed based on these loop oligopeptides.
多杀性巴氏杆菌是一种革兰氏阴性菌，可引起多种动物发生具有经济重要性的感染。由该细菌引起的肺炎型和败血型巴氏杆菌病在泰国的猪、牛和水牛中仍然是重要问题。本研究的目的是利用聚合酶链反应（PCR）、核苷酸测序以及序列和结构生物信息学分析，对1989年至2012年期间在泰国收集的191株牛和猪源多杀性巴氏杆菌分离株的毒力相关基因谱进行特征分析，并开发一种OmpA分子分型方案用于分类。
PCR筛选成功鉴定了所有分离株中25个毒力相关基因的谱型。基于8个基因（hgbB、hsf1、tadD、nanH、pfhA、plpE、pmHAS和tbpA），基因谱型将这些分离株分为牛源和猪源簇。对编码主要外膜表面蛋白的ompA基因对应的核苷酸和蛋白质序列进行系统发育分析，显示出两个主要的牛源和猪源簇。对OmpA跨膜结构域的结构预测和dN/dS比率分析揭示了四个高变细胞外环。利用这四个环开发了一种OmpA分型方案。该方案将186株分离株分为五种主要的环序列类型（LST8、LST12、LST15、LST18和LST19），与系统发育结果一致。预测牛源分离株的环区域比猪源分离株的环区域具有更强的抗原性。因此，OmpA蛋白的分子进化可用于将多杀性巴氏杆菌分离株分为不同的荚膜类型、宿主类型以及可能的致病水平。
本研究报道的分型方法与毒力相关基因谱型一起，能更好地了解多杀性巴氏杆菌的毒力。基于这些环寡肽可开发有效的监测方法和潜在的菌株特异性亚单位疫苗。
