Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Microbiology (Reading). 2013 Mar;159(Pt 3):580-590. doi: 10.1099/mic.0.063461-0. Epub 2013 Jan 17.
The aim of the present study was to use multilocus sequence typing (MLST) of a diverse collection of Pasteurella multocida with regard to animal source, place and date of collection, including all available serovars of Carter, Heddleston, Little & Lyon, Namioka, Cornelius and Roberts, to further investigate the evolution of this species with a focus on two lineages, A (P. multocida subsp. multocida and P. multocida subsp. gallicida) and B (P. multocida subsp. septica), previously reported. Isolates of P. multocida (n = 116) including reference strains of major serotyping systems were investigated by MLST based on partial sequences of the genes adk, est, gdh, mdh, pgi, pmi and zwf, and 67 sequence types (STs) were observed. Phylogenetic analysis of these concatenated sequences confirmed the separation of groups A (41 STs, 71 isolates) and B (22 STs, 38 isolates) out of the 67 STs. All Carter serovars, 12 Heddleston serovars, all three Little-Lyon types, six out of seven Namioka serovars, all five Roberts types and all four Cornelius serovars were allocated to the A group, while group B included the remaining four Heddleston serovars, 6, 7, 8 and 13, in addition to Namioka type 8 : A. The overrepresentation of reference strains of serotyping systems in the A group contrasts with the high number of isolates obtained from diseased birds in the B group, the effect of which should be addressed in future vaccine development. Isolates from birds (25) dominated the B group, which also included four isolates from Felidae, whereas group A included isolates from all types of hosts. The evolutionary implications of the lack of capsular type D, pig and bovine isolates in group B, as well as its association with Aves and Felidae that also applied to the whole Rural Industries Research and Development Corporation (RIRDC) MLST database, need further investigation. The combination of rpoB and 16S rRNA gene sequence comparison as well as the developed PCR test assigned isolates to lineage A, represented by the type strain of P. multocida subsp. Multocida, or lineage B represented by the type strain of P. multocida subsp. septica. It was not possible to circumscribe either the A or B lineages with a set of conserved phenotypic characters, calling into question the validity of subspecies within P. multocida. Phylogenetic analysis carried out on individual MLST genes showed deviations as to single or multiple genes for 17 % of group A and 43 % of group B, indicating that lineage A probably developed from lineage B, and that major changes are ongoing. From a genotypical point of view, we conclude that P. multocida subsp. gallicida represents an artificial unit.
本研究的目的是使用多种序列分型（MLST）方法对不同来源的多杀巴斯德氏菌进行研究，包括动物来源、采集地点和日期，包括卡特、赫德尔斯顿、利特尔和莱昂、波多黎各、科尼利厄斯和罗伯茨所有可用的血清型，以进一步研究该物种的进化，重点关注两个谱系，A 谱系（多杀巴斯德氏菌亚种多杀巴斯德氏菌和多杀巴斯德氏菌亚种加利福尼亚）和 B 谱系（多杀巴斯德氏菌亚种败血性）。以前报道过。对包括主要血清分型系统参考株在内的 116 株多杀巴斯德氏菌（n=116）进行了 MLST 研究，基于 adk、est、gdh、mdh、pgi、pmi 和 zwf 基因的部分序列，并观察到 67 个序列型（ST）。这些串联序列的系统发育分析证实了 A 组（41 个 ST，71 个分离株）和 B 组（22 个 ST，38 个分离株）的分离。所有卡特血清型、12 个赫德尔斯顿血清型、三个利特尔-莱昂型、七个波多黎各血清型中的六个、五个罗伯茨型和四个科尼利厄斯型都被分配到 A 组，而 B 组包括其余四个赫德尔斯顿血清型，6、7、8 和 13，以及波多黎各型 8:A。A 组中血清分型系统的参考菌株的过度表现与 B 组中从患病鸟类中获得的大量分离株形成对比，这一现象应该在未来的疫苗开发中得到解决。来自鸟类的分离株（25）主导 B 组，其中还包括来自猫科动物的 4 个分离株，而 A 组包括来自所有宿主类型的分离株。B 组缺乏荚膜型 D、猪和牛分离株以及与鸟类和猫科动物的关联的进化意义，这些关联也适用于整个农村工业研究和发展公司（RIRDC）MLST 数据库，需要进一步调查。rpoB 和 16S rRNA 基因序列比较的组合以及开发的 PCR 试验将分离株分配到 A 谱系，由多杀巴斯德氏菌亚种多杀巴斯德氏菌的代表菌株代表，或由多杀巴斯德氏菌亚种败血性的代表菌株代表。不可能用一组保守的表型特征来划定 A 或 B 谱系，这对多杀巴斯德氏菌亚种的有效性提出了质疑。对单个 MLST 基因进行的系统发育分析显示，A 组中有 17%的分离株和 B 组中有 43%的分离株在单个或多个基因上存在偏差，表明 A 谱系可能是由 B 谱系进化而来的，并且正在发生重大变化。从基因型的角度来看，我们得出结论，多杀巴斯德氏菌亚种加利福尼亚亚种代表了一个人为的单位。
