89株副猪嗜血杆菌临床分离株血清型、基因型鉴定与分析

Identification and Analysis of Serotype and Genotype of 89 Clinical Isolates of Haemophilus parasuis

旨在明确副猪嗜血杆菌(HPS)临床分离株分离部位的组织分布,进而探讨其血清型和基因型的流行分布特点及相关性,为猪格氏病(Glässer’s disease)的有效防控提供科学依据。针对临床分离鉴定的89株HPS,利用PCR技术鉴定血清型,统计各血清型HPS在不同分离部位的菌株分布数量;应用多位点序列分型(MLST)方法进行序列类型(ST)鉴定分析、位点多态性分析、BURST分群统计和UPGMA系统发育树聚类分析。89株HPS临床分离株共鉴定出9种血清型(1、2、4、5、7、11、12、13、14)以及未定型(NT),血清4、13、7和5型为优势血清型,分别占比28.09%、22.47%、13.48%和10.11%,有64株HPS分离于肺,占比71.91%;24种ST型,ST267、ST268、ST387和ST365为优势基因型,分别占比26.97%、21.35%、8.99%和5.62%,每个基因位点存在3~13个等位基因,多态性位点从3(g3pd)到71(6pgd)不等,BURST分析中划分为2个单个ST型和11个克隆群(CC),UPGMA系统发育树被分为4个分支,优势ST型处于2、3、4三个分支中,均对应具有毒力血清型的HPS分离株。HPS临床分离株流行血清型和基因型呈现多元化,具有明显的遗传异质性,同时,ST型与血清型存在一定的交叉性,且与HPS临床致病力相关。

In this study,we aimed to determine the tissue distribution of clinical isolates of Haemophilus parasuis(HPS)to explore the epidemic distribution and correlation of its serotypes and genotypes,and to provide scientific basis for the effective prevention and control of Glässer’s disease.According to the 89 strains of HPS isolated from the clinic,the serotypes of HPS were identified by PCR,and the number of HPS strains in different parts of the isolates was counted.Sequence type(ST)identification analysis,site polymorphism analysis,BURST cluster analysis and UPGMA phylogenetic tree cluster analysis were performed by using the multilocus sequence typing method.Nine serotypes(1,2,4,5,7,11,12,13,and 14)and undetermined serotypes(NT)were identified from 89 HPS clinical isolates.Serotypes 4,13,7 and 5 were the predominant serotypes,accounting for 28.09%,22.47%,13.48%and 10.11%,respectively,and sixty-four strains of HPS were isolated from the lung tissues.ST267,ST268,ST387 and ST365 were dominant genotypes,accounting for 26.97%,21.35%,8.99%and 5.62%,respectively,there were 3-13 alleles at each locus,and the polymorphisms ranged from 3(g3pd)to 71(6pgd).The BURST analysis showed that the 89 HPS were divided into 2 single ST types and 11 clonal groups(CC).The phylogenetic tree of UPGMA had 4 branches,the dominant ST type was found in 2,3 and 4 branches,which was corresponded to the HPS isolates with virulent serotype.The epidemic serotypes and genotypes of HPS are diversified and have obvious genetic heterogeneity.At the same time,ST types and serotypes have some crossover and are related to HPS clinical pathogenicity.