2014—2017年河南省副猪嗜血杆菌分离菌株的PCR鉴定及分型

PCR Identification and Serotyping of Haemophilus parasuis Isolated from Henan Province in 2014-2017

为了解河南省猪场副猪嗜血杆菌(Haemophilus parasuis,HPS)病的血清型流行情况,本研究于2014-2017年从河南省不同县市采集813份发病猪的肺、关节液等病料样品进行流行病学调查。首先通过TSA培养基分离病原,然后采用PCR法进行分子鉴定及分型鉴定。选取3个不同血清型分离株作为代表毒株进行外膜蛋白OMP基因的扩增及序列分析。结果显示HPS总分离率为7.75%(63/813),不同年份的分离率为5.49%(13/237)~11.31%(19/168),血清4型、5型、14型、不可分型(NT)的分离率分别为38.10%(24/63)、33.33%(21/63)、7.94%(5/63)、20.63%(13/63),血清4型、5型为优势血清型,其次是14型和不可分型的菌株。三个代表毒株与国内外毒株核苷酸相似性为98.6%~99.9%,氨基酸相似性为99.0%~100.0%。遗传进化树分析表明,Henan-HPS-ZK5和Henan-HPS-KF4、Henan-HPS-LY14分别处于两个分支,其中代表血清4型和14型的毒株Henan-HPS-KF4和Henan-HPS-LY14位于同一亚支,而血清5型的毒株Henan-HPS-ZK5与前两者距离较远。OMP氨基酸序列分析显示,Henan-HPS-ZK5第57位发生了T57(Thr)→A57(Ala)的突变;血清4型、9型和14型HPS第89位、115位、233位和239位氨基酸分别为Gln(Q)、Glu(E)、Arg(R)和Gly(G),而其他血清型均为Arg(R)、Lys(K)、Gly(G)和Arg(R)(未定型菌株除外)。本研究数据在一定程度上反映了河南省HPS病发病情况及血清型流行情况,从而为河南地区HPS病的疫苗研发、控制提供了科学客观的参考依据。

In order to investigate the prevalence of Haemophilus parasuis(HPS)and its serotype in Henan province during 2014-2017,813 suspected HPS samples were collected from different areas,such as Shangqiu,Luoyang,Zhoukou,etc.Firstly,pathogens were isolated by TSA medium,then,the isolates were identified based on 16 S rDNA sequences and were serotyped by type-specific primers.Three different serotype isolates were selected as representative strains,which outer membrane proteins(OMP)were cloned and analyzed.The results showed that the total isolation rate of HPS was 7.75%(63/813),and the isolation rate was from 5.49%(13/237)to 11.31%(19/168)in different years.In these isolates,the serotype 4,5,14 and nontypeable strains were 38.10%(24/63),33.33%(21/63),7.94%(5/63),20.63%(13/63),respectively.Serotype 4 and serotype 5 were the most predominant serotypes,followed by serotype 14 and non-typeable strains.Based on OMP sequence analysis,it showed that the homology of nucleotides and amino acids were 98.6%-99.9%and 99.0%-100.0%,respectively.Phylogenetic analysis indicated that all the strains were divided into two groups,Henan-HPS-KF4 and Henan-HPS-LY14 lied in the same branch,Henan-HPS-ZK5 was located in another branch.The comparison of amino acid sequence showed that the 57th amino acid of Henan-HPS-ZK5 was mutated from T57(Thr)to A57(Ala).The 89th,115th,233th and 239th amino acid of serotype 4,serotype 5 and serotype 14 HPS were Gln(Q),Glu(E),Arg(R)and Gly(G),respectively,while the other serotypes HPS were Arg(R),Lys(K),Gly(G)and Arg(R),except for the nontypeable strains.These data could respond the epidemiological situation of HPS and its serotypes in Henan province,which will contribute to vaccine research and provides a scientific and objective basis for prevention and control HPS disease.