猪源多杀性巴氏杆菌的分离与鉴定

Isolation and identification of Pasteurella multocida from swine

2014年3月至2015年2月从来源于全国15个省份的180份临床猪肺组织样品中共分离到71株多杀性巴氏杆菌,分离率为39.4%。利用PCR技术对所分离的多杀性巴氏杆菌进行荚膜血清型鉴定,其中A型菌株41株（占57.8%）,D型菌株27株（占38.0%）,F型菌株3株（占4.2%）,71株多杀性巴氏杆菌中包括1株D型产毒素多杀性巴氏杆菌（占1.4%）,说明当前在我国猪群中流行的多杀性巴氏杆菌主要为A型和D型菌株。应用PCR检测多杀性巴氏杆菌23种毒力基因,结果显示：多杀性巴氏杆菌毒力基因的数量大多分布在15～19之间,平均携带个数为17.5个;23种毒力基因中,ptfA,fimA,hsf-2,sodA,sodC,ompA,ompH,oma87,plpB,exbB,tonB,Fur,hgbB的检出率在90%以上,而toxA基因的检出率极低（1.4%）,并且在猪源多杀性巴氏杆菌中未检测到t bpA的存在;分析毒力基因在不同血清型菌株中的携带及分布情况发现,A型的平均毒力基因携带数为18.2个,D型为16.4个;不同毒力基因在不同血清型的多杀性巴氏杆菌中的分布也存在一定的差异,其中唾液酸代谢酶基因nanB、透明质酸酶基因pmHAS及黏附基因tadD和pfhA在A型多杀性巴氏杆菌中的检出率高于其在D型多杀性巴氏杆菌中的检出率（P〈0.01）;而黏附基因hsf-1在D型多杀性巴氏杆菌的检出率显著高于其在A型多杀性巴氏杆菌中的检出率（P〈0.01）。对所分离菌株的16SrRNA基因进行测序,并与NCBI上公布的10株多杀性巴氏杆菌16S rRNA基因进行进化树分析,结果显示：71株多杀性巴氏杆菌主要分为2个亚群,亚群Ⅰ和HB03及3480的亲缘关系较近,而亚群Ⅱ则与HN06、Pm70、36950等菌株的亲缘关系较近;遗传进化的结果说明,多杀性巴氏杆菌的亲缘关系与其来源、血清型及其导致的疾病种类并没有表现出特定的亲缘关系。

A total of 71 Pasteurella multocida were isolated from 180 lungs of pigs originating from approximately 15 provinces in China from March 2014 to February 2015.The isolation rate of P.multocida was 39.4%.PCR was used for serological analysis,the isolates were 41 serotype A strains（57.8%）,27 serotype D strains（38.0%）and 3serotype F strains（4.2%）,suggesting that P.multocidaserotypes A and D strains were the primarily prevalent in pig herds in China.Only 1toxigenic P.multocida was isolated（1.4%）.PCR was used for detecting 23virulence-associated genes harboured in those isolates,demonstrated that each P.multocidacarried 15 to 19virulence-associated genes and the average number of the virulence-associated genes was 17.5.Of the total 23 examined genes,the detection rate of ptfA,fimA,hsf-2,sodA,sodC,ompA,ompH,oma87,plpB,exbB,tonB,Fur,and hgbB was more than 90%,respectively,while that of toxA was 1.4%.Moreover,we did not examine the tbpAgene in the 71 isolates.In addition,the number of virulence-associated genes harboured in P.multocidaserotype A strains was 18.2,while that in the serotype D strains was 16.4.Detection rates of nanB,pmHAS,tadDand pfhAin P.multocidaserotype A strains were higher than those in P.multocidaserotype D strains,respectively（P〈0.01）,while the detection rate of hsf-1in serotype D isolates was significant higher than that in serotype A isolates（P〈0.01）.Phylogenetic tree based on the 16 SrRNA gene sequences of the 71 isolates revealed that those strains could be divided into two subgroups.Strains in subgroupⅠ showed a nearer relationship with the representative strains HB03 and 3480,and those in subgroupⅡ were close to the references HN06,Pm70 and 36950.Furthermore,the finer phylogenetic relatedness of the P.multocidastrains suggested that there appeared to be little or no correlation between the phylogenic relatedness of the strains and the country of isolation,the serotype,the disease or the host predilection.