鸡源多杀性巴氏杆菌的分离鉴定及其毒力基因的检测

Isolation,identification and virulence genes detection of Pasteurella multocida from chicken

取病死鸡肝接种于血清琼脂平板,挑取符合巴氏杆菌特征的单菌落进行纯化培养和染色镜检;对纯培养菌提取核酸,进行16S r RNA、kmt1和cap A基因的PCR鉴定,7个管家基因的PCR扩增和多位点序列分型,以及12个毒力基因的PCR检测;并进行分离菌的动物回归试验。结果显示,分离的2株病原菌(HN-1和CZ-6)均为两极浓染的革兰阴性短杆菌,16S r RNA和kmt1基因序列与多杀性巴氏杆菌的同源性均高于99%,荚膜血清型鉴定为A型,基因型为ST129型,除tox A、ptf A、nan H之外的9个毒力基因均被检出,动物回归试验表明分离菌对鸡的致病性较强。结果表明,这2株鸡源多杀性巴氏杆菌的分离鉴定为我国禽源菌株的遗传特性研究提供了一定参考。

The liver of dead chickens was inoculated on serum agar plates,and single colonies were subsequently selected in accordance with Pasteurella characteristics for purification,staining and microscopy.DNA extracted from pure cultures was used for PCR amplification of 16 S r RNA,kmt1 and cap A,7housekeeping genes for multi-locus sequence typing（MLST）,and 12 virulence genes. Finally,animal pathogenicity experiment was carried out.Two strains of Gram negative bacilli were isolated with bipolar staining.Sequences of 16 S r RNA and kmt1 genes shared homology higher than 99%with Pasteurella multocida,capsule serotype was identified as type A,and genotype were ST129.Except for tox A,ptf A and nan H,9virulence genes were detected,and animal pathogenicity experiment showed high pathogenicity for chicken.Identification of two P.multocida strains can provide references for genetic characteristics study of avian isolates in China.