【摘要】 多杀性巴氏杆菌（Pm）可感染多种动物，引起肺炎或全身性败血症，造成严重的经济损失。为鉴定河北昌黎县某肉牛养殖场导致牛大批病死的病原，本研究从病牛肺脏中分离到疑似Pm的菌株，对其进行革兰氏染色观察，16S rDNA基因的PCR扩增与序列分析，并对其荚膜和脂多糖进行PCR分型，PCR扩增其7个管家基因后分析分离菌的多位点序列分型（MLST）。结果显示，分离株为革兰氏阴性菌，与Pm的16S rDNA基因序列同源性达99.87%，表明分离到一株Pm。PCR分型鉴定结果显示其为血清B型和脂多糖L2型，MLST分型为ST44型，将该Pm分离株命名为PmB-1。将PmB-1以4.88 cfu/只经腹腔注射感染小鼠，进行致病性试验，结果显示PmB-1能引起小鼠内脏出血病变。采用IlluminaHiseq二代结合PacBio三代测序平台对PmB-1进行全基因组测序及生物信息学分析。结果显示，PmB-1基因组含1条染色质，长2 331 836 bp，编码2 141个基因，并含19个rRNA,58个tRNA和42个sRNA；含有3个前噬菌体、5个基因组岛，5个获得性免疫（CRISPR-Cas）系统、3个插入序列以及1个转座子元件。致病性预测分析结果显示，PmB-1含244个毒力相关基因，224个耐药基因，495个基因参与病原与宿主的互作，14个基因编码分泌系统蛋白，535个基因编码转运蛋白，190个基因编码分泌蛋白，499个基因编码跨膜蛋白，17个基因编码双组分调控系统。本研究分离到一株Pm，并对其进行小鼠致病性试验，全基因组测序和生物信息学分析，为预防Pm的感染、流行和研究其致病机制提供了参考依据。更多还原

【Abstract】 Pasteurella multocida can infect a variety of animals,causing pneumonia or systemic sepsis,resulting in serious economic losses.To identify the pathogen of a large number of cattle deaths in a beef cattle farm in Changli County of Hebei province,a strain of suspected Pm was isolated from the lungs of diseased cattle in this study,and was subjected to Gram staining and PCR amplification and sequence analysis of 16S rDNA gene.PCR amplification was further used for typing of the capsules and lipopolysaccharides.The 7 house keeping genes were amplified by PCR and uploaded to MLST network database for multi-locus sequence typing.The results showed that the isolated strain was Gram-negative with 99.87%homology to the 16S r DNA gene sequence of Pm,indicating that a strain of Pm was obtained.The results of PCR typing showed that it was serotype B and lipopolysaccharide L2,and MLST typing was type 44.The Pm isolate was named Pm B-1.The isolate was intraperitoneally injected into mice at a dose of4.88 cfu/mouse for pathogenicity test,and the results showed that Pm B-1 could cause visceral hemorrhage in mice.The whole genome sequencing and bioinformatics analysis of PmB-1 were carried out by IlluminaHiseq second generation combined with PacBio third generation sequencing platform,and it showed that the Pm B-1 genome contained 1 chromatin,2331836bp in length,2141 coding genes,19 r RNA,58 t RNA and 42 s RNA.It contains three prophages,five genomic islands,five acquired immunity (CRISPR-Cas) systems,three insertion sequences,and one transposon element.The results of pathogenicity prediction analysis showed that Pm B-1 contained244 virulence related genes;224 drug resistance genes;495 genes involved in pathogen and host interaction,including 14 genes encoding secretory system protein,535 genes encoding transporter protein,190 genes encoding secretory protein,499 genes encoding transmembrane protein,and 17 genes encoding two-component regulatory system.In conclusion,a strain of Pasteurella multocida was isolated,and its pathogenicity was tested in mice,and whole genome sequencing and bioinformatics analysis were performed in this study,providing a reference for preventing Pm epidemic and exploring its pathogenic mechanism.更多还原