一株鹅源多杀性巴氏杆菌强毒株的分离鉴定及其血清型鉴定相关基因的克隆

摘要

Goose pasteurellosis, caused by the bacterium Pasteurella multocida( Pm) , was a major infectious disease of goose in China in recent years. To identify the pathogen of goose disease in a goose-breeding farm, the causes of epidemic situation were investigated. A gram-negative bacillus was isolated and purified from the livers and hearts samples of the sick geese that preliminary diagnosed as pasteurellosis based on clinical symptoms and pathological changes, and it was identified as goose Pasteurella multocida by morphological and cultured characteristics, biochem-ical tests, animal pathogenic tests, drug sensitivity tests, species and serotype PCRs and so on. Then its serotype as-sociated genes for serotype PCRs were cloned and analysed. The results showed that the goose Pm isolate had typical morphology and biochemical characteristics of P. multocida subsp. multocida, and it belonged to capsular type A and serotype Ⅰ. This Pm isolate had a strong pathogenicity, and its minimus lethal dose ( MLD) to mice was 5 cfu. Drug sensitivity tests showed that this isolate was only sensitive to cefatrizine, cefalotin, ceftazidime, ceftizoxime and florfenicol. A capsular biosynthesis associated gene hyaD-hyaC about 4155 bp in this isolated Pm strain was ampli-fied and sequenced, which shared a 99% homology with the published gene sequences. The serotypeⅠPCR target gene fragments about 303 bp of this goose Pm strain and the reference strain C48-1 were purified and sequenced, the sequencing results were exactly the same. All these results indicated that the goose Pm isolate which had a strong pathogenic and belonged to capsular type A and serotypeⅠ, was the pathogen of goose disease to be diagnosed. And the amplified hyaD-hyaC gene sequences and serotypeⅠPCR target gene fragments of this Pm isolate both exhibited relative stability.%为探索某鹅场鹅巴氏杆菌病疫情发生的原因,采集初诊为鹅巴氏杆菌感染病例的肝脏、心脏等样品进行细菌分离纯化,经过染色镜检、培养特性、生化试验、种与血清型PCR鉴定、致病性试验和药敏试验等进行鉴定,并根据GenBank中多杀性巴氏杆菌血清型相关基因设计引物进行其血清型相关基因的克隆分析.结果显示,分离到1株荚膜A型鹅源多杀性巴氏杆菌,属于多杀亚种,血清型Ⅰ型,具有较强致病性,对实验小鼠最小致死量为5 cfu.该分离菌株具有多重耐药性,仅对头孢三嗪、头孢噻吩、头孢他啶、头孢唑肟和氟苯尼考5种药物敏感.成功克隆的该分离菌株荚膜合成相关基因hyaD-hyaC的开放阅读框4155 bp,与已发布基因序列同源性高达99%;血清型Ⅰ型PCR的扩增基因片段303 bp,与巴氏杆菌C48-1株扩增基因片段序列完全相同.综上,该株血清型Ⅰ型荚膜A型的鹅源多杀性巴氏杆菌强毒株,是鹅场疫情发生的病原,克隆的荚膜合成相关基因和菌体血清型特异性基因遗传相对稳定.