羊源多杀性巴氏杆菌的分离鉴定及药敏试验

Isolation,Identification and Drug Sensitivity Test of Pasteurella multocida from Dairy Goats

为确定陕西某规模化奶山羊场部分羊出现精神沉郁,食欲减退,采食量减少,呼吸困难甚至出现死亡的病因,以病羊病变组织为研究对象,采用细菌分离培养、生化试验、16S rRNA PCR扩增测序、药敏试验、致病性试验等方法对分离菌株进行鉴定和分析。结果显示,从病死羊肝脏组织中分离到1株革兰氏阴性、无芽孢、无鞭毛且两极浓染的短杆菌,在TSA培养基上呈现出贫瘠的细小、透明、露滴状菌落;在麦康凯培养基上不生长;在血琼脂平板上长出灰白色、圆形、光滑、湿润、不透明的露滴样菌落;分离菌株能分解葡萄糖、蔗糖、甘露醇、山梨醇、触酶、鸟氨酸脱羧酶,不能分解麦芽糖、乳糖、吲哚、枸橼酸盐、硫化氢、尿素、甲基红反应,与多杀性巴氏杆菌生化特性基本一致;分离菌株16S rRNA序列与GenBank中登录的多杀性巴氏杆菌的16S rRNA相似性达99.9%以上;分离菌对羧苄西林、哌拉西林、头孢唑林、头孢他啶、头孢曲松、头孢哌酮、庆大霉素等抗菌药敏感,对青霉素、苯唑西林、氨苄西林、头孢氨苄、头孢拉定、四环素、多西环素、红霉素等抗菌药耐药;致病性试验结果显示,在24 h内攻毒小鼠全部死亡,说明分离菌有较强的致病力。研究结果丰富了羊多杀性巴氏杆菌的流行病学资料,并为该病的防治提供了参考。

In order to determine the cause of mental depression,decreased appetite,reduced feed intake,dyspnea and even death of some goats in a large-scale dairy goat farm in Shaanxi province,this study took diseased tissues of sick goats as the research object.Bacterial isolation culture,biochemical test,16S rRNA PCR amplification and sequencing,drug sensitivity test,pathogenicity test and other methods were used to identify and analyze the isolated strains.The results showed that a Gram-negative,spore-free,no flagella and heavily stained short bacillus was isolated from the liver of the dead goats.It showed a thin,transparent and dewdrop-like colony on TSA medium,did not grow on MacConkey medium;On the blood agar plate,the pale,round,smooth,moist,opaque dew drops like colonies grew.The isolated strain could decompose glucose,sucrose,mannitol,sorbitol,thiase,ornithine decarboxylase,but could not decompose maltose,lactose,indole,citrate,hydrogen sulfide,urea,methyl red,which was basically consistent with Pasteurella multocida biochemical characteristics.The 16S rRNA sequence of isolated strain was more than 99.9%similar to that of Pasteurella multocida recorded in GenBank.The isolated bacteria were sensitive to carbobenzicillin,piperacillin,cefzolin,ceftazidime,ceftriaxone,cefoperazone,gentamicin,and resistant to penicillin,benzacillin,ampicillin,cefalexin,cefradine,tetracycline,doxycycline,erythromycin and other antibacterial drugs.The results of pathogenicity test showed that all mice died within 24 h after being challenged,indicating that the isolated bacteria had strong pathogenicity.The results of this study enrich the epidemiological data of Pasteurella multocida in goats,and lay a foundation for the prevention and control of the disease.