1株羊源溶血性曼氏杆菌的分离鉴定、药敏试验及致病性研究

[目的] 对某养殖场由呼吸道症状引发的山羊死亡病例进行病原学诊断,并对分离到的病原进行药敏试验及致病性分析。[方法] 无菌条件下剖检并采集死亡羊只肺脏及气管组织,接种于TSA固体培养基,37 ℃培养22~24 h;挑取疑似菌落,纯化后进行革兰染色镜检和生化鉴定;对经表型鉴定的分离菌株进行16S rDNA PCR扩增、测序,并进行遗传进化树构建和同源性分析;采用微量肉汤稀释法对分离菌株进行药敏试验;利用动物攻毒试验确定分离菌株对羔羊的致病性。[结果] 从临床样本中分离到1株细菌,命名为DMQ-Y-Mh。纯化后的分离菌株镜检为革兰阴性短杆菌,经生化鉴定以及16S rDNA PCR扩增、测序、同源性分析,将分离菌株鉴定为溶血性曼氏杆菌（Mannheimia haemolytica）。分离菌株对环丙沙星、阿奇霉素、庆大霉素敏感,对左氧氟沙星、土霉素、氟苯尼考中度敏感,对替米考星、恩诺沙星、泰乐菌素耐药。攻毒羔羊于72 h内死亡,剖检眼观气管及肺脏支气管内有黏性分泌物;肺脏组织有深红色病灶,与正常肺脏组织界限明显,可再次从死亡羔羊肺脏及气管组织分离到溶血性曼氏杆菌。[结论] 溶血性曼氏杆菌是造成该养殖场发生羊只死亡的病原菌,药敏试验结果为指导临床用药提供了参考。

Isolation,Identification,Antimicrobial Susceptibility Test and Pathogenicity of a Mannheimia haemolytica Strain from Goat

[Objective] A goat death case caused by respiratory symptoms in a farm was etiologically diagnosed, and the antimicrobial susceptibility and pathogenicity of the isolated bacterial pathogen were characterized. [Method] The dead goat was dissected and the lung and trachea tissues were collected in sterile conditions. The clinical samples were incubated on TSA solid medium and cultured at 37 ℃ for 22-24 h. After purification, the suspected bacterial colonies were subjected to Gram staining, microscopic examination and biochemical identification. The phenotypically identified strain was molecularly comfirmed by 16S rDNA PCR amplification, sequencing, phylogenetic tree construction and homology analysis. The antimicrobial susceptibility test of the isolate was performed with broth micro-dilution method. The pathogenicity of the isolate to goat kid was determined by using an animal challenge test. [Result] A bacterial strain designated as DMQ-Y-Mh was isolated from the clinical samples. The isolate was observed as Gram-negative bacilli under microscope, and was identified as Mannheimia haemolytica following biochemical identification as well as 16S rDNA PCR amplification, sequencing and homology analysis. It was sensitive to ciprofloxacin, azithromycin and gentamicin, moderately sensitive to levofloxacin, oxytetracycline and florfenicol, and resistant to tilmicosin, enrofloxacin and tylosin. Within 72 h of the challenge with the isolate, the goat kid perished, and the trachea and lung bronchi contained viscous secretions. There were dark red lesions in the lung tissue, and the boundary with the normal lung tissues was obvious. Mannheimia haemolytica could be isolated from the lung and trachea tissues of the dead goat kid again. [Conclusion] Mannheimia haemolytica was the pathogenic agent that caused the death of goat in the farm. The results of the antimicrobial susceptibility test served as a guide for clinical medication.