羊溶血性曼氏杆菌的分离鉴定及药敏试验

对陕西省关中某奶山羊养殖场呼吸道症状引发死亡的羔羊进行病原检测。无菌采集死亡羔羊肺脏组织接种50 mL/L绵羊血琼脂平板,分别置于恒温培养箱与厌氧培养箱中36℃±1℃培养24 h,厌氧培养平板无菌生长,恒温培养血平板上可见大量溶血的灰白色、半透明的圆形菌落,挑取单菌落纯化培养后对分离株进行染色镜检、生化鉴定、16S rRNA的PCR扩增和测序及遗传进化分析、血清型分型、药敏试验和小鼠致病性试验。结果显示,纯化后细菌在血琼脂平板上为圆形露珠样菌落;革兰氏染色镜检为阴性球杆菌;16S rRNA测序结果为溶血性曼氏杆菌,进一步分型为血清2型;分离株对头孢噻呋钠、多西环素等药物敏感,对氨苄西林、庆大霉素中度敏感,对青霉素、替米考星等药物耐药;感染小鼠于24 h内死亡,剖检可见胸腔黏连和化脓性肺炎,由死亡小鼠肺脏中可再次分离到溶血性曼氏杆菌。表明造成这次羔羊死亡的病原菌为溶血性曼氏杆菌,结果对羊溶血性曼氏杆菌的病原检测和临床防治提供了参考。

Isolation,Identification and Antimicrobial Susceptibility Test of Ovine Mannheimia haemolytica

The pathogen detection was conducted for lambs that died of respiratory symptoms in a Guanzhong dairy goat farm of Shaanxi province.Lung tissues of dead lambs were collected aseptically to inoculate 5%sheep blood agar plates and placed in a constant temperature incubator and an anaerobic incubator at 36±1℃respectively.After 24 hours of cultivation,the anaerobic culture plate grew aseptically,and a large number of hemolytic off white,translucent circular colonies can be seen on the constant temperature culture blood plate,a single colony was selected,the isolate was examined by staining microscopy,biochemical identification,16S rRNA gene PCR amplification and sequencing,genetic evolution analysis,serotyping,antimicrobial susceptibility test and mouse pathogenicity test.The results showed that the purified bacteria were round dewdrop-like colonies on the blood agar plate;Gram stained microscopy showed negative coccobacteria;the 16S rRNA gene sequencing result showed Mannheimia haemolytica,which was further classified as serotype 2;The antimicrobial susceptibility test showed that the isolates were sensitive to drugs such as ceftiofur sodium and doxycycline,moderately sensitive to ampicillin and gentamicin,and resistant to drugs such as penicillin and tilmicosin;After infecting the mice,the mice died within 24 hours.The necropsy showed thoracic adhesions and purulent pneumonia.The lungs of the dead mice could again obtain Mannheimia haemolytica.The test results indicated that the bacterium obtained from the lungs of dead lambs was Mannheimia haemolytica,which may be the pathogen causing the death of lambs.This test can provide important reference for the pathogenic research of Mannheimia haemolytica and the application of clinical prevention and treatment technology.