新疆石河子地区牛支原体的分离鉴定及药物敏感性分析

【目的】确定引起新疆石河子地区集约化牛场常发性肺炎的主要病原同时进行病原的体外药物敏感性分析。【方法】采集有典型咳嗽、流涕症状的牛鼻拭子10份和病死牛肺脏组织1份，用牛支原体特异性引物进行PCR检测，将检测为阳性的样本进行病原培养纯化，对纯化后的分离株菌落进行形态学观察、Dienes染色、生化试验及16S rRNA测序和进化分析，通过测定颜色变化单位(CCU)测定分离株生长曲线，并对分离株进行药物敏感性试验。【结果】PCR结果显示，10份鼻拭子中检测出7份牛支原体阳性样本，1份病死牛肺脏组织也检测为阳性；在涂有肺脏组织研磨液培养液的PPLO固体培养基上长出针尖状的菌落，纯化后分离株菌落形态为典型的煎蛋状；Dienes染色可见明显的深蓝色中心脐；生化试验结果显示，分离株不水解明胶、精氨酸、七叶苷，不发酵乳糖、葡萄糖和甘露醇，不分解尿素，可还原氯化三苯基四氮唑；16S rRNA测序结果显示，分离株与牛支原体国际标准株PG45相似性为99.7%,与国内牛支原体地方流行株XBY01、Ningxia-1、NM2012、Tibet-10的相似性最高，均为99.9%;生长曲线测定结果显示，分离株在培...

Isolation,Identification and Drug Sensitivity Analysis of Mycoplasma bovis in Shihezi Region of Xinjiang

【Objective】 The purpose of the test was to identify the primary pathogen causing recurrent pneumonia in intensive cattle farms in Shihezi region of Xinjiang,and in vitro drug sensitivity analysis of this pathogen was performed simultaneously.【Method】 Nasal swabs from 10 cattle with suspected symptoms and 1 lung tissue from dead cattle were collected and tested by PCR using primers specific for Mycoplasma bovis (M.bovis),and the positive samples were cultured and purified.Morphological observation,Dienes staining,biochemical test,16S rRNA sequencing and evolutionary analysis were carried out on the colony of the purified isolate.The growth curve of the isolate was determined by measuring the color change unit (CCU),and drug sensitivity test was carried out on the isolate.【Result】 The PCR results indicated that 7 M.bovis positive samples were detected in the 10 nasal swabs,and 1 of the lung of dead cattle collected was also positive.Needle like colonies grew on PPLO solid medium coated with lung tissue grinding fluid culture medium,after purification,the colony morphology of the isolates was typical fried egg like.A distinct dark blue central umbilicus was visible with Danies’ staining.Biochemical experiments showed that the isolate did not hydrolyze gelatin,arginine,esculin,nonfermentable lactose,glucose and mannitol,did not break down urea,and could reduce triphenyltetrazolium chloride.16S rRNA sequencing showed that the isolate shared 99.7% identity with the M.bovis international standard strain PG45 and 99.9% identity with the domestic M.bovis endemic strains XBY01,Ningxia-1,NM2012 and Tibet-10.The results of growth curve measurement showed that the isolate entered the logarithmic growth period after 6 h in the medium,reached the peak at 54 h and entered the stable period,and entered the decline period after 72 h.Susceptibility test revealed that the isolate was sensitive to nitrofurantoin,tetracycline and doxycycline,gentamycin and kanamycin,and moderately sensitive to norfloxacin and ofloxacin,whereas resistance developed to ciprofloxacin,lomefloxacin,azithromycin and erythromycin.【Conclusion】 In this study,a strain of M.bovis isolate was successfully identified from lung tissue,and 54 h was the optimal harvest time for this isolate,which was less different from most of the endemic strains of M.bovis in China,the overall genetic evolution was relatively stable,and there was certain drug resistance.The result of this study provided a reference for local prevention and control of M.bovis,and also laid a foundation for the research on the pathogenic mechanism of M.bovis and the development of vaccines.