1株鸡毒支原体的分离鉴定及体外敏感中药的筛选

Isolation and Identification of One Strain of Mycoplasma gallisepticum and Screening of Sensitive Traditional Chinese Medicine in vitro

本研究旨在对山东泰安地区鸡毒支原体的流行菌株进行分离培养及鉴定和纯化,并针对该分离株筛选体外敏感的中药,为后续进一步研究提供材料。采用液体培养法与固体培养法从病鸡组织样品中分离、培养和纯化鸡毒支原体,并通过瑞士染色和姬姆萨染色、血清学方法及分子生物学方法对分离株进行初步鉴定,在此基础上,采用微量稀释法测定8种中药对分离菌株的最小抑菌浓度(MIC),从而筛选到对分离菌株敏感的中药。结果显示,分离菌株在鸡毒支原体液体培养基中增殖后,培养基颜色由红变黄且呈半透明状态,在固体培养基中培养后呈典型的“煎蛋”样儿菌落,均符合鸡毒支原体培养特性;瑞士染色和姬姆萨染色结果显示,菌体形态符合鸡毒支原体的特征;血清学鉴定结果显示,分离株与鸡毒支原体阳性血清发生凝集;分子生物学鉴定结果显示,所扩增的核酸序列与鸡毒支原体匹配度高达99%;MIC测定结果显示,中药黄连和黄柏对分离的鸡毒支原体的抑菌活性较强,属敏感范畴,其MIC分别为≤0.98和0.39 mg/mL,而中药金荞麦和鱼腥草对鸡毒支原体中度敏感,MIC均≥125 mg/mL,板蓝根、白鲜皮、当归、艾叶对鸡毒支原体均不敏感。综上所述,本研究成功分离到1株鸡毒支原体,并且筛选出4种对该菌株敏感的中药,分别为黄连、黄柏、金荞麦和鱼腥草,为后期进一步研究防治鸡毒支原体病的中药组方时提供参考依据。

The study was aimed to isolate,cultivate,identify and purify the prevalent strains of Mycoplasma gallisepticum in Tai’an,Shandong,and to screen sensitive traditional Chinese medicines against the isolates in order to provide materials for further research.The methods of liquid culture and solid culture were used to isolate,culture and purify the Mycoplasma gallisepticum strain from the tissue samples of diseased chickens,the isolate was preliminarily identified by Swiss staining and Giemsa staining,serological methods and molecular biological methods.On this basis,the micro-dilution method was used to determine the minimum inhibitory concentration(MIC)of the eight Chinese medicines against the isolated strains,in order to screen the Chinese medicines sensitive to the isolated strains.The results showed that after the isolated strains were proliferated in the liquid medium of Mycoplasma gallisepticum,the color of the medium changed from red to yellow and was translucent,after cultured in the solid medium,it showed a typical‘fried egg’-like colony,which were consistent with the culture characteristics of Mycoplasma gallisepticum.The Swiss staining and Giemsa staining results indicated that the morphology of the bacteria was consistent with the characteristics of Mycoplasma gallisepticum.The serological identification results showed that the isolate had agglutinated with the positive serum of Mycoplasma gallisepticum.The molecular biology identification results showed the matching degree of the amplified nucleic acid sequence with Mycoplasma gallisepticum was as high as 99%.The result of MIC showed that the antibacterial activity of traditional Chinese medicines Coptis chinensis and Corcagium were sensitive to the isolated Mycoplasma gallisepticum,the MIC were≤0.98 and 0.39 mg/mL,respectively.While the traditional Chinese medicine Fagopyrum dibotrys(D.Don)Hara and Houttuynia aucuparia were moderately sensitive to Mycoplasma gallisepticum,with MIC≥125 mg/mL.Isatidis Radix,Nova cortices album,Angelica sinensis and Et persequebantur Josue were not sensitive to Mycoplasma gallisepticum.In conclusion,a Mycoplasma gallisepticum strain was successfully isolated in this study,and screened out four kinds of sensitive traditional Chinese medicines to this strain,which were Coptis chinesis,Corcagium,Fagopyrum dibotrys(D.Don)Hara and Houttuynia aucuparia,respectively,it provided a reference basis for further study of traditional Chinese medicine prescriptions of prevention and treatment of Mycoplasma gallisepticum.