4种抗原定量方法观察牛支原体培养特征

doi:10.16431/j.cnki.1671-7236.2016.10.018

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (10): 2634-2639.doi: 10.16431/j.cnki.1671-7236.2016.10.018

4种抗原定量方法观察牛支原体培养特征

王展慧, 赵萍, 陈胜利, 郝华芳, 秦明明, 王绍伟, 刘永生, 储岳峰

中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 农业部草食动物疫病重点开放实验室, 兰州 730046

收稿日期:2016-03-02 出版日期:2016-10-20 发布日期:2016-10-28
通讯作者: 赵萍, 储岳峰 E-mail:zhaoping73@126.com;chuyuefeng@caas.cn
作者简介:王展慧(1990-),女,河南安阳人,硕士,研究方向:动物传染病及其病原分子流行病学,E-mail:wangzhanhui2016@163.com
基金资助:
国家科技支撑计划（2015BAD12B02）；甘肃省科技支撑计划（1204NKCA071）；兰州市城关区科技计划（2012-2-1）

Observation of the Growth Trends of Mycoplasma bovis with Four Different Antigen Quantitation Methods

WANG Zhan-hui, ZHAO Ping, CHEN Sheng-li, HAO Hua-fang, QIN Ming-ming, WANG Shao-wei, LIU Yong-sheng, CHU Yue-feng

Key Laboratory of Epizootic Diseases of Grazing Agricultural Sciences, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2016-03-02 Online:2016-10-20 Published:2016-10-28

摘要/Abstract

摘要：

为探索在疫苗研制过程中牛支原体抗原收获时间及抗原定量替代方法，将牛支原体08M株接种于含10%马血清的Thiaucourt's培养基，在110 h内同时监测其颜色变化单位（color change units，CCU）、菌落形成单位（colony forming units，CFU）、菌体蛋白浓度和核酸含量的变化，绘制相应曲线。活菌计数结果（CCU和CFU）显示，牛支原体生长可分为明显的4期，10 h进入对数期，30 h进入稳定期，活菌数最高可达1.0×10⁸CCU/mL和7.7×10⁷ CFU/mL，75 h进入衰亡期；蛋白浓度从15 h开始迅速增长，至35 h蛋白浓度最高，为72.06 μg/mL，此后维持在58.38~70.65 μg/mL；核酸含量从15 h开始增长，至25 h后Ct值维持在15.32~17.84。结果表明，牛支原体蛋白含量在稳定期初期与活菌数具有良好的相关性。因此，在牛支原体灭活疫苗生产中，稳定期初期是最佳抗原收获时间，可用蛋白浓度法代替活菌计数法进行抗原定量。

关键词: 牛支原体; 抗原定量; 颜色变化单位; 菌落形成单位; 蛋白浓度; 核酸含量

Abstract:

To explore the antigen harvest time of Mycoplasma bovis (M.bovis) and the antigen quantitation alternative method,M.bovis 08M strain was inoculated in the Thiaucourt's medium.Four growth curve plottings were made by measuring color change units (CCU),colony forming units (CFU),protein concentration and nucleic acid levels.Both the results of CCU and CFU counts showed that the growth of M.bovis was divided into four phases,the logarithmic phase appeared after being cultrured 10 h,the stationary phase appeared at 30 h with the highest number of viable cells up to 1.0×10⁸ CCU/mL and 7.7×10⁷ CFU/mL,and the decline phase started at 75 h.The protein concentration of M.bovis increased rapidly from 15 to 35 h,reached 72.06 μg/mL at 35 h,then maintained at 58.38 to 70.65 μg/mL.The nucleic acid levels of M.bovis increased rapidly from 15 h,and the cycle threshold (Ct) values were maintained between 15.32 to 17.84 after 25 h.These results indicated that there was a good correlation between the protein concentration and viable count of M.bovis at the early stationary phase,which was the best time period to harvest antigen.The protein concentration determination could be an alternative method to quantify antigen contents of M.bovis when preparing inactivated M.bovis vaccine.

Key words: Mcoplasma bovis; antigen quantitative; color change units; colony forming units; protein concentration; nucleic acid contents

中图分类号:

S852.62

王展慧, 赵萍, 陈胜利, 郝华芳, 秦明明, 王绍伟, 刘永生, 储岳峰. 4种抗原定量方法观察牛支原体培养特征[J]. 《中国畜牧兽医》, 2016, 43(10): 2634-2639.

WANG Zhan-hui, ZHAO Ping, CHEN Sheng-li, HAO Hua-fang, QIN Ming-ming, WANG Shao-wei, LIU Yong-sheng, CHU Yue-feng. Observation of the Growth Trends of Mycoplasma bovis with Four Different Antigen Quantitation Methods[J]. , 2016, 43(10): 2634-2639.

参考文献

[1] Nicholas R A,Ayling R D.Mycoplasma bovis:Disease,diagnosis,and control[J].Research in Veterinary Science,2003,74(2):105-112.
[2] Caswell J L,Archambault M.Mycoplasma bovis pneumonia in cattle[J].Animal Health Research Reviews,2008,8(2):161-186.
[3] Maunsell F P,Woolums A R,Francoz D,et al.Mycoplasma bovis infections in cattle[J].Journal of Veterinary Internal Medicine,2011,25(4):772-783.
[4] Hale H H,Helmboldt C F,Plastridge W N,et al.Bovine mastitis caused by a Mycoplasma species[J].Cornell Veterinarian,1962,52(4):582-591.
[5] Nicholas R A,Baker S E,Ayling R D,et al.Mycoplasma infections in growing cattle[J].Cattle Practice,2000,8(2):115-118.
[6] Ball H J,Nicholas R A.Mycoplasma bovis-associated disease:Here,there and everywhere[J].The Veterinary Journal,2010,186(3):280-281.
[7] Blackburn P,Brooks C.Isolation of Mycoplasmas bovis from cattle in Northern Ireland from 1999 to 2005[J].Veterinary Record,2007,161(13):452-453.
[8] Gevaert D.The importance of Mycoplasma bovis in bovine respiratory disease[J].Tijdschr Diergeneeskd,2006,131(4):124-126.
[9] 陈嘉棣,黎济申,张赋平,等.上海乳牛霉形体的分离与鉴定[J].畜牧兽医学报,1983,14(4):277-282.
[10] 辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.
[11] 韩猛立,康立超,钟发刚,等.细菌性牛呼吸道疾病的研究进展[J].中国畜牧兽医,2013,40(5):165-172.
[12] 冉智光,谢建华,骆璐,等.我国部分地区牛支原体肺炎和关节炎的病原体诊断[J].中国预防兽医学报,2010,32(1):40-43.
[13] 姚永进,剡根强,王静梅,等.致犊牛肺炎和关节炎牛支原体新疆株的分离与鉴定[J].中国畜牧兽医,2011,38(12):76-79.
[14] Burki S,Frey J,Pilo P.Virulence,persistence and dissemination of Mycoplasma bovis[J].Veterinary Microbiology,2015,179(1-2):15-22.
[15] 毕丁仁,王桂枝.动物霉形体及研究方法[M].北京:中国农业出版社,1998.
[16] Rurangirwa F R,Mcguire T C,Kibor A,et al.An inactive vaccine for contagious caprine pleuropneumonia[J].Veterinary Record,1987,121(17):397-400.
[17] 丁敏.实时荧光PCR技术检测猪肺炎支原体研究[D].北京:中国农业科学院,2008.
[18] 邵倩,储岳峰,何生虎,等.牛支原体生长曲线的测定和培养基的筛选[J].湖北农业科学,2012,51(3):549-551.
[19] Rossetti B C,Frey J,Pilo P,et al.Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR[J].Molecular and Cellular Probes,2010,24(5):321-323.
[20] 石磊,龚瑞,尹争艳,等.肉牛传染性牛支原体肺炎流行的诊断[J].华中农业大学学报,2008,27(5):629-633.

4种抗原定量方法观察牛支原体培养特征

Observation of the Growth Trends of Mycoplasma bovis with Four Different Antigen Quantitation Methods

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