奶牛隐性乳房炎致病性大肠杆菌AI-2信号分子检测

Detection AI-2 of pathogenic Escherichia coli isolated from bovine subclinical mastitis

为了探索奶牛隐性乳房炎大肠杆菌不同生长时期及不同培养条件下,AI-2信号分子的产生与lux S和pfs转录水平间的相互关系。应用哈维弧菌BB170,检测奶牛隐性乳房炎致病性大肠杆菌不同分离株、不同生长期及不同培养条件下AI-2的活性,应用实时荧光定量PCR检测其AI-2产生水平与lux S基因与pfs基因转录的相关性。结果表明,E285、E80814、W41、E30707和EA701205株均能产生AI-2分子,但产生的AI-2均低于阳性菌BB152; E285在迟缓期、对数前期基本没有AI-2的表达,从对数中期开始AI-2开始大量表达,到对数后期达到顶峰,是阴性对照的12倍,随后下降,到稳定期已下降为阴性对照的3. 6倍。当培养基中添加蔗糖、麦芽糖、葡萄糖、甘露醇、Na Cl及乳糖时,AI-2的活性均明显提高。实时荧光定量PCR结果显示,E285在不同生长期和不同培养条件时,其AI-2活性的高低与lux S基因转录水平一致,但与pfs基因的转录没有明显关系。提示奶牛隐性乳房炎致病性大肠杆菌分离株均能产生AI-2分子,其AI-2活性与lux S基因转录水平具有高度的相关性,与pfs基因的转录水平无明显相关性,蔗糖、麦芽糖、葡萄糖、甘露醇、Na Cl及乳糖可促进AI-2合成。

In this study, the relationship between the transcription level of luxS gene or pfs gene and AI-2 production of E. coliisolated from subclinical mastitis in different growth phases and culture conditions were detected. The AI-2 of pathogenic E. coli iso-lated from subclinical mastitis in different growth phases and culture conditions was analyzed by the V. harveyi bioluminescence as-say. And the mRNA of luxS gene and pfs gene were determined simultaneously with real-time quantitative PCR. The result showedthat E285, E80814, W41, E30707 ,and EA701205 all produced the AI-2,but the amount of AI-2 of each of them was lower thanthat of positive control. The AI-2 activity of E285 had no expression in the lag phase and the pre-log phase,and it began to expressfrom themid-log phase and reached the peak at the post log phase, which was 12 times of that of the negative control. The AI-2 wasthat of 3. 6 times of that of the negative control at slationary phase of E258. When the culture medium added with sucrose, maltose,glucose, mannitol, NaCl and lactose, the level of AI-2 was significantly increased. The real-time quantitativ PCR results indicatedthat the activity of AI-2 was consistent with mRNA of luxS gene in different growth phases and different culture conditions. However,there was no significant correlation between the activity of AI-2 and the transcription level of pfs mRNA. Sucrose, maltose, glucose,mannitol, NaCl e and lactos can improve the synthesis of AI-2.