16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化

用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。

Tracking of the development of fecal bacterial community of diarrhea piglets by 16S rDNA techniques

PCR and denaturing gradient gel electrophoresis (DGGE) were used to track the development of fecal bacterial community of 5 diarrhea piglets. Clone libraries were created from complete 16S rDNA of 42-day fecal sample of piglet 1, 2 and 5. From the library, clones had their V6-V8 regions matched predominant bands on the DGGE gel were then sequenced and their 16S rDNAs subjected to an online similarity search. Results revealed that DGGE profiles from fecal samples of 5 piglets changed form simple to complex, and then returned simple to complex, finally tended to be stable and diverse. DGGE profiles from day 2and 16 fecal samples of 5 piglets were simplest, with high similarity (84%). Their most predominant bands were E. coil. Their Shannon index were 1.38 +/- 0.55 and 1.97 +/- 0.26 respectively. DGGE profiles from day 10 of 5 piglets became quite complex, and had quite low similarity (10%) with DGGE profiles from day 2and 16 fecal samples of 5 piglets. The Shannon index was 2.12 +/- 0.24. E. coil still existed, but it was not the most predominant band. There were significant differences of predominant bands between DGGE profiles from day 27 fecal samples and those from day 35 and 42 fecal samples, with 26.28% and 39.2% of similarity respectively. The predominant bands from day 35 and 42 fecal samples of 5 piglets were tended to be stable, with 55% of similarity. The Shannon index of day 27, day 35 and 42 fecal bacterial communities were 2.19 +/- 0.29, 2.20 +/- 0.17 and 2.14 +/- 0.20 respectively. The fecal bacterial communities of day 42 health piglets were mainly consisted of species of Enterococcus, Streptococcus, Clostridium, Peptostreptococcus and Lactobacillus.