Comparative Research On Microbial Community Structure And Diversity Between Healthy Piglets And Diarrhea Piglets

Using fecal samples of healthy piglets and diarrhea piglets as research objects, microbial total DNA of the two groups the healthy group and the diarrhea group) was extracted by the best method according to our comparative results in this study. Moreover, Comparative researches of microbial community structure and diversity were undertaken by the method of PCR-DGGE, and changes of five microbial communities related to diarrhea were determined by Real-time fluorescence quantification.1. Fresh fecal samples were taken of weaning healthy piglets and diarrhea piglets at the same age. Eight methods, including directly enzyme cleavage method(DECM), indirectly enzyme cleavage method(IDECM), directly glass homogenizer method(DGHM), indirectly glass homogenizer method(IDGHM), directly grinding in liquid nitrogen method(DGLNM), indirectly grinding in liquid nitrogen method(IDGLNM), reported method(RM),E.Z.N.A.TM Stool DNA Kit method(EZNA), were used to extraction the microbial total DNA respectively and then compared them. Among the eight methods of extraction, the DNA yield was the highest when using DECM and the IDGHM obtained the lowest. Through the comparative analysis, lots of23kb or so the large fragment of complete genome DNA appeared when using RM, DGLNM, DECM and IDECM, while a large number of the small fragment DNA existed in almost all the samples. The fingerprint cluster analysis showed that the number of microflora species was between20～50with no significant differences among the eight methods, and the similarity was between53%～96%by quantity one. As that the trend showed that DGLNM> IDGLNM=DECM>IDECM=RM> DGHM>I DGHM> EZNA, DGLNM was eventually chosen as the most suitable method to extract the total DNA in this study.2. After analyzing the fingerprints of total DNA samples of both diarrhea group and healthy group by the method of PCR-DGGE, the special bands and the common bands were identified. At the same time, Enterobacteriaceae faimly,Clostridium cluster I,Bifidobacterium spp,Lactobacillus spp and Fusobacterium spp were detected by real-time fluorescence quantification, and significance testing was used to compare the differences of those microflora between each other. Most of the20～30flora showed in the PCR-DGGE fingerprints are the same of the two sample groups. The similarity was between50%～80%by quantity one, and those of extra-group showed the higher similarity than those of extra-group. In addition, when identifying part of the common bands and special bands,found the major common bands were normal intestinal flora,such as Lactobacillus rhamnosus,uncultured Firmicutes bacterium,uncultured Lachnospiraceae bacterium,Faecalibacterium prausnitzii, Phascolarctobacterium succinatutens sp. and so on.The special bands in the diarrhea group included Weissella paramesenteroides、Pseudobutyrivibrio ruminis and two unknown bacteria,which the special bands in the healthy group were Streptococcus. Compared with the healthy group, Lachnospiraceae bacterium and uncultured Firmicutes bacterium tended to decrease, Pseudobutyrivibrio ruminis tend to increase in the diarrhea group.Results of Real-time fluorescent quantitative detection showed that healthy groups had extremely significant higher numbers of Enterobacteriaceae family (P<0.01) and remarkablely lower numbers of Bifidobacterium spp (P<0.05), and no obvious changes were detected between the two groups of the numbers of Clostridium cluster Ⅰ,Lactobacillus spp and Fusobacterium spp(P>0.05)...