Analysis Of The Diversity Of Bacteria Communities Along The Gastrointestinal Tract Of Adult Healthy Sheep

To investigate the diversity of bacteria communities and the number of butyric acid producing bacteria along gastrointestinal tract, five adult healthy Chinese Mongolian sheep were used in this study. All fresh samples collected from stomach (rumen, reticulum, omasum, and abomasum), small intestine (duodenum, jejunum, and ileum) and large intestine (cecum, colon and rectum) were analyzed by PCR-DGGE, real-time PCR and Illumina MiSeq. Results were as follows:1. PCR-DGGE analysis indicated that there are rich kinds and large amount of bacteria in the gastrointestinal tract of sheep. Cluster analysis and principal constituent analysis (PCA) showed samples from stomach, small intestine and large intestine clustered together, respectively. The stomach and large intestine had a higher bacteria diversity with the average number of 22.55 and 27.80 bands, respectively. The Shannon diversity index, evenness and richness of samples from abomasum, cecum, colon and rectum were higher, the abomasum were 3.19,0.82, and 25.20; the cecum were 3.27,0.84, and 26.40; the colon were 3.28,0.84, and 26.80; the rectum were 3.40,0.87, and 30.2. While the bacteria diversity of small intestine were lower with the average number of 16.20 bands, and the Shannon diversity index, evenness and richness of jejunum were lowest, which were 2.27, 0.58, and 10, respectively. In addition, eleven sequenced bands were belonged to Bacteroidetes and Firmicutes, which were uncultured Bacteroidetes bacterium, uncultured rumen bacterium, uncultured bacterium, uncultured Veillonella bacterium, Acinetobacter, and Lactococcus piscium.2. Real-time PCR demonstrated that a large number of butyric acid producing bacteria were detected along gastrointestinal tract of sheep, and the number of which were higher in the stomach and large intestine compared to small intestine. In addition, Firmicutes, Enterococcus, Lactobacillus,and Bifidobacterium were differences along the GIT (P<0.05). While Bacteroidetes, Clostridium cluster XIVa, Clostridium cluster IV, Faecalibacterium prausnitzii, Megasphaera elsdenii, and Butyrivibrio fibrisolvens had no differences along the GIT (P>0.05). And the number of Firmicutes was significant differences between duodenum and jejunum (P<0.01). The number of Enterococcus and Bifidobacterium was significant differences between abomasum and ileum (P<0.01). However, the number of Lactobacillus was significant differences in ileum (P<0.01)3. Illumina MiSeq revealed that 557 656 reads were generated with an average of 933 reads per sample, and the total number of operational taxonomic units(OTUs) detected reached 15933,with an average 464 OTUs per sample. In addition, mean length of the pyrosequencing reads was 252. There were eleven phyla detected in the GIT with Firmicutes(44.37%), bacteroidetes(38.73%),Proteobacteria(4.22%), Spirochaeates(3.36%), Euryarchaeota(1.84%), Actinobacteria(1.60%), Verrucomicrobia(1.57%), Lentisphaerae (1.29%), Unclassified(1.23%), Tenericutes (1.08%), and Fibrobacteres (0.58%). The Firmicutes and bacteroidetes were dominant in each sample. And Fibrobacteres were not detected in the sample from ileum. Cluster analysis and principal constituent analysis (PCA) of OTUs showed samples from stomach (rumen, reticulum, omasum, and abomasum), small intestine (duodenum, and jejunum), and large intestine (ileum, cecum, colon, and rectum) clustered together, respectively. Gap analysis of between-group showed that the bacteria in the rumen and abomasum, omasum and abomasum, abomasum and jejunum, jejunum and ileum, jejunum and rectum, ileum and rectum were differences along the GIT (P<0.05). However, a large range of alpha diversity index was detected, with species observed index of a range from 157 to 749, Chao index of a range from 190.44 to 882.38, Ace index of a range from 200.28 to 863.39, Shannon index of a range from 2.55 to 5.33, and Simpson index of a range from 0.01 to 0.27. This revealed that the bacteria were differences in the GIT, and the alpha diversity were higher in the stomach and large intestine, compared to small intestine.Conclusions:1) There are rich kinds and large amount of bacteria in the gastrointestinal tract of sheep, with the dominant phyla of Firmicutes and bacteroidetes. With rearward moving of position of intestinal tract, the diversity increased firstly and then decreased and finally increased.2) The similarity of flora in the stomach and large intestine was high, and the bacteria in the rumen and abomasum, omasum and abomasum, abomasum and jejunum, jejunum and ileum, jejunum and rectum, ileum and rectum were differences along the GIT (P<0.05).3) The butyric acid producing bacteria of Enterococcus, Lactobacillus, and Bifidobacterium were differences along the GIT (P<0.05).