Based On The Probiotics Regulation The Cyprinus Carpio Intestinal Microflora Flora Response Law Of The Pilot Study

Probiotics can be planted on the surface of the intestinal mucosa of fish, to activate the immune function, improve the intestinal flora balance, enhance immunity, inhibit pathogenic bacteria colonization plant and growth, and then prevent the occurrence of bacterial diseases. In this study, using activity origin Bacillus subtilis and Lactobacillus acidophilus as basic feed additives, Aeromonas hydrophila as indicator bacteria, the study on the response of intestinal microflora of fish was carried out based on probiotics, which is significant for the development of micro-ecological preparation of fish and prevention and control of bacterial diseases.Firstly, in order to research the influence of activity origin Bacillus subtilis and Lactobacillus acidophilus on intestinal flora of the Cyprinus carpio, we randomly divided the Cyprinus carpio into two groups, the first group was the test group, fed with basic feed of 0.5 per 1000 activity origin Bacillus subtilis and Lactobacillus acidophilus; The second group fed with basic feed as the control group. Intestinal contents was collected from the Cyprinus carpio of each group at 0d, 14 d, 28 d and 42 d, total DNA was extracted and conducted by PCR-DGGE. DGGE map was observed and conducted the matrix, system tree and strip identification analysis. 21 difference bands were obtained and analyzed by sequencing, indicated that they belonged to Proteobacteria, Actinobacteria, Firmicutes, Fusobacteria and Bacteroidetes. The highest proportion of them was Proteobacteria. The common bands sequencing result of each group was Proteobacteria, showed that Proteobacteria was the dominant bacteria in the intestinal flora of Cyprinus carpio. Secondly, in order to detemine the resistance of the Cyprinus carpio infected with Aeromonas hydrophila after added probiotic in the feed, the test group and control group were challenged after 42 d. Intestinal contents were collected after 96 h challenged, and conducted by PCR-DGGE. Compared challenged test group and challenged control group, Actinobacteria in test group significantly reduced, Proteobacteria was predominant, explained added probiotic in the feed could significantly improve the intestinal flora structure, which could promote the quantity increase of dominant bacteria in the intestinal tract, and then improve the resistance of the Cyprinus carpio infected with Aeromonas hydrophila.At the same time, using high throughput sequencing analysis, 159535 sequences with high quality were obtained, and classified into 17 gates at the phylum level, four bacteria doors accounted for 98.03% of total sequences, including Proteobacteria, 93.57%, Actinobacteria,2.99%, Firmicutes,1.32% and Fusobacteria, 0.15%. At the genus level, the test group and control group with the prolonged feeding were compared, Shewanella, Aeromonas and Leuconostoc significantly increased in the gut. It explained Bacillus subtilis and Lactobacillus acidophilus could promote the number of intestinal bacteria increase, and then enhance immunity.In this study PCR-DGGE combined with metagenomics were used and analyzed the influence of probiotics on intestinal flora of the Cyprinus carpio, confirmed intestinal microbial diversity of the Cyprinus carpio, found the inherently dominant bacteria of the Cyprinus carpio intestinal flora, and clarified the response rules base on the regulation of Bacillus subtilis and Lactobacillus acidophilus in the Cyprinus carpio intestinal microflora, which would provide the necessary theoretical basis and practical basis to the research and development of micro-ecological agents for fish, and new insights into the effective prevention and control of bacterial diseases in freshwater fish.