Effects Of Bacillus Subtilis HH2 On Isolated Fecal Microbiota Of Giant Pandas And The Diversity Of Canine Intestinal Microbiota

The giant panda source Bacillus subtilis HH2?B.subtilisHH2?is a strain isolated and identified in the laboratory from the panda feces.It can secrete a variety of antibacterial substances.It has a good inhibitory effect and can balance the micro-ecology in the intestine;it can regulate its own decomposition ability and reduce the consumption of non-essential energy under different carbon sources;it can form spores in harsh environment and has strong resistance to temperature and acidity.B.subtilis HH2 exhibits good resistance to stress.In order to study whether the giant panda source B.subtilis HH2can be artificially fed and added as a probiotic of giant panda in the future.Conduct two experiments around the above issues:1.In order to investigate the effect of B.subtilis HH2 isolates on intestinal microbiota of sub-adult giant pandas,this research collected 8 fresh sub-adult giant panda faeces,and then equal mixing prepared into giant panda fecal medium.Afterwards,fecal medium assigned to the following groups:the experimental group added B.subtilis HH2(5.0×10⁸cfu mL^-11 mL),the control group added the same amount of PBS.Samples were obtained at 0h,24h,48h,72h,respectively,after cultivating in the shaking table at constant temperature?37??.DNA extraction,PCR amplification were implemented and then used16S rRNA for high-throughput sequencing analysis.The results showed that when the fecal medium of giant panda was cultured to 24hours,the intestinal microecological colony diversity of giant pandas was increased in B.subtilis HH2 group,no significant change at other times.In species abundance analysis,finding that at the level of phylum,Firmicutes and Proteobacteria of the experimental group were increased,and Gammaproteobacteria,Clostridia,Pseudomonadales were increased in the class level,afterwards Enterobacteriales,Aeromonadales,were increased at the level of order classification,then Enterobacteriaceae,Aeromonadaceae,were increased at the level of family classification.At the genus level,the relative abundance of Aeromonas,Pseudomonas and Sphingobacterium was higher than that of the control group.B.subtilis HH2 is able to enrich the dominant microbiota for transient action.2.To further explore the effects of giant panda source B.subtilis HH2 strains on related animal dogs of intestinal microbiota.11 beagle canines?3-4 months?were randomly divided into two groups?experimental group:6,control group:5?and were keep routine husbandry.Afterwards,the experimental group were treated with B.subtilis HH2(5.0×10⁸ cfu mL^-110 mL)via gavage,and as a control,the same volume of saline was administered until the end of the 14d.On day 15,all canines were received the same amount of Escherichia coli(2.0×10⁹ cfu mL^-1).Bloods and anus swabs were collected from each dog at 0h,24h,48h and 72h,respectively.Anus swabs were subjected to DNA extraction,PCR amplification,and high-throughput sequencing analysis using 16S rRNA.The results showed that based on 16S deep sequencing with Illumina MiSeq technology,finding that the diversity of intestinal microbiota increased in experimental group of 24h after received ETEC and then the change of other times tended to flatten.We analysed the species abundance of beagle canines in experimental group at 24h,finding that Firmicutes,Proteobacteria and Bacteroidetes were dominant species at the phylum level;and at the class level,the relative abundance of proteus and Bacteroides was dominant,and the dominant microbiota tended to be abundant.At the 24h at the order level,the relative abundance of Aeromonadales and Enterobacteriales was higher in the control group.At 48h,the abundance of Bacteroidia and Clostridia was higher than that of the control group,and the abundance of Campylobacterales and Aeromonadales was lower than that of the control group.At the family level,the relative abundance of Helicobacteraceae was higher than that of the control group,the abundance of Peptostreptococcaceae was lower than that of the control group.At the genus level,the relative abundance of Helicobacter and Lactobacillus was higher than that of the control group,the abundance of Peptostreptococcus was lower than that of the control group,and the pathogenic bacteria group in the experimental group showed a decreasing trend.In summary,preliminary investigation found that B.subtilis HH2 can effectively enrich the dominant microbiota?Firmicutes,Proteobacteria?of sub-adult giant pandas in vitro feces and canine intestinal tract,reduce the proliferation of conditional pathogens.It is the foundation for the giant panda source B.subtilis HH2 to prevent intestinal diseases,improve health and develop micro-ecological preparations for giant pandas.