| The interaction between the gut microbiota and the host plays a key role in the development of intestinal diseases.Lactic acid bacteria is one of the main probiotic bacteria in the intestinal flora.It has the functions of optimizing the structure of the intestinal microecological flora and regulating the intestinal microbial ecosystem.It is the main line of defense against intestinal pathogens.A comprehensive understanding of the diversity and function of chicken intestinal flora has an important guiding role in the development of intestinal bacterial resources and the prevention and control of pathogenic bacterial infections.Salmonella enteritidis infection is a common cause of gastroenteritis in humans,and its infection can cause gastrointestinal dysfunction and microecological disorders.It is an important public health problem worldwide.Lactic acid bacteria can effectively prevent bacterial infections of the intestinal tract,but its mechanism of action in the intestinal tract has not yet been fully clarified.Therefore,this study took chicken as the research object,analyzed the structure and function of chicken intestinal flora,explored the diversity and function of lactic acid bacteria in chicken intestine,and aimed to screen lactic acid bacteria with good characteristics and resistant to Salmonella enteritidis from healthy chicken intestines.From the perspective of lactic acid bacteria regulating broiler intestinal flora and host gene expression against pathogen infection,the selected excellent lactic acid bacteria were tested against S.enteritidis ATCC13076 infection function and its mechanism of action.Provided new ideas and research basis for regulating the stability of intestinal infection microecological flora through lactic acid bacteria,improving the disease resistance of broilers and reducing the use of antibiotics.The main research contents are as follows:1.Diversity and function analysis of chicken gut flora and screening of lactic acid bacteriaThis chapter used metagenomics sequencing to explore the structure and function of the cecal intestinal flora of free-range(FRS)and large-scale commercial farming(CRS)chickens in different breeding environments,as well as white feather broilers(BC)and Roman layer hen(LC)chickens chickens of different genotypes.The results of the study found that Bacteroidetes,Firmicutes and Proteobacteria are the main flora of cecum bacteria in FRS and CRS.Deferribacteres were found in FRS,but almost did not exist in CRS.In FRS,Parabacteroides,Prevotellaceae_Ga6A1,Unclassified Proteobacteria and unclassified Spirochaetaceae are more abundant,while in CRS,Faecalibacterium,Ruminococcaceae and Helicobacter are more abundant.The main functional annotations of pecies are replication,recombination and repair,energy generation and transformation,cell wall/membrane/envelop biogenesis and amino acid transport and metabolism related functions.Analysis of metagenomic data of different kinds of chicken caecal flora showed that the main bacterial groups of BC and LC caecal bacteria were Bacteroidetes,Firmicutes and Proteobacteria.In BC,Faecalibacterium,Ruminococcus,and Alisipes are more abundant,while in LH,Bacteroides and Rikenellaceae_RC9_gut_group are more abundant.The main functional annotations of species are replication,recombination and repair,energy generation and transformation,cell wall/membrane/envelop biogenesis and amino groups.These results indicated that differences in the genotypes of chickens of the same species can cause changes in the number of intestinal flora,but do not change the structural composition and main functional differences of intestinal flora.Analysis of the data of lactic acid bacteria in chicken intestines found that lactic acid bacteria in chicken intestines are mainly subordinate to the four genera of Lactobacillus,Pediococcus,Sharpea and unclassified_f__Lactobacillaceae.L.reuteri belongs to Lactobacillus,and its abundance accounts for 14.6% of lactic acid bacteria.Its functions are mainly involved in replication,recombination and repair,amino acid transport and metabolism,carbohydrate transport and metabolism,coenzyme transport and metabolism,energy production and transformation,and defense mechanisms.Combined with the method of pure microbial isolation and culture,a strain of Lactobacillus reuteri S5(L.reuteri S5)was successfully isolated from the intestines of broilers.It was grown in MRS medium for 20 h as the optimal inoculation time and produced at 24 h.The maximum amount of lactic acid is 1.42 mmol/L,which is well tolerated.The activity of T.AOC,GSH-PX and T-SOD in the cell-free fermentation supernatant of L.reuteri S5 strain is higher than that of the bacteria.Strong in vitro antibacterial activity,the dominant carbon source and nitrogen source of this strain are glucose and soybean meal.2.L.reuteri S5 whole genome sequence analysis and anti-S.enteritidis ATCC13076 effect study in vitroLactic acid bacteria are strain-specific,which is particularly important for comprehensive analysis of L.reuteri S5 strain information and probiotic functions.L.reuteri S5 wholegenome sequencing analysis results showed that the L.reuteri S5 whole genome contains 1 ring chromosome,the genome length is 2,962,865 bp,the average length of the gene is 905 bp,a total of 2,787 genes,59 t RNA genes,19 r RNA gene,30 other nc RNA genes.It has a complete primary metabolic pathway,encoding the main functional enzymes of the glycolysis pathway and the enzymes of the pentose phosphate pathway.Its genome contains enes encoding antioxidants,inorganic salt ions,and acid and bile salt resistance,confirming L.reuteri S5 is a probiotic strain with excellent probiotic characteristics.Related molecular experiment results showed that L.reuteri S5 inhibits the formation and movement of S.enteritidis ATCC1307 biofilm by destroying the adhesion and invasion of S.enteritidis ATCC13076,the expression of virulence and cell membrane integrity genes,destroying the bacterial structure,and inhibiting intracellular protein synthesis,which in turn inhibits or kills the growth of S.enteritidis ATCC13076.These results laid the foundation for the discovery of the probiotic properties of L.reuteri S5 and its anti-S.enteritidis ATCC13076 effect.3.Regulation effect of L.reuteri S5 intervention on intestinal flora of chickens against S.enteritidis ATCC13076 infectionThe probiotic strain L.reuteri S5 was selected to construct an animal model of S.enteritidis ATCC13076 infected broiler chickens,and the regulation effect of L.reuteri S5 intervention on the structure of intestinal microflora in chickens infected with S.enteritidis ATCC13076 was analyzed.The results of the study showed that L.reuteri S5 intervention regulated the intestinal flora of chickens against S.enteritidis ATCC13076 infection.The L.reuteri S5 strain can be stably colonized in various intestinal segments of chicken intestine,especially in the cecum intestine segment.The colonization of L.reuteri S5 strain will lower the p H value of the intestinal environment and inhibit the colonization of S.enteritidis ATCC13076 in the intestine.It can enhance the intestinal barrier and reduce the bacterial displacement of S.enteritidis ATCC13076.Colonization of L.reuteri S5 in the intestine will increase the number of chicken caecum microbial flora OUT,increase the relative abundance level of Lactobacillaceae,and reduce the relative abundance level of Enterobacteriaceae family.Based on the analysis of the distance between groups,the composition of the chicken intestinal microflora in the L.reuteri S5 intervention infection group(LS group)was closer to the control group(C group).These results proved that the selected excellent lactic acid bacteria L.reuteri S5 can regulate the intestinal flora against S.enteritidis ATCC13076 infection.4.Effect of L.reuteri S5 intervention on intestinal transcriptome of chickens against S.enteritidis ATCC13076 infectionUsing animal in vivo experiments combined with transcriptome sequencing technology,the effect of L.reuteri S5 intervention on the expression of intestinal genes in chickens infected with S.enteritidis ATCC13076 was analyzed.The results showed that L.reuteri S5 can promote chicken growth performance,promote the secretion and expression of chicken ut immunoglobulin SIg A,and also promote the expression of inflammatory factors TNF-a,IFN-γ and IL-6,reduce chicken intestine histopathological damage.Transcriptome sequencing data analysis results showed that L.reuteri S5 can promote the regulation of many biological processes and cell metabolism of chickens,and can participate in the regulation of cell adhesion molecules(CAMs),amino acid synthesis and metabolism,vitamin metabolism,fatty acid metabolism,and sterol hormones.The synthesis,etc.,thereby promoting the growth performance of chickens.The main immune-related signaling pathways that L.reuteri S5 regulates intestinal infection against S.enteritidis ATCC13076 infection are the intestinal immune network that produces Ig A,the RIG-I-like receptor signaling pathway,the NOD-like signaling pathway,and the cytoplasmic DNA sensing pathway.These results laid a foundation for the investigation of L.reuteri S5 against Salmonella enteritidis infection.In summary,a probiotic strain L.reuteri S5 with excellent probiotic characteristics was successfully screened from healthy chicken intestines,its mechanism of action against S.enteritidis ATCC13076 was elaborated,and lactic acid bacteria regulated broiler intestinal flora and host gene expression against pathogenic bacteria From the point of view of infection,it proves its anti-S.enteritidis ATCC13076 intestinal infection regulation effect,which lays a foundation for further research on the prevention and treatment of S.enteritidis ATCC13076 infection by lactic acid bacteria. |
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