Protective Effect Of Calf Derived Lactic Acid Bacteria On Intestinal Inflammatory Injury Caused By Escherichia Coli In Mice

Purpose and background:Colibacillosisin calf,also known as calf pullorum,is a global acute infectious disease caused by Diarrheagenic Escherichia coli.The main clinical symptoms are calf diarrhea,Dehydration,acidosis,and death.At present,the abuse of antibiotics and antibiotic feed additives has led to the emergence of antibiotic-resistant bacteria,and the problem of antibiotic residues in meat has become increasingly serious,which poses a serious threat to human health.Lactic acid bacteria,a kind of probiotic bacteria,have an inhibitory effect on pathogenic bacteria in the intestine,which can maintain intestinal mucosal barrier function,regulate immune function,promote the metabolic absorption of nutrients,and have a good effect on intestinal diseases such as intestinal flora disorders,gastroenteritis,diarrhea.Therefore,the use of lactic acid bacteria microbial preparations to replace antibiotics has become an important way to safely and effectively prevent gastrointestinal diseases.The purpose of this study was to isolate and identify a calf-derived lactic acid bacteria that inhibits pathogenic bacteria and is tolerant to harsh environments,and explores its protective effect on inflammation and immune damage to the small intestine of mice caused by Escherichia coli.The application of bovine-derived lactic acid bacteria in the prevention of diarrheal diseases in actual breeding and production lays a theoretical foundation.Experiment 1:Isolation,identification and prebiotic characteristics of lactic acid bacteria from calvesThe isolates were identified by colony morphology,Gram stain morphology,16s r DNA gene sequence alignment and homology analysis.The results showed that a strain of Lactobacillus salivarius was successfully isolated from the feces of healthy calves,which was uploaded to NCBI and named WZ1.The probiotic properties of Lactobacillus salivarius WZ1 were explored through the determination of the growth curve of Lactobacillus salivarius WZ1,the determination of acid resistance,bile salt resistance,salt resistance and in vitro antibacterial test.The test results showed that Lactobacillus salivarius WZ1 grew well in MRS medium,reached the logarithmic phase in 6-8 h,and had a certain tolerance to p H 4,bile salt concentration 0.15%,and salt concentration 6%.And it has a good inhibitory effect on the growth of ETEC K88,Salmonella,Clostridium perfringens,Staphylococcus aureus and Aspergillus flavus.Experiment 2:Effects of Lactobacillus salivarius WZ1 on ETEC K88-induced intestinal structure and inflammatory injury in miceForty SPF Kunming mice were randomly divided into four groups:CON group(blank control group;n=10);L.S group(Lactobacillus salivarius WZ1 group;n=10);E.coli group(ETEC K88 group;n=10);L.S+E.coli group(Lactobacillus salivarius WZ1+ETEC K88 group;n=10).For the first 14 days,the L.S group and the L.S+E.coli group were intragastrically administered Lactobacillus salivarius WZ1 1×10~8 CFU/m L every day,and on the 15th day,the E.coli group and the L.S+E.coli group were intragastrically administered ETEC K88 1×10~9CFU/m L,sacrificed after 24 h.By HE staining in mouse jejunum,the ratio of villus length to crypt depth(V/C)was measured and calculated;the m RNA expression levels of TNF-α,IL-1β,IL-6 and IL-4 in mouse jejunum were detected;The expression of TLR4,NF-κB,My D88 and intestinal epithelial tight junction-related proteins Occludin,ZO-1 and Claudin-1 in mouse jejunum to evaluate the inflammatory injury effect of ETEC K88 on mouse jejunum and Lactobacillus salivarius WZ1 protective effect.The results showed that due to the stimulation of ETEC K88,the intestinal villi were disordered and had obvious breakage,and the crypt depth was greatly increased,resulting in a decrease in V/C(P<0.01).Lactobacillus salivarius WZ1can effectively prevent ETEC K88 Structural damage to mouse jejunum.In addition,infection with ETEC K88 also increased the m RNA expression of TNF-α,IL-1βand IL-6 in mouse jejunum(P<0.01),and decreased the m RNA expression of IL-4(P<0.01);The protein expressions of TLR4,NF-κB and My D88 in mouse jejunum were increased(P<0.01),and the expressions of intestinal epithelial tight junction-related proteins Occludin,ZO-1 and Claudin-1 were decreased(P<0.01),while the L.S+E.coli group showed the opposite trend.Infection with ETEC K88 also altered the intestinal flora,resulting in a very significant increase in the relative abundance of harmful bacteria such as Ralstonia and Helicobacter(P<0.01),and a significant decrease in the abundance of Lachnospiraceae.However,Lactobacillus salivarius WZ1 increased the abundance of beneficial bacteria genera such as Lactobacillus and Bifidobacterium in the gut,thereby better protecting the gut environment.Conclusion:A calf-derived Lactobacillus salivarius WZ1 was successfully isolated that was resistant to acid,bile salts and salt,and had good inhibitory effects on ETEC K88,Salmonella,Clostridium perfringens,Staphylococcus aureus and Aspergillus flavus.Lactobacillus salivarius WZ1 inhibits ETEC K88-induced intestinal inflammatory injury in mice by regulating gut microbiota and TLR4/NF-κB/My D88 pathway.