| Lactobacillus reuteri is a normal flora in the intestine of animals.It can produce bacteriocin to inhibit the growth of pathogenic bacteria in the intestine,and has the ability to adhere.It can be colonized in the intestine of animals and caused by intestinal tract.The bacteria compete for the adhesion site of the intestinal epithelial cells,which reduces the risk of the animal being attacked by intestinal pathogenic bacteria and is often used as a feed additive.This study intends to isolate Lactobacillus reuteri in the intestines of healthy piglets,and to screen out the strains with excellent performance by measuring the biological characteristics of the strains,to study its effects on the growth of newborn piglets and the anti-F4+enterotoxin-producing Escherichia coli(Enterotoxigenic Escherichia coli,ETEC)analysis of the effect of infection,laying the foundation for further application of Lactobacillus reuteri in production.In this experiment,12 strains of Lactobacillus reuteri were isolated from the intestines of healthy piglets of three months old,including 3 strains from the jejunum,7 strains of the cecum and 2 strains of the colon.It indicates that the Lactobacillus reuteri is widely distributed in the intestinal tract of piglets and can be colonized in the small intestine and large intestine.The growth curve and acid production capacity of 12 strains of Lactobacillus reuteri showed that 12 strains were slow in 0-2h after inoculation,logarithmic growth period in 4-10 h,and plateau in 12-20 h.The recession began to appear in 22-24 h.The 12 strains were able to reduce the initial pH of the culture solution from 5.75 to 4.0 after 24 h of culture.Resistance to acid and bile salts,gastrointestinal transport capacity,high temperature tolerance and antioxidant capacity of Lactobacillus reuteri were screened for excellent performance of Lactobacillus reuteri as CE2,CE3,CE8 and C021 Strain.In the in vitro adhesion experiment,C021 strain had the highest adhesion rate to Caco-2 cells and the highest extracellular polysaccharide yield.Antibacterial experiments showed that 12 strains had higher growth of F4+ETEC,Salmonella typhimurium and Staphylococcus aureus.Good inhibitory effect,and only CE2 strain and CE8 strain have inhibitory effect on Salmonella pullorum;drug sensitivity test showed that 12 strains of Lactobacillus reuteri mostly to erythromycin,clarithromycin,clindamycin,and chloramphenicol The sensitivity of the pigment and penicillin G was the highest.The surface hydrophobicity of CE1 strain was the highest,but the C021 strain was slightly lower than the surface hydrophobicity of the other strains The C021 strain had the strongest Auto-aggregation ability,indicating that it may have better adhesion properties.Based on the above experimental results,four strains of Lactobacillus reuteri with excellent performance were selected as CE2,CE3,CE8 and C021 strains,and the CO21 strain was selected for subsequent experimental research.The Lactobacillus reuteri C021 strain was selected for its adhesion and colonization ability in piglets,and the in vivo colonization experiment of newborn piglets was carried out to determine the colonization effect of CFDA-SE labeled Lactobacillus reuteri C021 after feeding piglets.The colonization rate of each intestine was detected by flow cytometry on the 4th,7th,10th and 14th day.The results showed that the intestines of the piglets could be colonized on the 4th day after feeding,and the duodenum in piglets could be detected on the 7th to 10th day.The colonization of jejunum,ileum and cecum is better.On the 14th day,there is still strong colonization ability in the small intestine,and the colonization effect is best in the jejunum segment.In order to explore the protective effect of Lactobacillus reuteri CO21 strain on F4+ETEC infected piglets,24 newborn 3 d old piglets were divided into normal control group and Lactobacillus reuteri + ETEC infection group(feeding group).And did not feed the Lactobacillus reuteri + ETEC infection group(infected group),the feeding group piglets were fed Lactobacillus reuteri(5×109 CFU/mL,2 mL/piglet/day),the other two groups were daily The same amount of normal saline.After 7 days of continuous gavage,the infected group and the feeding group were orally infected with F4+ETEC(1×108 CFU/mL,4 mL/piglet/day)for 3 days.The body weight of each group before and after infection and the diarrhea of the piglets were observed.To analyze the intestinal intestine villi and crypt depth of each group of piglets after infection,the main microbial flora in the cecal and colon contents of piglets,and the number of F4+ETEC in each intestine,the immune organs(thymus and spleen)index of piglets and serum And the expression of immune-related molecules in intestinal mucus.The results showed that feeding newborn piglets before infection with Lactobacillus reuteri significantly increased daily gain.After piglets infected with F4+ETEC,the feeding group significantly reduced the diarrhea rate of F4?ETEC-infected piglets(from 84.5%to 55.2%)compared with the infected group;significantly increased piglet thymus and spleen index;significantly increased The ratio of jejunum and ileal villus length/crypt depth in piglets indicates that feeding Lactobacillus reuteri can promote the development of immune organs in piglets and protect the intestinal environment of jejunum and ileum of F4+ETEC infected piglets.The feeding group significantly up-regulated the number of Lactobacilli and Bifidobacteria in the cecum and colon,and reduced the total number of E.coli in the cecum and colon,but the difference was not significant,which significantly reduced the number of F4+ETEC in each intestine.The detection of intestinal mucus and serum related indicators in infected piglets showed that the thymus and spleen index of the immune organs in the feeding group increased significantly compared with the infected group;the serum IgG was significantly up-regulated and the duodenum,jejunum and ileum were significantly up-regulated.The content of sIgA in mucus;inflammatory factors in serum and intestinal mucosa were detected by ELISA and real-time fluorescent quantitative PCR.The results showed that the feeding group significantly increased the anti-inflammatory factor IL-10 and down-regulated the pro-inflammatory factors IL-1?,IL.-6 and IL-12;mRNA expression levels of tight junction proteins Claudin-1,Occludin and Zo-1 were significantly up-regulated in duodenum,jejunum and ileum;TLR4,NF-?B and MyD88 in duodenum There was no significant difference in gene expression.Compared with the infected group,the feeding group could significantly down-regulate the mRNA expression levels of TLR4,NF-?B and MyD88 in jejunum and ileum of piglets.This study showed that feeding newborn piglets with Lactobacillus reuteri C021 can promote the growth of piglets and the development of immune organs,regulate intestinal flora,reduce the infection of piglets,increase the content of immunoglobulin,and enhance the intestinal barrier function.It has a certain protective effect on piglets infected with F4+ETEC.In summary,12 strains of Lactobacillus reuteri were isolated from the intestines of healthy piglets.The C021 strain with excellent probiotic and stress resistance was selected for research.It was found that the strain has strong colonization ability in piglets and can promote the growth and development of piglets,protection against F4+ETEC infection.It indicates that the isolated Lactobacillus reuteri has certain application prospects as a candidate strain for probiotics in piglet production. |
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