| Probiotics are safe feed additives that can replace antibiotics.Adding probiotics to fodder can prevent diseases and promote growth performance.Lactobacillus rhamnosus is one of the most widely studied probiotics that has been widely used to prevent and treat diarrhea,dental caries and other diseases in humans.This study proved that Lactobacillus rhamnosus GG(LGG)could tolerate the physical and chemical environment of the gastrointestinal tract,inhibit the growth of some pathogenic bacteria,colonize in the intestines and inhibit the adhesion of Escherichia coli(E.coli)to epithelial cells in vitro.The feeding experimental was carried out with broilers,the results showed that dairy with LGG could improve the growth performance,serum immunoglobulin,intestinal homeostasis and immune response of chickens,as well as its antibacterial infection.It confirmed that LGG has good application value as a fodder additive which can reduce the use of antibiotics.The main contents of the experiment are as follows:1.Assessment for probiotic properties of Lactobacillus rhamnosus in vitroThe survival rate of LGG cultured in artificial gastric acid and intestinal fluid for 2 h were both 100%,and the survival rate of LGG cultured in 0.3%bile salt for 24h was 76.5%.The adhesion of LGG to Caco-2 cells was 19%,indicating that LGG could tolerate the intestinal physicochemical environment and colonize in the intestinal tract LGG could reduce the adhesion of E.coli by 65.4%by competitive inhibition.LGG can reduce the adhesion of E.coli to Caco-2 cells by 26.3%and 34.6%through inhibition and displacement of E.coliIn this study,the antimicrobial activity of LGG lysate,LGG supernate,LGG culture and LGG cells against Escherichia coli,Staphylococcus aureus,Salmonella and Salmonella enteritis was determined.The results showed that LGG lysate,LGG supernate and LGG culture had strong inhibitory effects on the four pathogens,and the diameter of inhibition zone reached more than 12 mm.However,the LGG cells showed no bacteriostatic effect.It has been proved that LGG can tolerate the physical and chemical environment of the gastrointestinal tract,colonize the intestinal tract and inhibit the adhesion of E.coli to epithelial cells and the growth of some pathogen in vitro experiments.2.The effect of Lactobacillus rhamnosus on growth performance,immunoglobulin and intestinal health was evaluated in vivo80 1-day-old Ross 308 broilers were randomly divided into two groups with 40 chickens in each group.The chickens in trial group were fed with 108 CFU LGG every day for 3 weeks.At the second week,the average daily weight gain of the chickens in trial group was 19±2.6 g,which was significantly higher than that of the controls(P<0.05).On the 21st day of feeding,the body weight of chickens in the trial group was 423.7±3.6 g,which was significantly higher than that of the controls(P<0.05).The concentrations of serum IgA,IgG and IgM of chickens in LGG group were 84.4±12.4 g/mL,726.7±80.1 g/mL and 58.1±5.9 g/mL respectively at 3 weeks(P<0.05).Fed with LGG for 3 weeks,the duodenum,jejunum and ileum of chickens were collected for making tissue sections.The length of intestinal villus and the ratio of intestinal villus to crypt of the chickens in LGG group were higher than those of the controls,and the intestinal crypt was shorter than those of the control group(P<0.05).After 3 weeks of feeding with LGG,the cecum contents of the chickens were collected for 16S rRNA sequencing.The number of OTU of the chickens in the LGG group was 170,which was nine more than that of the controls,The increase of bacterial diversity could improve the immunity of body and promote the ability of absorbing nutrients.The relative abundance of Ruminococcaceae in the cecum of the chickens in LGG group was higher than that of the controls(P<0.05),and the relative abundance of Lactobacillaceae in the cecum of the chickens in LGG group was significantly higher than that of the controls(P<0.05).Ruminococcaceae can promote the ability of absorbing cellulose in fodder and Lactobacillus can produce antibacterial substances and digestive enzymes.In summary,feeding with LGG could improve the growth performance,the concentration of serum immunoglobulin,the diversity of intestinal flora and the relative abundance of some beneficial bacteria.To explore the influence of LGG on innate immunity of chickens,the expression levels of immune-related genes in spleen,liver and cecum were detected by qPCR after 3 weeks of feeding with LGG.The expressions of MHC ?-?,MyD88,and NF-?B in spleen of the chickens in LGG group increased 5.9-fold,2.6-fold and 2.3-fold respectively(P<0.05).The expressions of the downstream pro-inflammatory factors IL-6 and IL-8 in the spleen were up-regulated by 6.7-fold and 5.1-fold(P<0.05).After 3 weeks of feeding with LGG,the chickens were challenged with E.coli.The survival rate of chickens in LGG group was 66.7%,which was higher than that of the controls(16.7%)The number of E.coli in chickens' tissues was detected at 1dpi(days post infection)and 3dpi.The number of E coli in heart,lung and kidney of chickens in LGG group was significantly lower than that of the controls(P<0.05).At 1dpi and 3dpi,the expressions of TLR4 were up-regulated by 11.8-fold and 15.5-fold respectively,and the expressions of downstream immune-related factors were significantly up-regulated in kidney(P<0.05).The above results suggest that LGG can stimulate the expression of TLR4 in the body after infection with E.coli,which would induce the expressions of downstream pro-inflammatory factors.In summary,feeding LGG can improve the growth performance of chickens,the concentration of immunoglobulin in serum,increase the diversity of intestinal flora and the relative abundance of some beneficial bacteria,and improve the chicken's resistance to E.coli infection. |
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