Effects Of Bacillus Subtilis BYS2 On Intestinal Health And Probiotics Of Broilers

With the prohibition of antibiotics,probiotics have great potential to be added to feed as an antimicrobial agent.Bacillus subtilis(B.subtilis),as an outstanding probiotic,can resist stomach acid,bile salts and intestinal fluids to reach the gastrointestinal tract,thus playing its excellent role.In order to better assess the potential of B.subtilis previously isolated from the laboratory in production,we evaluated the adhesion performance of 3 strains of B.subtilis and2 strains of lactobacillus by BATH and coaggregation experiments,and evaluated the effect of 5 probiotics strains on inhibiting the adhesion of Escherichia coli(E.coli)to caco-2 cells by competitive,exclusion and replacement experiments.In order to evaluate the specific effect of B.subtilis in animal experiments,broilers were used as subjects in this study and fed for 6 weeks.Subsequently,this study for the first time designed a specific fluorescent probe based on the 16S rRNA sequence of bacillus and used the method of Fiuorescence in situ hybridization(FISH)to preliminarily determine the colonization of B.subtilis in broiler chickens after feeding.Then,the absolute quantitative standard curve of B.subtilis was constructed for the first time in this study to further determine the colonization of B.subtilis.At the same time,we examined the growth performance,intestinal villi morphology,immune organ index,serum immunoglobulin levels,cecum bacterial diversity,spleen and jejunum innate immune-related genes of broilers fed B.subtilis.In addition,this study examined the changes of innate immune-related genes and the survival rate of broilers fed B.subtilis after the challenge of E.coli to analyze the feasibility of B.subtilis as an alternative to antibiotics.This study is mainly divided into the following three parts:1.Adhesion of 5 probiotics and inhibition of E.coli adhesion to caco-2 cellsIn this study,the adhesion of 3 strains of B.subtilis(BYS2,BD17,BQ3)and 2 strains of lactobacillus(1.557,23174)was evaluated by BATH and coaggregation experiments.The results showed that BYS2,BD17,1.557 and 23174 had strong surface hydrophobicity,indicating that the four strains had strong adhesion.Subsequently,we investigated the ability of five probiotics to inhibit the adhesion of E.coli to caco-2 cells.In the competitive experiments,the adhesion inhibition rate of the five probiotics on E.coli adhesion to caco-2cells exceeded 35%,especially the adhesion inhibition rates of BYS2,BD17 and 1.557 were significantly higher than that of BQ3 and 23174(P<0.05).In the rejection experiment,the adhesion inhibition rate of the five probiotics on E.coli adhesion to caco-2 cells exceeded30%,especially the adhesion inhibition rates of BYS2,BD17 and 1.557 on E.coli adhesion were significantly higher than that of BQ3(P<0.05).In the replacement experiment,the adhesion inhibition rates of the five probiotics on E.coli adhesion to caco-2 cells were all more than 10%,especially the adhesion inhibition rates of BYS2,BD17 and 1.557 were significantly higher than that of BQ3 and 23174(P<0.05).The results showed that probiotics were more difficult to play their role after the pathogen infected the caco-2 cells,while the probiotics adhered to the caco-2 cells first,the bacteria adhered to the caco-2 cells later or the probiotics co-adhered to the caco-2 cells with the E.coli had a better inhibitory effect.Therefore,in subsequent experiments,we added probiotics to broiler 1-day-old feed.In summary,the effect of BYS2 and 1.557 was better.Considering that probiotics were needed for pelletizing later,however,lactobacillus were generally not able to withstand high temperature,so BYS2 was selected for subsequent experiments.2.The colonization of BYS2 in the intestinal tract of broilers and its effects on the immune performance,intestinal flora of broilers and growth performanceIn order to explore the distribution of B.subtilis in the intestinal tract of broilers after the use of BYS2,the process and probiotic mechanism were analyzed.In this study,a specific fluorescent probe was designed according to the 16S rRNA gene sequence of bacillus,and the number of major intestinal bacillus in broilers after feeding BYS2 was preliminarily detected by FISH.The results showed that the bacillus was mainly distributed in duodenum and cecum at 3 weeks,while the bacillus was mainly distributed in jejunum and duodenum at 5 weeks.Then,we further designed specific primers based on B.subtilis 16S rRNA gene sequence based on the results of FISH.After retrieving the target gene,we connected it to pClone007Simple Vector and constructed the absolute quantitative standard curve.The curve was y=-3.5497+37.543,the amplification efficiency was 91.29%,and the correlation coefficient R~2 was 0.995.The extracted intestinal mucosal bacterial genomic DNA was quantified by fluorescence,and then the data was substituted into the standard curve.The results showed that the copy numbers of duodenum,jejunum,ileum and cecum in the BYS2 group were significantly higher than those in the control group at 3 weeks or 5 weeks.B.subtilis was mainly distributed in the duodenum at 3 weeks,reaching 6.90�10~4 copy,which was significantly higher than that in the control group;B.subtilis was mainly distributed in the jejunum at 5 weeks,reaching 1.41�10~5copy,which was significantly higher than that in the control group(P<0.01).In summary,the increased number of bacillus in the 4 intestines may be B.subtilis,and B.subtilis is mainly distributed in the front of the small intestine(duodenum,jejunum).In addition,we explore the probiotic effect of BYS2 on broilers.When BYS2 was fed for5 weeks,the average weight of broilers in the BYS2 group was significantly increased by17.19%compared with the control group,and the height of villi in duodenum and jejunum and the ratio of villi to crypt were significantly increased(P<0.05).After feeding for 3 and 5weeks,the caecum contents of broilers in the BYS2 group and the control group were taken for 16S rRNA high-throughput sequencing,and the selected region was V3-V4.The results showed that the Shannon index(Alpha diversity)of both 3 and 5 weeks BYS2 groups was higher than that of the control group,indicating that the diversity of cecum flora increased.Whether it was at 3 or 5 weeks,the beneficial bacteria(Lactobacillus)in the BYS2 group showed an upward trend relative to the control group,and the harmful bacteria(Streptococcus)showed a downward trend relative to the control group,but the difference was not significant(P>0.05).Bacteroides in the BYS2 group had a significant decrease compared to the control group at 5 weeks(P<0.05).This means that the contents of pathogenic bacteria or conditioned pathogens are reduced,the composition of intestinal flora is improved,and the risk of infection is reduced.In summary,broilers can promote intestinal health after eating BYS2,adjust the proportion of beneficial bacteria and harmful bacteria,and improve the internal environment of the body.3.BYS2 promotes the innate immune response of broilers and enhances the disease resistance of broilersIn this study,to explore the effect of BYS2 on immune ability of broilers,we detected the expression of innate immune-related genes in the main immune organs(spleen)and main intestines(jejunum)of broilers.Compared with the control group,the main innate immune-related genes TLR4,IL-6,AvBD6 and AvBD12 in spleen and jejunum of broilers in the BYS2 group were significantly up-regulated at 3 weeks(P<0.01).Compared with the control group,the main innate immune-related genes TLR3,TLR4,IL-1?,IL-6 and AvBD12in spleen and jejunum of broilers in the BYS2 group were significantly up-regulated at 5weeks(P<0.05).The up-regulated Pattern recognition receptors(PPRs),inflammatory cytokines and defense molecules indicate that the body is in a state of immune prophylaxis,which plays an important role in preventing E.coli infection.Then we further tested the disease resistance of broiler chickens in the BYS2 group by attacking E.coli.Compared with the control group,the main innate immune-related genes TLR4 and IL-6 were down-regulated and AvBD3,AvBD6,AvBD9 and AvBD12 were up-regulated in the spleen and jejeum of broilers in the BYS2 group after 1 day of infection with E.coli.The results showed that at the early stage of infection,the level of inflammation in broilers was reduced,which helped broilers avoid immune damage,and the up-regulation of?-defensins was helpful for the clearance of E.coli.Compared with the control group,the main innate immune genes TLR4,IL-1?,IL-6 and AvBD9 were significantly up-regulated in spleen and jejunum of broilers in the BYS2 group after 3 day of infection with E.coli(P<0.01).Inflammatory cytokines and?-defensins up-regulated at the later stage of infection were helpful for the clearance of E.coli.In addition,compared with the control group,the serum IgG and IgM of broilers in the BYS2group increased by 31.60%and 30.52%respectively(P<0.05).Compared with the control group,broilers in the BYS2 group were significantly improved their ability to resist E.coli,and the survival rate was increased by 33.4%(P<0.05).In summary,BYS2 improves the growth performance and disease resistance of broilers.This study provides a theoretical reference for B.subtilis as a future antibiotic substitute.