The Effect Of Early Intervention By Clostridium Butyricum On Intestinal Immune And Microbial Community Structure Of Boiler Chicken Challenged By Escherichia Coli

Clostridium butyricum is an obligate anaerobic Gram-positive bacillus.Clostridium butyricum and its metabolites can stimulate the proliferation of Lactobacillus and other enteric probiotics,effectively inhibit the growth of E.coli,prevent and control the disorder of microbial flora in the intestine,as well as degrade polysaccharides.This paper studies on the impact of Clostridium butyricum and its metabolites on suppressing the growth of E.coli,on the expression of intestinal inflammatory factors and antimicrobial peptides,and on the structure of intestinal microorganism flora.It provides reference to the amelioration of intestinal micro-ecology in animals.This paper divides into three parts.Experiment 1,the impact of Clostridium butyricum and its metabolites on the genetic expression of E.coli flagellum.In this experiment,Clostridium butyricum MIYAIRI II 588 was adopted and cultivated in MRS media respectively under aerobic and anaerobic environment.The short-chain fatty acids were sampled and measured at 0 h,2 h,4 h,8 h,12 h,24 h,and 36 h,respectively.Meanwhile,The E.coli CVCC198 was cultured in LB medium,and then Clostridium butyricum in different concentrations(106 cfu/m L?107 cfu/m L?108cfu/m L)were added in for mixed cultivation.The mixed cultures were sampled at 1 h,2 h,4 h,6 h,and 12 h,and received real-time fluorescent quantitative PCR for inspection on the flagellin gene fae G.Meanwhile,the expression changes of est A and est B of E.coli in each group were detected at 2 h.The results showed that(1)there were no significant differentiation in acetic acid,propionic acid,isobutyric acid,valeric acid,and isovaleric acid in Clostridium butyricum culture media under anaerobic environment at different points in time(P>0.05).The butyric acid was 0.015 mmol/L-0.031 mmol/L by the end of 8h,and 0.462mmol/L at 12 h(P < 0.01).After 24 hours,the concentration of butyric acid extremely increased to 8.213-9.173mmol/L(P<0.01).Under aerobic environment,the butyric acid in the Clostridium butyricum culture media presented a rising trend but no significant difference during first 12 hours(P>0.05),while showed a remarkable elevation to 0.109 mmol/L at 24h(P<0.01),and an outstanding evaluation to 6.561 mmol/L at 36h(P<0.01);(2)The impact of Clostridium butyricum on the genetic expression of E.coli flagellum in vitro.In the E.coli control group,the relative expression amount of fae G were in a higher level at different time.After added with Clostridium butyricum in concentrations of 106 cfu/m L,107 cfu/m L,and 108cfu/m L,the fae G expression amounts all showed a significant decreasing trend(P<0.05),Clostridium butyricum significantly inhibited the production of est A and est B(P<0.01).Expriment 2,The impact of Clostridium butyricum on E.coli poising and attacking broilers' growth performances and intestinal inflammatory response related factors and on the antimicrobial peptide gene expression.In the experiment,200 healthy 1-day old broilers were randomly divided into 2 groups on the first 7 days,each group of 100 chickens,respectively,of which one group was CB Clostridium butyricum group and control group.On the 1-3 days,the Clostridium butyricum group was fed with 1m L Clostridium butyricum liquid with concentration of 109 cfu/m L,while the control group was fed with 1m L saline.At the 7th day,the Clostridium butyricum group was randomly divided into Clostridium butyricum group(CB)and Clostridium butyricum + E.coli group(CB+EC),while the control group was randomly divided into the E.coli group(EC)and the Control group(Control).At the 7th day,both Clostridium butyricum + E.coli group(CB+EC)and E.coli group(EC)were fed with 1 m L E.coli liquid with concentration of 108cfu/m L,while the other groups were fed with 1m L saline.The chickens were weighted at 3 d,5 d,7 d,10 d,14 d,and 28 d and fed of free food intake and free drink intake,and the daily food consumption and death of each group were recorded in conversion to ratio of weight to feed combined with increased weight.And the dysentery conditions of the group chickens were checked after poising and attacking.The obtained ileum tissue on the 10 d and 14 d to inspect the TNF?,IL-1?,IL-8,IL-10,and TLR4 gene expressions and related factor expression amounts of Av BD9 ? Av BD10 ?Cath-B1 endogenous host defense peptides.Cecum samples on 3d,7d and 10 d were selected to detect the expression amount of IL-1? gene,Av BD9,Av BD10 and Cath-B1 endogenous host defense peptide related factors.The results indicated,(1)after Clostridium butyricum was fed at 0 to 7 days,the weights of Clostridium butyricum group and control group were slight high but had no significant difference(P>0.05).On 14 d,in comparison of CB group and CB+EC group to the control group,these groups respectively increased with 19.78%(P ? 0.05)and 20.63%(P ? 0.05),and the ratio of feed to weight significantly decreased during 7th day to 10 th day(P<0.05).(2)In ileum tissue,TNF? and IL-1? had no significant differences on 10 th day(P>0.05),and the IL-8 and TLR4 expression significantly decreased compared to the EC group and CB+EC group(P<0.05);the IL-10 expression amount in E.coli group significantly decreased in comparison with that of CB group(P<0.05);There were no significant differences in TNF?,IL-1?,and IL-8 on 14 th day(P>0.05).Compared with CB + EC group,IL-10 expression amount significantly dropped in EC group(P<0.05).TLR4 expression amount in EC group and CB+EC group remarkably increased comparing to CB group(P<0.05).(3)In terms of ileum tissue,the Av BD9 and Cath-B1 expression amount had no significant difference and Av BD10 expression amount had no remarkable increase in Clostridium butyricum group(P<0.01)on the 10 th day,Av BD9 expression amount significantly increased in Clostridium butyricum group and E.coli group on the 14 th day(P<0.01),while Cath B1 expression amount in experimental group in comparison with control group(P<0.01).(4)In cecum tissue,the expression amounts of Av BD9 had no significant differences between groups(P>0.05),the expression of Cath-B1 in CB+EC group and Contol group were significant higher on the 10 th day(P<0.05).The Av BD10 expression amount in CB group showed a significant increase in comparison with other experimental groups(P<0.05).on the 14 th day,the expression of Av BD10 in Clostridium butyricum group and CB + EC group showed a significant increase on Control group and E.coli group(P<0.05),the expression of Av BD9 and Cath-B1 were no significant differences between groups(P>0.05).Experiment 3,The impact of Clostridium butyricum on E.coli infecting intestinal flora structure and short-chain fatty acid spectrum of broilers.The collected cecum contents from each group in the experimental 2 on 10 th day and 14 th day was treated with high throughput sequencing,and the contents of short-chain fatty acids in ileum and cecum contents were determined.The results suggested that(1)at the phyla level,firmicutes performed as the dominate phyla in each sample on the 10 th day,and the relative species richness was all above 93%.At the genus level,lactobacillus,spirillas,rumens,Weiss,bacillus played as the main bacteria flora in each sample,while the lactobacillus occupied the hightest species richenss.On the 14 th day,the firmicutes also took the domination in samples with a species richness above 76%;lactobacillu,ruminococcus,lachnospira,fusobactrium,Streptococcus,butyric cocci,Blaut S.,and bacillus were the main baceria groups in samples.And there was no structural differences in floras on the 10 th day and 14 th day.(2)in the ileum,the contents of short-chain fatty acids in each groups showed no significant differences on the 10 th day and 14 th day(P>0.05).In cecum,the acetic acid content in Clostridium butyricum group was significantly higher than that in other experimental groups on the 10 th day(P<0.01),and the acetic acid content in Clostridium butyricum group and Clostridium butyricum + E.coli group increased remarkably(P<0.01);the contents of acetic acid,propionic acid,butyric acid,and isobutyric acid in Clostridium butyricum group were all significantly higher than that in control group and E.coli group on the 14 th day(P<0.05).Based on abov results,Clostridium butyricum can inhibit the genetic expression of E.coli flagellin,and enacted a certain impact on the broilers growth performance and inflammatory factors and antimicrobial peptide gene expression amount,whole had an unobvious impact on the flora structure after poising and attacking on broilers.