The Research On Bacillus Amyloliquefaciens TL Affrcting The Expression Of Inflammatory Related Factors In Broilers

Bacillus amyloliquefaciens is an inspection-free strain of microbial fermentation in China and certified by FDA in the United States.It is an excellent plant biocontrol bacteria and animal probiotic.Bacillus amyloliquefaciens TL(B.A TL)isolated from breeding pigs in this laboratory is a probiotic strain with excellent performance.It’s been found that B.A TL can significantly improve the growth performance of broilers as well as the enhancement of disease resistance,which is very considerable on the application prospect.As we all know,inflammation is important to livestock and poultry,especially for the meat and poultry breeding of a short time.High level intestinal inflammation can hugely reduce the feed efficiency and production performance of individual animals,resulting in unnecessary waste of resources.So reducing intestinal inflammation within the normal threshold as much as possible is always an important scientific problem that always needs to be solved in broiler breeding.In recent years,the research of Bacillus amyloliquefaciens is still mainly focused on its antimicrobial peptides and biological enzymes to the plant crops field,but much less studies trying to clarify the key components of probiotics that affect the inflammatory response of animals.Therefore,this study aimed to explore the immunomodulatory effect between B.A TL and broilers’intestinal inflammation,and preliminarily identified the key components of probiotic immune activity and verified it at the cellular level.When Cobb500 broilers were fed with 4×10~9 CFU/kg B.A TL for 35 days,the growth performance and intestinal histomorphology of broilers were significantly improved,especially the average body weight,intestinal villus height and VCR from 14to 21 days.Transcriptome analysis of intestinal tissues showed that B.A TL could significantly down-regulate the expression of inflammation-related factors and receptors CCL4,CCL26,XCL1,CCR5,CX3CR1,IL1B,IL8,LITAF,TNFSF/RSF,IFNG and LYZ in 21 day’s broiler jejunum and ileum,and significantly down-regulate granulocyte migration(BP)and defense response to intestinal contents(BP).Cytokine-cytokine receptor interaction signaling pathway,JAK-STAT signaling pathway and NF-κB signaling pathway were also down-regulated.In 35 day,B.A TL also down-regulated the expression of cytokines including XCL1,IL6,IL8,LITAF and LYZ,and the synthesis of viral protein component(CC)in the intestinal tract of broilers,but could not significantly affect the expression and regulation of inflammation-related signaling pathways.In order to determine the main components of anti-inflammatory effect of B.A TL,chicken macrophage like cell HD11 was stimulated with bacteria body and supernatant respectively by pretreatment,simultaneous treatment and aftertreatment.It was found that pretreatment of probiotic supernatant with 1/10 OD1.0TL concentration could significantly inhibit the upregulation of inflammatory factors CCL4,IL1B,IL8 and LITAF(P<0.0001).The metabolite composition of B.A TL supernatant was analyzed,and found that the proportion of amino acids and sugars and their derivatives was up to68.54%.Given this,exopolysaccharide(EPS-TL)and extracellular proteins(EPro-TL)in the supernatant of B.A TL were isolated,the protective effects of these two components on high inflammation of HD11 cells under different treatment conditions were verified.The results showed that EPro-TL could not protect HD11 inflammation in any treatment conditions.Under the condition of pretreatment,EPS-TL can protect HD11 cells from inflammation more strongly than the supernatant of B.A TL.Thus,this study preliminarily determined that EPS-TL is closely related to the anti-inflammatory activity of B.A TL,which may be the key component for probiotic to exert inflammation regulatory activity.The gel-liquid chromatographic analysis of EPS-TL showed that EPS was a trisaccharide composed of gal,glc and man with a molar ratio of 1 to 4.3 and 7.5,its number-average molecular weight was about 4.7 k Da.In order to explore whether EPS-TL can exert stable anti-inflammatory activity in chicken intestinal epithelial cells,primary chicken embryo intestinal epithelial cells were successfully isolated by combined digestion of collagenase XI and neutral proteaseⅠ,and a hyperinflammatory model of primary cells was constructed with 10μg/m L O55 LPS.It was found that EPS-TL could still exert its anti-inflammatory activity stably and significantly inhibit the up-regulation of inflammatory cytokines CCL4,IL1B,IL8 and LITAF in primary chicken embryo intestinal epithelial cells(P<0.0001).To sum up,probiotic B.A TL can significantly improved the growth performance,intestinal morphology and the expression of intestinal inflammatory factors in broilers,which was better in the early day age of broilers,and showed a certain correlation with the growth performance.B.A TL has obvious inflammatory regulatory activity in broilers in vivo and in vitro,and EPS-TL may be the key component of this characteristic.It can show stable anti-inflammatory activity in chicken macrophage HD11 and primary cells of chicken embryo intestinal epithelium,which is positive to broiler breeding.