Effects Of Saccharomyces Boulardii And Bacillus Subtilis B10on Intestinal Immunity Of Broiler Mediated By Toll Like Receptors Signaling Pathway

Probiotics are defined as live microorganisms which, when administered in adequate amount exert beneficial effects on the host (FAO/WHO,2001). Recently, various probiotics were introduced in poultry industry as feed additive, including Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Propionibacterium, Bacillus, Enterococci, and prominent yeast Saccharomyces. However, a little information is available about the effect of Saccharomyces boulardii and Bacillus subtilis B10on chickens. Thus, present study was designed for evaluating the Effects of Saccharomyces bolardii and Bacillus subtilis B10on intestinal immunity of broiler, mediated by TLRs signaling pathway. The main results of the study are as follows:A total of300one-day-old Sanhuang broilers (a Chinese cross breed) were randomized into3groups, each group with five replications (n=20). Control group (Ctr) was fed a basal diet containing an antibiotic (virginiamycin,20mg/kg). Meanwhile, broilers in experimental groups received Saccharomyces boulardii (Sb) or Bacillus subtilis B10(Bs)(1×108cfu/Kg feed) in addition to basal diet, and the trial was conducted for72days. The results showed that experimental groups significantly improved the live BW. The activities of jejunal Na+K+ATPase (ATP), lipase (LP) and Gamma glutamyl transpeptidase (yGT) increased (P<0.05) in probiotic supplementary groups as compared to control group. Moreover, higher yGT activity was observed in ileum of Bs supplemented group only. The serum glutathione peroxidase (GSH-PX), peroxidase (POD), glutathione (GSH), glutathione reductase (GR) and catalase (CAT) activity were significantly higher, while malondialdehyde (MDA) content decreased (P <0.05) in probiotic groups. The blood biochemical analysis showed significant low level of uric acid and triglycerides in Sb and Bs groups. Conversely, albumin and low-density lipoprotein concentration increased in Sb and Bs groups as compared to control group (Ctr).The effect of Saccharomyces boulardii and Bacillus subtilis on intestinal mucosa structure and microbial composition were investigated. Compared to control group, the intestinal villus height, width and number of goblet cells increased in Sb and Bs groups. Meanwhile, modulation in the intestinal ultrastructure and increased mRNA expression levels of occluding, cloudin2and cloudin3(P<0.05) were observed in Sb and Bs groups. Intestinal microbiota rarefaction analysis showed that bacterial richness significantly increased in the jejunum and ileum of Sb and Bs groups, and Firmicutes appreared as a prominant microbial phylum and diversity of microbial community signigicantly decreased in the jejunum and Ileum of treatment groups.The effect of Saccharomyces boulardii and Bacillus subtilis B10on intestinal immunity and mRNA expression of TLR signaling pathway of broiler was obsered. Compared to control group, cytokines level in the jejunum and ileum, including IL-1β, TNFa, IL-17, IL-6, IL-4, IL-10and TGF-β increased in Sb group and TNFα, IL-17, IL-6and TGF-β were improved in Bs group. Conversely, jejunal INF-y and IL-8level decreased in probiotics group, and only INF-y decreased significantly in the ileum. In addition, IgA positive cell number noted higher in jejunum, meanwhile serum IgA and slgA level increased in both treatment groups, and IgG improved in Sb group only. Additionally, a significant improvement in mRNA expression of surface receptors TLR2, TLR4, TLR15and down streaming associated factors MyD88, TRAF6, TAB2and NF-κB in the jejunum and ileum were observed in the treatment groups.The effects of Saccharomyces boulardii and Bacillus subtilis B10on chi-BMDCs. Denderitic cells (DCs) play a central role in immune regulation and antigen presentation in animals, but quantitative description of chicken DCs culturing method employing rGM-CSF and optimal biological response was lacking in litrature. Foremost, the chicken recombinant granulocyte-macrophage colony-stimulating factor (chi-rGM-CSF) was expressed in E. coli BL21successfully and established a standard method to culture denderitic cells from chicken bone marrow by employing chi-rGM-CSF (lOng/ml) and (20ng/ml), co-culturing with interleukin-4(IL-410ng/ml), and treated with lipopolysachride (LPS,500ng/ml) to determin the biological response. Sruface markers CD40, CD80, CD83and CD86showed maturation of DCs and cytokine response was also noted in.Afterwards chi-BMDCs were stimulated with lipopolysachride (LPS), Saccharomyces boulardii (Sb), Bacillus subtilis B10(Bs) and co-culture of Sb+Bs at3h,6h and12h intervals and phosphate buffer saline (PBS) was added in control group (Ctr). The findings revealed that treatment groups modulated the phenotype and biological functions of chi-BMDCs. Scan electron microscopy showed attachment of probiotics on the surface of chi-BMDCs, and transmission electron microscopy (TEM) revealed efficiently engulfing and degradation of probiotics. Gene expression levels of MHC-Ⅱ, CD40, CD80and CD86up-regulated in stimulated groups. Furthermore, TLR1, TLR2, TLR4and chicken specific TLR15mRNA expression were increased. Downstream associated factors MyD88, TRAF6, TAB1and NFκ-B mRNA levels also increased in all treatment groups as compared to control. Conversely, NFκ-B response was noted significant higher in LPS group among all treatment groups. Cytokines IL-1β, IL-17, IL-4, TGF-β and IL-10production levels were found higher, and lower concentration of INF-y and IL-8were observed in Sb, Bs and Sb+Bs treatment groups. In contrast, LPS group showed prominent increase in IL-12, INF-y and IL-8concentration levels as compared to control group.Conclusively, these results emphasize the potentially significant role of Saccharomyces boulardii and Bacillus subtilis B10to modulating the intestinal immunological functions of broiler and also have improtant role to alter immunomodulatory effect of chi-BMDCs mediated by TLRs signaling pathway.