I. R. Rajput

Effect of Saccharomyces boulardii and Bacillus subtilis B10 on intestinal ultrastructure modulation and mucosal immunity development mechanism in broiler chickens

The recent ban on the use of antibiotics as a feed additive has led to the search for alternative sources of antibiotics in the feed industry. Presently, probiotics are considered as a potential substitute for antibiotic as a live biotherapeutic agent to improve animal health and performance. Accordingly, study was focused on evaluating the effect of Saccharomyces boulardii (Sb) and Bacillus subtilis B10 (Bs) on ultrastructure modulation and mucosal immunity development in broiler chickens. A total of three hundred 1-d-old Sanhuang broilers (a Chinese cross breed) were randomized into 3 groups, each group with 5 replications (n = 20). The control group (Ctr) was fed a basal diet containing an antibiotic (virginiamycin, 20 mg/kg). Meanwhile, broilers in experimental groups received Sb and Bs (1 × 10(8) cfu/kg of feed) in addition to the basal diet for 72 d. The results of the experimental groups revealed a significant improvement in live BW and relative weight of bursa of Fabricius and thymus. Also, intestinal villus height, width, and number of goblet cells increased in the Sb and Bs groups. Meanwhile, modulation in the intestinal ultrastructure and increased mRNA expression levels of occluding, cloudin2, and cloudin3 (P < 0.05) were observed in the Sb and Bs groups. Moreover, IgA-positive cells significantly increased in the jejunum of Sb- and Bs-supplemented groups (P < 0.05). Intestinal cytokines interleukin-6, tumor necrosis factor-α, interleukin-10, transforming growth factor-β, and secretory IgA concentrations were (P < 0.05) improved in the probiotic groups; however, Sb induced inflammatory and antiinflammatory cytokines (P < 0.05) in comparison with the Ctr group. The present findings conclusively revealed that Sb and Bs increased IgA-positive cells in the lumen of the intestinal villus and revealed that Sb and Bs could modulate intestinal ultrastructure through increasing occluding, cloudin2, and cloudin3 mRNA expression levels in broiler intestine.

Influence of dietary inclusion of Bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens

This experiment was conducted to evaluate the effects of dietary inclusion of Bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens. Hy-Line Variety W-36 hens (n = 540; 28 wk of age) were randomized into 6 groups, each group with 6 replications (n = 15). The control group received the basal diet formulated with maize and soybean meal. The treatment groups received the same basal diets supplemented with 0.01, 0.02, 0.03, 0.06, and 0.09% Bacillus licheniformis powder (2 × 10(10) cfu/g) for an 8-wk trial. The results showed that dietary supplementation with 0.01 and 0.03% B. licheniformis significantly increased egg production and egg mass. However, no significant differences were observed in egg weight, feed consumption, and feed conversion efficiency among the 6 groups. Supplementation with different levels of B. licheniformis was found to be effective in improvement of egg quality by increasing egg shell thickness and strength. Compared with control, d-lactate content, diamine oxidase activity, and adrenocorticotropic hormone level in serum decreased significantly, and the level of estradiol and follicle-stimulating hormone increased significantly in plasma of all the experimental groups. Dietary supplementation with B. licheniformis increased the intestinal villus height and reduced the crypt depth. In conclusion, dietary inclusion of B. licheniformis could improve laying performance and egg quality significantly in a dose-dependent manner by decreasing the stress response, upregulating the growth hormone, and improving intestinal health.

Saccharomyces boulardii and Bacillus subtilis B10 Modulate TLRs Mediated Signaling to Induce Immunity by Chicken BMDCs

Dendritic cells (DCs) are professional antigen‐presenting cells (APCs) that play a critical role to activate immune response. They may be targeted for immunomodulation by microbes, including probiotics. In this study, chicken bone marrow dendrite cells (chi‐BMDCs) were stimulated with lipopolysachride (LPS), Saccharomyces boulardii (Sb), Bacillus subtilis B10 (Bs), co‐culture of Sbu2009+u2009Bs and phosphate buffer saline (PBS) as a control group (Ctr) at 3, 6, and 12u2009h intervals. Results 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. Additionally transmission electron microscopy (TEM) revealed efficiently engulfing and degradation of probiotics. Gene expression levels of MHC‐II, CD40, CD80 and CD86 up‐regulated in stimulated groups. Furthermore, toll‐like receptors TLR1, TLR2, TLR4, and chicken specific TLR15 expressions were improved and downstream associated factors MyD88, TRAF6, TAB1, and NFκ‐B mRNA levels increased in all treatment groups as compared to control. Surprisingly, NFκ‐B response was noted significant higher in LPS treatment among all groups. Moreover, IL‐1β, IL‐17, IL‐4, TGF‐β, and IL‐10 production levels were found higher, and lower concentration of INF‐γ and IL‐8 were observed in Sb, Bs, and Sbu2009+u2009Bs treatment groups. In contrast, LPS groups showed prominent increase in IL‐12, INF‐γ, and IL‐8 concentration levels as compared to control group. Altogether, these results emphasize a potentially important role of Saccharomyces boulardii and Bacillus subtilis B10 in modulating immunological functions of chi‐BMDCs by targeting specific toll like receptors (TLRs) and associated factors. The role of probiotics on chi‐BMDCs functionality in a non‐mammalian species have been presented for the first time. J. Cell. Biochem. 115: 189–198, 2014.

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.

Effects of Bacillus subtilis B10 spores on viability and biological functions of murine macrophages

This study was conducted to investigate the effects of Bacillus subtilis B10 spores on the viability and biological functions of murine macrophage. RAW 264.7 cells were stimulated both with and without B. subtilis B10 spores for 12 h. Then cell viability was determined to evaluate the cytotoxic effect of B. subtilis B10 spores to the cells, and the activities of acid phosphatase (ACP) and lactate dehydrogenase (LDH), the production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and the secretion of inflammatory cytokines were measured to analyze the functions of macrophages. The results showed that B. subtilis B10 spores were not harmful to RAW 264.7 cells and they also strongly enhanced the activities of ACP and LDH (P < 0.01), remarkably increased NO and iNOS production (P < 0.01), and significantly stimulated the secretion of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-1 beta (IL-1β), IL-6, IL-8 and IL-12 (P < 0.01) while they reduced anti-inflammatory cytokine IL-10 (P < 0.01). The outcomes suggest that B. subtilis B10 spores are not only safe for murine macrophages, but also can activate these cells and enhance their immune function. The above findings suggest that B. subtilis B10 spores are potentially probiotic.