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1. The objective of this study was to investigate the potential immunomodulatory effects of yeast (Saccharomyces cerevisiae) and yeast-derived products treated with a cell wall lytic enzyme mixture on the gene expression of toll-like receptors and cytokines of chicken B cell line (DT 40) stimulated with lipopolysaccharide. 2. The effect of brewer's yeast (Y), yeast cell wall (YCW), distilled dried grains with solubles (DDGS) and a processed yeast + nucleotide-rich product (PY/N) treated with a yeast cell wall lytic enzyme (E) was assessed using a chicken B cells and LPS challenge model. 3. Relative gene expression of toll-like receptors (TLRs) and cytokines was investigated. Treatment of cells with Y, YCW, YCW + E and PY/N upregulated the expression of TLR2b following LPS challenge. Gene expression of TLR4 was downregulated in E, YCW and YCW + E treatments compared to control, while adding DDGS and PY/N upregulated the expression of TLR4 either before or after enzyme treatment. Following LPS challenge, expression of IL-4 and IL-10 was upregulated in cells treated with YCW and PY/N, both pre and post enzyme incubation. Adding YCW and PY/N to the cells challenged with LPS upregulated the expression of IFN-γ and IL-12 before and after enzyme treatment. Treatment of cells with YCW, DDGS and PY/N increased the expression of IL-6 prior to LPS challenge. 4. In conclusion, the results suggested that yeast-derived products affected immunomodulatory activities by changing the expression of cytokines involved in the innate immune response. [ABSTRACT FROM AUTHOR]

1. This study evaluated and characterised the effect of folic acid (FA) on chromosomal DNA methylation and the epigenetic result on gene expression control mechanisms in chicken B cells as a model of antigen presenting cells. 2. After FA supplementation, the methylation pattern on the proximal promoter area and mRNA expression of toll-like receptor (TLR) 2b, TLR4, B cell receptor (BCR) immunoglobulin (Ig) β and major histocompatibility complex (MHC) II β chain genes in chicken B cells was observed 3. Chicken B cell line (DT40) cultures were incubated with 0, 1.72 or 3.96 mM of FA for 4 and 8 h and samples were taken at specific time points. After 4 h of incubation, cells were challenged with 0, 1 or 10 µg/ml of lipopolysaccharide (LPS) and samples were collected 4 h post-challenge. 4. FA supplementation modified the methylation patterns of the proximal promoter regions of TLR4, Igß, and MHCII ß chain at 4 and 8 hours of incubation; however, the single CpG dinucleotide of TLR2b remained methylated regardless of the treatment. 5. A positive association was found between FA concentration and percentage DNA methylation on the promoter area of Igβ and TLR2b. However, there was a negative association between FA and MHCII β chain. 6. There were downregulatory effects in TLR4, Igß and MHCII ß chain gene expression after 8 h of incubation, nut not at 4 h. Although incubation time did not affect TLR2b gene expression, FA concentration did, whereby it increased TLR2b expression at 1.72 mM FA (P < 0.05). 7. LPS significant downregulated TLR2b expression, while an interaction between FA and LPS concentration affected TLR4 and Igβ gene expression. 8. In conclusion, the results showed that FA can have an immunomodulatory effect on chicken B cells, possibly affecting their ability to both recognise antigens through the TLR and BCR pathways, and to present it via the MHCII presentation pathway. [ABSTRACT FROM AUTHOR]

This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotidecontaining diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell-mediated immune response indicating the immunomodulatory activities of these products following immunization with non-inflammatory antigens. [ABSTRACT FROM AUTHOR]

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The turnover of intestinal epithelial cells is a dynamic process that includes adequate cell pro-liferation and maturation in the presence of microbiota and migration and seeding of immune cells in early gut development in chickens. We studied the effect of yeast-derived macromolecules (YDM) on performance, gut health, and immune system gene expression in the in-testine of broiler chickens. One thousand eighty 1-d-old birds, with 60 birds per pen and 6 pens per treatment, were randomly assigned to 3 treatment diets; a diet con-taining monensin (control), control diet supplemented with bacitracin methylene disalycylate (BMD), and BMD diet supplemented with YDM. Feed intake, BW, mortality, ileum histomorphology, and gene expression of Toll-like receptors (TLR2b, TLR4, and TLR21), cy-tokines [interferon (IFN)-γ, IFN-β, IL-12p35, IL-1β, IL-6, IL-10, IL-8, IL-2, IL-4, and transforming growth factor (TGF)-4β], and cluster of differentiation (CD)40 in the ileum, cecal tonsil, bursa of Fabricius, and spleen were assessed. No significant overall difference in performance in terms of feed intake, BW gain, and G:F was observed among treatments (P > 0.05). The YDM diet resulted in significantly higher villi height and villi heightxrypt depth ratio compared with BMD and con-trol diets (P < 0.05). A significantly lower mortality was observed in the YDM treatment compared with both control and BMD treatments. Compared with the control, gene expression analysis in YDM treat-ment showed no major change in response in the ileum, whereas higher CD40, IFN-β, IL-β, IL-6, TGF-β4, IL-2, and IL-4 in the cecal tonsil; TLR2b, TLR4, TLR21, and TGF-β4 in the bursa of Fabricius; and TLR4, IL-12p35, IFN-γ, TGF-β4, and IL-4 in the spleen was ob-served (P < 0.05). In conclusion, supplementation of YDM supports pro- and anti-inflammatory cytokine production via T helper type 1 and 2 (Thl and Th2) cell-associated pathways both locally and systemically with a stronger additive effect in the cecal tonsil in the presence of BMD in the diet of chickens. [ABSTRACT FROM AUTHOR]

The combined effects of probiotics {Lactobacillus acidophilus, Lactobacillus casei, Streptococcus faecium, and Saccharomyces cerevi-siae) and organic acids (sorbic and citric acid) on intestinal morphology and expression of immune-related genes were investigated. One-day-old chicks were randomly assigned to 1 of 3 groups: birds not receiving probiotic or organic acids (control; T1), or birds receiving an oral combination (1 g/L in water) of 108 CFU/g of each of the aforementioned probiotics and organic acids (1% sorbic acid and 0.2% citric acid) for 7 (T2) or 14 d (T3). Each group was divided into 5 replicate pens of 20 birds each, and 5 birds from each group (1 from each pen) were killed on d 11 and 22. Intestinal sections were collected for histological assessment, and reverse transcriptase-PCR analysis was used to assess defensin and cathelicidins expression. Quantitative real-time PCR was used to assess toll-like receptors (T1R) and cytokine expression. Duodenal villus height was greater in T2 and T3 at d 11 (P≤ 0.036) and 22 (P ≤ 0.015) compared with T1. At d 11, duodenal goblet cell/unit area was less in T3, whereas it was greater in T2 compared with T1 in the jejunum (P = 0.009). Ileal goblet cell/unit area was greater in T3 at d 22 compared with T1 (P < 0.001). Avian beta-defensin-3 was expressed in all tissues except the bursa of T3 birds at d 11, and T1R-2 was down regulated in the cecal tonsil of birds in T2 and T3 at d 11 compared with T1 (P = 0.020 and 0.003, respectively). Expression of IL-12p35 in the ileum at d 11 was down regulated in T2 and T3 compared with T1 (P = 0.030 and 0.012, respectively). Reduced expression of INF-y was observed in the ileum in T3 compared with T1 at d 11 (P = 0.047). Ileal IL-6 and IL-10 and cecal tonsil interferon-gamma INF-&ggr; expressions were greater T2 at d 22 (P < 0.047) than T1. In conclusion, supplementation of combined probiotics and organic acids resulted in inconsistent gut morphology associated responses, and avian beta-defensins and cathelicidins expression were not associated with combined probiotics and organic acids supplementation. Birds supplemented with combined probiotics and organic acids for 7 d showing similar responses in T1R-2, IL-12p35, and IFN-y compared with those supplemented for 14 d indicates that shorter periods of supplementation might be enough to elicit beneficial responses. [ABSTRACT FROM AUTHOR]

Tetracyclines, which represent one of the most commonly used antibiotics for poultry, are known to be deposited in bones, where they can remain, despite the observation of appropriate withdrawal times. The aim of the study was to determine the concentration of oxytretracycline (OTC) residues in the bone and muscle of chickens, following the oral administration of a commercially available liquid formulation, and to test their cytotoxic effects on an in vitro cell culture model. Seventy-two 1-day-old broiler chickens were randomly allotted into 2 groups (control and treated animals). OTC (40 mg/kg BW) was administered via drinking water during the 1 to 5 and 20 to 25 days of life periods. At the end of the trial, the birds were slaughtered and the OTC residues in the target tissues were measured by means of liquid chromatography (LC) - tandem mass spectrometry (MS/MS). Cytotoxicity was assessed by evaluating the pro-apoptotic effect of the bone residues on the K562 erythroleukemic line and on the peripheral blood mononuclear cells (PBMC). In all the animals, the OTC residues in the muscle were far below the established MRL of 100 µg/kg. The OTC levels in the bones of the treated animals were instead found in the parts per million (ppm) range. Cell cytotoxicity was assessed by evaluating the pro-apoptotic effect of OTC bone residues on the haematopoietic cell system. This in vitro system has revealed a significant pro-apoptotic effect on both the K562 cell line and PBMC cultures. This result suggests potential human and animal health risks due to the entry of tetracycline residues contained in the bones of treated livestock into the food-chain. This could be of concern, particularly for canine and feline diets, as meat, bone meal, and poultry by-products represent some of the main ingredients of pet foods, especially in the case of dry pet food. Further studies are needed to define the underlying mechanisms of cytotoxicity and to evaluate the in vivo toxicological implications due to the observed in vitro effects. [ABSTRACT FROM AUTHOR]

Necrotic enteritis (NE) caused by Cbstridium perfringens is a reemerging disease of economic importance in areas of the world where antibiotic growth promoters have been banned. The effect of mannan-oligosaccharide (MOS) supplementation in organic diets of broilers challenged with C. perfringens on performance, gut morphology, and innate immunity was investigated. Three hundred Ross-308 broilers were fed antibiotic-free certified organic starter and grower diets. On d 14, birds were orally challenged with 1 mL of C. perfringens culture at 3 x 1010 cfu/bird. Treatments consisted of a control no-challenge (CO; 0 g/kg of MOS in the basal diet), control challenge (COC, 0 g/kg of MOS in the basal diet), and MOS challenge (2 g/kg of MOS in the basal diet). Challenge of birds resulted in decreased feed intake and BW gain (P = 0.048 and P = 0.026, respectively). Even though supplementation of diet with MOS improved feed intake (P = 0.985), BW gain and G:F were not improved compared with those of the CO group (P = 0.026 and P = <0.001, respectively). There was no significant difference among treatments in jejunal and ileal villus height, crypt depth, and goblet cells/mm2 (P> 0.05). Quantitative real-time PCR showed that, in the ileum, the MOS diet resulted in an upregulation of toll-like receptor (TLR)2b, TLR~j, interleukin (IL)-12p35, and interferon (IFN)-'y compared with CO (P = 0.003, P = 0.018, and P = 0.024, respectively). In the cecal tonsil, challenging birds with C. perfringens resulted in an upregulation of TLR2b compared with CO (P = 0.036), and MOS resulted in an upregulation of TLR~j (P = 0.018). In conclusion, feeding a MOS-supplemented diet to C. perfringens-challenged broiler chickens did not improve performance and gut morphology-associated responses. However, MOS was capable of altering TLR and cytokine profiles, where dual TLR52 and TLR~ pathways were associated with MOS supplementation with subsequent upregulation of ileal IL-12p35 and IFN-'y, implying that MOS supplementation in C. perfringens-challenged chickens supports a proinflammatory effect via T-helper cell-i associated pathways. [ABSTRACT FROM AUTHOR]