Folic acid enhances proinflammatory and antiviral molecular pathways in chicken B-lymphocytes infected with a mild infectious bursal disease virus.

1. This study evaluated the effect of folic acid (FA) supplementation on the proinflammatory and antiviral molecular pathways of B-lymphocytes infected with a modified live IBDV (ST-12) mild vaccine strain during a timed post-infection analysis.2. A chicken B-lymphocytes (DT-40) cell line was cultured in triplicate at a concentration of 5 × 105 cells per well in 24-well plates; and was divided into three groups: 1: No virus, FA; 2: Virus, no FA; 3: Virus + FA at a concentration of 3.96 mM. The experiment was repeated three times.3. Cells in groups 2 and 3 were infected with a modified live IBDV (ST-12) mild vaccine strain at one multiplicity of infection (MOI: 1). After 1 hour of virus adsorption, samples were collected at 0, 3, 6, 12, 24 and 36 hours post-infection (hpi).4. The modified live IBDV (ST-12) mild vaccine strain triggered a B-lymphocyte specific immune response associated with the upregulation of genes involved in virus recognition (Igß), virus sensing (TLR-2, TLR-3, TLR-4 and MDA5), signal transduction and regulation (TRIF, MyD88 and IRF7), and the antiviral effector molecules (IFN-α, OAS, PKR, and viperin).5. FA supplementation modulated IBDV replication and regulated the proinflammatory and antiviral downstream molecular pathways.6. In conclusion, the low virulent pathotype serotype I modified live IBDV (ST-12) mild vaccine strain was able to trigger and mount an immune response in chicken B-lymphocytes without affecting B-cell viability. FA supplementation modulated B lymphocytes response and improved their innate immune proinflammatory and antiviral response molecular pathways.

Antimicrobial peptides as an alternative to relieve antimicrobial growth promoters in poultry.

1. This review describes different classes of antimicrobial peptides (AMP) found in the gastrointestinal (GI) tract of avian species, and their antimicrobial and immunomodulatory activities. The potential benefits of synthetic AMP in poultry production are examined, in the context of the use of AMP as alternatives to antimicrobial growth promoters (AGP).2. Since the mid-1950s, antibiotic growth promoters (AGP) have been used in feed at low prophylactic doses to modulate the homoeostasis of intestinal microbiota, decreasing the risk of intestinal dysbacteriosis and the growth of pathogens within the avian gut. Over the last three decades, AGP have faced major regulatory restrictions due to concerns of generating antimicrobial resistance (AMR). It is now well documented that the rate of infectious disease outbreaks is higher in flocks that are not fed prophylactic antibiotics, resulting in a compensatory increase in antimicrobial use for therapeutic purposes.3. Endogenous natural AMP production is associated with the presence of microbiota and their interaction with the intestinal epithelial and lamina propria lymphoid cells. Their antimicrobial activity shapes the beneficial microbiota population and controls intestinal pathogens such Clostridium and Salmonella spp., and stimulates the development and maturation of the local immune system.4. Similar to AGP, AMP can establish a well-balanced gut beneficial microbiota for adequate immune-competence, animal health and high growth performance parameters such as feed intake, daily weight, feed conversion and accumulated mortality.5. Antimicrobial proteins and peptides constitute an essential part of the innate immune system of all organisms and protect the host from invading pathogenic bacteria, viruses, fungi, and parasites by interacting with the negatively charged pathogen membranes.

Yeast cell wall polysaccharides enhanced expression of T helper type 1 and 2 cytokines profile in chicken B lymphocytes exposed to LPS challenge and enzyme treatment.

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.

Differential immunomodulatory effect of vitamin D (1,25 (OH)2 D3) on the innate immune response in different types of cells infected in vitro with infectious bursal disease virus.

It has been demonstrated that vitamin D (Vit D) included in diets offers a beneficial effect by improving innate immune responses in chickens. However, its mechanisms of action and the effect on immunosuppressive pathogens, such as infectious bursal disease virus, are not yet known. In the present study, we have studied the immunomodulatory effect of Vit D on the innate immune response in 3 cell lines: fibroblast cells (DF-1), macrophages (HD11), and B cells (DT-40) infected with IBDV (intermediate vaccine) at 2 multiplicity of infections (MOI) (1 and 0.1). Genes associated with innate immune responses (TLR-3, TLR-21, MDA-5, MyD88, TRIF, IRF-7, INF-α, INF-ß, PKR, OAS, viperin, IL-1ß, IL-6, and IL-12) were evaluated at different time points (3, 6, 12, 24, and 36 h after infection, h.p.i). Virus production reached a maximum at 24 h.p.i., which was significantly (P < 0.05) higher in DF-1 cells, followed by HD-11 and DT-40 cells. Mainly in HD-11 cells, there was a significant (P < 0.05) effect of Vit D supplementation on receptors TLR-3, TLR-21, and MDA-5 after 12 h.p.i, independent of MOI. DT-40 cells showed the highest antiviral activity, with a significant (P < 0.05) effect on IRF-7, IFN-ß, OAS, and PKR gene expression, where expression of IRF-7 and IFN-ß correlated positively with Vit D supplementation, while OAS and PKR were independent of Vit D. Proinflammatory cytokines were significantly (P < 0.05) upregulated and found to be Vit D and MOI dependent. In conclusion, this study demonstrated the capacity of IBDV to trigger a strong innate immune response in chicken cells and contributes to the understanding of the activation pathways of innate immunity induced by IBDV and further shows the benefitial effect of Vit D supplementation as an immunomodulator.

Epigenetic effect of folic acid (FA) on the gene proximal promoter area and mRNA expression of chicken B cell as antigen presenting cells.

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) ß and major histocompatibility complex (MHC) II ß 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 Igß and TLR2b. However, there was a negative association between FA and MHCII ß 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 Igß 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.

Gut microbiota modulates type I interferon and antibody-mediated immune responses in chickens infected with influenza virus subtype H9N2.

Commensal gut microbes play a critical role in shaping host defences against pathogens, including influenza viruses. The current study was conducted to assess the role and mechanisms of action of commensal gut microbiota on the innate and antibody-mediated responses of layer chickens against influenza virus subtype H9N2. A total of 104 one-day-old specific pathogen free chickens were assigned to either of the four treatments, which included two levels of antibiotics treatment (ABX- and ABX+) and two levels of H9N2 virus infection (H9N2- and H9N2+). At day 17 of age, chickens in the H9N2+ group were infected via the oral-nasal route with 400 µl of 107 TCID50/ml (200 µl/each route). Oropharyngeal and cloacal swabs at days 1, 3, 5, 7 and 9 post-infection (p.i.) for virus shedding, tissue samples at 12 h, 24 h and 36 h p.i. for mRNA measurement, and serum samples at days 7 and 14 p.i. for hemagglutination inhibition (HI) assay and IgG antibodies were collected. Virus shedding analysis showed that antibiotic treated (depleted)-H9N2 virus infected chickens showed a significantly higher oropharyngeal virus shedding at all time points, and cloacal shedding at days 3 and 5 p.i. compared to control treated (undepleted)-H9N2 infected chickens. Analysis of mRNA expression showed that infection of depleted chickens with H9N2 virus resulted in significantly down-regulated type I interferon responses both in the respiratory and gastrointestinal tracts compared to undepleted-H9N2 infected chickens. However, antibody-mediated immune response analysis showed a significantly higher HI antibody titre and IgG levels in the serum of chickens depleted with antibiotics and infected with H9N2 virus compared to undepleted-H9N2 infected chickens. In conclusion, findings from the current study suggest that the gut microbiota of chickens plays an important role in the initiation of innate responses against influenza virus infection, while the antibody-mediated immune response remains unaffected.

The association between subclinical mastitis around calving and reproductive performance in grazing dairy cows.

The objective of this study was to evaluate the effect of subclinical mastitis (SCM) on calving-to-first-service interval (CFS), calving-to-conception interval (CC), and on the number of services per conception (S/C) in grazing Holstein and Normande cows. Primiparous (n=43) and multiparous (n=165) cows were selected from five dairy herds. Two composite milk samples were aseptically collected from each cow at drying-off, and then every week during the first postpartum month. One sample was used for somatic cell count (SCC), and the other one for bacteriological analysis. Cows were followed up to 300 d after calving. Non-parametric and parametric survival models, and negative binomial regression were used to assess the association between SCM, evaluated by SCC and milk culture, and reproductive indices. Staphylococcus aureus, CNS, and Streptococcus uberis were the most frequent isolated pathogens. Subclinical mastitis in the first month of lactation was not associated with CFS; however, the CC interval was longer in cows with SCM compared to healthy cows, the former also had a higher number of S/C.

Short communication: Extended-spectrum cephalosporin-resistant Escherichia coli in colostrum from New Brunswick, Canada, dairy cows harbor blaCMY-2 and blaTEM resistance genes.

Dairy calves are colonized shortly after birth by multidrug resistant (MDR) bacteria, including Escherichia coli. The role of dairy colostrum fed to calves as a potential source of MDR bacteria resistance genes has not been investigated. This study determined the recovery rate of extended-spectrum cephalosporin-resistant (ESC-R) E. coli in colostrum from cows. The ESC-R E. coli isolates were further investigated to determine their phenotypic antimicrobial resistance pattern and the genes conferring ESC-R. Fresh colostrum was collected from 452 cows from 8 dairy herds in New Brunswick, Canada. The ESC-R E. coli was isolated from the colostrum by using the VACC agar, a selective media for extended-spectrum ß-lactamase producing Enterobacteriaceae. Minimum inhibitory concentration was determined for all the suspected ESC-R E. coli isolates using a commercial gram-negative broth microdilution method. Two multiplex PCR were conducted on all the suspected ESC-R E. coli isolates to determine the presence of the blaCTX-M (groups 1, 2, 9, and 8/25) blaCMY-2, blaSHV, and blaTEM resistance genes. The ESC-R E. coli were detected in 20 (4.43%) of the colostrum samples. At least 1 ESC-R E. coli isolate was detected in 6 (75%) of the dairy herds. All ESC-R E. coli had MDR profiles based on minimum inhibitory concentration testing. No blaCTX-M groups genes were detected; however, the blaCMY-2 gene was detected in 9 or 20 (45%) and blaTEM was detected in 7 of 20 (35%) of the ESC-R E. coli. No ESC-R E. coli had both blaCMY-2 and blaTEM resistance genes. This is the first report of blaCMY-2 and blaTEM genes found in E. coli isolates cultured from dairy colostrum to our knowledge.

Dietary supplementation with a nucleotide-rich yeast extract modulates gut immune response and microflora in weaned pigs in response to a sanitary challenge.

An experiment was carried out to evaluate the short-term effect of supplementing a nucleotide-rich yeast extract (NRYE) on growth performance, gut structure, immunity and microflora of piglets raised under sanitary and unsanitary conditions. A total of 84, 21-day old piglets were used in this study; 42 piglets were raised in a room designated as the clean room that was washed once per week, whereas the other 42 piglets were raised in a room designated as the unclean room in which 7 kg of manure from the sow herd was spread on each pen floor on day 1 and 7 and the room was not washed throughout the experiment. The pigs were fed a corn-soybean meal-based diet without or with 0.1% NRYE. Each treatment had 7 replicate pens in each room, and each pen housed 3 pigs. Feed disappearance and BW were recorded on day 1 and 14. On day 14, one pig per pen was euthanized to collect ileum, mesenteric lymph nodes and spleen tissues, and cecum and colon digesta. Overall, NRYE supplementation did not affect growth performance in both clean and unclean conditions, improved kidney weight in both clean (P=0.0002) and unclean room (P<0.0001) and tended to improve the villus height/crypt depth ratio in the clean room (P=0.073). Supplementing NRYE was associated with upregulation of Ileal programmed cell death gene-1 (P=0.0003), interleukin (IL)-1ß (P<0.0001), IL-6 (P=0.0003), IL-10 (P<0.0001) and tumor necrosis factor-α (TNF-α) (P<0.0001) in pigs raised in the unclean room. Supplementing the NRYE in pigs raised in the clean room suppressed growth of cecal Enterobacteriacea (P<0.0001) members and colonic Enterococcus spp. (P<0.019), improved proliferation of cecal Lactobacillus spp. (P<0.002) and colonic Clostridium cluster IV (P<0.011) and XVIa members (P<0.0002). Supplementing the NRYE in the unclean room improved proliferation of cecal Clostridium cluster IV (P<0.026) and suppressed proliferation of colonic Enterococcus spp. (P<0.037). In conclusion, supplementing the NRYE to piglets under unsanitary conditions improved ileal immune response by upregulating inflammatory cytokines, and positively modulated proliferation of beneficial gut bacteria and suppression of harmful ones in both clean and unclean rooms.

Maternal antibody decay and antibody-mediated immune responses in chicken pullets fed prebiotics and synbiotics.

Three experiments were conducted to evaluate the effect of yeast-derived carbohydrates (YDC), and a blend of probiotics and YDC (synbiotic, SNB) on serum IgG concentration, maternal-derived antibody (MDA) decay, and specific antibody-mediated immune response in chick pullets following immunization with T-cell dependent antigens. A total of 300 day-old pullet chicks were randomly assigned to 3 dietary treatments including: a basal diet (Control), and diets containing YDC, and SNB (Lactobacillus acidophilus, L. casei, Streptococcus faecium, and Bacillus subtilis, and YDC). In experiment one, on d 1 and wk 3, 4, 5, and 6, blood samples were collected and serum were analyzed by ELISA for total IgG (Y), and MDA against Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV). The second experiment examined the specific antibody against infectious bronchitis virus (IBV) in pullet chicks following vaccination against IBV at d 1. Finally, in experiment 3, on d 21 and 28 posthatch, 10 birds per treatment were immunized intramuscularly with both sheep red blood cells (SRBC) and bovine serum albumin (BSA), and 11 after immunization serum samples were analyzed by hemagglutination assay for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. The results demonstrated that diet containing SNB increased serum IgG at wk 3 posthatch. However, the decay rate of MDA against NDV and IBDV were not affected by dietary treatments. Birds fed YDC showed higher specific antibody response against IBV in wk 4, while both diets containing YDC and SNB decreased antibody response to IBV in wk 6. In addition, specific antibody response against SRBC and BSA was not affected by diets. In conclusion, supplementation of diet with SNB improved humoral immunity by increasing IgG concentration in serum, and modulated the adaptive antibody-mediated immune response against IBV.

The immunomodulatory effect of vitamin D in chickens is dose-dependent and influenced by calcium and phosphorus levels.

Vitamin D requirement is estimated to be higher than recommended values for the first two weeks of a broiler chicken's life, and is heavily dependent on the concentrations of Ca and P in the diet. There are data indicating the beneficial effect of higher vitamin D levels on performance and overall health of the chickens. However, data on the role of higher vitamin D levels on the innate immune response of chickens are limited. Therefore, in the current study, we examined the effect of higher doses of vitamin D supplementation on the innate immune response in broiler chickens receiving optimal or calcium (Ca) and phosphorus (P) deficient diets. Three hundred Ross-308 male broiler chicks were randomly allocated into 60 cages with 5 birds per cage in a 3 × 2 factorial design with three levels of vitamin D and two levels of Ca/P with each experimental diet fed to 10 cages (10 replicates). Quantitative reverse transcription PCR (n = 5) was used to assess Toll-like receptor (TLR2b and 4), cytokine/chemokine (IL-12, IFN-γ, IL-10, IL-4, IL-13, IL-18, CxCLi2) and cathelicidin (CATH1, CATHB1, CATH3) transcription levels in peripheral blood mononuclear cells (PBMCs), spleen, and bursa of Fabricius. Vitamin D supplementation of the Ca and P deficient diet considerably augmented transcription of TLR2b, TLR4, CATH1, and CATHB1 and predominantly Th2 cytokines in spleen. Supplementation of the control diet with vitamin D downregulated TLR4 transcription, and dose-dependently increased CATH1, CATHB1, Th1, and Th2 cytokine transcription (Th2>Th1). All diets downregulated CATH3 transcription. In conclusion, vitamin D or its derivative 25-OH-D3 both have a robust immunomodulatory property with a more favorable Th2 response, while at the same time enhancing observed Th2 cytokine responses under both optimal and lower Ca and P inclusion levels in the diets of broiler chickens.

Effect of yeast-derived products on systemic innate immune response of broiler chickens following a lipopolysaccharide challenge.

This study evaluated the effect of yeast-derived products on growth performance, serum antibody levels, and mRNA gene expression of pattern-recognition receptors, and cytokines in broiler chickens. Two hundred and sixteen one-day-old male broiler chickens (Ross-308) were randomly assigned to six dietary treatments with six replicates (cage) of 6 birds per cage. Dietary treatments consisted of a Control diet without antibiotics (C), 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 8% of distiller's dried grains with solubles (DDGS). On d 21 post-hatch blood samples were collected from 6 birds per treatment and serum sample were analyzed for antibody levels. After blood sampling, birds were injected intraperitoneally with 3 mg/kg of BW of lipopolysaccharide (LPS). The unchallenged group was fed the Control diet and injected with saline solution. Spleen samples were collected to measure the gene expression of toll-like receptors (TLR)2b, TLR4, and TLR21, macrophage mannose receptor (MMR), and cytokines including interleukin (IL)-12, IL-10, IL-4, IL-6, IL-18, and interferon (IFN)-γ. No significant difference in body weight gain, feed intake, and FCR were observed among treatments. Regarding humoral immunity, the diet supplemented with YCW increased serum immunoglobulin (Ig)A level compared with the antibiotic group; however, serum concentrations of IgG and IgM were not affected by dietary treatments. Relative gene expression of TLR2 and TLR4 was not affected by dietary treatments, whereas the expression of TLR21 and MRR was upregulated in diets containing YCW and DDGS. The diet supplemented with YCW increased the expression of all cytokines, and expression of IFN-γ was upregulated in the DDGS group. However, no significant difference was observed for cytokine gene expression in the antibiotic and nucleotide diets. In conclusion, supplementation of diet with YCW stimulated the systemic innate immune responses of broiler chickens following challenge with LPS.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance and local innate immune response of broiler chickens challenged with Clostridium perfringens.

This study evaluated the effect of yeast-derived products on growth performance, gut lesion score, intestinal population of Clostridium perfringens, and local innate immunity of broiler chickens challenged with C. perfringens. One-day-old broiler chickens were randomly assigned to eight dietary treatments providing six replicate pens of 55 birds each per treatment. Dietary treatments consisted of Control diets without and with C. perfringens challenge, and diets containing bacitracin methylene disalicylate (BMD, 55 g/tonne), nucleotides (150 g/tonne), yeast cell wall (YCW, 300 g/tonne), and a commercial product Maxi-Gen Plus (1 kg/tonne) fed to chickens challenged with C. perfringens. Diets containing 10% distillers dried grains with solubles without and with C. perfringens challenge were also used. Birds were orally challenged with C. perfringens (10(8) colony-forming units (cfu)/bird) on day 14. On day 21, intestinal samples were collected for gene expression analysis. Pathogen challenge significantly (P < 0.05) impaired feed intake, body weight gain, and feed conversion ratio (FCR) shortly after the challenge (14-21 days). Increased C. perfringens counts and intestinal lesion scores were observed for challenged birds except the BMD-containing diet. Over the entire trial (1-35 days), no difference in growth performance was observed except the BMD diet which improved FCR over the Control, challenged group. Birds receiving nucleotides showed increased expression of toll-like receptors and cytokines interleukin (IL)-4 and IL-18 compared to the Control, challenged group. Expression of macrophage mannose receptor and IL-18 was upregulated in birds receiving YCW. Increased expression of cytokines and receptors involved in innate immunity in broilers receiving nucleotides and YCW suggests the immunomodulatory properties of these products under pathogen challenge conditions.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

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 nucleotide-containing 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.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance, gut morphology, and gene expression of pattern recognition receptors and cytokines in broiler chickens.

An experiment was carried out to investigate the effect of yeast-derived products and distillers' dried grains with solubles (DDGS) on growth performance, small intestinal morphology, and innate immune response in broiler chickens from 1 to 21 d of age. Nine replicates of 5 birds each were assigned to dietary treatments consisting of a control diet without antibiotic (C), and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus, 0.025% of nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On d 21, 5 birds per treatment were euthanized and approximately 5-cm long duodenum, jejunum, and ileum segments were collected for intestinal morphology measurements. Cecal tonsils and spleen were collected to measure the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, macrophage mannose receptor (MMR), and cytokines IFN-γ, IL-12, IL-10, and IL-4. No significant difference was observed for growth performance parameters. However, diets containing 0.05% of nucleotides and YCW significantly increased (P < 0.05) villus height in the jejunum. Furthermore, the number of the goblet cells per unit area in the ileum was increased (P < 0.05) in diets supplemented with yeast-derived products. The expression of TLR2b in the spleen was down-regulated for diets supplemented with nucleotides and antibiotic. In addition, lower expression of TLR21 and MMR was observed in the spleen of birds receiving yeast-derived products and antibiotic. However, expression of TLR4 in the spleen was up-regulated in diets supplemented with YCW and nucleotides. The expression of IFN-γ and IL-12 was down-regulated in the spleen of birds fed diets supplemented with yeast-derived products. In addition, inclusion of YCW, Maxi-Gen Plus, or 0.05% of nucleotides down-regulated the expression of IL-10 and IL-4 in the cecal tonsils. In conclusion, down-regulation of receptors and cytokines in spleen and cecal tonsils of birds fed diets supplemented with yeast-derived products may suggest that yeast products do not exert immune stimulating effect under normal health conditions.

Organic trace mineral supplementation enhances local and systemic innate immune responses and modulates oxidative stress in broiler chickens.

The effect of organic trace mineral supplementation on performance, intestinal morphology, immune organ weights (bursa of Fabricius and spleen), expression of innate immune response related genes, blood heterophils/lymphocytes ratio, chemical metabolic panel, natural antibodies (IgG), and oxidative stress of broiler chickens was studied. A total of 1,080 day-old male broilers were assigned to 1 of 3 dietary treatments, which included basal diet with Monensin (control), control diet supplemented with bacitracin methylene disalicylate (BMD), and BMD diet supplemented with organic trace minerals (OTM). No difference in feed conversion ratio was observed among treatments; ileum histomorphological analysis showed a lower crypt depth, higher villi height/crypt depth ratio, and lower villi width in the OTM treatment compared to control. Furthermore, OTM treatment resulted in higher uric acid and lower plasma malondehaldehyde (MDA), indicating lower oxidative stress. Gene expression analysis showed that OTM treatment resulted in up-regulations of TLR2 bin the ileum, and TLR2b, TLR4, and IL-12p35 in the bursa of Fabricius, and down-regulation of TLR2b and TLR4 in the cecal tonsils. In the spleen, OTM treatment resulted in up-regulation of IL-10. In conclusion, OTM supplementation to broiler diets may have beneficial effects on intestinal development, immune system status, and survival by improving ileum histomorphological parameters, modulation of Toll-like receptors and anti-inflammatory cytokines, and decreasing level of MDA, which in conjunction could enhance health status.

Innate immune response of pullets fed diets supplemented with prebiotics and synbiotics.

Prebiotics and synbiotics are considered to be among the most promising replacements for in-feed antibiotic growth promoters (AGP) in poultry feed. The current study was designed to study the effect of Bacitracin methylene disalicylate (BMD) (Control), yeast-derived carbohydrates (YDC), and a blend of YDC and probiotics [Lactobacillus acidophilus, Lactobacillus casei, Streptococcus faecium, Bacillus subtilis, and YDC] (SNB) in the performance and innate immune response of pullets. Feed intake and BW were measured on a weekly basis. At the end of the study (d 21), 10 birds/treatment were sacrificed by cervical dislocation and ileum, cecal tonsil, and spleen samples were collected for gene expression analysis. No significant difference (P > 0.05) in feed intake and G:F was observed among treatments. In the second and third wk age, higher BW gain was observed in SNB treatment compared to control and both control and YDC treatments, respectively. Expression of TLR2b was upregulated in YDC and SNB in the ileum, and in SNB in the spleen (P < 0.05). Expression of TLR4 was downregulated in SNB in the cecal tonsil. Expression of TLR21 was downregulated in YDC in the ileum, while it was upregulated in SNB in the spleen (P < 0.05). In the ileum, YDC resulted in downregulated IL-12p35, CxCLi2, and IL-13, and SNB resulted in upregulated IL-6, interferon (IFN)-γ, and IL-4 (P < 0.05). In the cecal tonsil, YDC resulted in upregulated IL-12p35, IL-2, IL-13 and IL-10, and SNB resulted in downregulated IL-2 and upregulated IL-13 and IL-10 (P < 0.05). In the spleen, YDC resulted in dowregulated IL-2 and CxCLi2, and SNB resulted in upregulated IL-6, IFN-γ, IL-4, and IL-10 (P < 0.05). In conclusion, no change in performance was observed. Innate immune response analysis showed SNB with a more potent effect compared to YDC where the former showed a balanced T-helper (Th)-1/Th-2 response locally and a more Th-2-dependent response systemically; SNB might provide a more beneficial immune modulation with maintaining immune homeostasis, which was observed in a strong IL-10 response.

Evaluation of the systemic innate immune response and metabolic alterations of nonlactating cows with diet-induced subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) increases lipopolysaccharide endotoxin in the rumen, which might translocate into the systemic circulation, triggering a cascade of clinical and immunological alterations. The objective of this study was to characterize the clinical immune and metabolic responses to ruminal-derived lipopolysaccharide in nonlactating cows induced with SARA using 2 challenges, a grain-based SARA challenge (GBSC) or an alfalfa-pellet SARA challenge (APSC). Six dry, nonlactating Holstein cows were used in a 3 × 3 Latin square arrangement of treatments with 4-wk experimental cycles. All cows received the control diet containing 70% forage and 30% mixed concentrates (dry matter basis) for 3 wk. In wk 4, cows received a control diet, GBSC (38% wheat-barley pellets, 32% other mixed concentrate, and 30% forages), or APSC (45% mixed concentrate, 32% alfalfa pellets, and 23% other forages). Total plasma proteins and immunology-related proteins, acute phase proteins, blood cells, serum chemistry, mRNA gene expression of peripheral blood cell surface markers, and selected proinflammatory cytokines were evaluated. Ruminal pH was lower in both groups with induced SARA compared with a control group. Ruminal endotoxins were higher in GBSC; however, plasma endotoxin was not detected in any study group. No significant differences in feed intake, rectal temperature, white blood cell counts, or differentials were found between control and SARA challenge groups; changes in glucose, urea, Ca, and Mg were observed in SARA groups. Total plasma proteins were lower in both SARA groups, and acute phase proteins were higher in GBSC. The expression of CD14, MD2, and TLR4 mRNA in peripheral blood leukocytes was not affected by SARA induction. The induction of SARA as a result of GBSC or APSC challenge was successful; however, LPS was not detected in plasma. Changes in clinical, metabolic, and inflammatory responses were not observed in the SARA-challenged cows, suggesting that, in this study, SARA was not associated with a systemic response to inflammation.

Effect of supplementing direct-fed microbials on broiler performance, nutrient digestibilities, and immune responses.

Direct-fed microbials (DFM) are used to improve livestock health and performance. The effects of 2 DFM products, a blend of 3 Bacillus strains (DFMB) and a Propionibacteriumspp. (DFMP), on broiler performance, nutrient utilization, and immune responses were investigated. Day-old (n = 120) male broilers were divided into 24 groups of 5 birds and fed 3 wheat-based diets in mash form (8 groups per diet) from d 1 to 22. The control diet was fed without or with 7.5 × 10(4) cfu/g of either DFMB or DFMP. From d 19 to 21 fecal samples were collected for determination of total tract apparent retention (TTAR) of nutrients and AMEn. On d 21, feed intake and BW were determined. On d 22, 5 birds per treatment were killed by cervical dislocation to collect jejunal and ileal contents for determination of digesta viscosity and apparent ileal digestibility (AID) of nutrients, respectively, and ileum, cecal tonsil, and spleen tissues for Toll-like receptors (TLR) and cytokine expressions. Compared with the control, DFM did not affect BW gain and feed intake but DFMP reduced G:F (P < 0.01). Compared with the control (2,875 kcal/kg), birds fed on DFMB and DFMP had higher AMEn (2,979 and 2,916 kcal/kg, respectively; P < 0.05), whereas both DFM reduced the AID of DM (P < 0.001) and CP (P < 0.01). Furthermore, DFMP reduced TTAR of NDF (29.0 vs. 18.4%; P < 0.001), whereas both DFM increased TTAR of DM and fat (P < 0.001). Supplementing DFMP downregulated ileal expression of TLR-2b, IL-2, IL-4, IL-6, IL-10, and IL-13, whereas DFMB downregulated TLR-2b, IL-2, IL-4, and IL-6 in all 3 tissues, IL-10 in the spleen, and upregulated IL-13 in the spleen. In conclusion, the DFM did not improve performance but increased the AMEn of diet by possibly increasing DM and fat retention. Overall, both DFM showed an antiinflammatory effect in the ileum, but DFMB had more effects on local and systemic immunity than DFMP.

Effect of crowding stress and Escherichia coli K88+ challenge in nursery pigs supplemented with anti-Escherichia coli K88+ probiotics.

Under commercial conditions and during production periods, pigs are generally exposed to a number of stressors that may have direct or indirect influence on their performance and general health. As a result, environmental stressors can influence the productivity as well as the onset and severity of infectious diseases. The aim of this study was to investigate the effects of crowding stress on performance and some immunological responses in piglets challenged with enterotoxigenic Escherichia coli (ETEC) K88(+) and fed diets containing anti-ETEC probiotic strains derived from E. coli in a 3-wk trial. Ninety-six piglets at 20 ± 1 d of age were randomly assigned to 6 experimental treatments with 3 pigs/pen and 5 replicates/treatment and fed a basal mash diet. To model stress, piglets were housed in 2 kinds of pens: standard floor allowance (SFA; no stress) and half standard floor allowance (HSFA; stressed). The treatments were 1) control pigs with SFA, 2) control pigs with HSFA, 3) pigs with SFA and challenged with ETEC (ESFA), 4) pigs with HSFA and challenged with ETEC (EHSFA), 5) pigs with ESFA and supplemented with a cocktail of E. coli probiotics (PSFA), and 6) pigs with EHSFA and supplemented with a cocktail of E. coli probiotics (PHSFA). After 4 d acclimatization, crowding stress was started on d 5, E. coli probiotics were given daily from d 5, and ETEC challenge was administered on d 8 and 10. Body weight and feed disappearance were determined weekly and severity of diarrhea was characterized daily. Blood samples were collected for biochemistry and inflammatory analysis and pigs were euthanized to obtain digesta for bacterial enumeration. The ADG and ADFI decreased (P < 0.05) in stressed and ETEC-challenged pigs 5 d after ETEC challenge. The ETEC population was higher in both ileal and colon digesta of stressed pigs whereas ETEC-challenged and probiotic-fed pigs had higher fecal consistency scores 96 h and 7 d after ETEC challenge. Stressed pigs had higher (P < 0.001) levels of neutrophils, neutrophil:lymphocyte ratio, albumin:globulin ratio, fibrinogen, total protein:fibrinogen ratio, and cortisol. Generally, the levels of cortisol, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) differed over time among some treatment groups. In conclusion, reduced space allowance stress and ETEC challenge depressed certain aspects of performance and immunological responses and increased E. coli counts; however, probiotics feeding had little positive effects on these measures suggesting further research.