Commensal gut microbiota plays an important role in health and disease. The current study was designed to assess the role of gut microbiota of chickens in the initiation of antiviral responses against avian influenza virus. Day-old layer chickens received a cocktail of antibiotics for 12 (ABX-D12) or 16 (ABX-D16) days to deplete their gut microbiota, followed by treatment of chickens from ABX-12 with five Lactobacillus species combination (PROB), fecal microbial transplant suspension (FMT) or sham treatment daily for four days. At day 17 of age, chickens were challenged with H9N2 virus. Cloacal virus shedding, and interferon (IFN)-α, IFN-β and interleukin (IL)-22 expression in the trachea, lung, ileum and cecal tonsils was assessed. Higher virus shedding, and compromised type I IFNs and IL-22 expression was observed in ABX-D16 chickens compared to control, while PROB and FMT showed reduced virus shedding and restored IL-22 expression to levels comparable with undepleted chickens. In conclusion, commensal gut microbiota of chickens can modulate innate responses to influenza virus subtype H9N2 infection in chickens, and modulating the composition of the microbiome using probiotics- and/or FMT-based interventions might serve to promote a healthy community that confers protection against influenza virus infection in chickens.

Nucleotide-rich yeast extract (YN) was investigated for effects on growth performance, jejunal physiology, and cecal microbial activity in Eimeria-challenged broiler chickens. A total of 360-day-old male chicks (Ross × Ross 708) were placed on floor pens and provided a corn–soybean meal-based diet without or with YN (500 g/MT; n = 12). On d 10, 6 replicates per diet were orally administered with 1 mL of E. acervulina and E. maxima sporulated oocysts and the rest (non-challenged control) were administered with 1 mL of distilled water. On d 15, 5 birds/pen were then necropsied for intestinal lesion scores, histomorphology and cecal digesta pH, short chain fatty acids (SCFA), and microbial community using Illumina Miseq platform. Supplemental YN improved (P = 0.01) Feed conversion ratio (FCR) during the prechallenge phase (d 0 to 10). In the postchallenge period (d 11 to 15), Eimeria depressed (P < 0.05) Body weight gain (BWG) relative to non-challenged birds, whereas YN-fed birds had a higher (P = 0.05) BWG compared to that of non-YN-fed birds. There was an interaction between YN and Eimeria on jejunal villi height (VH) (P = 0.001) and expression of cationic amino acid transporter 1(CAT1) (P = 0.04). Specifically, in the absence of Eimeria, YN-fed birds had a shorter VH (892 vs. 1,020 μm) relative to that of control but longer VH (533 vs. 447 μm) in the presence of Eimeria. With respect to CAT1, YN-fed birds had a higher (1.65 vs. 0.78) expression when subjected to Eimeria than when not challenged. Independently, Eimeria decreased (P < 0.01) the jejunal expression of maltase, Na glucose transporter 1 and occludin genes, ceca digesta abundance of genus Clostridium cluster XlVa and Oscillibacter but increased (P < 0.01) jejunal proliferating cell nuclear antigen and interleukin 10. Interaction between YN and Eimeria was observed for ceca digesta pH (P = 0.04) and total SCFA (P = 0.01) such that YN increased SCFA in the absence of Eimeria but reduced SCFA and increased pH in the presence of Eimeria. In summary, Eimeria impaired performance and gut function and shifted gut microbiome; YN improved performance independently, attenuated Eimeria damage on indices of gut function, and modulated cecal microbiome.

A study was conducted to assess the effect of yeast-derived carbohydrates (YDC) on performance and innate immune responses of broiler chickens. In total, 1,080 one-day-old birds were randomly assigned to one of 3 dietary treatments (n = 360): a standard broiler diet containing monensin (control), control + bacitracin methylene disalycylate (BMD), and YDC treatment (control + YDC at 0.02%, 0.01%, and 0.005% for starter, grower, and finisher, respectively). Weekly BW, feed intake (FI), and feed conversion ratio (FCR) were recorded. Immune organ weights, gut morphology, gene expression, heterophil:lymphocyte (H:L), and serum IgG were determined at d 42. No significant difference in FCR, FI, and mortality was observed among treatments. However, BW gain in starter phase was higher in control and YDC treatments compared with BMD treatment. Ileal villi height, crypt depth, and their ratio were not significantly different among treatments, whereas villi width was lower in control and YDC treatments compared with BMD treatment. The number of goblet cells per unit area in the ileum was lower in BMD treatment compared with control and YDC treatments. Expression of TLR2b and IL-6 in the ileum and cecal tonsils was not significantly different among treatments (P > 0.05). Expression of TLR4 was downregulated in YDC treatment compared with control in the ileum. Expression of IL-12p35 and IFN-γ were downregulated in the YDC treatment only in the cecal tonsils. Compared with the control, the expression of IL-10 in both the ileum and the cecal tonsils was downregulated in YDC treatment. Serum IgG and H:L ratio were lower and higher, respectively, in the YDC treatment compared with control and BMD treatments. In conclusion, dietary inclusion of YDC affected intestinal cytokines anti-inflammatory profile on a gut location associated immune pathways manner, suggesting different immune pathways that require further studies in this field.

Necrotic enteritis (NE) caused by Clostridium 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 × 10(10) 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/mm(2) (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, TLR4, interleukin (IL)-12p35, and interferon (IFN)-γ 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 TLR4 (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 TLR2 and TLR4 pathways were associated with MOS supplementation with subsequent upregulation of ileal IL-12p35 and IFN-γ, implying that MOS supplementation in C. perfringens-challenged chickens supports a proinflammatory effect via T-helper cell-1 associated pathways.

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 TCID/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.