Avian Β-defensins Research Articles

Effect of probiotic supplementation on the expression of tight junction proteins, innate immunity-associated genes, and microbiota composition of broilers subjected to cyclic heat stress.

This study investigated the effects of probiotic on intestinal innate immunity-associated gene expression and cecal microbiota in heat-stressed broilers. A total of 180 21-day-old male broilers were randomly assigned to three treatment groups with four replicates per group. The thermoneutral group (TN) (23 ± 1°C) received a basal diet, and another two heat-stressed groups (28-35-28°C for 12 h daily) were fed the basal diet (HS) or the basal diet supplemented with probiotic at a dose of 1.5 × 108 CFU/kg (HS_Pro) for 21 consecutive days. Compared with the TN group, the abundance of beneficial bacteria was decreased (p < 0.05) in the caecum of heat-stressed broilers. Heat stress downregulated (p < 0.05) the expression of Toll-like receptor (TLR)2 and upregulated (p < 0.05) the expressions of TLR5, TLR15, avian β-defensin (AvBD)4, AvBD8, and AvBD14 in the ileum as compared with the TN group. Dietary supplementation of probiotic upregulated (p < 0.05) the occludin expression in the ileum, improved the microbiota balance in the caecum, and decreased (p < 0.05) the gene expressions of TLR5 and TLR15 in the ileum of heat-stressed broilers. Collectively, dietary probiotic supplementation could promote intestinal barrier function via improving gut microbiota community and regulating innate immunity-associated gene expressions in heat-stressed broilers.

Synergistic Induction of Chicken Antimicrobial Host Defense Peptide Gene Expression by Butyrate and Sugars.

Antimicrobial resistance is a major concern to public health demanding effective alternative strategies to disease control and prevention. Modulation of endogenous host defense peptide (HDP) synthesis has emerged as a promising antibiotic alternative approach. This study investigated a potential synergy between sugars and butyrate in inducing HDP gene expression in chickens. Our results revealed that sugars differentially regulated HDP expression in both gene- and sugar-specific manners in chicken HD11 macrophage cells. Among eight mono- and disaccharides tested, all were potent inducers of avian β-defensin 9 (AvBD9) gene (p<0.05), but only galactose, trehalose, and lactose obviously upregulated cathelicidin-B1 (CATHB1) gene expression. The expression of AvBD14 gene, on the other hand, was minimally influenced by sugars. Moreover, all sugars exhibited a strong synergy with butyrate in enhancing AvBD9 expression, while only galactose, trehalose, and lactose were synergistic with butyrate in CATHB1 induction. No synergy in AvBD14 induction was observed between sugars and butyrate. Although lactose augmented the expression of nearly all HDP genes, its synergy with butyrate was only seen with several, but not all, HDP genes. Mucin-2 gene was also synergistically induced by a combination of lactose and butyrate. Furthermore, lactose synergized with butyrate to induce AvBD9 expression in chicken jejunal explants (p<0.05). Mechanistically, hyper-acetylation of histones was observed in response to both butyrate and lactose, relative to individual compounds. Mitogen-activated protein kinase, NF-κB, and cyclic adenosine monophosphate signaling pathways were also found to be involved in butyrate- and lactose-mediated synergy in AvBD9 induction. Collectively, a combination of butyrate and a sugar with both HDP-inducing and barrier protective activities holds the promise to be developed as an alternative to antibiotics for disease control and prevention.

Baicalin ameliorates APEC-induced intestinal injury in chicks by inhibiting the PI3K/AKT-mediated NF-κB signaling pathway

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis. Baicalin (BA) possesses multiple pharmacological effects, but the mechanism underlying its activity in APEC-induced intestinal injury remains unknown. This study aims to investigate the protective effects and possible mechanism of BA against APEC-induced intestinal injury. Sixty 1-day-old chicks were randomly divided into 4 groups: the control group (basal diet), E. coli group (basal diet), BAI10 group (10 mg/kg BA), and BAI20 group (20 mg/kg BA). After pretreatment with BA for 15 d and subsequent induction of APEC infection by pectoralis injection, the ileum was collected and analyzed. The results showed that BA-pretreatment demonstrated an alleviation of chicks in diarrhea rate, mortality, and histopathological changes in intestinal tissues after APEC infection. Additionally, following APEC infection, BA improved the intestinal barrier by elevating zona occludens (ZO)s (ZO-1, 2, 3), Claudins (Claudin1, 2, 3), Occludin, avian β-defensin (AvBD)s (AvBD1, 2, 4), lysozyme (Lyz) mRNA levels and ZO-1, Claudin1, and Occludin protein levels. Besides, the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and the SOD-1 and CAT mRNA levels and SOD-1 protein level were elevated by BA pretreatment. BA pretreatment also decreased the malondialdehyde (MDA) content, heme oxygenase-1 (HO-1) and NADH quinone oxidoreductase 1 (NQO1) mRNA levels, and HO-1 protein level after APEC infection. BA alleviated the APEC-induced inflammatory response, including downregulating the mRNA levels of proinflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin [IL]-1β, IL-6, IL-8) and upregulating the mRNA levels of anti-inflammatory cytokines (IL-4, IL-10, IL-13, transforming growth factor-β [TGF-β]). Furthermore, BA decreased the mRNA and protein levels of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and nuclear factor kappa-B (NF-κB) as well as the expression of the phosphorylated forms of these proteins after APEC infection. Collectively, our findings indicate that BA exerts a protective effect against APEC-induced intestinal injury in chicks by inhibiting the PI3K/AKT-mediated NF-κB pathway, suggesting that BA may be a potential therapeutic approach for avian colibacillosis.

Upregulation of gut cathepsin L during Eimeria tenella infection

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial β-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian β-defensins that are key molecules of innate immunity.

Developmental and Tissue Patterns of the Basal Expression of Chicken Avian β-Defensins.

Defensins are a class of antimicrobial peptides in vertebrates that function as the first line of innate immunity with potent antimicrobial and immunomodulatory activities. Fourteen defensins, namely, avian β-defensin 1 to 14 (AvBD1-14), have been identified in chickens. Before characterizing the role of AvBDs in innate immunity during the early development of chickens, we collected tissue segments from the liver, spleen, and gastrointestinal (GI) tract including the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon from broilers at days 1, 3, 7, 14, and 28. After RNA isolation and reverse transcription, we determined the expression levels of the 14 AvBD genes in these tissues during the first 28 days after hatching by real-time PCR. The results suggested the AvBDs were widely expressed in the chicken liver, spleen, and gastrointestinal (GI) tract. Interestingly, we did not detect AvBD11 expressed in the GI tract, even in the liver and spleen. Additionally, AvBDs were differentially expressed in the chicken GI tract. AvBD5 and AvBD14 were expressed most abundantly in the proximal GI tract, especially the esophagus and crop. Moreover, AvBD5, AvBD7, AvBD9, and AvBD14 were expressed in an inverted-V pattern with the peak being the observed expression at days 3, 7, or 14 in the chicken spleen, esophagus, duodenum, and cecum. Other AvBDs presented biphasic or inverted-V expression patterns in different tissues. The expression levels of all detected AvBDs were strengthened after hatching rather than decreasing steadily. Therefore, AvBDs were found to be expressed widely in the chicken liver, spleen, and GI tract and their expression levels were primarily up regulated during the early development of chicken, implying the potential essential roles of AvBDs in early innate defense and infection resistance of chickens.

Effects of avian infectious bronchitis with Newcastle disease and Marek's disease vaccinations on the expression of toll-like receptors and avian β-defensins in the kidneys of broiler chicks

The aim of this study was to determine the effect of vaccinations for avian infectious bronchitis with Newcastle disease (IB/ND) and Marek's disease (MD) on the expression of toll-like receptors (TLR) that recognize viral RNA and microbial DNA, and AvBD in chick kidneys. Day-old chicks were vaccinated with MD or IB/ND vaccines or received no treatment (control group). The gene expression of TLR and AvBD in the kidneys of 3-day-old chicks and 10-day-old chicks was examined using real-time PCR. The localization of AvBD2 and AvBD4 was examined by immunohistochemistry at day three only. At 3 days of age, the expression of TLR7 and TLR21 was significantly higher in the IB/ND group (but not in the MD group) than in the control group. Conversely, at 10 days of age there was no significant difference in the expression of the three TLR between groups. In the 3-day-old chicks the expression levels of AvBD4, 5, 6, and 7 were higher in the MD group than in the control group. Furthermore, at this age, the expression levels of other AvBD were not significantly different between the control and vaccination (MD and IB/ND) groups. At 10 days of age, no AvBD expression was affected by MD and IB/ND vaccinations. Immunohistochemistry results localized AvBD2 in the leukocytes in the interstitial tissue and AvBD4 in the surface of microvillus epithelial cells of renal tubules, and in the epithelial cells of the collecting ducts and ureter. The localization of AvBD2 and AvBD4 was identified in all chicks. We suggest that the expression of innate immune molecules (including TLR and AvBD) in kidneys could be modulated by MD and IB/ND vaccination when performed at the day-old stage. Although the effects of both vaccinations may subside within 10 days, the enhanced expression of those innate immune molecules may support the innate immunodefense function in the kidneys of young chicks.

Analysis of the Diversity of the AvBD Gene Region in Japanese Quail.

The avian β-defensin (AvBD) gene region is an important component of the innate immune system, encoding a variety of antimicrobial peptides. The AvBD region forms a multigene cluster in a specific chromosomal region. Comparison of the AvBD region among various birds suggests the presence of defects, duplications, and pseudogenization at many loci. The AvBD region in certain galliform birds, namely chicken, turkey, and bobwhite quail, includes AvBD3, -6, and -7, with the latter exhibiting copy number variants (CNVs) in chickens. DNA for genomic analysis was extracted from the peripheral blood of 99 randomly selected quail (Coturnix japonica) from 6 inbred lines. Nine CjAvBD1 and 8 CjAvBD12 alleles were detected. Ten haplotypes, including three that were strain specific, were found in alleles from the quail AvBD1 (CjAvBD1) and -12 (CjAvBD12) loci. Next-generation sequencing was used to determine the nucleotide sequences of the CjAvBD gene region (56-70 kb) for 7 homozygous diplotypes of these 10 haplotypes. These 7 haplotypes contained between 12 and 16 CjAvBD genes and were composed of 11 common loci: CjAvBD1, -2, -4, -5, -8, -9, -10, -11, -12, -13, and -14, but lacked CjAvBD3 and -7. Furthermore, up to 5 CjAvBD101 (AvBD6 ortholog) CNVs were observed among the 7 haplotypes. In addition, we detected amino acid substitutions causing net charge mutations that could affect antimicrobial activity in CjAvBD4, -13, -14, and -101. These results suggest that the CjAvBD region is unique among the Galliformes and that its diversity results in potential functional variation in innate immunity.

Peptidoglycan derived from Lactobacillus rhamnosus MLGA up-regulates the expression of chicken β-defensin 9 without triggering an inflammatory response.

Defensins are critical components of the innate immune system and play an important role in the integration of innate and adaptive immune responses. Although information on the immunomodulatory properties of peptidoglycan from bacteria is abundant, little is known about the β-defensin induction effect of peptidoglycan from the probiotic Lactobacillus. This study investigated the effect of intact peptidoglycan from L. rhamnosus MLGA on the induction of avian β-defensin 9 in chicken immune cells and intestinal explants. Peptidoglycan from Lactobacillus rhamnosus MLGA dose dependently promoted avian β-defensin 9 mRNA expression in chicken PBMCs, splenocytes, thymocytes, hepatocytes, and chicken embryo jejunum, ileum, and cecum explants and increased the capacity of PBMC or splenocyte lysates to inhibit the growth of Salmonella Enteritidis. In contrast to the effect of L. rhamnosus MLGA-derived peptidoglycan, peptidoglycan derived from pathogenic Staphylococcus aureus reduced avian β-defensin 9 mRNA expression in chicken PBMCs and splenocytes. The inducible effect of peptidoglycan from L. rhamnosus MLGA on avian β-defensin 9 expression in PBMCs and splenocytes was observed without activation of the expression of associated pro-inflammatory cytokines IL-1β, IL-8, and IL-12p40, whereas these cytokine expressions were suppressed by peptidoglycan hydrolysate obtained by lysozyme digestion. The results of the present study show the capability of peptidoglycan derived from L. rhamnosus MLGA to induce the antimicrobial peptide defensin while simultaneously avoiding the deleterious risks of an inflammatory response.

Chicken avian β-defensin 8 modulates immune response via the mitogen-activated protein kinase signaling pathways in a chicken macrophage cell line

Defensins are antimicrobial peptides composed of 3 conserved disulfide bridges, a β-sheet, and both hydrophobic and cationic amino acids. In this study, we aimed to demonstrate the immunomodulation role of avian β-defensin 8 (AvBD8) in a chicken macrophage cell line. Chicken AvBD8 stimulated the expression of proinflammatory cytokines (IL-1β, interferon gamma, and IL-12p40) and chemokines (CCL4, CXCL13, and CCL20) in macrophages. Furthermore, by Western blotting and immunocytochemistry, we confirmed that AvBD8 activated the mitogen-activated protein kinase signaling pathway via extracellular regulated kinases 1/2 and p38 signaling molecules. Overall, AvBD8 plays a crucial role in host defense as not only an antimicrobial peptide but also an immunomodulator by activating the mitogen-activated protein kinase signaling pathway and inducing the expression of proinflammatory cytokines and chemokines.

Immunomodulatory effects of avian β-defensin 5 in chicken macrophage cell line

Antimicrobial peptides (AMPs) protect host from pathogens as first line of defense. Especially, β-defensins shows antimicrobial activity and immune modulation effects. Avian species also have β-defensins as avian β-defensins (AvBDs) from AvBD1 to AvBD14. In this study, we characterized chicken AvBD5 and demonstrated its immune modulatory functions in chicken macrophage cell line (HD11). Chicken AvBD5 is composed of a signal, pro, and mature peptides containing one α-helix, four β-sheet, and three disulfide bonds. Here, we also showed that chicken AvBD5 induced Th1, Th2, and Th17 cytokines in chicken macrophage cell line and stimulated MAPK signaling pathways through ERK1/2 and p38 molecules. In addition, AvBD5 stimulated MyD88 and CD40 to regulate immune systems. Taken together, chicken AvBD5 can modulate host immune systems by inducing cytokines expression and stimulating MAPK signaling pathway.

Effect of antibiotic treatment on microbial composition and expression of antimicrobial peptides and cytokines in the chick cecum

The aim of this study was to confirm whether the expression of innate immune molecules in the chick cecum is altered in association with changes in the composition of the intestinal microbiome that are regulated by treatment with antibiotics. Broiler chicks were administered with antibiotics (penicillin and streptomycin) daily, and the composition of the microbiota, expression of innate immune molecules, and localization of antimicrobial peptides in the chick cecum were examined at day 7 and day 14 using real-time PCR and immunohistochemistry. The oral administration of antibiotics caused an increase in the relative frequency of the Enterobacteriaceae family and a decrease in some gram-negative (Barnesiellaceae) and gram-positive bacterial (Clostridiaceae and Erysipelotrichaceae) families. The gene expression levels of immune molecules, including 4 Toll-like receptors (TLR) (TLR 2, 4, 5, and 21), inflammation-related cytokines (IL-1β, TGFβ3, TGFβ4, and IL-8), and antimicrobial peptides (avian β-defensins and cathelicidins) showed a tendency to decrease with antibiotic treatment at day 7. However, expression levels of TLR21 and some cytokines (IL-1β, TGFβ3, and IL-8) were higher in the cecum or cecal tonsils of the antibiotic-treated group than in those of the control at day 14. The immunoreactive avian β-defensin 2 and cathelicidin 1 proteins were localized in the leukocyte-like cells in the lamina propria, and they were aggregated in the form of small islands. We conclude that the expression of innate immune molecules, including TLR, inflammation-related cytokines, and antimicrobial peptides, in the cecum are altered in association with changes in the density or composition of the luminal microbiota during the early phase of life in chicks.

Tissue-, age- and dose-dependent changes in avian β-defensin and LEAP2 mRNA abundance in the intestines of Salmonella Typhimurium challenged broilers

Salmonella is a pathogen normally found in the gastrointestinal tract of poultry. The objective of this study was to determine changes in avian β-defensin (AvBD) and liver-enriched antimicrobial peptide 2 (LEAP2) mRNA following Salmonella challenge. Day of hatch chicks were challenged with 106, 107 or 108 colony-forming units (cfu) of Salmonella typhimurium. There were dose-, tissue- and age-specific changes in AvBD and LEAP2 mRNA. At 1-day post-infection (dpi) there was a transient upregulation of AvBD1, 8, 10 and 12 mRNA in the 108 cfu group. At 5 dpi, all seven AvBD mRNA were downregulated in the ileum, while only AvBD1, 6, 10 and 11 mRNA were downregulated in the jejunum and AvBD6, 8, 10, 12 and 13 were downregulated in the cecum. At 7 dpi, there was downregulation of all seven AvBD mRNA in the duodenum and downregulation of selected AvBD in the jejunum, ileum and cecum. LEAP2 mRNA was downregulated at all doses of Salmonella in the cecum at 1 dpi and in the ileum at 5 dpi. In summary, Salmonella infection caused an initial upregulation followed by a downregulation of AvBD mRNA.

Effects of Toll-like Receptor Ligands on the Expression of Proinflammatory Cytokines and Avian β-defensins in Cultured Chick Intestine.

Few studies have focused on the regulation of cytokine and avian β-defensin (AvBDs) expression for promoting immune defense in the avian intestine. The aim of this study was to investigate the effects of different Toll-like receptor (TLR) ligands (bacterial patterns) on the expression of proinflammatory cytokines (IL-1β and IL-6) and AvBDs (AvBD1, AvBD4, and AvBD7) in the chick intestine. The ileum and cecum of 3-day-old chicks were collected and examined histologically to identity the cells present in the intestinal mucosa. Other tissues were cultured with or without the TLR2, TLR4, and TLR21 ligands—Pam3CSK4, LPS, and CpG-ODN—for 1 or 3 h. The gene expression profiles of proinflammatory cytokines and AvBDs were determined in these tissues using real-time polymerase chain reaction (PCR). The mucosa of the ileum and cecum contained leukocytes, luminal and crypt epithelial cells, and other enterocytes. Pam3CSK4 tended to downregulate the expression of IL-1β, AvBD1, and AvBD7 in the ileum but upregulated their expression in the cecum. LPS downregulated the expression of IL-1β and IL-6 in both the ileum and the cecum, whereas it upregulated the expression of AvBD1, AvBD4, and AvBD7 in the cecum. CpG-ODN upregulated the expression of IL-6 and AvBD7 in the ileum and IL-1β in the cecum, and downregulated the expression of IL-1β and AvBDs in the ileum. We suggested that the expression levels of proinflammatory cytokines and AvBDs in the chick intestine are affected by TLR2, TLR4, and TLR21 ligands. Thus, these innate immune factors may be modulated by the luminal microbe complex in the intestine.

Effects of Probiotics Lactobacillus reuteri and Clostridium butyricum on the Expression of Toll-like Receptors, Pro- and Anti-inflammatory Cytokines, and Antimicrobial Peptides in Broiler Chick Intestine.

The aim of this study was to determine the effects of live probiotics Lactobacillus reuteri (LR) and Clostridium butyricum (CB) on the expression of genes of innate immune system in broiler chick ileum and cecum. Chicks were administered 500 µl water with or without LR or CB, daily from day 1 to 6 after hatching. The ileum and cecum were collected on day 7 for analysis of gene expression of Toll-like receptors (TLRs), pro- and anti-inflammatory cytokines, and antimicrobial peptides (AMPs) using real-time PCR. The expression of TLR2-1 was upregulated by CB in the ileum and that of TLR5 was upregulated by both LR and CB. Expression of IL-1β and TGFβ2 in the ileum and of TGFβ3 and TGFβ4 in the cecum was upregulated by both LR and CB. The gene expressions of avian β-defensin (AvBD) 1 and cathelicidin (CATH) 3 were upregulated by CB and that of AvBD4 was upregulated by LR in the cecum. However, the expression of CATH2 in the ileum was downregulated by LR. These results suggest that probiotic LR and CB treatments affect a part of the innate defense system in the ileum and cecum by modulating the expression of innate immune molecules including TLRs, pro- and anti-inflammatory cytokines, and AMPs.

Effects of Oral Administration of Lactobacillus reuteri on Mucosal Barrier Function in the Digestive Tract of Broiler Chicks.

Probiotic bacteria are known for their beneficial effects on the intestinal immune function of the host animal. However, their effects on mucosal barrier function in chicks are not completely understood. The aim of this study was to determine the effects of the probiotic bacterium, Lactobacillus reuteri (LR), on the gastrointestinal mucosal barrier function of broiler chicks. One day-old male broiler chicks were orally injected water (300 µL) with or without 1 × 108 cfu of LR (5 mg FINELACT, Asahi Calpis Wellness Co. Ltd.) every morning for 7 days (day 0 to 6). The crop, duodenum, ileum, and cecum were collected on day 7 and were used for histological analysis and RNA extraction. Then, the thickness of the mucosal structures and the number of goblet cells in the digestive tract were assessed using histological analysis. The expression of Mucin 2, factors related to the formation of tight junctions (Claudin1, 5, and 16, ZO2, and JAM2), cytokines (IL-6, CXCLi2, and IL-10), and avian β-defensin 10 (AvBDs) (AvBD2, 10, and 12) in the crop, duodenum, ileum, and cecum were analyzed using real-time polymerase chain reaction (PCR). Results showed that oral administration of LR increased ileal villus height and crypt depth, decreased Claudin16 level in the crop and increased JAM2 level in the crop and ileum, and decreased the expression of AvBD10 in the ileum and cecum and that of AvBD12 in the crop. It did not affect goblet cell number and Mucin 2 expression. These results suggested that LR used in this study may enhance mucosal barrier function by regulating tight junctions in the upper gastrointestinal tract.

Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-β-defensin family

Out of the 14 avian β-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian β-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to β-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed β-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-β-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

Fowl adenoviruse-4 infection induces strong innate immune responses in chicken

Fowl adenovirus (FAdV), as the causative agent of hepatitis-hydropericardium syndrome (HHS), poses a significant threat to the poultry industry in China in recent years. In this study, we investigated the immunopathogenesis of a FAdV-4 strain HN/151025 in 60-day-old chickens. The virus was highly virulent in chickens, with a broader tissue tropism in chickens, causing 60 % mortality. Postmortem findings of dead chickens showed mild HHS and liver degeneration and necrosis. Importantly, FAdV-4 infection induced significant upregulation of genes encoding most toll-like receptors, some cytokines (interleukin-1β, 2, 6, 8, and 18, and interferon-γ), most of avian β-defensins, myeloid differentiation primary response protein 88, p38 mitogen-activated protein kinases, and inducible nitric oxide synthase, in tissues of infected chicken, especially in spleen and bursa of Fabricius. There was also a significant positive correlation between FAdV-4 genome load and the mRNA expression levels of most of these factors in specific infected tissues. The results indicated the potential role of these proteins in host immune response against FAdV-4 infection. However, overexpression of these proteins might contribute to tissue damage of FAdV-4 infected chickens, and eventually lead to chicken death.

Effects of the routine multiple vaccinations on the expression of innate immune molecules and induction of histone modification in ovarian cells of layer chicks

The aim of this study was to determine whether vaccination affects the expression of Toll-like receptors (TLRs), cytokines, and avian β-defensins (AvBDs) in the chick ovary with or without lipopolysaccharide (LPS) stimulation. White Leghorn female chicks were administered vaccines for infectious bronchitis, Marek's disease, Newcastle disease, and infectious bursal disease during the first 14 D after hatching and ovarian tissues were collected on day 21. Control chicks received water or dilution buffer in place of vaccine. In Experiment 1, ovarian tissues were incubated with or without LPS, and the expression of innate immune molecules (TLRs, cytokines, and AvBDs) was examined by real-time PCR. In Experiment 2, the levels of histone modification in fresh ovarian tissues were examined by western blot analysis. The results of Experiment 1 showed that, in vaccinated chick ovaries, the expression of TLR1-1, 2-1, 2-2, and 21 was up-regulated, whereas that of TLR1-2, 4, and 7 was down-regulated under LPS stimulation. Among the examined 6 cytokines, only the expression of TNFSF15 was lower in the ovaries of vaccinated chicks than that in control with or without LPS stimulation. The expression of AvBD1, 2, 4, and 7 was lower in the ovaries of vaccinated chicks than in control without LPS stimulation, and that of AvBD1 and 2 was also lower even in ovaries incubated with LPS. In Experiment 2, the density of di-methyl histone H3 (Lys9) and acetyl histone H3 (Lys9) was significantly higher in the vaccine group than in the control, whereas di-methyl and tri-methyl histone H3 (Lys4) and acetyl histone H3 (Lys27) did not show differences between the groups. These results suggest that vaccination positively or negatively affects the expression of innate immune molecules in the chick ovary including TLRs, TNFSF15, and AvBDs, and it may be associated with epigenetic reprogramming by histone modifications in ovarian cells. Thus, in the future, it may be possible to develop or improve vaccination programs for the enhancement of the innate immune system in the hen ovary.

Lactobacillus reuteri Enhances the Mucosal Barrier Function against Heat-killed Salmonella Typhimurium in the Intestine of Broiler Chicks.

Salmonella Typhimurium (ST) infection in chickens inhibits their growth and can lead to food-borne diseases in humans. Probiotics are expected to enhance the function of host intestinal barrier against pathogen infection. The aim of our study was to determine the effect of viable Lactobacillus reuteri (LR) on the response of the mucosal barrier function to antigen stimulation in broiler chicks. Day-old male (n=8) and female (n=4) broiler chicks were orally administered either 1 × 108 LR or a water-only control, every day for 7 days. After 7 days, either 1 × 108 heat-killed ST (k-ST), or a buffer-only control, was administered via intra-cardiac injection. The ileum and cecum were collected 3 h post-injection, and paraffin sections were prepared for either mRNA extraction (males), or gut permeability tests (females). Villus and crypt lengths were determined via histological analysis. Real-time PCR was used to calculate expression levels of Toll-like receptors (TLRs), pro-inflammatory cytokines, anti-inflammatory cytokines, avian β-defensins, and tight-junction-associated molecules. Gut permeability was assessed using the inverted intestine method. We found that (1) expression of TLR2-1, TLR21, TGF-β2 and TGF-β3 were reduced following k-ST stimulation, but were unaffected by LR-treatment; (2) oral administration of LR led to increased Claudin1, Claudin5, ZO2, and JAM2 expression following k-ST stimulation; (3) cecal permeability was reduced by co-treatment with LR and k-ST, but not by treatment with LR or k-ST alone. These results suggest that LR, as used in this study, may enhance the intestinal mucosal physical barrier function, but not the expression of other immune-related factors in newly hatched chicks.

Transcriptome analysis reveals differentially expressed genes and pathways for oviduct development and defense in prelaying and laying hens.

The oviduct plays an indispensable role in the formation of eggs, especially the magnum and uterus. The identification of oviduct development in different stages will help to target candidate genes and pathways in regulation of albumen and eggshell formation, as well as defense mechanism in oviduct and egg. To identify the function differences and the molecular defense mechanism of the oviduct and egg, we performed transcriptome sequencing analysis of the magnum and uterus in 120-d-old and 300-d-old Lohmann layers, three birds in each group. With fold changes (log2 ratio)≥2 and false discovery rate (FDR)<0.01, RNA-Seq revealed 1,040 genes expressed differentially in the magnum and 595 genes in the uterus. By combining GO enrichment and KEGG pathway analysis, it served to show that gene activities of the magnum and uterus in prelaying chickens were more likely to concentrate on growth and development, and after egg-laying, they were mainly inclined to enhance the substances transmembrane transport and secretion activities. We further characterized 1579 new genes, while only 803 of them were functionally annotated. A complex mixture of proteins related to defense was measured in this study. A subset of avian β-defensins (AvBDs) and ovodefensins (OvoDs), that is, AvBD12, AvBD11, AvBD10, OvoDA1, OvoDB1, OvoDA2, OvoDA3, and OvoDBβ, was detected to express in the magnum of laying hens at high levels. Collectively, the identification and functional analysis of these differentially expressed genes (DEGs), as well as specific expression of avian defensins, may contribute to understand the development and defense mechanisms of oviduct and eggs.