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Coccidiosis in chickens is an intestinal parasitic disease caused by protozoan parasites named Eimeria spp. In some Eimeria infections, intestinal lymphocytes are known to highly express chicken NK-lysin (cNK-lysin), an antimicrobial peptide with anticoccidial activity. Therefore, this study aims to investigate the expression of cNK-lysin in E. necatrix-infected chickens and its role in E. necatrix infection. The expression of cNK-lysin transcript was significantly increased in E. necatrix sporozoites-treated lymphocytes. In E. necatrix infection, cNK-lysin transcript was induced in intestinal lymphocytes but not in the spleen. The recombinant cNK-lysin exhibited anticoccidial activity against E. necatrix sporozoites as well as immunomodulatory activity on macrophages by inducing proinflammatory cytokines. These results indicated that E. necatrix infection induces high local expression of cNK-lysin and the secreted cNK-lysin helps protect coccidiosis.

Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P

Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-β1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13–induced expressions of chIL-13Rα2 and TGF-β1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-β1 secretion. Importantly, the newly developed anti–chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.

R. anatipestifer (RA) is one of the most harmful bacterial pathogens affecting the duck industry, and infection is associated with the production of proinflammatory cytokines, including IL-17A. Another proinflammatory cytokine, IL-23, is critical for the development of Th17 cells, which produce IL-17. However, IL-23 roles have not been studied in this infection. Here, we describe the identification and mRNA expression analysis of duck IL-23p19 (duIL-23p19) in splenic lymphocytes and macrophages stimulated with killed RA and in spleens of RA-infected ducks. Expression of duIL-23p19 transcript identified in this study was relatively high in livers of healthy ducks and was upregulated in mitogen-activated splenic lymphocytes as well as in splenic lymphocytes and macrophages stimulated with killed RA. In spleens of RA-infected ducks, expression levels of duIL-23p19 transcript were unchanged at all time points except on days 4 and 7 post-infection; however, duIL-17A and IL-17F expression levels were upregulated in both spleens of RA-infected ducks and splenic lymphocytes and macrophages stimulated with killed RA. In sera collected at 24 h after this infection, duIL-23p19 expression levels were unchanged, whereas IL-17A significantly upregulated. These results suggest that IL-23p19 does not play a critical role in the IL-17A response in early stages of RA-infected ducks.

Multiple challenges confront the increasing demand for wholesome poultry food products, including governmental restrictions on the use of antibiotic growth promoters (AGPs), nutritional requirements to obtain maximum growth potential, understanding crosstalks among the immunity–microbiota–neuroendocrine system in the gut to maximize intestinal efficiency, high-density production conditions, waste management, and the emergence of infectious pathogens, particularly those that emerge in the antibiotic-free animal production environment. Although in-feed antibiotics have dramatically increased the efficiency of commercial poultry production over the last 50 years, we are now faced with an increasing global crisis concerning the heightened use of antibiotics in animal agriculture and the emergence of multidrug-resistant superbugs that threaten disease management in animals and humans. Therefore, much interest has focused on the development of alternative, antibiotic-free methods of commercial poultry production. Initially, alternatives to antibiotics included any strategies that replace AGPs, but now include any feed additives or treatment that will allow antibiotic-free animal production to prevent and/or treat diseases. These newer disease control strategies can be classified broadly into those that are directly cytotoxic against infectious agents or remove pathogenic toxins, including vaccines, hyperimmune antibodies, antimicrobial peptides, and bacteriophages, and those that augment non-specific host immunity and gut health, including phytochemicals, adjuvants, prebiotics, and probiotics. Furthermore, because the gut microbiota influences various physiological aspects of the immune response, brain function, and gut health, most antibiotic alternatives are expected to promote beneficial microbes that will benefit host physiological responses. However, there is a timely need to better understand the role of the microbiota in gut health if we want to use microbes to modulate the host response to enhance growth performance. This review will highlight current knowledge of host immunity in poultry, and various strategies to modulate host immunity, growth performance, and disease responses to guide the development of alternatives to reduce the use of antibiotics, using a few selected alternatives and a description of their efficacy and modes of action.

Chicken NK-lysin peptide 2 (cNK-2) is a natural lytic peptide with direct cytotoxicity against many apicomplexan parasites including Eimeria. Developing an effective oral delivery strategy to express cNK-2 in the intestine, where Eimeria parasites interact with the host's gut epithelial cells, may effectively reduce the fecundity of parasites and minimize intestinal damage. Furthermore, cNK-2 modulates gut immune responses to decrease local inflammation elicited by Eimeria infection in the intestine. Therefore, we developed a stable strain of Bacillus subtilis (B. subtilis) that carries cNK-2 to the gut to determine its effectiveness in ameliorating the negative impacts of coccidiosis and to replace the use of antibiotics in controlling coccidiosis in commercial broiler chicken production. Chickens were randomly allocated into eight treatment groups: two control groups (NC: E. acervulina infected non-B. subtilis control; CON: non-infected control); three B. subtilis-empty vector (EV) groups (EV6: 106 cfu/day/bird; EV8: 108 cfu/day/bird; EV10: 1010 cfu/day/bird), and three B. subtilis-cNK-2 groups (NK6: 106 cfu/day/bird; NK8: 108 cfu/day/bird; NK10: 1010 cfu/day/bird). All chickens, except those in the CON group, were challenged with 5,000 freshly sporulated E. acervulina oocysts through oral gavage on day 15. Chickens were given an oral dose of B. subtilis on days 14, 15, and 16. Body weight, weight gains, and fecal oocyst shedding were measured. To investigate the efficacy of oral B. subtilis-cNK-2 against coccidiosis, gene expression of gut health-related biomarkers was measured using RT-PCR. Markers included SOD1, CAT, and HMOX1 for oxidative stress in the spleen and intestinal mucosa, OCLN, ZO-1, and JAM2 for tight junction proteins, and MUC2 for mucin gene expression in the gut. The results showed that oral treatment of young chickens with B. subtilis-cNK-2 improved growth performance, enhanced gut integrity, and reduced fecal oocyst shedding. Altogether, these results confirm B. subtilis-cNK-2 treatment as a promising and effective alternative strategy to replace antibiotics against coccidiosis based on its ability to reduce parasite survival, to reduce coccidiosis-induced body weight loss, and to decrease gut damage based on the enhanced expression of proteins associated with gut integrity and intestinal health.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G, which affects global poultry industry by compromising the performance, health, and welfare of chickens. The causative main virulent factor responsible for NE pathogenesis has been shifted from a phospholipase C portion of an α-toxin, to an NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, in NE development. Therefore, the ability to detect NetB toxin will enable early diagnosis of field NE. Because the NetB protein can only be detected by western blot analysis with polyclonal anti-NetB antiserum, we developed a NetB-specific monoclonal antibody (mAb)-based capture enzyme-linked immunosorbent assay (ELISA). Twenty mAbs reacting with Escherichia coli-expressed NetB protein were selected, isotyped, and conjugated with horseradish peroxidase for antibody pair tests. Multiple mAb pairs were found to detect E. coli NetB protein and native NetB protein secreted by netB-positive C. perfringens isolates. The developed capture (sandwich) ELISA could be useful to identify in vitro production of native NetB protein secreted from netB-positive field C. perfringens isolates and to conduct a large field test of commercial chickens undergoing NE infection. Here, we first report that native NetB toxin can be detected in C. perfringens NetB-specific mAb-based capture ELISA.

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed a sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast was used to immunize the mice. Five mAbs which specifically recognize chIFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing assay to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were identified as optimum pairs of antibodies for detection of chIFN-α. The sandwich ELISA effectively detected native chicken IFN-α in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus (VSV) was evaluated in chicken embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize the activity of chicken IFN-α against VSV were identified. The newly developed antigen capture sandwich ELISA for the detection of chIFN-α will be a useful tool to monitor IFN-α production in chickens during viral infections.

In mammals, alternatively activated macrophages (AAMs) are well-recognized and are produced by stimulation with Th2 cytokines such as interleukin 4 (IL-4) and IL-13. On their mammalian counterparts, AAMs in chickens has neither been reported nor the functionality of chicken IL-4 (chIL-4) has been studied till date. Therefore, present study developed mouse monoclonal antibodies (mAbs) against chIL-4 and used these antibodies to investigate whether chIL-4 induces activation of HD11 chicken macrophage cell line. Upon characterization of mAbs using western blot, immunocytochemistry (ICC), flow cytometry and capture ELISA, activation of HD11 cells was investigated by measuring nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, arginase activity and gene expressions of iNOS, CD80 and CD86 (associated with mammalian M1 phenotype) and chemokine (C-C motif) ligand 17 (ccl17) and mannose receptor C-type1 (MRC1L-A) (as possible chicken M2 markers) in HD11 cells treated with chIL-4, lipopolysaccharide (LPS), chIL-4+LPS, chIL-4+LPS + mAbs. The newly developed mAbs displayed wide applicability in detecting chIL-4 by capture ELISA, ICC and flow cytometry with no cross reactivities with human or mouse IL-4 and other chicken cytokines. Further, our results showed that chIL-4 inhibited NO production by LPS-stimulated HD11 cells and primary monocyte/macrophage cells with reduced iNOS expression and increased arginase activity and, induced robust expression of genes associated with M2 phenotype than M1-related genes. All these effects were neutralized by anti-chIL-4 antibodies. In summary, present study results showed a possible application of anti-chIL-4 mAbs as valuable immune reagents to explore chIL-4 functionality. In addition, our results demonstrated that chIL-4 may override LPS functionality and regulates alternative activation of HD11 cells in chicken via increased arginase activity and expression of M2 associated markers and thus may indicate the possible existence of M1/M2 paradigm in chickens.

With the increase in regulations regarding the use of antibiotic growth promoters and the rise in consumer demand for poultry products from ‘Raised Without Antibiotics’ or ‘No Antibiotics Ever’ flocks, the quest for alternative products or approaches has intensified in recent years. A great deal of research has focused on the development of antibiotic alternatives to maintain or improve poultry health and performance. This review describes the potential for the various alternatives available to increase animal productivity and help poultry perform to their genetic potential under existing commercial conditions. The classes of alternatives described include probiotics, prebiotics, synbiotics, organic acids, enzymes, phytogenics, antimicrobial peptides, hyperimmune egg antibodies, bacteriophages, clay, and metals. A brief description of the mechanism of action, efficacy, and advantages and disadvantages of their uses are also presented. Though the beneficial effects of many of the alternatives developed have been well demonstrated, the general consensus is that these products lack consistency and the results vary greatly from farm to farm. Furthermore, their mode of action needs to be better defined. Optimal combinations of various alternatives coupled with good management and husbandry practices will be the key to maximize performance and maintain animal productivity, while we move forward with the ultimate goal of reducing antibiotic use in the animal industry.

Avian coccidiosis is caused by Eimeria, which is an intracellular apicomplexan parasite that invades through the intestinal tract to cause devastating disease. Upon invasion through the intestinal epithelial cells, a strong inflammatory response is induced that results in complete villous destruction, diarrhea, hemorrhage, and in severe cases, death. Since the life cycle of Eimeria parasites is complex and comprises several intra- and extracellular developmental stages, the host immune responses are diverse and complex. Interferon-γ-mediated T helper (Th)1 response was originally considered to be the predominant immune response in avian coccidiosis. However, recent studies on other avian T cell lineages such as Th17 and T regulatory cells have implicated their significant involvement in maintaining gut homeostasis in normal and disease states including coccidiosis. Therefore, there is a need to understand better their role in coccidiosis. This review focuses on research findings concerning the host immune response induced by avian coccidiosis in the context of T cell immunity, including expression of T-cell-related cytokines and surface molecules that determine the phenotype of T lymphocytes.

Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.

Riemerella anatipestifer causes infectious disease and considerable economic loss in the duck industry worldwide. Our previous studies demonstrated an association between proinflammatory cytokine interleukin (IL)-17A and R. anatipestifer infection. Here, we provide evidence for IL-17A involvement in R. anatipestifer infection using a mouse model. Mice showed higher resistance to R. anatipestifer infection than ducks, with median lethal doses (LD50) of 3.5 × 1010 and 5 × 107 colony-forming units (CFU), respectively. Twenty-four hours after infection, mice with a sub-lethal dose (3.5 × 109 CFU) exhibited levels of IL-17A and IL-23 expression similar to uninfected mice. Thus, we hypothesized that exogenous IL-17A or IL-23 administration affects susceptibility of mice to R. anatipestifer. Mice pretreated with IL-17A or IL-23 prior to sub-lethal dose infection of R. anatipestifer exhibited increased bacterial burden and spleen weights compared to untreated infected mice, confirming the involvement of IL-17A in susceptibility to R. anatipestifer infection in vivo.

Indoles, as the ligands of aryl hydrocarbon receptor (AhR), have been shown to possess immune-modulating property in terms of the balancing between regulatory T cells (Treg) and T helper 17 cells (Th17) activities. In the present study, we examined the effects of dietary indoles, 3,3′-diindolylmethane (DIM) and indole-3-carbinol (I3C), on CD4+T cell population and functions in chickens. Furthermore, the effects of dietary DIM treatment on chicken coccidiosis caused by an apicomplexan parasite were investigated. Dietary treatment of healthy chickens with DIM and I3C induced increased CD4+CD25+ (Treg) cells and the mRNA expression of IL-10, while decreasing number of CD4+IL-17A+ (Th17) cells and Th17-related cytokines transcripts expression in the intestine. In addition, we explored the role of AhR in indole-treated splenic lymphocytes by using AhR antagonist and our results suggested that DIM is a ligand for chicken AhR. In chicken coccidiosis, treatment of DIM increased the ratio of Treg/Th17 cells and significantly reduced intestinal lesion although no significant changes in body weight and fecal oocyst production were noted compared to non-treated control group. These results indicate that DIM is likely to affect the ratios of Treg/Th17 reducing the level of local inflammatory response induced by Eimeria or facilitate repairing process of inflamed gut following Eimeria infection. The results described herein are thus consistent with the concept that AhR ligand modulates the T cell immunity through the alteration of Treg/Th17 cells with Treg dominance. To our knowledge, present study is the first scientific report showing the effects of dietary indole on T cell immunity in poultry species.

IL7 is a hematopoietic growth factor required for development and maintenance of lymphocytes including T cells, B cells, and natural killer cells. Recently, chicken IL7 (chIL7) has been cloned and studied in viral and parasite infection models. However, no monoclonal antibodies (mAb) that specifically detect chIL7 have been developed so far. In this study, recombinant chIL7 that expressed for immunization and mAb against chIL7 were developed and characterized to assess their immunologic properties. Five mAb exhibiting specific binding to chIL7 were generated and investigated for their applicability by Western blot, ELISA, and neutralization assays. A sandwich ELISA mAb pair that enables the measurement of chIL7 protein levels in biological samples from Eimeria-infected chickens was identified and several mAb neutralized chicken primary thymocyte proliferation mediated by chIL7. The mAb developed in this study will be valuable reagents for fundamental and applied immunological studies in poultry.

Although IL-17 cytokines play critical roles in host defense immunity, dysregulated expression of these cytokines is associated with inflammation and autoimmune diseases. Riemerella anatipestifer is the most important infectious bacterium in the duck industry. Interestingly, not all avian species are equally susceptible to R. anatipestifer infection. This paper reports the first description of mortality rate, bacterial burden, and expression profiles of immune-related genes between ducks and chickens infected with R. anatipestifer. Ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Comparative expression analyses of immune-related genes in R. anatipestifer-infected tissues obtained from both species revealed that TLR3, TLR7, IL-2, IL-4, and IFN-γ transcript levels were higher in chickens, whereas TLR4 and IL-17A transcript levels were higher in ducks. Marked increases in expression of IL-17A and IL-6, but not TGF-β, were associated with Th17 cell differentiation in duck splenic lymphocytes, but not in chicken splenic lymphocytes, stimulated with R. anatipestifer. Moreover, upregulation of IL-1β, IL-6, and IL-17A mRNA expressions, but not TGF-β, was confirmed in the liver and spleen of ducks infected with R. anatipestifer, indicating that IL-17A is strongly associated with Riemerella infection in ducks.

Interleukin (IL)-17D is a proinfiammatory cytokine with currently largely unknown biological functions. Here we provide the description of the sequence, bioactivity, and mRNA expression profile of duck IL-17D homologue. A full-length duck IL-17D (duIL-17D) cDNA with a 624-bp coding region was identified from the large intestine. dull-17D shares approximately 94.7% identity with its chicken counterpart, which is also identified in this work. duIL-17D exhibits 62.6-68.4% and 52.1-53.1% identity with mammalian and piscine homologues. Recombinant duIL-17D promoted the expression of proinfiammatory cytokines such as IL-6, IL-8, and IL-1 beta in duck embryo fibroblast cells. Very low levels of dull-17D transcript were observed in healthy lymphoid tissues, including bursa, thymus, and spleen, while duIL-17D expression was relatively high in the heart. The dull-17D expression profiles were examined in mitogen-stimulated splenic lymphocytes, as well as tissues affected by Riemerella anatipestifer infection. The levels of duIL-17D were mostly upregulated in mitogen-activated splenic lymphocytes but downregulated in the liver and spleen of R. anatipestifer-infected ducks. These results provide new insights into the roles of IL-17D in host protective immune responses to Riemerella infection, which can therefore lead to further studies of its biological functions in different disease models of ducks and other avian species. Basic Science Research Program through the National Research Foundation of Korea (NRF) - Ministry of Education [2015R1D1A1A02059953] This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A02059953). Public domain – authored by a U.S. government employee

3,3’-Diindolylmethane (DIM) is found in cruciferous vegetables and is used to treat various inflammatory diseases because of its potential anti-inflammatory effects. To investigate effects of DIM in Riemerella anatipestifer-infected ducks which induce upregulation of inflammatory cytokines, ducks were treated orally with DIM at dose of 200 mg/kg/day and infected the following day with R. anatipestifer. Infected and DIM-treated ducks exhibited 14% increased survival rate and significantly decreased bacterial burden compared to infected untreated ducks. Next, the effect on the expression level of inflammatory cytokines (interleukin [IL]-17A, IL-17F, IL-6, IL-1β) of both in vitro and in vivo DIM-treated groups was monitored by quantitative reverse-transcription PCR (qRT-PCR). Generally, the expression levels of the cytokines were significantly reduced in DIM-treated splenic lymphocytes stimulated with killed R. anatipestifer compared to stimulated untreated splenic lymphocytes. Similarly, the expression levels of the cytokines were significantly reduced in the spleens and livers of DIM-treated R. anatipestifer–infected ducks compared to infected untreated ducks. This study demonstrated the ameliorative effects of DIM in ducks infected with R. anatipestifer. Thus, DIM can potentially be used to prevent and/or treat R. anatipestifer infection via inhibition of inflammatory cytokine expression.

Aims To assess the production level of interleukin (IL)-10 inClostridium perfringens (CP)-stimulated intestinal epithelial cells (IECs) and CP-infected chickens using an antigen capture ELISA developed by mouse monoclonal antibodies against chicken IL-10. Also, to investigate which CP toxins induced IL-10 in chicken IECs stimulated with CP. Methods and results Chicken IECs were stimulated with CP toxin in the absence or presence of antibodies (α-toxin or NetB) for 18 h. Expression of chicken IL-10 and IL-6 was monitored by quantitative real-time polymerase chain reaction. Serum and jejunum samples were collected from CP-infected chickens at 9 days post-infection and expression of IL-10 and IL-6 was analyzed. IL-10 production was detected by antigen capture ELISA in chicken IECs stimulated with CP and in serum samples collected from CP-infected birds. The mRNA levels were consistent with the results of antigen capture ELISA. Conclusion CP induced IL-10 production in chicken IECs. Increased IL-10 production was detected in CP-stimulated chicken IECs and in serum collected from CP-infected birds, using antigen capture ELISA. Antigen capture ELISA could be a useful tool to monitor IL-10 production in chicken disease.

Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37–46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL-17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1β, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions.

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.

As the dysregulation of IL-17 is implicated in the pathogenesis of various autoimmune and inflammatory diseases, the suppression of IL-17 production by Th2 cytokines could alleviate the development of these diseases. Previously, we confirmed that inflammatory cytokines including IL-17A are strongly associated with R. anatipestifer infection, which is one of the most important bacterial pathogens in the duck industry. Here, we found that IL-4 treatment downregulated the expression of IL-17A and IL-17F transcripts in splenic lymphocytes stimulated with R. anatipestifer. Moreover, duck IL-4 (duIL-4) treatment in R. anatipestifer-stimulated lymphocytes suppressed the expression of IL-23p19 and IL-12p40 transcripts compared to untreated and stimulated lymphocytes. Conversely, duIL-4 increased levels of IFN-γ and IL-10. We identified a full-length duIL-4 cDNA encoding 136 amino acids from ConA-activated splenic lymphocytes that shares 49.3–50% amino acid sequence identity with chicken and quail IL-4 and 21–29.7% with mammalian and piscine homologues. Low or moderate levels of duIL-4 transcript were observed in healthy tissues, including the spleen, bursa, and thymus, whereas duIL-4 expression was higher in the kidney and lung. Levels of duIL-4 were generally upregulated in mitogen-activated splenic lymphocytes but lower in the liver and spleen of R. anatipestifer-infected ducks compared to those of infected chickens. Recombinant duIL-4 promoted nitric oxide synthesis in duck macrophages stimulated by R. anatipestifer compared to untreated and stimulated control macrophages. These results demonstrate that IL-4 is an important Th2 cytokine that inhibits inflammatory responses in splenic lymphocytes stimulated with R. anatipestifer.

Riemerella anatipestifer, an important infectious bacterium affecting the duck industry, has 5–75% mortality, depending on strain virulence. We previously demonstrated that proinflammatory cytokines are involved in inflammation during, and regulating susceptibility to, R. anatipestifer infection. We investigated the effects of the anti-inflammatory compound berberine in duck splenic lymphocytes stimulated with killed R. anatipestifer, and in R. anatipestifer- infected ducks. IL-17A, IL-17F, and IL-1β transcripts were downregulated, and IFN-γ and IL-10 transcripts enhanced, in berberine-treated stimulated splenic lymphocytes, compared to stimulated untreated splenic lymphocytes. Similarly, IL-17A, IL-17F, IL-6, and IL-1β expressions were significantly reduced, and IFN-γ and IL-10 expressions significantly upregulated, in spleens and livers of R. anatipestifer- infected berberine-treated ducks, compared to infected untreated birds. Moreover, infected and treated birds showed increased survival rates and significantly decreased bacterial burdens compared to infected untreated birds, confirming that inflammatory cytokines are strongly associated with R. anatipestifer infection in ducks.

Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) that is produced by cytotoxic T cells and natural killer cells. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core α-helical region of cNK-lysin, have antimicrobial activity against apicomplexan parasites such as Eimeria spp., via membrane disruption. In addition to the antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their interactions with host cells is increasingly recognized. Thus, in this study, we investigated whether cNK-lysin derived peptides modulate the immune response in the chicken macrophage cell line HD11 and in chicken primary monocytes by evaluating the induction of chemokines, anti-inflammatory properties, and activation of signalling pathways. cNK-2 induced the expression of CCL4, CCL5 and interleukin(IL)-1β in HD11 cells and CCL4 and CCL5 in primary monocytes. We also determined that cNK-2 suppresses the lipopolysaccharide-induced inflammatory response by abrogating IL-1β expression. The immunomodulatory activity of cNK-2 involves the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as the internalization of cNK-2 into the cells. These results indicate that cNK-2 is a potential novel immunomodulating agent rather than an antimicrobial agent.

Interleukin-17F (IL-17F) is a proinflammatory cytokine, which plays an important role in gut homeostasis. A full-length chicken IL-17F (chIL-17F) cDNA with a 510-bp coding region was identified from ConA-activated chicken splenic lymphocytes. ChIL-17F shares 53% amino acid sequence identity with the previously described chicken IL-17 (chIL-17A) and 38-43% with mammalian homologues. The locus harboring chIL-17 and chIL-17F displayed inverted order compared to those of mammals. ChIL-17F transcript expression was high in lymphoblast cell line CU205 and at moderate levels in small and large intestines and liver. ChIL-17F and chIL-17 expression profiles were examined by quantitative real-time RT-PCR in mitogen-stimulated splenic lymphocytes and intestinal areas affected by Eimeria maxima and Eimeria tenella infections. Expression levels of chIL-17F, like chIL-17, were elevated in mitogen-activated splenic lymphocytes. ChIL-17F, but not chIL-17, expression was upregulated in intestinal tissues affected by E. maxima and E. tenella infections. Recombinant chIL-17F biological activities were similar to that of chIL-17 in primary chicken embryonic fibroblasts. These results suggest that chIL-17F is a unique member of the IL-17 family of cytokines.

We identified low-molecular weight compounds derived from the antimicrobial peptide human neutrophil peptide-1 that bind to Lipid II, an essential precursor of bacterial cell wall biosynthesis. These compounds act as antibacterials on multiple biosynthesis pathways with specificity against gram-positive organisms. Here, we have tested a small subset of our most promising leads against the bacteriumEfectos anti infecciosos pleiotrópicos de los compuestos antimicrobianos derivados de la defensina. Se identificaron compuestos de bajo peso molecular derivados del péptido antimicrobiano, péptido neutrófilico humano 1 que se unen al lípido II, que es un precursor esencial de la biosíntesis de la pared celular bacteriana. Estos compuestos actúan como antibacterianos en múltiples vías de biosíntesis con especificidad contra organismos Gram positivos. En este estudio se probó un pequeño subconjunto de los proyectos más prometedores contra la bacteria Clostridium perfringens y los esporozoitos de Eimeria tenella, que es un parásito protozoario intracelular que causa enfermedad intestinal en aves comerciales. Se encontró un compuesto, 1611-0203 (2-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(2-hidroxifenoxi)fenil]fenoxi}fenol), específicamente para inhibir el crecimiento de ambos agentes de todos los compuestos probados. Además, el compuesto 1611-0203 inhibe a Staphylococcus aureus y Enterococcus spp. Los estudios del mecanismo de acción revelan además que 1611-0203 afecta la biosíntesis de la pared celular e inhibe las vías biosintéticas adicionales. Estos resultados combinados indican que los compuestos como el 1611-0203 tienen potencial terapéutico para actuar como anti infecciosos contra varios organismos simultáneamente.

Epidemiology of Eimeria species in poultry flocks is important to increase the effectiveness of vaccinations and prophylactic strategies on chicken farms. In this study, fecal samples from 356 chicken farms were collected randomly and examined for the prevalence of Eimeria species. Through microscopic examination, it was determined that 78.7% of the tested farms were positive in Eimeria-infection. Seven Eimeria species were detected in all the positive farms by PCR amplification of the internal transcribed spacer 1 (ITS-1) region with species-specific primers. E. acervulina and E. tenella were the most prevalent, followed by E. brunetti and E. praecox (87.5, 62.5, 59.3, and 37.5% of the farms, respectively). Each of E. maxima, E. mitis, and E. necatrix was identified in 31.3% of the farms. Individual positive fecal samples contained multiple Eimeria species (mean=3.4). Since E. maxima is known to generate antigenic variants, cross-immunity was investigated for four isolates of E. maxima from the poultry farms in different regions of Korea. The extent of cross-protection varied from 54.3 to 100% against the heterologous isolates. The results obtained from this large-scale survey will be a useful reference for controlling coccidiosis in the poultry industry.

Both interleukin-17A (IL-17A) and IL-17F are proinflammatory cytokines that have an important role in intestinal homeostasis via receptor signaling. These cytokines have been characterized in chickens, but very little is known about their receptors and their functional activity. We provide here the first description of the sequence analysis, bioactivity, and comparative expression analysis of chicken IL-17RA (chIL-17RA) in chickens infected with Salmonella and Eimeria , two major infectious agents of gastrointestinal diseases of poultry of economic importance. A full-length chIL-17RA cDNA with a 2,568-bp coding region was identified from chicken thymus cDNA. chIL-17RA shares ca. 46% identity with mammalian homologues and 29.2 to 31.5% identity with its piscine counterparts. chIL-17RA transcript expression was relatively high in the thymus and in the chicken macrophage cell line HD11. The chIL-17RA-specific small interfering RNA inhibits interleukin-6 (IL-6), IL-8, and IL-1β mRNA expression in chicken embryo fibroblast cells (but not in DF-1 cells) stimulated with chIL-17A or chIL-17F. Interaction between chIL-17RA and chIL-17A was confirmed by coimmunoprecipitation. Downregulation of chIL-17RA occurred in concanavalin A- or lipopolysaccharide-activated splenic lymphocytes but not in poly(I·C)-activated splenic lymphocytes. In Salmonella - and Eimeria -infected chickens, the expression levels of the chIL-17RA transcript were downregulated in intestinal tissues from chickens infected with two Eimeria species, E. tenella or E. maxima , that preferentially infect the cecum and jejunum, respectively. However, chIL-17RA expression was generally unchanged in Salmonella infection. These results suggest that chIL-17RA has an important role in mucosal immunity to intestinal intracellular parasite infections such as Eimeria infection.