Hyun S. Lillehoj

Role of chicken IL-2 on γδ T-cells and Eimeria acervulina-induced changes in intestinal IL-2 mRNA expression and γδ T-cells

Abstract Continuous culture of concanavalin A (Con A)-activated spleen cells in the presence of chicken recombinant IL-2 (rIL-2) promoted preferential growth of γδ T-cells. These cells displayed a high level of spontaneous cytotoxicity against LSCC-RP9 tumor cells, an avian NK cell target. Stimulation of IL-2-dependent γδ T-cells with Con A induced IFN-γ and IL-2 mRNA transcripts, whereas stimulation with rIL-2 induced only IFN-γ mRNA. Subcutaneous injection of 3-week-old chickens with IL-2 DNA increased splenic cells, expressing the CD8 and γδ TCR antigens. To investigate the role of IL-2 and γδ T-cells in parasitic infection, chickens were orally infected with Eimeria acervulina and the expression of IL-2 mRNA transcripts in the spleen and duodenum and the percentage of γδ T-cells in the duodenum were examined. Following both, the primary and secondary infections, a significant enhancement of IL-2 mRNA transcripts in the spleen and intestine and increased percentage of intraepithelial γδ T-cells in the duodenum were observed. These results indicate that host immune responses to E. acervulina involve an up-regulation of IL-2 secretion and an increased duodenum γδ T-cells.

Molecular and functional characterization of chickenIL-15

Abstract A cDNA encoding chicken interleukin-15 was cloned from a CD4 + T cellhybridoma expression library by screening with a rabbit antibody against a protein fraction ofconditioned medium containing T cell growth promoting activity. The chicken IL-15 cDNAcontains an open reading frame of 143 amino acids with a single potential N-linked glycosylationsite. The predicted m.w. of the encoded protein [16] matched the size of animmunoreactive band on Western blots of E. coli expressing the recombinant IL-15.Amino acid and nucleotide sequence analyses of chicken IL-15 revealed 31% and 46% identitywith bovine IL-15 respectively and lesser homologies to other mammalian IL-15s. Chicken IL-15contained all 4 highly conserved cysteine residues present in mammalian IL-15 sequences.RT-PCR demonstrated that the chicken IL-15 gene is expressed in many tissues including spleen,intestine, and muscle and in established macrophage, T lymphoma and fibroblast cell lines.Activation of spleen cells with Con A enhanced the expression of IL-15 gene transcripts in atime-dependent manner. CHO-K1 cells transfected with the chicken IL-15 cDNA secreted abiologically active protein supporting the growth of Con A activated spleen lymphocytes.Continuous culture of spleen Con A lymphoblasts with chicken IL-15 over two months resulted inan enriched T lymphocyte population expressing the gdTCR, CD8a, and CD3 cell surfaceantigens.

Antigen-Specific Lymphocyte Proliferation and Interleukin Production in Chickens Immunized with Killed Salmonella enteritidis Vaccine or Experimental Subunit Vaccines

Abstract SUMMARY. Lymphocyte proliferation and interleukin (IL)-2 and IL-6 levels in serum were measured as indicators of cell-mediated immunity after immunization of chickens with a commercial killed Salmonella enteritidis (SE) vaccine or experimental subunit vaccines of crude protein (CP) extract or the outer membrane protein (OMP). Significantly increased proliferative responses to SE flagella, but not lipopolysaccharide, porin, CP, or OMP, were observed at 1 wk postimmunization in the three vaccination groups. The responses to flagella were specific because flagella-induced proliferation was not seen in chickens immunized with adjuvant alone. Of the three immunization protocols, use of the killed SE vaccine appeared most effective because it induced higher flagella-stimulated lymphocyte proliferation at 1 and 2 wk postvaccination compared with the CP- and OMP-vaccinated groups. Significantly increased IL-2 and IL-6 levels in serum were seen at 1 wk postimmunization in the three vaccination groups compared with adjuvant alone, but there were no differences between the killed vaccine and the subunit vaccines at this time, and the levels of both lymphokines returned to baseline at 2 wk postimmunization. We conclude that cell-mediated immunity to SE after vaccination with the killed bacterial vaccine or subunit vaccines is transient and mainly limited to flagella.

Clostridium perfringens α-Toxin and NetB Toxin Antibodies and Their Possible Role in Protection Against Necrotic Enteritis and Gangrenous Dermatitis in Broiler Chickens

SUMMARY. Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C. perfringens are its four major toxins (&agr;, &bgr;, &egr;, &igr;) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C. perfringens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C. perfringens &agr;-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C. perfringens &agr;-toxin and NetB toxin that were increased in birds experimentally co-infected with Eimeria maxima and C. perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

Impact of Fresh or Used Litter on the Posthatch Immune System of Commercial Broilers

SUMMARY. This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.

Molecular cloning, characterization and mRNA expression of duck interleukin-17F

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.

Effects of Various Field Coccidiosis Control Programs on Host Innate and Adaptive Immunity in Commercial Broiler Chickens

Coccidiosis control programs such as vaccines or in-feed anticoccidials are commonly practiced in the poultry industry to improve growth performance and health of commercial broiler chickens. In this study, we assessed the effects of various coccidiosis control programs (e.g., in ovo vaccination, synthetic chemicals, and antibiotic ionophores) on immune status of broiler chickens vaccinated against infectious bronchitis virus and Newcastle disease virus (ND) and raised on an Eimeriacontaminated used litter. In general, the levels of α-1-acid glycoprotein, an acute phase protein, were altered by the treatments when measured at 34 days of age. Splenocyte subpopulations and serum antibody titers against ND were altered by various coccidiosis control programs. In-ovo-vaccinated chickens exhibited highest mitogenic response when their spleen cells were stimulated with concanavalin A (Con A) at 7 days of age. It is clear from this study that the type of coccidiosis control program influenced various aspects of innate and adaptive immune parameters of broiler chickens. Further studies will be necessary to delineate the underlying relationship between the type of coccidiosis control program and host immune system and to understand the role of other external environmental factors such as gut microbiota on host-pathogen interaction in various disease control programs.