1a.Objectives (from AD-416)
Develop chicken intestinal microarray chips which contains 10,000 non-redundant EST sequences and investigate the molecular basis for the macrophage-mediated innate immune responses in the gut to Salmonella and Eimeria. Identify the nature of host genes which control the protective immunty against Salmonella and Eimeria by conducting high throughput gene expression assays using chicken intestinal and macrophage chips.

1b.Approach (from AD-416)
Chicken intestinal microarray chips will be first developed by PCR reaction of 10,000 non-redundant intestinal genes selected from chicken intestinal cDNA library. Chickens will be infected with coccidia or salmonella, and RNA samples will be prepared from infected intestinal lymphocytes. Primary macrophages will be prepared from broiler chickens and infected with Eimeria or Salmonella for probe RNA preparation. Molecular basis for the protective innate immune responses in the gut to Salmonella and Eimeria infections will be investigated by carrying out high-throughput gene expression analysis using intestinal and macrophage cDNA microarrays. The role of host immune factors which were identified in the Objective 1 will be validated by real time PCR and northern blot analysis and host factors which are responsible for the intracellular killing of Salmonella and Eimeria will be identified.

3.Progress Report
This report documents research conducted under a Reimbursable Agreement between ARS and NRI. Additional details of the research can be found in the report for the associated in-house project 1265-31320-072-00D, “Development of Functional Genomic and Immunologic Approaches to Control Avian Mucosal Pathogens”.

1) To identify host genes which control coccidiosis resistance, we compared two commercial, pure broiler lines with different susceptibility to coccidiosis to fine map quantitative trait loci (QTL) associated with the previously identified marker LEI0101 located on chromosome 1. Multipoint analysis showed a maximum LOD score between LEI0071 and LEI0101 at 254 cM (LOD score = 3.74). Further studies to determine the physical locations of these and other markers will allow additional application association mapping techniques using single nucleotide polymorphism (SNP) markers. 2) NK-lysin is an anti-microbial and anti-tumor protein expressed by NK cells and T lymphocytes. Chicken cDNA encoding NK-lysin was isolated from a library prepared from chicken intestinal intraepithelial lymphocytes (IELs). Recombinant chicken NK-lysin expressed in COS7 cells exhibited anti-tumor cell activity. We conclude that chicken NK-lysin plays important roles during anti-microbial and anti-tumor defenses. 3) Coccidiosis, a major intestinal parasitic disease of poultry, induces a cell-mediated immune response against the etiologic agent of the disease, Eimeria. Following E. maxima infection. transcripts of the pro-inflammatory and Th1 cytokines IFN-gamma, IL-1-beta, IL-6, IL-12, IL-15, IL-17, and IL-18 were increased. In contrast, IFN-gamma, TGF-beta-4, and K60 transcripts showed no increased expression, and only the level of the Th2 cytokine IL-13 was increased following secondary E. maxima infection. Taken together, these data indicate a global chicken intestinal immune response is produced following Eimeria infection involving multiple cytokines, chemokines, and T cell subsets. Monitoring activity for this grant was in submitting timely progress reports.

Review Publications
Hong, Y.H., Lillehoj, H.S., Dalloul, R.A., Miska, K.B., Tuo, W., Lee, S., Han, J., Lillehoj, E.P. 2006. Molecular cloning and characterization of chicken NK lysin. Infection and Immunity. 110:339-347.