2006 Annual Report 4d.Progress report. This report serves to document research conducted by ARS under a grant from NRI. Additional details of research can be found in the report for the parent project 1265-31320-070-00D:Genomic, Proteomic, and Immunologic Approaches to Controlling Avian Coccidiosis. This is a two-year NRI grant funded by CSREES and the project started in September, 2004. The short-term goals of this project include expanding our basic understanding of the function of macrophage-mediated innate immunity in avian salmonellosis and coccidiosis, and characterizing the underlying immunological mechanisms associated with protection against Salmonella and Eimeria infections at the cellular and molecular levels. Coccidiosis is recognized as the major parasitic disease of poultry and is caused by the apicomplexan protozoa Eimeria. Increasing evidence shows the complexity of the host immune response to Eimeria and microarray technology presents a powerful tool for the study of such an intricate biological process. Using an avian macrophage microarray containing 4,906 unique gene elements, we identified important host genes whose expression changed following infection of macrophages with sporozoites of E. tenella, E. acervulina, and E. maxima. This approach enabled us to identify a common core of 25 genetic elements whose transcriptional expression is induced or repressed by exposure to Eimeria sporozoites and to identify additional transcription patterns unique to each individual Eimeria species. Besides inducing the expression of IL-1, IL-6, and IL-18 and repressing the expression of IL-16, Eimeria treated macrophages were commonly found to induce the expression of the CCL chemokine family members MIP-1 (CCLi1), K203 (CCLi3), and ah221 (CCLi7). However, the CXCL chemokine K60 (CXCLi1) was found to be induced by macrophage exposure to E. tenella but was repressed upon macrophage exposure to E. maxima and E. acervulina. Fundamental analysis of avian chemokine and cytokine expression patterns offers insight into the unique avian immunological responses to these related but biologically unique pathogens. A comparison of elements exhibiting significant expression changes in each treatment found that each strain of Eimeria induced significant (> 2-fold) expression changes in a unique set of approximately 60 elements. This comparison also showed that treatments with E. acervulina and E. tenella induced changes in almost no common genes with the exception of the 25 core elements. However, E. acervulina and E. maxima commonly induced expression changes in a set of 22 genes uniquely different from the set of 26 genes exhibiting expression changes when macrophages were exposed to E. tenella and E. maxima. Many important immune effector genes appear among these most highly induced elements lists, including complement component C1q, MIP-1 (CCLi1), and the chemokines K60 (CXCLi1) and ah221 (CCLi7). While some genes are highly induced following two of the three treatments (AFABP, MIP-1, JTAP-1), no common induced genes are found in all three treatments. However, the quiescence specific protein precursor, CH21, is among the most highly repressed in all three treatments, being the most highly repressed following E. acervulina and E. tenella exposure and the second most highly repressed following E. maxima exposure, where an element of unknown function was highest repressed (> 11-fold). The AMM contains a significant number of avian cytokine and chemokine genes. Many of these elements demonstrated significant expression changes following stimulation with different Eimeria species. IL-1beta and MIP-1beta (CCLi1) show the most dramatic and consistent expression inductions, while IL-16 expression was consistently repressed. Several of the cytokine/chemokine elements also show differential expression following treatment with different Eimeria species, including chemokine K60 (CXCLi1), which is highly induced at 48 hr in E. tenella exposed macrophages and is repressed at 48 hr in E. acervulina and E. maxima exposed macrophages. The expression patterns of four of these 10 genes (K60, ah221, IL-18, and MIP-1) were confirmed by quantitative real-time reverse transcription PCR (Q-RT-PCR). Although the amplitude of theses changes was generally found to be higher by Q-RT-PCR than by array analysis, the temporal regulation patterns found by the two methods were nearly identical. Review Publications Lillehoj, H.S., Ding, X., Dalloul, R.A., Sato, T., Yasuda, A., Kukkegih, E.P. 2005. Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants. Journal of Parasitology. 91:666-673.