2011 Annual Report 1a.Objectives (from AD-416) Develop immune reagents for veterinary animals of economic importance including cattle, swine, poultry, horses, and aquaculture species. 1b.Approach (from AD-416) Immune reagents will be developed for basic and applied studies in veterinary animal species. They will be developed into multiplex assays to measure cytokine and chemokine production during infections and host immune responses. As a member of a large consortium grant to develop immunological reagents for five different animal species, we will identify major cytokines and cell surface markers of poultry, express them in suitable expression vectors and validate their function using in vivo and in vitro assay systems. 3.Progress Report This is a reimbursable agreement with the University of Massachusetts to advance veterinary immunology. The main goal of this agreement is to develop immunological reagents specific for poultry, ruminants, swine, equine and aquaculture species. Veterinary immunological reagents that will be developed in this grant period include monoclonal antibodies (mAb) and polyclonal antibodies which can identify the major leukocyte subpopulations. Sequence of 5 genes (IL1R1, IL7R, IL10R-beta, IL17R, and IL21R) were verified and these genes were sent to Cornell Univ. for the construction of chimeric proteins with equine IgG1 heavy chain constant region. Eleven chicken cDNA homologs (CXCL4, GM-CSF, IFN-gamma, IL12p40, IL17A, IL17D, IL21, IL22, LITAF, Lymphotactin, and TNFSF) were amplified from Con-A stimulated chicken spleen cell RNA using gene-specific primers and sent to the Kingfisher Laboratory for protein expression. Chicken IL4, IL12, GM-CSF, IFN-gamma, and TNFSF, and turkey IGF-1 and IGF-2 were biologically active. CD25 and CD80 expressed at Univ. of Mass and IL18 expressed at ARS were biologically active. Four hybridomas (IL-17A, IL17D, IFN-gamma, and TL1A) have been developed against each chicken cytokines, showed preferential binding to its respective immunizing antigen, and were single cell cloned. Twelve Polyclonal Abs (Ch IL2, IL4, IL8, IL17A, IL17D, TGF-ß4, TL1A, cMIF, EaMIF, Net B, a-toxin, and Gam82) were developed. This agreement is monitored by monthly teleconferences, annual progress reports, and bi-annual meetings among the participating scientists. This research relates to NP103, C2, P.S.2c