Brief Summary of Minutes of Annual Meeting

Extensive collaboration among members was evident and much meeting discussion focused on genetic resources and conservation of living stocks. A special tribute to Dr. W. Elwood and Mrs. Ruth Briles was held in conjunction with the meeting and included an evening dinner celebration. The tribute recognized the enormous contributions of the Briles to the NE-1016 (formerly NE-60) group and to the field of avian immunogenetics. Studies continued to elucidate the molecular and cellular basis for resistance and susceptibility to Mareks disease, Salmonella, Rous sarcoma virus, autoimmune disease, and general immune function. Much progress was made in profiling of genes involved in resistance, exploration of new genes and determination of the contribution of genetic variation to disease resistance and susceptibility. Work continues in these areas. Auburn University (AL). Quantitative differences in mRNA expression of BF1 and BF2 loci alleles for broiler breeder lines were established. BF1 of four haplotypes have the same sequence of alpha 1 and alpha 2 regions, one haplotype has an additional nucleotide difference leading to two different amino acids. Locus specific probes were designed for BF1 and BF2 for real time qPCR. Transcript differences were evident and closely related BV1 alleles may exhibit wide differences in expression; regulatory elements could contribute. A new area of research is the analysis of the functional polymorphism in the Mx gene. Mx has antiviral function (mice) and Mx1 possesses a major impact on resistance and susceptibility to influenza; Mx proteins block viral replication. A PCR test was developed to identify polymorphisms in the Mx gene. Beckman Research Institute (CA). Work continues on defining functions of B and Y regions of the MHC. Major sequencing effort with international collaborators continues. To date, 15 Ylec loci are found intertwined w/16 non classical Class I loci in 255 Kb. An overall objective is to search for the gene(s) impacting MD and tumor growth. Studies of Ylec molecules are in progress; these may be involved with NK cell activities and may be used by viruses as decoys to evade NK cell activation. Work continued on isolated peptides to establish features of the Class I binding site domains; amino acid motifs (dominant to infrequent) and models with folded protein structure were described. Motifs can be used within a web-based alogorithm to search what other proteins (and their sources) have similar motifs, e.g., from RSV and MDV (candidate proteins among viral proteins). Prior research showed that BR2 and BR4 recombinants, which are serologically identical, differ in Mareks disease incidence (BR2 incidence is 14% and BR4 is 48%) this was further confirmed and these were found to differ in crossover breakpoint defining a candidate gene region for influence of MHC in MD (UCD, Wakenell; UNH, Taylor). Cornell University (NY). SPF flocks were remarked on, and the priority over the next 5 yr is to conserve these including P2a (B19, C2, MDV susceptible), N2a (B21,C2 MDV resistant). Cornell also maintains a White Peking duck flock including 20 females 8 drakes. CIAV latency and reactivation studies continued. Four hormone response elements sites were identified in the promoter region along with a SP1 local site. These were tested for hormonal control, using various constructs, and transfections of DF-1, LMH, LMH/2A, theca and granulose primary cells. Downstream elements down regulate transcription, and long promoters resulted in fewer transcripts by qPCR. vvMDV impact on cytokine levels in the N2a and P2a SPF flocks was studied. Lines exposed to virulent JM-16 or vv+RK1 were examined and virus replication studied by qPCR, cytokine transcripts were measured (spleen and cerebellum), nitrous oxide (NO), and also histopathology for lesions. High responses were seen in the N (resistant line). Conclusion is that vv+ causes a strong proinflammatory response in brain, independent of MHC type. Clemson University (SC). Hardarian gland (HG) studies continued to establish cytokine expression with specific emphasis on IL-6 involvement using a bioassay for IL-6 activity. Proteomic analysis (S. Burgess) of HG supernatant fraction was conducted and protein sequences compared. Several proteins were cloned and sequenced including H-Prostaglandin GD2Synthetase. The C1 Vick line was studied and the enzyme system was found to be present. Studies contribute to understanding mast cell biology; intermediate and end products of mast cell metabolism (hypersensitive immune response) involves immediate and latent effects due to mobilization of arachidonic acid pathway and production of prostaglandins, PGE2 is of prime importance given most symptoms. Iowa State University (IA). Gene expression tools were established (microarray and qPCR) to study variation in the founder lines for the ISU resource population. Following Salmonella exposure, mRNA was examined at 2 and 18 hr, using chicken 13K cDNA array from the Hutchinson Cancer Research Ctr; spleen and cecum were studied. A mixed model approach (rather than threshold change) was the approach for data analysis. Applied consistency criteria to reduce number of genes to consider for qPCR as 100s of differentially expressed genes were identified at threshold at both times or for all lines. Genes included those one would expect, MHC class II up-regulated, Ig light chain down-regulated, in addition to novel genes such as quiescence specific protein precursor (A30230). The goal is to use data from advanced intercross (F8) to identify important genes, segregating SNPs, develop immunohistochemistry and proteomics profiling. ISU Genetic Resources at ISU for Chicken Biotechnology Research include the following: 20 inbred line, many MHC congenic sets; 4 partially inbred Leghorn line (B X IrGAT); 1 non-inbred broiler breeder male line; 2 adv intercross line (broiler x inbred) (now F8). Genetic line priorities include the Advanced Intercross, G-B1 and G-B2 Leghorn lines, M5.1 and M15.2, Fayoumi, a broiler line, and the lines from 1925 inbreeding program. Northern Illinois University (IL). An exacting search for recombinants involving crossover between B19 and B21 was conducted; over 1000 chicks were examined, but no recombinants were recovered indicating B system recombination is a rare event. Haplotyping of five Hy-Line commercial stocks was undertaken (closed WL female breeder lines) for A,B, C, D, H, I, L, P and M systems. On average 1-2 alleles were found per system (and three for P in one line). LEI0258 microsatellite marker was used for MHC allele detection (Fulton); B13 and B21 are serologically different but exhibit the same LEI0258 amplicon size; by SSCP a 1-2 nt difference was detected. Ring Necked Pheasants were dropped from the NIU collection, the Northern and masked bobwhite colonies remain. BL polymorphisms are being examined by SSCP to categorize patterns. The objective is to maintain 12 MHC haplotypes from the original colony (1999). Evidence was found for a trisomic condition in Line 4 for GGA 16. Typing is ongoing w/ non cross reactive alloantisera, and LEI0258 wherein three amplicon sizes were identified (195, 264 311 bp sizes for B1, B2/B29, B10). Texas A&M University (TX). Enhancement of immune response of commercial poultry to eimera species was studied. Mechanisms were explored for new vaccine strategies and sensitization. Three experimental strategies were explored: Drinking water antigen (BSA) exposure, CpG oligonucleotides, and enhancement via live oocyst vaccination. Increased IgG levels were dependent on route of administration. For protein antigens drinking water may be an effective route. Also examined immunity boost with non-methylated CpG oligonucleotides as an adjuvant to BSA using a single oral administration to explore the resulting Ig status. Results indicated that, given orally, CpG can increase Ig secretions. University of Arkansas (AR. Smyth line chicken is an animal model for human autoimmune vitilligo, a pigment disorder, wherein pigment is lost in patchesthis is the only animal model for the human disease. Recent work focused on the issue of whether there exists a defect in anti-oxidative capacity in the melanocytes and their ability to respond to stress. Smyth cultured cells were found to possess an inherent susceptiblity to lipid peroxidation (higher in Smyth cells) vs Brown Line (parental stock) or Light Brown Leghorn (control) lines. Catalase activity was similarly heightened in Smyth line cells. In vivo, in the feather tissue, heightened levels of lipid peroxidation correlated to onset of disease. Other autoimmune diseases are found in Smyth line, alopecia and autoimmune thyroiditiis (as in human). University of CaliforniaDavis (CA). A new survey is in progress for listing poultry and avian genetic research resources, to update that published within the Avian Genetic Resources Task Force Report in 1999. The information will be presented within a searchable website. Results were reviewed from a collaborative study (Miller, Bacon) exploring MHC expression in the early chicken embryo at 0-48 hr of incubation. Results indicate that B-F and B-L are expressed on cells of early differentiating embryos at each of the stages examined whereas B-G was found at only at one stage (12 hr blastula stage). These results show that the avian embryo, like the mammalian has MHC expression prior to the elaboration of the immune system. University of California-Davis (CA). Reviewed studies of MDV resistance of R2 and R4 birds after challenge w/ RB1B (vv strain of MDV). Serum chemistry (dried format) can provide good insight to health status of birds and provide correlates with typical MD phenotype (e.g., lesions, etc.). Liver enzyme analysis was utilized (alkaline phosphatase (AP)). Lesion profiles of R2 and R4 birds were typical (kidney, liver, gonad etc.) although thymus and lung tumors were observed (which are not typical). Interestingly, in the inbred/congenic lines bearing liver tumors, retrospectively a drop in AP levels could be detected three to four weeks prior and so one could pick up initial disease before the animal succumbs to virus. Such predictability is of great value for future. Note this drop in AP level is opposite to what happens in mammals with liver disease; the decrease remains to be explored. University of Delaware (DE). F-box proteins are involved in the ubiquitization of proteins which marks them for degradation (e.g., viral proteins). Database ESTs were searched to identify proteins for motifs and 20 were identified allowing the categorization of three classes. Analysis keyed on specific proteins that might be involved in cancer, tumor transformation, and viral presentation and a subset will be studied for their expression in immune-activated tissues. Proteins were characterized of MD-stunted birds (commercials) in families with high incidence of growth defect during MD challenge. Unusual proteins were identified in spleen including annexin 5 (involved in apoptosis, protein metabolism). DNA of birds was archived to retroactively examine for correlated alleles. University of New Hampshire (NH). Characterization continued of responses to RSV of the B1 haplotype from a commercial line. B1/B2 and B1/B5 haplotypes were tested by exposure to RSV subgroup D and tumor growth was monitored. Pattern of tumor growth and analysis of genotypes are underway. LEI0258, a microsatellite marker is being used as a marker to discriminate the haplotypes. Studies continued in producing congenic MHC recombinants on UCD 003 including R1-R6, and R13, R7, 10, 11, 12. Although for some lines, only created one cross w/UCD 003 and then discontinued due to financial constraints. Wagenigen Agricultural University (NL). Immunomodulation studies were conducted involving primary and secondary immunization w/HuSA. The Ig, IgM, IgG responses of various treatments of LPS, PBS, KLH were studied and it was observed that these homotopes enhanced responses, likely a non-antigen specific recall response. Non-steady state situations (complex stress simulations: starvation, long term cold stress) were examined to explore if chickens respond/adapt to these stressors. Antibody responses were not affected, specific antibody responses were normal; however, natural antibody levels were affected by cold stress. Over time, adapted responses were observed for continuous cold stress (using both high and low antibody lines). Body weights were reduced, at half rate of expectation. Studies show that immune response has a high priority as compared to growth. Research speaks to homeostasis: IR has low energy requirements (as compared to growth), but gets priority under stressed situations. Wagenigen Agricultural University (NL). Studies of QTLs for KLH response in commercial feather pecking population (behavioral differences) indicated three QTLs detected on GGA 12, 14, 16. GGA14 (~80 cM) fine mapping was conducted to find candidate genes using SNP markers in one family. The QTL was narrowed toward the end of the chromosome, with the greatest effect at the end of the available markers, ~78/80 cM. In early generations a paternal allele effect was observed i.e., parent of origin effect on the QTL, in downstream generations observed lack of effect when allele was inherited from female in downstream generations. Results provide preliminary evidence for parent of origin effect for immune traits.