Brief Summary of Minutes of Annual Meeting

Minutes of Annual Meeting Recorded by Pat Wakenell, 2007 secretary NE1016 business meeting opened 4:15 PM Present: Pat Wakenell, Sue Lamont , Tom Scott, Henk Parmentier, Jan van der Poel, Renee Kopulos, Bob Taylor, Mohammad Heidari, Gisela Erf, Shayan Sharif, Fritz van Ginkel, Marcia Miller, Ton Schat, Mark Parcells, Narayan Rath, Sandy Ewald, Kirk Kerr Kirk Kerr: explained requirement for renewal of grant every 5 years  old is closed out and new begun  time line was handed out in hard copy format. Request for renewal must be submitted by 10/31/07, proposal must be submitted by 2/29/08 AFTER peer-review by at least 3 reviewers  group can suggest the reviewers  get to peer reviewers by 2/1/08. From todays meeting, need minutes, annual report and a person to do the summary report of end of project (due 90 days from 10/1/08). Pat responsible for minutes and Ton responsible for annual report. There was no annual report from last year  Bob will take care of that. Some of new members did not submit proper forms for membership  Huaijun Zhou - Bob will set up a secondary list of invited but not formal members and ask them to submit the required forms if they still plan to be members. No budget information available with absence of Muquarrab Qureshi. Kirk plans to work on revision but will be stepping down July 1. The technical committee commends Kirk for the years of service to the NE1016 committee. Kirk asked if there are any suggestions of a person in the Northeast that would replace him. Bob is in charge of the renewal of the true NE-60 / NE1016 project. Bob had emailed two requests prior to this meeting with an incredible dearth of responses. He will send 1 reminder 1 week later. After that, you will be on the tardy list that will be distributed to all members for maximum effect. Changes of objectives: New suggestions: Objective 1. Identify and characterize genes and their relationships to disease resistance in poultry with an emphasis on the MHC as well as other genes encoding alloantigens, communication molecules and their receptors and other candidate systems. Objective 2. Identify and characterize environmental, dietary and physiologic, factors that modulate immune system development, optimal immune function and immune system related disease resistance and welfare in poultry genetic stocks. Objective 3. Develop, evaluate and characterize methodologies, reagents and genotypes to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry Publications lists needed by Fri  from 2003 no in press, format of Poultry Science, list only # of abstracts, no Endnote formats! Based on above new objectives, need a max 2000 limit total of which objective and what will be done in new project by 11/1/07.

Extensive collaboration among members continues as evidenced by the frequent exchange of reagents, birds and virus strains. Studies continued on the identification of candidate genes influencing resistance to pathogens, fine mapping of the MHC region, and on understanding vaccine induced immunity. Auburn University (AL). Broiler flocks are being established that segregate for the MX gene Asn631 and Ser631. The MX gene with Asn631 has been linked with antiviral activity against avian influenza virus (AIV) after activation by type 1 interferon (IFN-I). Chicken embryo kidney (CEK) cells were treated with IFN-± and challenged with low pathogenic (LP) AIV. Thus far few results have been analyzed to determine the antiviral effects of MX Asn631 (MX+) with our without IFN-± treatment. There was very little antiviral affect in the Mx+/- IFN-±- compared with the Mx-/- IFN-±-treated cultures, although there may be a slight antiviral affect in MX+/- versus MX-/- cells in the absence of IFN-±. A similar approach was used to determine if MX gene activation would reduce infectious bursal disease virus (IBDV) replication in chicken embryo fibroblasts (CEF). IFN-± was a strong inhibitor of IBDV replication, but this is likely to be independent of the MX genotype. Beckman Research Institute (CA). The gene map encompassing 242 kb of the MHC region has been completed. The BG2 gene is separated from the BL and BF genes by 28 other genes, many of which are most likely to be important for immune responses. The new map also allowed locating more precisely the breakpoints for some of the recombinants which differ in the resistance to Mareks disease (MD). The epitopes were defined for BF2*13 and BF2*21. The former consisted mostly of 10 amino acids with 4 anchors (positions 2, 3, 5, and 8), while the epitopes for BF2*21 consisted mostly of 10 amino acids with anchors at position 8 and 10. The knowledge of the epitopes may facilitate the development of designer vaccines. Proteomic profiling of functional studies of embryonal natural killer (NK) cells has indicated that expression of the nonclassical MHC YF7.1 inhibits NK cell activity. In addition BF1 alleles from BF13 and B21 also inhibit NK cell activity while B2*21 promotes killing. Studies are in progress to examine the specificity of the NK receptor encoded by the Blec2 gene. Preliminary data suggest that this receptor recognizes specific nonclassical MHC-I (e.g., Y7.1) alleles. Clemson University (SC). Studies were continued on the importance of thrombocytes as a source of cytokines. Broiler chickens were fed a control chick starter feed, starter feed supplemented with corticosterone, or starter feed supplemented with corticosterone and Vitamin C. These diets did not influence the production of pro-inflammatory cytokines IL-1beta, -6 and -12 in thrombocytes. The effects of in vitro LPS treatment on thrombocytes were examined using thrombocytes from 8-12 week old white leghorn hens. LPS treatment increased significantly mRNA expression of TLR4, IL-6, and COX-2 and the release of PGE2 into the culture media. Treatment with the inhibitor PD98059 did not affect expression of IL-6 and irrespective of PD98059 treatment LPS significantly elevated the expression of IL-6. However, the LPS-stimulated expression of COX-2 and release of PGE2 into culture media were dramatically decreased by PD98059 pre-treatment followed by LPS treatment. Treatment with BMS345541 significantly inhibited LPS stimulation of IL-6 and COX-2 mRNA and had a slight effect on reduction of PGE2 release from thrombocytes into culture media. The importance of the NF-ºB pathway on the expression of IL-8 in thrombocytes was also examined. Inhibition of IKK² significantly reduced Il-8 mRNA expression. These findings suggest that thrombocytes are important cells for elaboration of inflammatory mediators which can be cytokines, prostanoids and chemokines. Cornell University (NY). The specific-pathogen-free P2a (B19B19) and N2a (B21B21) flocks are continued to be maintained and fertile eggs are available for research. The MD virus (MDV) polypeptide pp38, but not the recently described splice variants A and B, is responsible for the alteration of metabolic activity in the QTP32 (dox-inducible quail cell line QT35). Because of the difference in the activity of mitochondrial succinate dehydrogenase an up-regulation in the number of transcripts of key mitochondrial enzymes in the oxidative phosphorylation pathway was expected after Dox-induction Real-time RT-PCR (qRT-PCR) assays for Japanese quail Cytochrome C oxidase-1 (Cox-1), Cytochrome B, and ATP synthase A chain showed unexpectedly that Cox-I and ATP synthase A-chain were significantly down regulated in Dox-induced cells only expressing full length pp38. Transcription of cytochrome B was more variable but not significantly different between induced and control cells. ATPase enzyme activity in isolated mitochondria was also significantly decreased after dox-induced expression of pp38. The potential use of immune complex (Ic) vaccines consisting of chicken anemia virus (CAV) with predetermined antibody levels was examined in SPF chickens. Viremia was delayed in chickens inoculated with Ic preparations and hematocrit values were not affected with the optimal dose of antibodies. Differential qPCR analysis indicated that birds were protected against challenge. Iowa State University (IA). Seventeen highly inbred lines (50  100 generations of sib matings) of defined MHC type and a broiler line were maintained. A resource population for analyzing the host genetics of early response to Salmonella (Iowa Salmonella Response Resource Population, ISRRP) has been developed over the years by crossing outbred broiler males with females of two distinct, highly inbred lines (Leghorn and Fayoumi). The ISRRP is now maintained in advanced intercross line (AIL) form, with the F12 generation being hatched in the past year. In total 2700 SNPs were analyzed for association with Salmonella enteritidis (SE) burden levels in poultry in two F8 AILs. A total of 21 SNPs were significant at a false discovery rate of 25% for SE bacterial levels (13 associated with cecal bacterial burden and 8 with spleen bacterial burden), representing 19 new candidate genes for host response to SE with a wide variation in molecular function, including apoptosis, cell signaling, and DNA repair. ARPP-21 and MLH1 were the only genes that were associated with both spleen and cecal bacterial burden. Ten SNPs were in pathways that have been previously associated with immune response to SE bacterial challenge (toll-like receptor [TLR] signaling, apoptosis, and MAPK signaling pathways). We have conducted microarray analysis of tissue samples from the ISRRP AIL F8 generation from 1-week-old birds exposed to Salmonella at one day of age, and characterized the SE-exposed tissues as relatively resistant or susceptible to bacterial colonization based upon bacterial count. Two publications outlining the results from early (< 1d) spleen responses of three genetic lines and one on F8 cecal tissue are in preparation. qRT-PCR assays were used to investigate TLR expression in cecum and spleen of three genetic chicken lines and in two AIL (8th generation) lines after challenge with Salmonella. Genetic line effect was significant on TLR RNA expression in the spleen of Salmonella infected birds, with higher TLR2 and TLR4 expression in the Fayoumi line and higher TLR5 expression in broiler line. In Salmonella challenged birds, TLR2 expression in both cecum and spleen and TLR4 expression in spleen were significantly higher at 18 h post-inoculation compared to 2 h PI. Infection of the AIL chickens resulted in significant upregulation of TLR2 and TLR5 RNA expression in spleens and upregulation of TLR4 RNA expression in both cecum and spleen. The increase in TLR2, TLR4 and TLR5 RNA expression level was found in spleens from male but not female chicks. Bacterial burden of Salmonella serovar Enteritidis in challenged birds was not correlated with TLR RNA expression level. The impact of SE infection on lymphocytes was examined by immunohistochemistry, infection was associated significantly associated with an increased area staining for macrophages and decreased apoptosis in cecal tissue sections compared to uninfected animals. North Carolina State University (NC). Previously, the effect of the probiotic Primalac on the physiology of the gut was examined but the biological relevance of minor differences was not clear. Current studies are examining a potential influence on the immune response to sheep red blood cells (SRBC). Preliminary data suggest that Primalac may influence the kinetics and class switching of the antibody response. Type-2 turkey astrovirus (TAstV-2) is associated with the poult early mortality syndrome (PEMS), but there is no clear adaptive immune response to TAstV-2. To better understand the local immune responses to this gut pathogen qRT-PCR assays are being developed for 14 genes believed to be expressed in mucosal immunity. Northern Illinois University (IL). The RFLP patterns of Y haplotypes vary between lines to the extent that each chicken population seems to have unique Y haplotypes. Currently there are at least 30 different YF haplotypes segregating in the lines that have been analyzed. Four additional recombinant lines have been partially characterized using the LEIO258 marker. Four commercial lines, C1, C2, C3 and C6 have been tested for several of the non-MHC alloantigen systems in addition to B and Y system genotyping for progeny evaluation. These lines are currently used to produce line specific typing reagents. Proteins representing the L alloantigen have been submitted to Dr. Shane Burgess at Mississippi State University for analysis. Specific matings are conducted to supply collaborators Lillehoj and Collisson with hatching eggs for projects on coccidiosis and AIV, while Wakenell is evaluating 6 allotypes for potential resistance to MD. Texas A&M University. Tools and assays have been developed to start the evaluation of T lymphocyte responses to AIV. MHC-defined kidney cells from 10-day old naive birds will be used as a source of MHC compatible and mismatched antigen presenting cells (APC). Immune cells are collected from the peripheral blood mononuclear cells (PBMC) of AIV infected chickens 2 to 25 weeks of age and 10 days to 5 months p.i. Infected APCs and T cells are co-cultured for 24 to 48 hours prior to adding macrophage generated NO containing supernatant fluids and assayed for interferon-gamma production. Attwaters prairie chickens are a federally endangered grouse species. Captive breeding programs are hampered by the high incidence of reticuloendotheliosis virus (REV). In infected birds as much as 50% of the CD8+ or CD4+ cells in a preparation of PBMC can be actively infected with REV. Using nested PCR approaches rather than single PCR it was possible to generate a small nucleus of negative birds for breeding. However, infection could be reintroduced perhaps through poxviruses carrying REV. To prevent reinfection an experimental DNA vaccine has been developed which will be tested in the related, but not endangered, greater prairie chicken. University of Arkansas (AR). The onset of vitiligo in the Smyth line chickens is preceded and accompanied by increases in IFN-³, IL-1², IL-8 and IL-12 expression but not IL-4, iNOS and IL-6 expression based on qRT-PCR studies. These observations are in line with reports of a Th1 polarized response in human vitiligo. Moreover, NALP1, an intracellular pattern recognition receptor which detects danger signals such as microbial products and HSP and forms part of the inflammasome, is required for the cleavage of IL-1² into its active form and was recently identified as a candidate gene involved in human vitiligo and other autoimmune/immunoinflammatory diseases. The expression of NALP1 in feathers from SL chickens with vitiligo is increased 3-fold above levels observed in feathers from SL chickens without vitiligo and parallels the increase in IL-1². Studies are in progress to determine the potential involvement of macrophages, complement and autoantibodies in vitiligo. The latter may be used as indicators of antigens involved in the melanocyte destruction. A time course study on the role of herpesvirus of turkeys (HVT) in vitiligo is in progress. Feather tips were found to be a good source for the study of immune cells and antibody responses. Antigen can be injected in the feather tips and the tips as well as blood samples can than be used to monitor local as well as systemic immune responses. University of California-Davis (CA). The resistance to MD of an additional recombinant, BR3, was compared to the MD resistance of BR2 and BR4. The percentage of MD positive BR3 homozygous birds (12) was intermediate between that of the BR2 homozygotes (8) and the BR4 homozygotes (16), but histopathology is still pending. The pathogenicity of QMDV, a MDV isolate rescued from QT35 cells, was compared with JM, RB1B, and NC584A (chickens only) in both SPF chickens and quail. Preliminary results indicate that QMDV is comparable to RB1B in chickens and JM in quail. Serum chemistry was performed on SPF chickens challenged with Eimeria tenella. Serum AP, LK aspartamine aminotransferease (AST), GGT and LDH were significantly lower in E. tenella challenged birds at day 5 PC then in control birds. When serum chemistries were compared between birds which died during this period and those that survived, AP and AST were significantly depressed in moribund birds at day 5 post infection but there were no differences with CK, GGT and LDH. University of Delaware (DE). The MDV Meq gene has been linked to transformation, but may have also other functions. Meq genes in lower virulence MDVs (notably JM102, CU-2, BC-1, etc.) have an expansion of the proline-rich repeat region, which is associated with transcriptional repression. In vv+MDVs Meq has specific mutations in this proline rich repeat region with functional consequences for the transcriptional activation function of this protein. Research is focused on the identification of actin-regulating genes as a class of Meq target genes and to determine if Meq-induced changes can influence the susceptibility of tumor target cells to cell-mediated immune responses. The importance of MDV glycoprotein L (gL) was examined by using a gL deletion mutant (TK-1a) of the TK strain. Antibodies to gL, gH and the gH/gL complex (generated against MDV-1, 584c p80, or CVI988 strains) cross-react strongly with gL and gH of HVT and SB1. These data suggest that not only did the vaccines present similar epitopes to the gL/gH complex, but that HVT expresses a higher-density of these antigens per infected cell than MDV-1 strains. The gL deletion was observed in all field isolates of MDV-1 (n=30) obtained from vaccine breaks in 2005 and 2007. To examine the imporetance of this deletion for vaccine-induced protection, commercial broiler embryonated eggs were vaccinated in ovo at ED 18 with HVT (5,000 PFU), with bivalent vaccine (5,000 PFU HVT/ 2,500 PFU SB1) or left unvaccinated and inoculated with 200 PFU of TK-1a (gL ), TK-2a (gL non-deleted) or a 1:1 mix of these strains (Mix) at hatch. The incidence of MD + Mortality was decreased by vaccination (HVT and HVT/SB1 groups) indicating that even with challenge at day of age, vaccination provides a level of protection. Despite having a decreased incidence of MD + Mortality in vaccinated groups, the TK-1a strain caused high levels of MD + Mortality in both HVT and HVT/SB1 vaccinated groups. Challenge with TK2a showed the expected pattern of incidence with HVT providing some protection, but the bivalent vaccination provided higher levels of protection. Birds infected with theTK-2a and the Mix of TK-1a +TK-2a experienced a greater level of MD + Mortality than the TK-1a strain alone. Analysis of virus in the birds inoculated with Mix showed predominantly TK-1a virus. University of Guelph (CANADA). Immune-specific Microarrays have been used to compare immune system gene responses in the Cornell P2a and N2a chickens at 4-21 days post infection with the RB1B strain. Genes associated with T and B cell and perhaps NK cells were differentially expressed during this period. T cell subsets from RB1B-infected P2a and N2a chickens were prepared at 4, 10 and 21 days post infection and analyzed for expression of cytokine mRNA by qRT-PCR. IL-18 was upregulated at 4 days in CD4+ cells and at 21 days in CD8+ cells in the susceptible line and at 4 and 21 days in CD8+ cells in the resistant line. Il-6 was upregulated at 4 days in both lines, while IL-10 was expressed at all time points in both lines. CD4+ and CD8+ cells infiltrate in the feather pulp of MDV-infected chickens starting at 4 days post infection with peak infiltration at 10 days. The expression of IL-18, IL-6, interferon-gamma and MHC-I was significantly enhanced in the feather pulp of MDV-infected chickens. University of New Hampshire (NH). Six-week-old R13R13 chickens were compared to other MHC congenic recombinant (R1, R2, R4, R5) lines for Rous sarcoma virus (RSV) tumor outcome after challenge with subgroup C RSV. Tumors grew in >90% of inoculated chickens. The tumor size increased in all genotypes through week 3 post-inoculation. Tumors in R2R2, R4R4, and R5R5 chickens plateaued thereafter while tumors in R1R1 chickens continued to increase in size. The mean tumor profile index (TPI) for R5R5 chickens was significantly lower than the TPI of R2R2, R4R4, and R13R13, but not R1R1. USDA, Avian Disease and Oncology Laboratory. A formal report was not presented due to the change in personnel associated with the NE-1016 project. Wagenigen Agricultural University (NL). The 20K SNP set (Illumina Infinium platform) are being used to genotype a broiler chicken population and several research chicken lines from the IAH (Compton, UK), INRA and the Wageningen selection lines (high, low and control). The sequences of the first domain of the B-F locus and the BLb2 locus were analyzed in the Wageningen lines showing variation. Thus, sequencing of the BF1 or BLb2 gene exon seems a necessary addition to the molecular MHC typing using the LEI0258 microsatellite marker. Based on the DNA sequence analysis two B24 subtypes could be defined in animals, which seemed homozygous using the LEI0258 marker. One of the Wageningen B24 subtypes is identical to B24 defined by Briles, the other is clearly distinct. LEI0258 homozygous chickens from the INRA lines also showed polymorphism in TAP1, TAP2, tapasin, BF and BL genes. A SNP set representing 1536 SNP focusing on the QTL region on GGA 3, 4, 5, 7, 26 and Z has been used to genotype 960 animals, the resulting genotypes are being are being used for associations studies with previously recorded immunological and behavioral parameters. PAMP-like components present in organic feed may modulate immune responses. The proposed working hypothesis is that organic feed may restore immune functions to a normal species-restricted average level. To determine if alpha-GAL has an adjuvant function, layers were inoculated with alpha-GAL Human serum albumin (HuSA), beta-GAL HuSA, or naïve HuSA. Unexpectedly, the addition of alpha-GAL, but not beta-GAL, decreased antibody responses to HuSA significantly. In preliminary studies the addition of dietary probiotics did not influence immune competence in layers. Studies were initiated to identify physiological (behavioral, immunological) mechanisms and parameters that predict and/or underlie health status and zootechnical characteristics but also abnormalities (feather pecking, cannibalism, and unidentified death causes) in various layer breeds kept under commercial conditions. The first results suggest a relation between level of binding of natural antibodies to LPS or KLH and risk of mortality. Additional data suggest different levels of (heat-) stress sensitivity in layer breeds which were established at the neuron-endocrine as well as the immunological level, and which were reflected at the growth and production level. The data suggest that birds that are able to mount higher corticosteron responses to stress are more able to respond and/or adapt to stress than birds with lower corticosteron responses.