Brief Summary of Minutes of Annual Meeting

OBJECTIVE 1. Several NE-60 stations (CA, IA, NC, NH, NYC, NIU, ARS-ADOL) maintained important genetic resource populations despite budgetary constraints. Attempts were made to identify MHC haplotypes in commercial broiler lines. Genotyping was done using restriction fragment length polmorphism (RFLP) for B-G, and RT-PCR followed by cloning and nucleotide sequence determination for B-L and B-F genes (AL). Eight haplotypes were characterized in Line C, and two haplotypes in Line E (AL). In combination with the 10 haplotypes previously characterized in broiler breeder Line A, seven haplotypes were identified that encoded novel B-F and B-Lb alleles not previously reported in Leghorns (AL). The influence of two MHC haplotypes (BA4 and BA5) on disease resistance in broiler breeder Line A was also tested (AL). BA5 haplotype was associated with relative resistance to cellulitis induction by E. coli, by comparison with BA4 haplotype. The reverse was seen in collaborative studies (AL and DE) in which chicks were challenged with RB-1B strain Mareks disease virus (MDV). DE has characterized the molecular MHC genotypes in a commercial broiler population. At least 4 class II and 8 class IV RFLP genotypes were discovered whose frequencies varied among three lines within the population. At least one more class II allele that was not previously reported was identified. An MHC gene, B11, that appeared to confer a significant degree of MDV resistance was studied by CA. Eight non-MHC alloantigen systems were examined for their effects on chicken mononuclear phagocytic system cells (NC in collaboration with NIU). Chicks of P allotype carrying P13 and P1 allele were high responders for LPS-induced nitric oxide synthase (iNOS) expression and activity, whereas macrophages from I8 allotype chicks produced highest levels of IL-6. This suggested that alloantigen systems can exert significant influence on immune endpoints independent of the B complex (NC). Non-MHC alloantigen systems had significant effects on Rous sarcoma virus (RSV) tumor outcome in a progeny of matings between B2B5 sires and B5B5 dams derived from a cross of Modified Wisconsin Line 3 and Line NIU 4 White Leghorns. (NH). The influence of B and L haplotypes on immunity to E. tenella was evaluated. The results support prior studies showing lower lesion scores in the B5B5 genotype compared to B2B2 chickens (NH). Identification of candidate genes for response to Salmonella enteritidis (SE) was of interest to IA. Associations of the following genes were found with one or more traits of SE response: MHC class I, NRAMP, IAP1, caspase, and prosaposin (IA). Several groups also examined gene systems with involvement in immunocompetence. A chicken genomic DNA clone was found to contain a region of chicken chromosome 1 and it was syntenic with human chromosome 22 (DE). This clone was determined to contain the genes, Synapsin 3 and FBX07. To identify other non-MHC genes that affect MD resistance in broilers, DE established an F2 population, challenged it with RB1B, and recorded MD clinical signs, lesions and mortality. A collaborative project between (DE) and ARS-PBESL sought to identify genes affecting coccidiosis resistance. At least one quantitative trait locus (QTL) was identified on chromosome 1. Furthermore, a genetic component was identified (DE) that influences body weight during a MDV challenge in broilers Other studies compared the iNOS expression and activity between B15B15 (Cornell K-strain), B6B6 (G-B2) and B13B13 (G-B1) genotypes (NC). Macrophages from these chicken lines can be hyper- or hypo-responders for iNOS gene expression and activity regardless of the source of bacterial lipopolysaccharide. NH studied major histocompatibility (B) complex effects. The outcome of Rous sarcomas was assessed in second backcross generation BQB17 chickens with a genomic composition of 87.5% inbred Line UCD 003 and 12.5% inbred Line UCD 001. The results of this study suggested complementation between the BQ and B17 haplotypes. OBJECTIVE 2. PEMS is a multifactorial disease of turkeys that is characterized in part by immune dysfunction. NYC and NC have isolated of a novel reovirus (ARV-CU98). This astrovirus induced significant growth suppression, diarrhea, thymic and bursal atrophy and altered B-, T-, and macrophage functions. The finding that chicken infectious anemia virus (CIAV) was present in SPF chicken flocks had a major impact on NYC research. In order to generate quantitative data on the effects of experimental infection with CIAV on CTL generation, NYC developed real-time, Taqman-based, PCR and RT-PCR assays. These assays can also differentiate between two CIAV strains, Cux-1 and CIA-1. NYC found that CIAV can be an important immunosuppressive agent that can modulate immune responses. Polychlorinated biphenyls (PCB) are important environmental contaminants. Chickens treated in ovo with 0.39 ng/g 2,2-dichlorobiphenyl (2,2¢-DCB) had significantly lower primary antibody titers against SRBC compared with the vehicle-treated and control chickens (NH). Dietary factors were shown to have significant impact on performance and immunocompetence of chickens. Mycotoxins were shown to significantly suppress several immune parameters in direct-fed chickens or in progeny chicks (NC). Dietary addition of other compounds such as beta-glucans improved various immune endpoints in chickens (NC). AR also conducted several studies on nutritional supplements demonstrating beneficial immunomodulatory effects of dietary supplementation with vitamin E or bacterial cell wall products from both Gram + and Gram- bacteria. FRAMINGHAM revealed that including mannanoligosaccharide in poultry diets increases levels of both natural and acquired antibody. The importance of nitric oxide (NO) as a modulating factor for Mareks disease was examined. The most likely cells producing NO are macrophages or their precursors present in the CEF cultures. In most experiments, N2a chickens showed an increase in plasma NO levels at 7 dpi compared to the susceptible lines P2a. The very virulent (vv)+ strains of MDV caused significant higher levels of NO in the plasma than the less virulent strains. NYC also examined the importance of rIFN-a for the activation of natural killer (NK) cells by inoculating chickens with rMDV- IFN-a or by feeding rIFN-a in the drinking water. In both instances IFN-a caused a decrease in NK cell activity in spleen cells. AR focused studies on the mutant Smyth line chicken which exhibits genetically controlled spontaneous autoimmune vitiligo. AR has demonstrated that pigment cell death in Smyth line chickens involves an inherent melanocyte defect, an environmental factor , and immunological factors. One study evaluated primary and secondary antibody response to BA and SRBC in eight different genetic groups consisting of four different MHC types on two different line backgrounds. The overall results indicated complex interactions between specific MHC alleles and the non-MHC background of the lines in which they were studied (IA). Other experiments aimed at identifying genomic regions and specific genes controlling antibody kinetics in adult hens. Several candidate gene fragments were sequenced, including MHC class I and II, IgL, IAPI, ZOV3, IFN-g, TGF-b2 and b3 (IA). Thyroid hormone and thymulin were studied extensively by NYC. The station found that low level (0.1 ppm) thyroid hormone (i.e., triiodothyronine or T3) supplementation enhances both IL-2 activity from culture supernatants and IL-2R expression while higher levels (1.0 ppm) decrease the expression of both of these activities. Thymulin was found to modulate both the percentage of CD4+ and CD8+ cells and the mean fluorescence intensity (MFI) of these co-receptors. (NYC). The initial thyroidal status of a strain or of an individual appears to correlate with baseline natural killer (NK) cell activity. T3 supplementation may enhance NK cell activity, particularly in the T3-deficient SLD and this appears to be at least partially due to an enhanced responsiveness of NK cells to IFNg (NYC). Dietary T3 supplementation significantly increased abdominal exudate cell (AEC) responsiveness to rcIFNg in the hypothyroid SLD strain while not affecting AEC responsiveness in the K strain. T3 levels affect avian IFNg activity by influencing both the responsiveness to and the production of IFNg. Direct in vitro exposure of chicken splenocytes to thymulin suggests that one function of thymulin is to enhance the responsiveness of these cells to IFNg, perhaps by enhancing the expression of IFNg receptors. The ability of thymulin to act directly on macrophages was examined by testing the effect of in vitro thymulin treatment on HD-11 (a macrophage cell line) responsiveness to rcIFNg (NYC). rcIFNg treatments alone consistently increased NO production in a dose-dependent manner. NYC most recent work has focused on the ability of thymulin to stimulate in vivo NK responsiveness to a viral infection and suggests that we can enhance the resistance to infection with avian infectious bronchitis virus (IBV) with short-term thymulin supplementation. Matrix metalloproteinases (MMP) were examined in RSV tumors from NH congenic lines 6.6-2 (B2B2) and 6.15-5 (B5B5). B5B5 birds had significantly higher mean tumor profile index (TPI) than did B2B2 chickens. Congenic Line 6.15-5 tumors had significantly more RSV tumor sections that stained positively for MMP-2 and MMP-9 than did congenic line 6.6-2 tumors. PA investigated the role of cytokines and hormones involved in the initiation of humoral immunity, using lipopolysaccharide (LPS) [T-independent antigen] and bovine serum albumen (BSA) [T-dependent antigen]. It was concluded that although LPS and BSA injection can induce a humoral antibody response in chickens, the mechanism of antibody response initiation involving cytokine network and neuroendocrine system activation are different for each antigen. PA also found a possible recirculation of lymphocytes from circulation to different lymphoid compartments during septic response and to the local site of injection during the cutaneous hypersensitivity response. In studying the role of the photoperiod and melatonin on broiler performance and on the immune responses, PA demonstrated that using intermittent lighting (increasing the dark period), is advantageous over using 23 hrs of light for enhancing both production performance and immune responses. OBJECTIVE 3. Non-gene-specific genetic factors related to the response to Salmonella enteritidis (SE) in young chicks were investigated. In one experiment, using layer chicks, line was demonstrated to be highly significant on survival. In surviving chicks, the genetic line effect was significant on cecal SE burden but not on spleen, and these two measures were under independent genetic control (IA). In broiler breeder chicks, a significant negative correlation of vaccine antibody response and bacterial burden in cecum indicated that chicks with greater genetic potential to respond to vaccine are better able to control and eliminate the pathogenic bacteria (IA). Genomic regions associated with vaccine or challenge response to SE were identified in a resource population formed by crossing broiler sires and inbred (Leghorn or Fayoumi) dams. Microsatellites were used as the marker system. Associations of four microsatellites were confirmed for vaccine response and SE bacterial burden in spleen and cecal content (IA). The AL station developed a PCR with sequence-specific primers (PCR-SSP) to type alleles of the B-LbII locus. Serological reagents to identify new haplotypes discovered in broiler breeder chicken lines were produced. Another project was development of quantitative measures of bursal damage and viral load in chickens challenged with infectious bursal disease virus, for the purpose of assessing MHC associations with disease resistance. Finally, analysis demonstrated that broiler MHC haplotypes varied dramatically in expression of MHC class I molecules on lymphocyte cell surface; furthermore, MHC class I expression correlated with proliferative response to Concanavalin A. To map quantitative trait loci (QTL) associated with disease resistance to avian coccidiosis and growth, two commercial broiler lines with different degrees of resistance to the disease were crossed to generate F1 generation that were intercrossed to produce 314 F2 generation offspring (ARS-PBESL). Inter-trait comparisons revealed that oocyst shedding was a good parameter for evaluating disease resistance/susceptibility. A locus on chromosome 1 associated with oocyst shedding (LOD = 3.46) was revealed. The genetic mechanism of this locus appeared additive. The genomic scan also identified three potential growth QTL on Chromosomes 1, 6, and 8 (ARS-PBESL). NYC developed an approach to study CMI responses to MDV using the REV-based system. MHC-defined REV cell lines that can be lysed by syngeneic REV-sensitized CTL were stably transfected with MDV genes and used as target cells for the detection of MDV-specific CTL recognizing gB, pp38, meq, and ICP4 epitopes, although the latter one was only detected using CTL from the resistant N2a line. The effector cells were characterized as CD8+CD4- CTL expressing TCRab1. During the current NE-60 project period, NYC found that REV cell lines expressing the immediate early protein ICP27 are lysed by syngeneic effector cells from both P2a and N2a chickens. More recently a large study was completed in which CTL responses to MDV glycoproteins gC, D, E, H, I, K, L, and M were examined. Cell lines expressing gI were lysed by effector cells from N2a and P2a chickens, while N2a-derived CTL also lysed cell lines expressing gC and gK and to a lesser extent gH, gL, and gM. P2a-derived effector cells did not lyse cell lines expressing these glycoproteins, but did lyse cell lines expressing gE. The importance of these studies is that the differential recognition of MDV proteins by CTL from chickens with different MHC genes contributes to the understanding of differences in genetic resistance to MDV.