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The resolution of genes that determine resistance to disease is described using chicken lines maintained at the Avian Disease and Oncology Laboratory (ADOL). This description includes a summary 1) of existing selected and inbred lines differing for resistance to viral-induced tumors, i.e., Marek's disease (MD) and lymphoid leukosis (LL), and of the use of inbred and line crosses to define relevant disease-resistant genes, e.g., TV, ALVE, B, R, LY4, TH1, BU1, and IGG1; 2) of the development of TVB*/ALVE congenic lines to establish the affects of endogenous virus (EV) expression on resistance to avian leukosis virus (ALV), and methods to detect ALVE expression; 3) of the development of B congenic lines to define the influence of the MHC on MD resistance and vaccinal immunity, for producing B antisera, and for evaluating DNA sequences of Class I and II genes; and 4) of the current development of 6C.7 recombinant congenic strains (RCS) to define the role of non-MHC genes influencing susceptibility to MD and LL tumors, immune competence, and epistatic effects of genes. The procedures of pedigree mating, to avoid or maintain inbreeding, and of blood-typing, to ensure genetic purity of the lines, are also described.

Chickens of the B2, B5B15B19 and B21B-congenic haplotypes differ in disease resistance. Complementary DNA from B-congenic chicken strains have been analyzed for allelic diversity of expressed Class II MHC genes. The predicted amino acid sequences of eight genes from five haplotypes were subjected to Wu-Kabat variability analysis. The B-L gene polymorphic regions and conserved regions are highly similar to the human leukocyte antigen Class II genes. Therefore, the present analysis reveals candidate polymorphisms important in determining the spectrum of antigenic peptides presented to T helper cells, and allelic differences possibly important in resistance to avian disease.

The process of RNA interference (RNAi) has been exploited in cultured chicken cells and in chick embryos to assess the effect of specific gene inhibition on phenotypes related to development and disease. We previously demonstrated that avian leukosis virus-based retroviral vectors are capable of delivering effective RNAi against Marek's disease virus (MDV) in cell culture. In this study, similar RNAi vectors are shown to reduce the replication of MDV in live chickens. Retroviral vectors were introduced into d 0 chick embryos, followed by incubation until hatching. Chicks were challenged with 500 pfu of strain 648A MDV at day of hatch, followed by assays for viremia at 14 d postinfection. Birds were monitored for signs of Marek's disease for 8 wk. A stem-loop PCR assay was developed to measure siRNA expression levels in birds. Delivery of RNAi co-targeting the MDV gB glycoprotein gene and ICP4 transcriptional regulatory gene significantly reduced MDV viremia in vivo, although to lesser extents than were observed in cell culture. Concomitant reductions in disease incidence also were observed, and the extent of this effect depended on the potency of the MDV challenge virus inoculum. Successful modification of phenotypic traits in live birds with retroviral RNAi vectors opens up the possibility that such approaches could be used to alter the expression of candidate genes hypothesized to influence a variety of quantitative traits including disease susceptibility.

Alloantisera directed against chicken class I MHC (BFIV) antigens were produced by using transfected cell lines expressing cloned BFIV sequences. The cloned BFIV sequences were from haplotypes *12, *13, and *21. Two laboratory-derived class I mutant sequences (BFIV13m126 and BFIV21m78) were developed to analyze cross-reactive epitopes and to induce specific alloantisera. Antisera were tested in hemagglutination and flow cytometry assays. The antisera produced were highly specific and had minimal cross-reactivity. The antisera induced by the BF1V21m78 mutant confirmed the significance of amino acids 78 and 81 in cross-reactivity between haplotypes B*21 and B*5. The highly specific antisera were tested by hemagglutination on red blood cells of 31 different MHC haplotypes. The consistency of hemagglutination patterns and minimal cross-reactivity demonstrated that these BFIV antisera are extremely valuable in defining MHC haplotype in various chicken lines. Because of the extreme low level of recombination between the chicken class I and class II loci, identification of BFIV allele can be used to define MHC haplotype within a line. Complete identity between the transfected cell line and the chicken used to produce the antiserum is required to ensure the monospecificity.

Current poultry vaccines are based either on live attenuated organisms or on killed organisms. Future vaccines also may be based on deletion mutants, live viral or bacterial vectors that express foreign genes, and naked DNA. Vaccines have different purposes, depending on the disease, which govern their intrinsic characteristics. Improvement of vaccine efficacy can be addressed by modifications of the vaccine and its administration, modifications in the capacity of the host to mount an immune response, and modifications of environmental factors. The concept of "designer vaccines" for matching vaccines that deliver specific antigenic peptides to chickens with the MHC haplotype that best presents those peptides to T cells is discussed.