FUNCTIONAL GENOMICS AND GENETIC ANALYSIS OF THE INNATE IMMUNE RESPONSE REQUIRED TO RESIST FOOD-BORNE BACTERIAL INFECTIONS IN POULTRY
Location: Food and Feed Safety Research
Project Number: 6202-32000-021-00
Start Date: Jan 03, 2006
End Date: Jan 02, 2011
Poultry producers suffer major economic losses both from mortality due to infectious diseases, and from contamination of food products due to transfer of microorganisms. As a result, they have a keen interest in breeding birds capable of resisting infection by enteric food-borne pathogens such as Salmonella and Campylobacter, and the development of novel immunologically based strategies to control pathogenic microorganisms in the gastrointestinal tract of poultry. Understanding how the immune system is regulated and responds to infectious agents requires whole system approaches given that single immunological parameters have been unable to unlock immune system complexity. It is increasingly important to be able to measure changes in the expression of multiple genes in a tissue or animal in response to a single physiological change. The availability of the chicken genome sequence provides the opportunity to resolve questions concerning the molecular components of the innate immune system. Key developments in molecular, genetic, and cellular biological techniques provide us with new approaches to use the genome to investigate the functional genomics (the study of the how and why a given gene behaves in a certain way under specific conditions) and pathogenomics (the study of how genes behave when pathogens interact with their host) of the avian innate response to Salmonella and Campylobacter. Our primary goal is the use of genomic technologies to understand prospective control points for modulating innate immunity, thus providing the poultry industry with novel, pre-harvest intervention tools to control food-borne pathogens and provide safe food products to the consumer.
1) Assess innate immune variability in chickens and turkeys by examining the frequency of genetic polymorphism in genes related to innate immunity (cytokines/chemokines, toll-like receptors [TLR]). Initial analysis will be undertaken in 2 pedigree lines of chickens that we have characterized in terms of innate immune function and disease susceptibility and commercial lines of turkeys and their wild turkey counterparts; 2) Identify differentially expressed or genetically altered genes in heterophils from the innate immune functionally divergent lines of chickens and turkeys by use of suppressive subtractive hybridization (SSH). Normalized, directionally cloned avian heterophil cDNA libraries will be constructed from pools of mRNA purified from resting, inflammatory, and activated heterophils following either the in vitro stimulation with inflammatory agonists or in vivo following infection with Salmonella or Campylobacter; 3) Develop new modulators of innate immunity such as pathogen associated molecular patterns (PAMPs) as immune modulators of early colonization, PAMPs as adjuvants for commercial live vaccines, and the development of new live vaccines that specifically induce a heterophil-mediated innate immune response; 4) Apply anti-sense and RNAi technologies in poultry by introducing anti-sense oligonucleotides and RNAi to available cell lines (the macrophage HD-11 cell line) by transfection, using western-blot and Real Time-PCR to assess the effectiveness of gene silencing, and characterizing the function of the target gene by stimulating the cells with PAMPs and measuring immune responses. The in vivo experimental approach is similar to that stated above, except that in ovo route will be used to introduce anti-sense oligonucleotides and RNAi; and 5) Evaluate anti-microbial peptides from chicken and turkey heterophils that have potential as new biotherapeutics for food-borne bacteria.