Genetic Basis for Resistance and Immunity to Avian Diseases

NON-TECHNICAL SUMMARY: Poultry diseases are the most problematic management problem in the poultry industry. Prevention and damage control of diseases cause major revenue losses to the poultry industry. Our project is aiming at gaining a better understanding of immune function in poultry and more specifically how the immune system is being regulated by the nervous system and by hormones. <P>

APPROACH: The specific aims of our contribution to this project include: (1) determining the nature of the secretory products of the thymic and bursal neuroendocrine (NE) cell populations we have recently identified in the chicken; and (2) determining the putative paracrine effects of these NE messengers on developing B- and T-lymphocytes in vitro. NE cell populations will be isolated by FACS or microdissected directly from immunostained bursal and thymus tissue sections, and their specific mRNA profiles will be determined by cDNA microarray analysis and by PCR-based subtractive hybridization. While cDNA microarray analysis using existing microarrays will yield a fast first inventory, not all genes of interest may be represented in the respective EST libraries. Therefore, PCR-based subtractive hybridization will be employed as an independent parallel approach identifying genes differentially expressed by the NE cells, but not by surrounding immunonegative cells. The immunomodulatory effects of the identified secretory factors will be further analyzed by testing biosynthetically produced NE molecules in vitro on chicken B- and T-cell lines and on lymphocytes from primary organ explants. Effects, such as lymphocyte proliferation, differentiation or cell death, will be measured by FACS analysis and by gene expression profiling with a cDNA microarray that was specifically composed and produced for this purpose at the Fred Hutchinson Cencer Research Center.<P>
PROGRESS: 2003/10 TO 2009/09<BR>
OUTPUTS: The overall objective of our share in this project was to develop an antibody-based biosensor for the rapid and specific detection of avian influenza virus H5N1. The contribution of the Berghman lab to this project was the production of monoclonal antibodies and monospecific polyclonal anti-peptide antibodies against hemagglutinin (HA) 5 and neuraminidase (NA) 1. The following methodology was applied: (1) production of polyclonal rabbit antisera against selected synthetic peptides from HA5 and NA1. Testing of these antisera was done by ELISA, initially against commercially available HA (Protein Sciences Corporation, Meriden, CT) and then against inactivated virus, which was made available through the Poultry Health Laboratory (Dr. Billy Hargis, University of Arkansas). Synthetic peptides were selected so as to yield antibodies with maximal specificity from a region that is not prone to mutation. (2) Production of monoclonals against commercially available HA proteins. Monoclonal antibodies were produced according to previously published protocols. In view of the considerable cost of commercially available antigens, primary screening was performed against the inactivated virus attached to ELISA plates and for confirmation, a secondary screening was performed against the commercially available recombinant proteins attached to ELISA plates. (3) Production of monoclonals against inactivated virus. Screening against virus attached to ELISA plates, followed by screening against commercially available HA proteins to discriminate between anti-HA and anti-NA antibodies. Currently four papers are in preparation by Dr. Yanbin Li, who is the PI of this project. <BR>PARTICIPANTS: Collaborators from the University of Arkansas: Dr. Yanbin Li. Professor of Biological Engineering, and his collaborators, Dr. Billy Hargis, Professor of Poultry Science, Dr. Steve Tung, Associate Professor of Mechanical Engineering, and Dr. Walter Bottje, Professor and Head of Poultry Science, <BR>TARGET AUDIENCES: The results of this research are of interest to the poultry industry, but also to the public at large, given that avian influenza can potentially cause a human pandemic. Dr. Li is currently working on four publications about the biosensor and has also written a news release about it, meant for broader dissemination of our research efforts. <BR><BR>
IMPACT: 2003/10 TO 2009/09<BR>
The interdisciplinary research team at the University of Arkansas led by Dr. Yanbin Li, Professor of Biological Engineering, and his collaborators, Dr. Billy Hargis, Professor of Poultry Science, Dr. Steve Tung, Associate Professor of Mechanical Engineering, and Dr. Walter Bottje, Professor and Head of Poultry Science, in collaboration with Dr. Luc Berghman at Texas A&M University, with their expertise in biosensors, virology, immunology and microfluidics and experiences in biological detection, poultry diseases and microelectromechanical systems has successfully developed an impedance biosensor for infield rapid screening of avian influenza viruses. The biosensor technology is based on three novel concepts: (1) magnetic bio-nanobeads for highly efficient and rapid separation of target virus in a poultry swab sample; (2) a microfluidic biochip with embedded interdigitated array microelectrode for precise delivery and sensitive measurement of the target virus; and (3) the complexes of red blood cells and nanobeads for great amplification of impedance signal. The biosensor is able to specifically detect avian influenza H5N1 as low as 100 EID50/ml in a poultry cloacal or tracheal swab sample in less than 30 min. The impact of this biosensor is potentially enormous. It is a fast and inexpensive tool to quickly and unequivocally identify the Avian Influenza virus H5N1 in the field. Early detection is invaluable in taking the necessary measures to prevent the disease from spreading and causing gigantic damage. In addition, the concept of this biosensor can in the future be applied to the detection of other microorganisms.