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<OL> <LI> Development of Genetically Engineered Live Attenuated Infectious Laryngotracheitis Virus (ILTV) Strains: "Knock out / Knock in" System to Genetically Manipulate the Viral Genome. The primary objective is to produce gene deletion mutants of ILTV that can serve as safe live vaccines. Expected outcome is a new family of ILTV vaccine candidates. <LI> The specific goals of this project are to determine the role of newly identified viruses from the RSS viral metagenome analysis, as well as evaluate recombinant protein vaccination strategies that can be used by our industry to mitigate the health and economic effects of runting/stunting syndrome (RSS). Our specific research objectives for this project are to: Generate experimental recombinant ANV-1, ANV-2 and chicken parvovirus capsid protein vaccines. Generate experimental recombinant ANV-1, ANV-2 and chicken parvovirus capsid protein vaccines. Expected outcomes include vaccine candidates for RSS. <LI> The long-term goal of this research is to create an in vitro system to use continuous cell lines of avian origin expressing immunogenic proteins of fastidious microorganisms or viruses without the need of embryonated eggs for vaccine production. Specifically, immunogenic proteins of infectious bursal disease virus (IBDV) and avian influenza virus (AIV) expressed in continuous cell lines of avian origin will be used to inoculate specified pathogen free chickens (SPF) to demonstrate specific antibody production in vivo. Expected outcome is a new platform for production of poultry vaccines. <LI> Characterize selected recent avian Mycoplasma isolates. This will include in vitro and in vivo studies to determine genotype, pathogenicity and transmissibility of isolates as well as their potential use as live vaccines. Expected outcome is new live vaccine candidates.
NON-TECHNICAL SUMMARY: This project will address several important poultry diseases, infectious laryngotracheitis, avian mycoplasmosis, runting/stunting syndrome of broilers, and infectious bursal disease. These diseases represent current important challenges for the US poultry industry and are diseases for which current control methods are inadequate. The components of this project focus primarily on the development of new vaccines and development of novel methods to produce these vaccines. The expected outcome of this research is the generation of new vaccines candidates for these important diseases. <P>
APPROACH: <BR> 1) Molecular virology methods will be used to delete specific genes in the ILTV genome. Gene deletion mutants will be evaluated in-vitro and in-vivo to determine suitability as live vaccine candidates. <BR> 2) Based on data collected from the metagenome analysis performed in 2008, three vaccine candidates have been selected - ANV-1, ANV-2 and a chicken parvovirus. Using a baculovirus expression system, immunogenic proteins from these viruses will be cloned and expressed. Supernatant from baculovirus expressed proteins will be emulsified in a water-in-oil adjuvant routinely used in our laboratory for preparation of inactivated oil emulsion vaccines. The vaccination/protection study outlined in research objective 2 with the 3 described vaccines will be performed simultaneously in colony houses maintained on PDRC property. One negative control group will be maintained as well. The experimental protocol will be the same for each treatment group with the exception of vaccine treatment. <BR> 3) The methods of this research include (not exclusively): a) the verification of immunogenic protein expression in vitro for all proteins of interest in this research project; and b) evaluation of serological responses in vivo against immunogenic proteins of IBDV (namely, the VP2 protein from the D78, Lukert and Variant E IBDV strains) and AIV (H5 type). <BR> 4) The ts-11 like isolate will be further characterized to make every effort to differentiate it from the vaccine. We plan to target more areas of the genome for sequence analysis. The MS isolate associated with recent outbreaks will be tested for virulence and transmissibility. Broilers will be challenged with the recent MS isolates and evaluated at 10 days post challenge for severity of lesions. Broilers will also be infected with the MS isolates and the rate of spread of the MS within a pen, between pens and via fomites will be compared to well characterized MS strains. A potential live MS vaccine will be identified and tested. Chickens will be challenged with the potential vaccine strains and evaluated at 10 days post challenge to determine the virulence of the strains. Chickens at 6 weeks post infection with the potential vaccines will be challenged with virulent MS and evaluated at 10 days post challenge to determine the efficacy of the strains.