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<OL> <LI> Identify reservoirs of infectious respiratory disease agents in wild birds and poultry. <LI> Develop improved diagnostic capabilities including real-time PCR as well as other rapid on-farm tests for economically important respiratory diseases. <LI> Develop new prevention and control strategies for poultry respiratory diseases.</ol> Outputs: <UL> <LI> Collect and maintain new isolates of respiratory disease agents for research. <LI>Develop primer sets for DNA-based diagnostic test applications such as real-time PCR assays. <LI>Develop disease control strategies including vaccines and specific bio-security measures. </ul> Research outputs information will be disseminated through refereed publications, symposia, workshop and informal discussions at national and international meetings. Research will produce greater understanding of disease reservoirs, processes and control interventions. Reduced mortality and condemnation will result through use of improved vaccines and bio-security measures. Consumers will enjoy safe, healthy and competitively priced eggs and poultry meat products
NON-TECHNICAL SUMMARY: Objective 1. To identify reservoirs of infectious respiratory disease agents in wild birds and poultry. Clinical samples from commercial poultry, backyard poultry and live bird markets will tested by serological and real time PCR specific for matrix, H1, H5, and H7 genes of AIV. <BR><BR> Objective 2. Develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases. PrimerHunter ensures to select specific and sensitive primers used in real time PCR assays. Validation experiments will be carried out with avian influenza HA and NA subtypes, and to confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences. Multiplex-real-time PCR will be developed using the pooling regiments of the primers specific for HA and NA in order to detect and differentiate AIV subtypes in one assay. <BR><BR>Objective 4. Develop new prevention and control strategies for poultry respiratory diseases. Modified reverse genetic vaccines will be developed for H5 and H7 subtypes of AIV. Modified attenuated vaccines viruses will be a good candidate for protection against subtype H5 and H7 infections. We will study immunogenicity of M2e-nanoparticles constructs as a possible future avian influenza vaccine candidate in poultry industry. In-ovo DNA immunization may become one of the most important innovations in the DNA vaccination of poultry against Infectious bronchitis virus (IBV), allowing it to be used in commercial in-ovo vaccination as a much safer vaccine than the attenuating live IBV vaccines used currently.
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APPROACH: <BR>Avian Influenza virus (AIV) Objective 1. Identify reservoirs of infectious respiratory disease agents in wild birds and poultry. Throughout New England, clinical samples from commercial poultry, backyard poultry and live bird markets will tested by serological and real time PCR specific for matrix, H1, H5, and H7 genes of AIV. <BR><BR>Objective 2. Develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases. a)Primer design tool for PCR-based virus subtype identification. Avian Influenza virus Primer design tool for PCR-based virus subtype identification. Rapid and reliable virus subtype identification is critical for accurate diagnosis of animal and human infections, effective response to epidemic outbreaks, and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase Chain Reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this study we will use new tool developed in our laboratory, called PrimerHunter, which can be used to select highly sensitive and specific primers for virus sub-typing. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest-neighbor model via an efficient fractional programming algorithm.<BR><BR> Objective 4. Develop new prevention and control strategies for poultry respiratory diseases. a) Development of Modified Attenuated Avian Influenza Vaccine Viruses Using Reverse Genetic Technique. The H, N, and M genes of AIV will be used to develop DNA-based and vector-based molecular vaccines. Attenuated DIVA (differentiating infected from vaccinated animals) vaccines for AIV will be developed by selecting M2, NS deletion variant using site directed mutagenesis. Reassortants will be engineered for HA subtypes H5 and H7 by incorporating a selected NS gene into low pathogenicity strains of H5 and H7 using reverse genetics. b)Preliminary study of immunogenicity of M2e nanoparticles as a future vaccine against avian influenza. Four weeks of SPF chickens will be inoculated with M2e nanoparticles construct, immune responses to M2e specific antibodies will be checked by ELISA tests at various intervals. <BR><BR>Infectious Bronchitis Virus (IBV). Objective 1. Novel genotypes are known to arise in layers raised in multi-age flock management system. Periodic blood and tracheal samples will be collected for IBV serology and identification of IBV by RT-PCR assay. <BR><BR>Objective 4. Recombinant DNA vaccines specific for S1 subunit and S genes of IBV developed earlier will be evaluated for better protection in in-ovo application with cytokines as an adjuvant.