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2011 Annual Report

1a. Objectives (from AD-416)
1. Investigate modified simple slaughter methods to reduce aerosol generation during home slaughter process and determine the impact of such improvements on reducing virus transmission. 2. Improved methods of evaluating vaccine efficacy in poultry AI vaccines. 3. Determining infectivity, transmissibility, and pathogenicity of 5 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) for pigs.

1b. Approach (from AD-416)
Aerosol generation and virus levels will be measured in rooms conducting simulated home processing of asymptomatic H5N1 HPAI virus infected chickens. A simple, plastic-bag method for processing will be evaluated for reducing aerosol and virus generation and reducing transmission to chicken and ferret models. Develop attenuated H5 AI viruses that will be used to produce reference antibodies from chickens for testing cross neutralization of H5 HPAI viruses in a chicken egg neutralizing test as a means to assess vaccines seed strain efficacy against drift variant field viruses. Five H5N1 HPAI viruses will be intrabronchally inoculated into pigs and examined for contact transmission. Pigs will be examined for infection by serology, virus isolation, and RRT-PCR assays.

3. Progress Report
This research is related to inhouse objective 1: Identify determinants of virulence, tissue tropism and host range of avian influenza viruses. 1) A study was performed to examine the pathogenesis of infections of ferrets exposed via intranasal or aerosol-generated exposure. Brain lesions and H5N1 avian influenza viral antigen were found in ferrets exposed to virus by either aerosol or intranasal (IN) routes. The animal exposed to 1log10 virus particles by aerosol demonstrated evidence of systemic disease, with lesions in liver and spleen tissues at 5 days post inoculation. In contrast, the animal that received the same dose by IN route developed neurologic signs seven days later, but did not have liver or spleen lesions 12 days post-inoculation (p.i.). Lung lesions were apparent only in the animal that inhaled a dose of 4log10 aerosolized virus particles. Interestingly, gross examination revealed external evidence of multilobar pneumonia only in the lungs of ferrets receiving doses of 4log10 virus particles by aerosol or IN routes, consistent with histology and immunohistochemistry results. No lesions were present in the negative control animals that were administered only phosphate-buffered saline (PBS) (IN group) or PBS + antifoam agent (aerosol group). 2) A slaughter study was conducted to validate the ability of slaughter of high pathogenicity avian influenza (HPAI) virus infected chickens to produce airborne virus and the containment of the new flexible film enclosures. 9-week-old White Leghorn chickens were intranasally inoculated with A/chicken/Chile/184240-1(4322)/2002 (H7N3). On days 4 and 5 post inoculation, chicken that has died were necropsied and air samples (collected by negative air-ionizing sampler) were collected: 1) 2 feet from necropsy table and 2) outside the bioenclosure, but within the room. The bioenclosure high efficiency particulate air (HEPA) filtered fan speed was reduced to 1/4, 1/8 and 1/16 of normal velocity when necropsying 2, 2, and 1 bird, respectively. Oral swabs from all 5 birds necropsied contained H7N3 HPAI virus, but no virus was detected in the air collected during the necropsy procedure either inside or outside of the bioenclosure. All virus detection was performed by virus isolation in embryonating chicken eggs. Eight adult White Leghorn chickens were inoculated intranasally with 5.9log10 mean embryo infectious doses of A/chicken/Chile/184240-1(4322)/2002 (H7N3). At 24 hours post challenge, all birds had HPAI virus in oral swabs. 4 birds were subjected to standard home slaughter procedure and 4 were subjected to the slaughter in plastic bag method with ventilation at 1/16 of velocity of fans. Air samples did not detect virus inside or outside the bioenclosure. Conclusion – the necropsy or slaughter of H7N3 HPAI virus infected chickens did not produce airborne virus detected by negative ionizing air samplers as was demonstrated previously with H5N1 HPAI virus infection. 3) Protocols for respiratory challenge of pigs with six HPAI viruses have been developed. Six viruses were selected for challenge and sent to USDA/ARS National animal Diseases Center for pig challenge studies.

4. Accomplishments