Project Number: 8072-42000-065-00
Start Date: Jan 31, 2011
End Date: Jan 30, 2016
Under objective 1, we will determine if differences in seawater salinity and pH significantly affect the growth and persistence of the human pathogens Vibrio parahaemolyticus and V. vulnificus in seawater; whether algae (Tetraselmis chui) will affect vibrio blooms in seawater or the levels of uptake in shellfish; and if vibrio persistence in oysters (Crassostrea virginica) varies depending on vibrio species, strain, or the presence of virulence genes. Oysters will be obtained from the Univ. of Delaware Marine Lab in Lewes, DE. Bacteriological analyses and titering of vibrio inocula, oysters, and seawater will be performed according to our newly developed and quantitative pour plate method which detects streptomycin-resistant mutants of the virulent and avirulent strains of V. parahaemolyticus and V. vulnificus. Oysters, vibrios, and algae will be added to tanks of seawater containing shellfish, both of which will be collected daily, serially diluted, and each dilution will be tested to enumerate specific pathogens. Under objective 2a, we will identify bacteriophages against V. tubiashii; isolate and characterize them biochemically and morphologically; propagate and quantify the phages using methods developed in this lab; and apply phage cocktails (multiple phage strains) in shellfish hatcheries to determine if they can significantly reduce larval shellfish mortalities. In addition, lytic phages against V. parahaemolyticus and V. vulnificus will be evaluated as a potential processing intervention to reduce human pathogenic vibrios in commercially harvested oysters. Under objective 2b, we will determine under what conditions high pressure processing (HPP), electronic-beam irradiation, and other processing techniques can eliminate viruses from shellfish. Various concentrations of acidic flavorings and ethanol will be evaluated to add novel flavors, develop altered product forms, and to increase the efficiency (reducing required pressure) of HPP against norovirus and hepatitis A virus. Hepatitis E virus (HEV) studies will be performed to evaluate the ability of HPP to inactivate HEV using a chicken model. Under objective 3, we will evaluate the uptake and persistence of viruses by oyster blood cells (hemocytes) through fluorescent microscopy or strepavidin-labeling and histological techniques. Intervention methods that specifically target, destroy, or eliminate theses hemocytes, or the pathogens within the hemocytes, will be evaluated. Biogenic silver nanoparticles will be evaluated for possible use in targeting viruses within lysosomal compartments in hemocytes. Under objective 4, we will explore hemocytes as a concentrated source of viruses within shellfish; determine if hemocytes are a suitable target for improvement of virus assay, extraction from virus-contaminated shellfish, and automated testing on a microscale format; and evaluate whether virus receptor interactions may be used to discriminate between potentially infectious and non-infectious viruses. We will automate extraction and detection methods, exploiting magnetic beads and/or chip-type formats.