Investigating Uptake and Colonization of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) in Relation to Phytoplankton Presence

Vibrio parahaemolyticus is a human pathogen that is a frequent cause of seafood-associated illness. It is a bacterium that lives in seawater and a common contaminant of shellfish. The virulent pandemic serotype 03:K6 strain of V. parahaemolyticus was first observed in the United States in the late 1990s, and was the cause of a number of outbreaks. The goal of this project is to determine the differences in the colonization of wild-type O3:K6, toxRS, and rpoS deletion mutants in Eastern oysters (Crassostrea virginica). The wild-type strain is the strain that is naturally found in the environment. The toxRS deletion mutant strain lacks a proposed tdh virulence gene, and the rpoS deletion mutant strain lacks a regulatory gene. In order to perform this experiment oysters were acclimated to room temperate in a laboratory setting in 20 liters of seawater, and fed 1 mL of a commercially prepared algal diet consisting of 30% Isochrysis, 20% Pavlova, 20% Tetraselmis, and 30% Thalassiosira weissflogii. They were acclimated in different tanks in order to test each strain separately. Once the oysters were actively pumping they were then contaminated with approximately 2 x 10⁸ CFU of the vibrio strain of interest. An oyster homogenate was prepared from each set of oysters. The homogenate was diluted in 0.1% peptone water, and pour plated on tryptic soy agar-1% NaCl plates with added streptomycin. The plates were then allowed to incubate for no longer than 24 hours at 26^oC, and the colonies were enumerated. There were very low amounts of cells bioaccumulated by the oysters. The same experiment will be performed using a live algal diet consisting of Isochrysis. This will demonstrate the difference in uptake of the V. parahaemolyticus by the oyster according to their diet. These studies will increase our knowledge of the influence of the oyster diet on V. parahaemolyticus uptake and potential colonization within the Eastern oysters.