Vibrio parahaemolyticus is a Gram-negative, halophilic, human pathogenic bacterium ubiquitous in the marine environment. Like many Vibrio species, V. parahaemolyticus commonly associates with shellfish, particularly oysters. Ingestion of a raw or under cooked oysters contaminated with V. parahaemolyticus can cause gastroenteritis, which is typically self-limiting and rarely causes death. Globally, oyster production is highly lucrative, especially on the West Coast of the United States where approximately 60% of oyster production occurs each year. Outbreaks of V. parahaemolyticus can result in a significant public health problem as well as an economic burden for the oyster farms implicated in the outbreak. With the increase in overall V. parahaemolyticus outbreaks, improved post-harvest processing strategies have been developed to reduce this natural contaminant. Depuration was developed to allow shellfish to purge contaminants from their tissues into the clean, flowing seawater where they are held. This post-harvest processing technique can typically reduce fecal contaminants from the oyster tissues but is relatively ineffective at eliminating V. parahaemolyticus and other Vibrio species.. Thus, improved methods for reducing this and other human pathogenic Vibrio are needed to effectively produce safer oysters for the consumer. To develop more effective and novel V. parahaemolyticus intervention strategies, first we must identify the factors that are involved in V. parahaemolyticus colonization of the oyster, allowing them toresist depuration. This study sought to investigate specific factors utilized by V. parahaemolyticus and, in the process, determined that various strains of V. parahaemolyticus have different alleles of the Type IV pili, mannose-sensitive hemagglutinin (MSHA)and chitin-regulated pilus (PilA). In addition, we expanded our investigations into the allelic diversity of MSHA and PilA from Vibrio cholerae and Vibrio vulnificus and found that V. cholerae strains that possess the Type IV toxin co-regulated pilus (TCP) maintained highly conserved MSHA and PilA sequences while strains of V. cholerae without TCP, and all of the V. vulnificus and V. parahaemolyticus strains examined, had highly divergent sequences with no discernable connection to isolation source or observed phenotype. Following that discovery, we determined that Type I, and Type IV pili, as well as polar and lateral flagellar systems contribute to V. parahaemolyticus persistence in the Pacific oyster during depuration, while Type III secretion systems and phase variation do not. Overall, we have identified factors involved in colonization of the Pacific oyster by V. parahaemolyticus. Future studies investigating conditions that affect pili and flagella production in V. parahaemolyticus may provide novel depuration conditions that could easily and effectively increase the efficiency of oyster depuration, ultimately reducing the risk of seafood-borne illness by V. parahaemolyticus associated with oysters. / Graduation date: 2013

Live holding systems are used as temporary storage facilities for shellfish. The potential for mishandling of shellfish stored in these systems is high. The objective of the project was to examine the effects of storing oysters in a spray and an immersion systems on the survival of Escherichia coli and Vibrio vulnificus within the oysters. The effects of physiological stress imposed on oysters, as a result of interstate shipping, were examined by monitoring the level of E. coli in these oysters during storage in a spray tank. The survival rates of naturally-present E. coli and V. vulnificus in oysters were also observed. The research examined the distribution of artificially- and naturally-present V. vulnificus in oyster tissues during storage in an immersion system. There was no significant difference (p = 0.12) in the artificially-inoculated bacterial population of oysters after 120 hr of storage in a spray live holding tank. The level of E. coli in oysters which were subject to physiological stress did not change significantly (p = 0.30) after 96 hr in the spray tank. Naturally-present E. coli and V. vulnificus in oysters at harvest persisted during the 72 hr storage in the spray tank. V. vulnificus was loosely associated with mucus on the surfaces of the adductor and the mantle tissues in artificially-inoculated oysters. As a result, the bacterial level was reduced on these surfaces during the 72 hr of depuration. V. vulnificus on the gills and the digestive system of artificially-inoculated oysters may become entrapped in cilia and mucus. There was no significant reduction in the bacterial population on the gills (p = 0.11) and on the digestive system (p =0.21). There was no significant difference in the population of V. vulnificus in the adductor muscle (p = 0.37), the mantle (p = 0.16), the gills (p = 0.5), and the digestive system (p = 0.5) of summer oysters naturally-infected with the bacterium. It seems unlikely that depuration of V. vulnificus from oysters naturally harboring the bacterium may be effective. / Ph. D.

Vibrio parahaemolyticus is a Gram-negative, halophilic pathogen that occurs naturally in coastal and estuarine environments. This human pathogen is frequently isolated from a variety of seafood, particular oysters, and is the leading cause of gastroenteritis associated with seafood consumption. Several outbreaks of V. parahaemolyticus infections linked to consumption of raw oysters have been documented. Contamination of oysters with V. parahaemolyticus is a concern for public health. This study investigated the efficacy of high pressure processing (HPP) in inactivating V. parahaemolyticus in raw Pacific oysters (Crassostrea gigas) and identified a process condition capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in raw oysters for commercial application. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10⁴⁻⁵ cells per gram and processed at 293 MPa (43K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. A minimum HPP of 293 MPa for 120 s at groundwater temperature (8±1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. The HPP (293 MPa for 120 s at 8±1 °C) was validated at a commercial scale according to the FDA's National Shellfish Sanitation Program Post Harvest Processing (PHP) Validation/Verification Interim Guidance for Vibrio vulnificus and Vibrio parahaemolyticus. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). Oysters processed at 293 MPa for 120 sec had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This validated HPP was accepted by the FDA as a post harvest process to eliminate V. parahaemolyticus in raw oysters. / Graduation date: 2012