Development of Methods for the Detection and Characterisation of Pathogenic Vibrio parahaemolyticus in Seafood.

The aim of this project was to investigate both molecular and classical approaches to the detection and characterisation of pathogenic Vibrio parahaemolyticus in seafoods, and standardise a characterisation tool that could be harmonised throughout laboratories in Europe. A standardised, controlled protocol for PFGE typing was established for V. parahaemolyticus and used successfully to establish clonal relationships between serologically, geographically and temporally unrelated isolates from both clinical and environmental sources. The method was rapid taking up to 72 hours, and demonstrated 100% typeability for the V. parahaemolyticus strains examined in this study. In addition, a MLST scheme was developed utilising four genes (mdh, gnd, recA, and dnaE) as a potential further sub-characterisation tool. The MLST scheme was successful in discriminating between isolates of environmental and clinical origin but was unable to further separate clonally discrete strains as judged by macro-restriction. These methods were applied to investigations of V. parahaemolyticus in bivalve molluscan shellfish in the environment and Chinese mitten crabs (Eriocheir sinensis) from the River Thames to elucidate the distribution, prevalence, geographical spread and transmission of clonally related strains over time. The work presented here indicated that V. parahaemolyticus was present in ~30% of indigenous UK shellfish samples. Furthermore, genes encoding for expression of the thermostable direct (TDH) and thermostable direct related haemolysins (TRH), that are strongly associated with pathogenicity, were found in small proportions of samples. V. parahaemolyticus was also unusually isolated in high numbers and all year round from Chinese mitten crabs found in the River Thames. The clinical significance of seafood isolations was assessed in terms of their potential human health significance. Additionally application of PFGE clearly indicated that two travel related isolates belonged to the same European clone of V. parahaemolyticus O3:K6 linked to outbreaks in Spain in 2004. The MLST scheme confirmed strains belonging to the V. parahaemolyticus O3:K6 pandemic clonal complex however, it was not possible to further discriminate between strains using this approach. The methods developed in the scope of this project will be applied to future European epidemiological and surveillance studies thus advancing the knowledge base and providing important information on future decisions on controls