Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May–October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard Most Probable Number-PCR (MPN-PCR) assay and a direct plating (DP) method on CHROMagar™ Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+) V. parahaemolyticus genes, and total (vvhA) and pathogenic (vcgC) genes for V. vulnificus. The Colony Overlay Procedure for Peptidases (COPP) assay was evaluated for total Vibrionaceae. DP had high false negative rates (14–77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+ V. parahaemolyticus and vvhA+ V. vulnificus in oysters (85.7–90.9%) and seawater (81.1–92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+ V. parahaemolyticus were ∼8-fold higher in Delaware than in Maryland oysters, whereas abundances of vcgC+ V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+ compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay compared to the Chesapeake Bay. IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor intensive, and relatively expensive. In this study, the rapid, simple and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

No abstract

This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.