Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods

Abstract

Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to 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) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 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 to 90.9%) and seawater (81.1 to 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 oysters 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 than in 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.

Keywords: COPP assay; Chesapeake Bay; Delaware Bay; MPN-PCR; Mid-Atlantic; Vibrio; Vibrio parahaemolyticus; Vibrio vulnificus; direct plating; oysters.

FIG 1

Principal components (PCs) representing the environmental gradients encountered in Maryland (closed circles) and Delaware (open circles) in this study in surface waters (A) and bottom waters (B). Abbreviations: S and Sw, surface water; B and Bw, bottom water; Chla, chlorophyll a; do, dissolved oxygen; temp, temperature; sal, salinity; tur, turbidity; MD, Maryland; DE, Delaware.

FIG 2

Average surface water physicochemical parameters for eight sampling sites. Abbreviations: LE, Lewes; BO, Bowers; SB, Slaughter Beach; BC, Broad Creek; CR, Chester River; MR, Manokin River; TS, Tangier Sound; OX, Oxford; Temp, temperature; DO, dissolved oxygen.

FIG 3

Average abundances of total Vibrionaceae in oysters (A and C) and seawater (B and D) determined using the COPP assay. Abbreviations: DE, Delaware; MD, Maryland; LE, Lewes; BO, Bowers; SB, Slaughter Beach; BC, Broad Creek; CR, Chester River; MR, Manokin River; OX, Oxford; TS, Tangier Sound.

FIG 4

Average abundances of total Vibrio parahaemolyticus in oysters (A and C) and seawater (B and D) determined using MPN-PCR. Abbreviations: DE, Delaware; MD, Maryland; LE, Lewes; BO, Bowers; SB, Slaughter Beach; BC, Broad Creek; CR, Chester River; MR, Manokin River; OX, Oxford; TS, Tangier Sound.

FIG 5

Average abundances of total Vibrio vulnificus in oysters (A and C) and seawater (B and D) determined using MPN-PCR. Abbreviations: DE, Delaware; MD, Maryland; LE, Lewes; BO, Bowers; SB, Slaughter Beach; BC, Broad Creek; CR, Chester River; MR, Manokin River; OX, Oxford; TS, Tangier Sound.

FIG 6

The five sample collection sites in Maryland and three sample collection sites in Delaware.