Table 1.
Bacterial strain, source information and virulence characteristics of Vibrio parahaemolyticus used in this study.
Table 2.
Depuration trial conditions.
Fig 1.
Pilot-scale depuration trial configuration.
A) Overall system configuration, with seawater flowing from left (through filter and chiller unit, not shown). B) Side view of holding chamber, showing seawater drain tube. C) Overhead view of holding chamber, showing random distribution of oysters.
Fig 2.
Growth curves of individual pathogenic (left column) and non-pathogenic (right column) Vibrio parahaemolyticus strains in A) alkaline peptone water (APW), B) sterile seawater, and C) tryptic soy broth with 1.5% sodium chloride with incubation at 35°C for 24 h.
Lines represent growth in individual wells (n = 3/strain).
Fig 3.
Concentrations of Vibrio parahaemolyticus in oyster tissue (n = 5 oysters/species/cocktail/replicate) after 24 h of holding in artificial or natural seawater inoculated with non-pathogenic or pathogenic V. parahaemolyticus cocktails (6-7 log CFU/mL).
Data are shown from three biologically independent inoculations with artificial seawater inoculations in Waite-Cusic laboratory and autoclaved natural seawater inoculations in Schubiger laboratory. Statistical differences between inoculation procedures (One-way ANOVA with post-hoc Tukey’s HSD; p < 0.05) are represented by different capital letters within each oyster species and V. parahaemolyticus cocktail. P-values are displayed for treatments that were not considered to be statistically significant. The graphical inlay shows the stability of non-pathogenic V. parahaemolyticus cell density in C. gigas oysters stored at 4°C for up to 7 d.
Fig 4.
Comparison of oyster inoculation procedures conducted on individual or batches of oysters using the non-pathogenic Vibrio parahaemolyticus cocktail.
Both inoculation methods achieved the minimum inoculation threshold for validation studies (5 log CFU/g) for all oyster species. Brackets indicate statistical comparisons between batch and individual oyster inoculation procedures by oyster species; numbers reported are p-values from two-sample t-tests. * indicates a significant difference (p < 0.05) between inoculation methods.
Table 3.
Non-Vibrio parahaemolyticus bacterial species recovered on Thiosulfate-citrate-bile salts-sucrose (TCBS) from oysters received from commercial grow beds in Washington State. Isolates were speciated by Sanger sequencing of the 16S rRNA gene.
Table 4.
Vibrio parahaemolyticus reduction estimates and predicted depuration times to achieve targeted microbial clearance goals for each depuration trial as modeled using the nonlinear mechanistic growth function with inverse prediction. NA = Not achieved; the model estimated a maximum reduction that was less than the desired target reduction.
Fig 5.
Change in non-pathogenic and pathogenic Vibrio parahaemolyticus populations in (A) Crassostrea gigas and (B) Crassostrea sikamea during depuration at 5°C for up to 5 days (Trial 1).
Figures of the left indicate the V. parahaemolyticus survivors. Figures on the right indicate V. parahaemolyticus reduction with the colored lines indicating the fit model for the pathogenic and non-pathogenic V. parahaemolyticus clearance. Dashed lines indicate 3.0- and 3.52-log reduction targets for depuration validation.
Fig 6.
Change in non-pathogenic and pathogenic Vibrio parahaemolyticus populations in (A) Crassostrea gigas, (B) Crassostrea sikamea, and (C) Crassostrea virginica during depuration at 11°C for up to 5 days (Trial 2).
Figures of the left indicate the V. parahaemolyticus survivors. Figures on the right indicate V. parahaemolyticus reduction with the colored lines indicating the fit model for the pathogenic and non-pathogenic V. parahaemolyticus clearance. Dashed lines indicate 3.0- and 3.52-log reduction targets for depuration validation.
Fig 7.
Change in non-pathogenic and pathogenic Vibrio parahaemolyticus populations in (A) Crassostrea gigas, (B) Crassostrea sikamea, and (C) Crassostrea virginica during depuration at 13°C for up to 7 days (Trial 3).
Figures of the left indicate the V. parahaemolyticus survivors. Figures on the right indicate V. parahaemolyticus reduction with the colored lines indicating the fit model for the pathogenic and non-pathogenic V. parahaemolyticus clearance. Dashed lines indicate 3.0- and 3.52-log reduction targets for depuration validation.
Fig 8.
Change in non-pathogenic Vibrio parahaemolyticus populations in Crassostrea gigas, Crassostrea sikamea, and Crassostrea virginica during depuration at 13°C for up to 7 days (Trial 4).
Inoculation was performed using the batch method. Figures of the left indicate the V. parahaemolyticus survivors. Figures on the right indicate V. parahaemolyticus reduction with the colored lines indicating the fit model for the pathogenic and non-pathogenic V. parahaemolyticus clearance. Dashed lines indicate 3.0- and 3.52-log reduction targets for depuration validation.