Life history of oysters influences Vibrio parahaemolyticus accumulation in Pacific oysters (Crassostrea gigas)

Abstract

Vibrio parahaemolyticus infection in humans is associated with raw oyster consumption. Evaluation of V. parahaemolyticus presence in oysters is of most interest because of the economic and public health issues that it represents. To explore V. parahaemolyticus accumulation and depuration in adult Crassostrea gigas, we developed a GFP-tagged V. parahaemolyticus strain (IFVp201-gfp⁺ ), as well as a rapid and efficient quantification method in C. gigas oysters haemolymph by flow cytometry. Impact of the life history of C. gigas on accumulation and depuration of V. parahaemolyticus IFVp201 was subsequently investigated. We found that naive oysters, i.e. grown in controlled facilities with UV treated seawater, accumulated significantly more IFVp201 than environmental oysters, i.e. grown in intertidal environment. We hypothesized that environmental oysters could have been immune primed, thus could limit V. parahaemolyticus accumulation. Meanwhile, both naive and environmental oysters had similar depuration rates.

Fig. 1

Detection of IFVp201‐gfp ⁺ by FCM. Results of overnight culture of IFVp201‐gfp ⁺ in LBS diluted up to 10⁵ CFU m⁻¹ in buffered‐physiological water based on OD600. Properties of green fluorescence (FL1, A and B), Forward Scatter (FSC, A and C) and Side Scatter (SSC, C) are shown.

Fig. 2

Bland and Altman representation for IFVp201‐gfp ⁺ enumeration in different media by FCM and PE. X‐axis: average enumeration by FCM and by PE for one sample. Y‐axis: difference between the enumeration by FCM and by PE for one sample. Red solid line: bias; red dashed line: limits of agreement/95% confidence interval.

Fig. 3

IFVp201‐gfp ⁺ quantification by FCM in haemolymphs of oysters after an overnight accumulation (black circles) and after 24 h of depuration (blue triangles). Oysters exposed overnight to 10⁵ CFU mL⁻¹ of IFVp201‐gfp ⁺ then depurated for 24 h, A1‐A2‐A3: naive oysters, E1‐E2‐E3: environmental oysters. Data represent the mean of two pools of five oysters (two analytical replicates per pool) for overnight accumulation and the mean of three pools of five oysters (two analytical replicates per pool) for 24 h depuration ± standard error. Statistical analysis: one‐way ANOVA and post hoc Tukey tests (p < 0.05).