3.1 Seasonal densities and survival after transportation
As shown in Table 1, significant differences in V. vulnificus vvha+ densities between seasons were observed, with higher (P < 0.05) mean levels during windy (0.720 log10 MPN/g) and the lowest in rainy (−0.523 log10 MPN/g) seasons. During windy season, the average water temperature in the MLS-Mata Grande bank was 25.6°C, nevertheless mean V. vulnificus vvha+ densities decreased during rainy season when the average water temperature increased (P > 0.05) to 27.4°C. However, salinity was higher (P < 0.05) in windy (25.8‰) than in rainy (7.3‰) seasons (Table 5).
Seasons | Vibrio vulnificus vvha+ (log10 MPN/g mean and range) | V. parahaemolyticus tlh+ (log10 MPN/g mean and range) |
---|
At-harvest | At-market | At-harvest | At-market |
---|
Windy | 0.720 ± 0.344a,x (0.477–0.964) | 3.351 ± 0.041a,y (3.322–3.380) | 0.477 ± 0.001a,x (0.477–0.0.477) | 3.041 ± 0.001a,y (3.041–3.041) |
Dry | −0.483 ± 0.056b,x (−0.523 to −0.444) | 1.055 ± 0.129b,y (0.964–1.146) | 0.686 ± 0.0.149a,x (0.580–0.792) | 3.210 ± 0.239a,y (3.041–3.380) |
Rainy | −0.523 ± 0.001c,x (<−0.523)* | 3.351 ± 0.041a,y (3.322–3.380) | 0.713 ± 0.221a,x (0.556–0.869) | 3.380 ± 0.001a,y (3.380–3.380) |
Table 1.
Seasonal variations of V. vulnificus vvha and V. parahaemolyticus tlh densities (log10 MPN/g) in Crassostrea virginica samples from the MLS during 22-h supply-chain transportation in windy, dry, and rainy seasons, respectively, from MLS to Mexico City.
Means with different letter (a, b, c) are significantly different (P ˂ 0.05) between seasons.
Means with different letter (x, y) are significantly different (P ˂ 0.05) between hours of transportation within each season.
In contrast, V. parahaemolyticus tlh+ density levels were high (P > 0.05) in rainy (0.713 log10 MPN/g) and low in windy (0.477 log10 MPN/g) seasons. After 22 h of supply-chain transportation, V. vulnificus vvha+ and V. parahaemolyticus tlh+ densities increased (P < 0.05) in all seasons probably due to the high ambient temperatures observed during transportation (20.1, 25.6, and 24.4°C). Table 2 shows that no V. vulnificus vvha+ vgcE densities were detected at-harvest and remain unculturable after 22-h transportation during dry season. A seasonal trend was observed, as higher (P > 0.05) V. vulnificus vvha+ vgcC density (0.469 log10 MPN/g) in oysters harvested from Mata Grande bank was found during windy season, and no densities were detected during dry and rainy seasons. Similarly, V. parahaemolyticus tdh+ density in oysters increased (P > 0.05) in windy season (−0.020 Log10 MPN/g), but no densities were detected during dry and rainy seasons. In contrast, no V. parahaemolyticus trh+ density was detected in all seasons. After 22 h of supply-chain transportation, a slight increase (P > 0.05) in V. vulnificus vgcE (−0.483 log10 MPN/g) in windy and rainy seasons was observed. V. vulnificus vgcC density in oysters increased (P < 0.05) in windy (0.781 log10 MPN/g) and rainy seasons (0.469 log10 MPN/g) as well. An increase in densities of V. parahaemolyticus tdh+ and trh+ (−0.484 log10 MPN/g) in oysters was observed in rainy season, probably due to the high ambient temperature observed (24.4°C) in rainy season. Our results were lower than those reported in oysters harvested from the U.S. Gulf of Mexico during dry season (3.36 log10 MPN/g), which were higher than those detected during windy season (1.0 log10 MPN/g) [37]. V. vulnificus proliferates in areas or during months where the water temperature exceeds 18°C as in MLS, and culturable concentrations of V. vulnificus are generally lower when water temperatures are cooler.
Seasons | Vibrio vulnificus vgcE (log10 MPN/g mean and range) | Vibrio vulnificus vgcC (log10 MPN/g mean and range) |
---|
At-harvest | At-market | At-harvest | At-market |
---|
Windy | −0.523 ± 0.001a,x (<−0.523) | −0.483 ± 0.056a,x (−0.523 to −0.444) | 0.469 ± 0.010a,x (0.462–0. 477) | 0.781 ± 0.005a,y (0.778–0.785) |
Dry | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001b,x (<−0.523) | −0.523 ± 0.001b,x (<−0.523) |
Rainy | −0.523 ± 0.001a,x (<−0.523) | −0.483 ± 0.056a,x (−0.523 to −0.444) | −0.523 ± 0.001b,x (<−0.523) | 0.469 ± 0.010c,y (0.462–0. 477) |
| V. parahaemolyticus tlh+/tdh+ | V. parahaemolyticus tlh+/tdh−/trh+ |
Windy | −0.020 ± 0.707a,x (<−0.523–0.477) | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001a,x (<−0.523) |
Dry | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001a,x (<−0.523) | −0.523 ± 0.001a,x (<−0.523) |
Rainy | −0.523 ± 0.001a,x (<-0.523)* | −0.484 ± 0.056a,x (−0.523 to −0.444) | −0.523 ± 0.001a,x (<−0.523) | −0.484 ± 0.056a,x (−0.523 to −0.444) |
Table 2.
Seasonal variations of pathogenic V. vulnificus (genotypes E and C) and V. parahaemolyticus (tlh/tdh, tlh/trh) densities (log10 MPN/g) in Crassostrea virginica samples from the MLS during 22-h supply-chain transportation in windy, dry, and rainy seasons, respectively, from MLS to Mexico City.
Means with different letter (a, b, c) are significantly different (P ˂ 0.05) between seasons.
Means with different letter (x, y) are significantly different (P ˂ 0.05) between hours of transportation within each season.
In other study, V. vulnificus was isolated from oyster samples collected from Pueblo Viejo Lagoon, Veracruz, and 27% (39/143) of the oyster samples were vvha+. Although positive samples were found during all seasons of a 1-year period, a seasonal fluctuation was observed. Isolation rates from oysters were significantly higher in June than in the period from November to February (P < 0.0002), indicating that water surface temperatures >24°C and salinity conditions >18‰ are more favorable for V. vulnificus [13]. In our study, we found higher (P < 0.05) V. vulnificus vvha+ densities during windy (December to March) and dry seasons (April to July) when water temperature and salinity were 25.6°C/25.8‰ and 28.7°C/29.8‰, respectively. However, a decrease was observed during rainy season when water temperature and salinity were 27.4°C and 7.3‰, respectively. Thus, V. vulnificus colonization of oysters in MLS may be influenced by water parameters such as temperature or salinity as previously reported [38]. An important finding in our study was the isolation of pathogenic V. vulnificus vgcC strains. This is the first study to report the presence of V. vulnificus vgcC in oysters from the Mexican coastline of the Gulf of Mexico. It is unclear if levels of the two V. vulnificus genotypes are unique to certain environmental conditions. As with previous studies of total V. vulnificus levels, a significant negative correlation with salinity was observed for the vgcC strains from oysters (r = −0.35, P = 0.008) [39]. In our study, there was a significant increase in the population of V. vulnificus vgcC in oysters in winter season when MLS water salinity levels were high. These results seem to indicate that V. vulnificus vgcC strains have evolved to cope with the stresses associated with changing environment. The fact that oysters have vgcC strains as the dominant strain type further suggests the possibility that those oysters harboring larger densities of this genotype would likely to be more infective to humans as V. vulnificus vgcC type is more infectious [29].
Regarding V. parahaemolyticus, our results demonstrated that V. parahaemolyticus tlh+ strains are present almost throughout the year as V. parahaemolyticus abundance in the Gulf of Mexico is almost constant because temperature is warmer (>11.6°C) [40]. The seasonal trends in V. parahaemolyticus densities observed in our study agree with previous studies since the seasonal cycle of the pathogen has been correlated with dry and rainy seasons in tropical waters, being salinity the major factor. V. parahaemolyticus tlh+ density was detected at 3.26 log10 MPN/g in oysters (Crassostrea brasiliana) harvested from Sao Paulo, Brazil during the dry season when mean water temperature was 29°C and salinity 29‰ [41]. Our previous studies have shown that there is a seasonal variation in the survival and virulence of V. parahaemolyticus, probably caused by a response of gene expression to stress. V. parahaemolyticus tlh+/tdh+ densities in oysters harvested from the MLS were observed during windy and dry seasons (0.97 and − 0.18 log10 MPN/g), respectively, and V. parahaemolyticus tlh+/tdh−/trh+ (−0.37 log10 MPN/g) was only detected during dry season. Meanwhile, during rainy season only, −0.509 log10 MPN/g was identified [42]. The presence of pathogenic V. parahaemolyticus strains raises important health issues and may be indicative of high risk in usual consumers of oyster from Mandinga lagoon during windy season where the maximum densities are found. These data suggest that V. vulnificus and V. parahaemolyticus populations in oysters are controlled by different factors. Moreover, the oyster, as a living host, may have contributed to the variation in these pathogen densities because of fluctuations in physiology resulting from reproductive status, diet, and health [11]. Densities above Mexican limits (absence in 50 g of sample) [19] for V. parahaemolyticus tlh+ and V. vulnificus vvha+ were detected in oyster samples at-harvest and at-market. In Mexico, these pathogens are not currently included in the microbiological surveillance programs of shellfish from harvesting areas and they are also excluded from the Mexican communicable disease surveillance plans. Thus, the presence of pathogenic strains is a public health concern, as these strains are not covered by current regulations.
The values for the kinetic growth parameters and performance statistics of the modified Gompertz model for V. parahaemolyticus (tlh+, tlh+/tdh+, and tlh+/tdh−/trh+) and V. vulnificus vvha+ and genotypes E and C, at ambient temperatures during 22 h transportation of oysters are shown in Tables 3 and 4, respectively. The average R2 value of the model fitted to V. parahaemolyticus growth was 0.9999 for nonpathogenic tlh+ and for pathogenic tdh+ and trh+ strains. Similarly, R2 value of the model fitted to V. vulnificus growth was 0.9999 for vvha+ and for vcgE and vcgc strains, indicating that this model was able to describe both pathogens growth. As shown in Table 3, the predicted lag time values of the nonpathogenic tlh strains were 4.2909, 4.3582, and 4.2484 h in windy, dry, and rainy seasons, respectively; meanwhile, the predicted lag time values for both pathogenic tdh+ and trh+ strains were 6.3439 during rainy season, indicating faster growth and better adaptation of the nonpathogenic strain to ambient temperatures during transportation.
V. parahaemolyticus | μmax (h−1) | λ (h) | A | G (h) | R2 | Syx |
---|
Windy | | | | | | |
tlh+ | 1.0242 | 4.2909 (257.5 min) | 2.564 | 0.6767 (40.6 min) | 0.9999 | 0.00067 |
Dry | | | | | | |
tlh+ | 1.0117 | 4.3582 (261.5 min) | 2.520 | 0.6851 (41.1 min) | 0.9999 | 0.00073 |
Rainy | | | | | | |
tlh+ | 1.0736 | 4.2484 (254.9 min) | 2.670 | 0.6456 (38.7 min) | 0.9999 | 0.00073 |
tlh+/tdh+ | 0.0096 | 6.3439 (380.6 min) | 0.039 | 72.0207 (4321.2 min) | 0.9999 | 0.00018 |
tlh+/tdh−/trh+ | 0.0096 | 6.3439 (380.6 min) | 0.039 | 72.0207 (4321.2 min) | 0.9999 | 0.00018 |
Table 3.
Parameter values using the modified Gompertz model for V. parahaemolyticus (tlh+, tlh+/tdh + and tlh+/tdh−/trh+) growth in oysters transported for 22 h at 20.1, 25.6, and 24.4°C (windy, dry, and rainy seasons) from MLS to Mexico City.
V. vulnificus | μmax (h−1) | λ (h) | A | G (h) | R2 | Syx |
---|
Windy | | | | | | |
vvha+ | 1.0592 | 4.2838 (257.0 min) | 2.630 | 0.6544 (39.3 min) | 0.9999 | 0.00757 |
vcgE | 0.0098 | 6.3022 (378.1 min) | 0.040 | 70.7009 (4242.1 min) | 0.9999 | 0.00021 |
vcgC | 0.0836 | 5.9274 (355.6 min) | 0.31 | 8.2914 (497.5 min) | 0.9999 | 0.00013 |
Dry | | | | | | |
vvha+ | 0.5280 | 4.5347 (272.0 min) | 1.540 | 1.3126 (78.8 min) | 0.9999 | 0.00621 |
Rainy | | | | | | |
vvha+ | 1.7885 | 4.3926 (263.6 min) | 3.870 | 0.3876 (23.3 min) | 0.9999 | 0.00730 |
vcgE | 0.0098 | 6.3022 (378.1 min) | 0.04 | 70.7009 (4242.1 min) | 0.9999 | 0.00021 |
vcgC | 0.3063 | 4.9150 (294.9 min) | 0.990 | 2.2633 (135.8 min) | 0.9999 | 0.00005 |
Table 4.
Parameter values using the modified Gompertz model for V. vulnificus (vvha+, vcgE, and vcgC) growth in oysters transported for 22 h at 20.1, 25.6, and 24.4°C (windy, dry, and rainy seasons) from MLS to Mexico City.
Pathogenic strains were detected in oysters after 22 h of transportation only during rainy season. These results indicated that nonpathogenic tlh+ and pathogenic tdh+ and trh+ strains reached a maximum growth rate and the maximum density (6.670, 0.039, and 0.039 log10 MPN/g, respectively) after 22-h transportation at ambient temperature during rainy season. The values of lag time observed in this study were lower than those previously reported for nonpathogenic tlh+ (24.6 h) and pathogenic trh+ (38.7 h) strains of V. parahaemolyticus isolated from raw Korean oysters [35]. In the present study, the longer lag time of pathogenic strains may be due to the time required for colonization of the oyster tissue. It has been reported that V. parahaemolyticus colonized oyster tissues according to the change of time as it is digested by oysters [43]. The maximum specific growth rate (max) predicted for pathogenic tdh+ and trh+ strains (0.0096 h−1) was lower than that for nonpathogenic tlh+ (1.0242, 1.0117, and 1.0736 h−1) in windy, dry, and rainy seasons, respectively; generation times (G) of nonpathogenic (0.6767, 0.6851, 0.6456 h) in windy, dry, and rainy seasons, respectively, were shorter than that for pathogenic strains (72.0207 h). These results indicated that nonpathogenic V. parahaemolyticus strains reached a maximum growth rate faster by storage temperatures. However, both pathogenic and nonpathogenic V. parahaemolyticus grew during storage in rainy season, although it was not detected in at-harvest oysters. This finding suggests that the bacterium was most likely present in numbers below the limit of detection, or perhaps in a viable but nonculturable state. In addition, it has been also observed that V. parahaemolyticus multiplied rapidly in live oysters held at 26°C after harvest [20]. It has been reported that higher concentrations of V. parahaemolyticus are present in market oysters than in at-harvest oysters [44]. In our study, pathogenic V. parahaemolyticus strains had the ability to adapt and survive at 24.4°C during transportation in rainy season, prior to marketing. However, the growth characteristics of V. parahaemolyticus might vary by strain variation.
According to Table 4, the predicted lag time values of V. vulnificus vvha+ strains were 4.2838, 4.3547, and 4.3926 h in windy, dry, and rainy seasons, respectively. The predicted lag time values were 6.3022 for vcgE and 5.9274 and 4.9150 for vcgC during windy and rainy seasons, respectively, indicating faster growth and better adaptation to ambient temperatures during transportation of the vvha+ than vcgC strains. No vcgC and vcgE strains were detected in oysters after 22 h of transportation during dry season. V. vulnificus vcgE strains lag time values were similar to those of V. parahaemolyticus tdh+ and trh+ strains, but higher than those of V. vulnificus vcgC strains. The maximum specific growth rate (max) predicted for vcgE (0.0098 h−1) and vcgC strains (0.0836 and 0.3063 h−1) were lower than that for vvha+ (1.0592, 0.5280, and 1.7885 h−1) in windy, dry, and rainy seasons, respectively. Generation times (G) of vvha+ (0.3876 h), vcgE (70.7009 h), and vcgC strains (2.2633 h) in rainy season were shorter than that observed in windy and dry seasons. These results indicated that V. vulnificus vvha+, vcg, and vcgC strains reached a maximum growth rate and the maximum density (3.870, 0.04, and 0.990 log10 MPN/g, respectively) after 22-h transportation at ambient temperature during rainy season. It has been reported a maximal growth rate of 0.175/h and a 1.3 log10 increase in V. vulnificus levels in oysters stored at 28°C [45]. Recently, a predictive growth model for V. vulnificus in Pacific oysters was developed [46], where growth rate and lag time of V. vulnificus in shucked oyster meat at 24°C were 0.0138 h−1 and 5.38 h, respectively. Overall, this growth rate is much lower than those observed in V. vulnificus vvha+ strains in our study. However, the lag time value is higher than our V. vulnificus vvha+ strains values. V. vulnificus and V. parahaemolyticus densities in shell oysters at the stage of distribution were greater than those observed in oysters at-harvest. Moreover, both V. vulnificus and V. parahaemolyticus grew during storage, although they were not detected at-harvest oysters. During transport and storage of raw oysters, adverse conditions (low oxygen levels, accumulation of waste, and feeding interruption) are able to disrupt a variety of cellular processes and can promote the development of more stress-resistant cells, modulating the fitness and virulence of bacterial pathogens.
Studies have suggested that pathogenic strains have low levels of detection compared with nonpathogenic strains and are more sensitive to dystrophic conditions, rapidly becoming nonculturable [47]. Furthermore, differences in regulated genes between strains may more likely due to be a response against environmental stressors. Harvest and transport techniques used in this study were typical for the commercial oyster industry in the MLS and Alvarado Lagoon zones.
Therefore, these bacteriological findings in the commercial handling portion of the study are representative of the industry in Veracruz state and throughout perhaps the entire Mexican Gulf Coast oyster industry. These results indicate that the safety of raw oysters for consumption depends upon their initial degree of contamination, mainly due to the quality of seawater from which they are extracted or cultured, and to postharvest storage conditions. Because temperature abuse during postharvest handling and storage may increase the risk of illness due to the consumption of oysters, it is very important to predict the risk of V. vulnificus and V. parahaemolyticus to consumers. The infectious dose of V. vulnificus for the high-risk group is 2 log CFU/g [6]; therefore, for the protection of consumers, careful storage and consumption guidelines for oysters at retail markets and restaurants must be emphasized.
3.2 Risk assessment
According to Table 5, the results indicate that the risk of consuming a typical meal of 12 raw oysters contaminated with V. vulnificus would be higher in dry and rainy seasons. V. vulnificus levels in oysters and the corresponding consumer risk at the vending site are strongly influenced by climate, especially water and air temperatures. The findings indicate that the risk of oyster consumption from Veracruz, Mexico is slightly lower than those estimated by WHO/FAO [48] for V. vulnificus predicted to be associated with month- and year-specific water temperatures in the Gulf of Mexico, which were 3.37 × 10–5 and 4.28 × 10–5 during dry and rainy seasons, respectively. However, the risk of oyster consumption during windy season (2.9 × 10–6) was similar to that reported by WHO/FAO (1.26 × 10–6).
Season | Air temperature (°C) | Water temperature (°C) | Salinity (‰) | Risk for at-risk population (cases per 100,000 servings; 95% confidence interval) |
---|
Windy | 20.1 | 25.6 | 25.8 | 2.9 × 10−6 (2.0 × 10−6–3.8 × 10−6) |
Dry | 25.6 | 28.7 | 29.8 | 4.7 × 10−6 (3.8 × 10−6–5.8 × 10−6) |
Rainy | 24.4 | 27.4 | 7.3 | 4.3 × 10−6 (3.5 × 10−6–5.4 × 10−6) |
Table 5.
Estimated risk assessment to V. vulnificus associated to consumption of raw oyster cocktail expended at-harvest at the MLS and at-market in Mexico City during windy, dry, and rainy seasons.
It is important to point out that seasonal expansion of V. vulnificus illnesses associated with oysters harvested from the Gulf of Mexico corresponds with warmer water temperatures (>20°C). The evidence indicates that climate anomalies have already greatly expanded the risk for vibrio illnesses [49]. WHO/FAO [48] reported a risk assessment for primary septicemia cases associated with consumption of raw oysters from the Gulf Coast of USA with mean densities of 57,000 and 80 MPN/g during summer and winter harvest seasons, respectively. In this context, variation in water and air temperatures and the characteristics and temperature of transportation and storage time have the effect of increasing the variation of V. vulnificus numbers at each point along the harvest-to-consumption continuum.
Table 6 summarized the results of risk to V. parahaemolyticus. Results indicated that the contamination rates of virulent V. parahaemolyticus (tdh+ and trh+) in raw oysters at-harvest and at-market, and the transportation temperatures significantly influence the probability of illness. The risk of recently harvested oysters during windy season in Veracruz-Boca del Río oyster bars and restaurants where oysters harvested at the MLS are sold was 1.1-fold higher than the mean risk of consuming oysters during the rainy season. These results indicate that the risk of raw oyster consumption in Veracruz, Mexico is lower than those of the U.S. which were 4.4 × 10–4 [50], similar to those reported in Australia (6 × 10–8–6.1 × 10–6), higher than those of Canada (7.5 × 10–10–1.1 × 10–6) and New Zealand (8.6 × 10–8–3.2 × 10–7), but lower to that in Japan during autumn (1.2 × 10–4) [51] and Taiwan (8.56 × 10–5) [52]. The estimated risk in our study is similar to that reported in Malaysia (1.76 × 10–6) [53], but lower than the average risk associated with the consumption of raw oysters contaminated with pathogenic V. parahaemolyticus marketed at Sao Paulo, Brazil of 4.7 × 10–4, 6.0 × 10–4, 4.7 × 10–4, and 3.1 × 10–4 for spring, summer, fall, and winter, respectively [36]. As the microbial risk assessment was conducted, several limitations were identified. The estimation did not include the growth model of V. vulnificus and V. parahaemolyticus during the time gap from markets to consumption.
Season | Vibrio parahaemolyticus density (log10 NMP/g) | Pathogenic rate (%) | Risk for at-risk population (cases per-100,000 servings; 95% confidence interval) |
---|
tdh+ | trh+ | tdh+ | trh+ |
---|
Windy | | | | | |
At-harvest | −0.020 | 10.0 | ND | 8 × 10−6 (6.4 × 10−7–1.0 × 10−4) | ND |
At-market | ND | ND | ND | ND | ND |
Rainy | | | | | |
At-harvest | ND | ND | ND | ND | ND |
At-market | −0.484 | 0.2 | 0.2 | 7.8 × 10−7 (6.2 × 10−8–9.9 × 10−6) | 7.8 × 10−7 (6.2 × 10−8–9.9 × 10−6) |
Table 6.
Estimated risk assessment to V. parahaemolyticus associated to consumption of raw oyster cocktail expended at-harvest at the MLS and at-market in Mexico City during windy, dry, and rainy seasons.
However, the model’s assumption can be referred for retail outlets that serve fresh raw oysters where there is minimal time for the growth of both pathogens to occur. There is a growing body of evidence to suggest that V. vulnificus and V. parahaemolyticus infections are increasing and tend to follow regional climatic trends, with outbreaks following episodes of unusually warm weather. Moreover, several epidemiological factors, such as growing consumption and international trade of seafood produce, may increase the incidence of these pathogens [12]. In Mexico, there is currently a lack of detailed surveillance information regarding V. vulnificus and V. parahaemolyticus infections, which probably disguises their real clinical burden. However, there have been some reports of outbreaks and deaths caused by consumption of oysters contaminated with these pathogens. Recently, a patient with hepatic cirrhosis and hepatic carcinoma suffered fulminant sepsis and necrohemorrhagic bullae secondary to a V. vulnificus infection. The patient had ingested oysters in Mexico City 36 h before [54]. Along Veracruz state in Mexican Gulf Coast, 18 V. parahaemolyticus infections were reported. Of 18 patients, 27.7% (5/18) consumed raw oysters at oyster bars and restaurants located in Boca del Río and Veracruz Port [55]. The information provided in this study is important for preventing public health problems as pathogenic genes such as vcgC, tdh+ and trh+ were detected. Moreover, the distribution and variation in numbers of virulent V. vulnificus and V. parahaemolyticus in oysters may need to be determined before harvest as these data should be valuable for assessment of the human health risk due to consumption of raw oysters which represents a significant threat to human health and seafood safety.