Occurrence of Vibrio parahaemolyticus in oysters (Crassostrea gigas) and mussels (Perna perna) of the seacoast of Santa Catarina, Brazil

This research aimed to identify and quantify Vibrio parahaemolyticus in fresh oysters (Crassostrea gigas), mussels (Perna perna) and seawater from different regions of cultivation of bivalve shellfishes in the seacoast of Santa Catarina, Brazil. Samples were collected between October 2012 and December 2013 and 130 oysters samples (Crassostrea gigas), 215 mussels samples (Perna perna) and 222 seawater were collected. The occurrence of V. parahaemolyticus in oysters and mussels was low, 10.76 and 11.62% of the samples tested. Higher incidences of V. parahaemolyticus were observed in seawater (18%). The density of V. parahaemolyticus in summer (December to March) was significantly greater than those in the other 3 seasons (P < 0.01). The occurrence of pathogenic V. parahaemolyticus in oyster, mussels and seawater was very low (<10%). It is recommend that control measures should be considered, including the establishment of an intensive and continuous monitoring of potentially pathogenic V. parahaemolyticus from all oyster-growing areas, the environmental parameters, and the assessment of the region-specific human health risk due to consumption of oyster.


INTRODUCTION
Seafood is recognized as a nutritious food choice, and is liked by increasing numbers of consumers worldwide (Hellberg et al., 2012).For the last two decades, there has been a fourfold growth in commercial aquaculture worldwide (Cabello, 2006).In Brazil, the production of bivalve shellfishes occurs mainly in the state of Santa Catarina, in the southern region of Brazil, due to the excellent geographical conditions of this area for the cultivation of marine organisms, such as the presence of a large number of bays, which facilitates the establishment of marine farms (Coelho et al., 2003;Corrêa et al., 2007).
Despite the increase, the main obstacles in the consumption of seafood are its high perishability and risk to health due to contamination by pathogens (Reyhana and Kutty, 2014).In addition to the indicators of faecal contamination, which are used to assess the microbiological quality of bivalve molluscs in Brazil, different species of the Vibrio genus occur naturally in marine, coastal and estuary environments, where some species such as Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are potentially pathogenic for men, and may be present in fishes and raw shellfishes or partially subjected to cooking (Thompson et al., 2004).The possibility of seafood consumers to be infected by pathogenic vibrios by eating oysters depends on the microbiological quality of the marine habitat, as well as on the practices of handling and processing of these shellfish (Vieira et al., 2011).The occurrence of these bacteria is not related to the counts of Escherichia coli or thermotolernat coliforms, therefore the specific constant monitoring is required (Pereira, 2002;Oliver, 2006;Suffredini et al., 2014).
Pathogenic strains of Vibrio parahaemolyticus can be differentiated from non-pathogenic strains with its ability to produce thermostable hemolysin (TDH), whose production is called the Kanagawa phenomenon.The pathogenicity of Vibrio parahaemolyticus is associated with the presence of the tdh and trh gene in oysters (Nishibuchi and Kaper, 1995).
The concentration of V. parahaemolyticus, in oysters and mussels is directly related to water temperature, with a higher concentration being present when the bivalve molluscs are in warm water.Because of this, these microorganisms are rarely isolated when the water temperature is below 15°C (Pruzzo et al., 2005;Su and Liu, 2007).In Brazil, the temperature of sea waters is above 20°C in most of the year, favouring the occurrence of these microorganisms in the different stations.
This research aimed to identify and quantify V. parahaemolyticus in fresh oysters (Crassostrea gigas) and mussels from different regions of cultivation of bivalve shellfishes in the seacoast of Santa Catarina, Brazil.
da Silva et al. 1323

Collection and preparation of the samples
Between October, 2012 and December, 2013, 130 oysters samples (Crassostrea gigas), 215 mussels samples (Perna perna) and 222 seawater samples were collected directly from three geographical regions in Santa Catarina where there is shellfish farming in Brazil (Figure 1).Each oysters and mussels sample consisted of 12 units.The oysters and mussels were transported to the laboratory in an isothermal box with packaged potable ice, and analyzed within 6 h of sampling.
The oysters and mussels were scrubbed under tap water to remove debris, allowed to dry, disinfected with 70% ethanol, and opened aseptically using a sterilized knife.The flesh and intervalve liquid were aseptically transferred to sterile bags and were homogenized for 1 min, forming the pool of 12 units.

Isolation and enumeration of Vibrio parahaemolyticus in oyster and mussels samples
Enumeration of V. parahaemolyticus was performed using most probable number (MPN) technique (Kaysner and DePaola, 2004).Approximately 25 g of the homogenate was added to 225 ml of phosphate buffered saline (PBS).Serial 10-fold dilutions were prepared up to 1:106 and three aliquots of each dilution were inoculated into alkaline peptone water tubes and incubated overnight at 37°C.After incubation, a loopful from the top 1 cm, approximately, of each broth tube with turbid growth was streaked onto thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates (Oxoid, UK) and incubated at 37°C for 24 h.Five to ten typical colonies from each plate were selected and isolated for identification.Sucrose-negative (blue-green on TCBS agar) colonies were submitted to confirmation as Gram-negative and oxidasepositive.Further biochemical differentiation for identification and confirmation of isolated items were performed using the API 20E system (bioMérieux, France).Total populations of V. parahaemolyticus in oysters and mussels were determined by converting numbers of APW tubes that were positive for V. parahaemolyticus to MPN g-1 using the MPN table.All strains of V. parahaemolyticus were confirmed genotypically through the detection of the tlh gene by multiplex qPCR.

Multiplex PCR for the detection of rox, tdh and trh genes
The extraction of bacterial DNA was made in QiaCube equipment (Qiagen) using the DNeasy Blood and Tissue kit (Qiagen) with specific protocol for the equipment.Real time multiplex PCR was performed using the kit V. parahaemolyticus multiplex kit (Biotecon).The target genes were the Rox to confirm the species, and tdh and trh genes of pathogenicity.The protocol used was indicated in the kit manual.

Statistical analysis
Results of microbiological tests were transformed into log values and were assumed to be normally distributed; statistical analyses were performed in the Statistica 7.0 ® software (Stat-Soft, Inc., USA).To facilitate statistical analyses of quantitative data obtained by most probable number for counts V. parahaemolyticus when levels were below the limit of detection, there was substitution for 2 MPN g -1 and test of significance of the observed differences in V.  parahaemolyticus levels, environmental parameters in oysters and mussels across the 22 samplings sites was conducted using a oneway analysis of variance (ANOVA), an alpha level of 0.05 was considered using the minimum level for statistical significance.

RESULTS AND DISCUSSION
The occurrence of V. parahaemolyticus in oysters and mussels was low, 10.76% and 11.62% of the samples tested (Table 1).Higher incidences of V. parahaemolyticus were observed in seawater (18%).
The densities of V. parahaemolyticus in oyster, mussels and seawater samples are listed in Table 1.They were higher in the summer months, especially in February and March.The density of V. parahaemolyticus in summer (December to March) was significantly greater than those in the other 3 seasons (P < 0.01).The occurrence of pathogenic V. parahaemolyticus in oyster, mussels and seawater was very low (<10%).Only 4 of 130 oysters, 5 of 215 and 5 of 220 seawater samples contained detectable levels of pathogenic strains.These results indicated that most V. parahaemolyticus in the environment were nonpathogenic to humans.Although, the levels of V. parahaemolyticus in oysters reported in this study were much lower, postharvest processing conditions and storage temperatures could allow contaminated V. parahaemolyticus to multiply to a higher level in market oysters.Studies have shown that the populations of V. parahaemolyticus in unrefrigerated oysters could increase rapidly to reach 50-fold to 790-fold its original level within 24 h after harvest if oysters were exposed to an elevated temperature (Gooch et al., 2002).
Epidemiological data from CDC on association with V. parahaemolyticus gastroenteritis with tdh-carrying strains in the period 2001-2004 and US risk assessment studies on oysters (FDA, 2005), support the assumption that V. parahaemolyticus risk is proportional to exposure to different levels of pathogenic V. parahaemolyticus (WHO, 2011).According to some studies, pathogenic V. parahaemolyticus levels may be reliably estimated from total V. parahaemolyticus levels (Miwa et al., 2003;Nordstrom et al., 2007).On the other side, other studies showed that the ratio between total and pathogenic V. parahaemolyticus in the environment may be quite variable over time, as in the case of the monitoring performed in Alaskan waters, where percentage of potentially pathogenic strains in two consecutive summers (2004 and 2005) changed from 74 to 30% (WHO, 2011).Such variability, together with the limited number of quantitative data on V. parahaemolyticus levels in the environment and in shellfish harvested in regions as Europe (Cantet et al., 2013), Asia (Deepanjali et al., 2005), South America (Garcia et al., 2009), which are occasionally involved in outbreaks, underline the need for analytical assays which allow the enumeration of both total and potentially pathogenic (tdh and/or trh positive) V. parahaemolyticus strains.Trouble variables for the presence of V. parahaemolyticus in seafood have been shown in studies by many researchers around the world, using conventional bacteriological methods.The results found in this study are in agreement with the results reported by Nordstrom et al. (2007), a study conducted in Alaska (USA), Cabello et al. (2007) in Chile, Gil et al. (2006) in Peru and Quintoil et al. (2007), India.
Higher incidence of V. parahaemolyticus, however, was found using conventional methods of wild mullet in Italy (Serracca et al., 2011), cockles in Indonesia (Zulkifli et al., 2009), various seafood in India (Chakraborty et al., 2008) and in the USA mussels (Lu et al., 2006).Furthermore, the lower incidence of 8% (Hassan et al., 2012), were reported in the Netherlands seafood.Ramos et al. (2014) found an incidence of V. parahaemolyticus, 30.0% in samples of oysters and 33.3% in water samples from cultivation sites in Bahia Sul in Florianopolis, in the study region of this work.
Several factors are involved in the distribution and survival of microorganisms in estuarine ecosystems such as biotic and abiotic parameters of water, such as temperature, salinity, pH and turbidity (Ristori et al., 2007;Strom and Paranjpye, 2000).The concentration of V. parahaemolyticus in seawater increases with increasing temperature and is correlated with the seasonal increase in the occurrence of sporadic cases of infections in months with higher temperature (Hlady and Klontz, 1996).
da Silva et al. 1325 The presence of V. parahaemolyticus seems to be constant where the sea water temperature is >10°C, unlike what occurs in Europe, where isolation of this pathogen decreases during the winter months (Baker-Austin et al., 2013).Hence, V. parahaemolyticus can be considered ubiquitous in the marine environment.The World Health Organization (WHO, 2011) listed the optimum temperature for V. parahaemolyticus growth as 37°C, with a wide growth range of 5-43°C.Several studies have shown a positive correlation between contamination of raw shellfish by V. parahaemolyticus and water temperature with higher frequencies being detected during warmer months in spring and summer seasons than in winter (DePaola et al., 2003;Parveen et al., 2008;Johnson et al., 2012;Ceccarelli et al., 2013).
The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus and seasonal distribution, will help in defining appropriate monitoring programs and post-harvest policies for this hazard.The acquisition of further quantitative information on V. parahaemolyticus distribution in production areas and marketed products (exposure assessment), together with studies on the effectiveness of post-harvest treatments, will help in the definition of codes of practice for vibrios in shellfish and improve the safety of products.

Conclusion
In conclusion, these results demonstrate greater seasonal variations in total and pathogenic V. parahaemolyticus densities in oysters.Hence, there may be more uncertainty in the use of densities of total V. parahaemolyticus organisms as alternative for risk predictions as was previously recognized.These findings can provide a reference for the comprehensive management and control of the harvesting areas.Therefore, it is recommended that control measures should be considered, including the establishment of an intensive and continuous monitoring of potentially pathogenic V. parahaemolyticus from all oyster-growing areas, the environmental parameters, and the assessment of the region-specific human health risk due to consumption of oyster.Thus, more research is needed to assess differences in virulence among various toxigenic strains and to assess and manage the risk of illness due to human exposure to oysters harvested in contaminated environments under the light of the climate change.

Figure 1 .
Figure 1.The coastal region which is the cultivation area of bivalve molluscs in the Santa Catarina.

Table 1 .
Occurrence of Vibrio parahaemolyticus in oyster, mussels and seawater samples.