Introduction
The genus Vibrio currently contains 147 species (Parte 2018), some of which are pathogenic to humans, for example Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae (Kaysner and DePaola 2004, Johnson 2013). The members of this genus are free-living microorganisms that are found in marine and estuarine water and sediment habitats, but they can also be associated with the fishes, bivalves, or plankton in an ecosystem (Cariani et al. 2012, Ottaviani et al. 2013, Givens et al. 2014).
Vibrio parahaemolyticus (Fugino et al. 1951) is a major causative agent of gastroenteritis in humans following ingestion of contaminated raw shellfish (Paranjpye et al. 2012, Wang et al. 2015). The symptoms of V. parahaemolyticus infection include diarrhea with abdominal pain, nausea, vomiting, headache, and fever (FAO/WHO 2011, Letchumanan et al. 2014). Thermostable direct haemolysin (tdh) and tdh-related haemolysin (trh) are the primary pathogenic factors that have been identified in V. parahaemolyticus (Zhang and Orth 2013, Ceccarelli and Colwell 2014). Since 1996 outbreaks of tdh+/O3:K6 V. parahaemolyticus infections have been recorded in Asia (Miyoshi 2013, Chung-Saint et al. 2016), Europe (Martinez-Urtaza et al. 2016), the United States (Xu et al. 2015), and Latin America (Gavilán and Martínez 2011, García et al. 2013, Velazquez-Roman et al. 2014).
In Mexico, toxigenic V. parahaemolyticus has been reported primarily in the state of Veracruz, near the Gulf of Mexico. Flores-Primo et al. (2014) and López-Hernández (2015) reported the presence of V. parahaemolyticus in oysters growing in the coastal lagoons of Veracruz. Coastal lagoons along the Pacific coast of Mexico, on the other hand, have been less studied than those along the shoreline of the Gulf of Mexico. In 2004, more than 1,230 V. parahaemolyticus O3:K6 clinical gastroenteritis cases were recorded after the consumption of raw shrimp from the Huizache-Caimanero lagoon system (Sinaloa, Mexico) (Cabanillas-Beltrán et al. 2006). The Caimanero and Huizache lagoons support an important artisanal shrimp fishery, the products of which are distributed to restaurants in tourist areas in the state of Sinaloa and other states in Mexico. The Huizache-Caimanero lagoon system is characterized by extreme salinity fluctuations (between 3 and 60), a seawater temperature range of 20 to 30 ºC, and an intermittent input of non-native nutrients (De la Lanza and Rodríguez 1990). Overfishing and eutrophication by effluents loaded with nutrients from shrimp farms in the Huizache and Caimanero areas have contributed to changes in the natural ecological conditions of the lagoons and have thus affected the distribution and abundance of microbial populations, including those of bacteria (Beltrán-Pimienta and Retamoza-Leyva 2003, Romero-Beltrán et al. 2014). Bacterial associations with nutrient-rich effluents have also been observed in agricultural activities in other parts of Sinaloa (Ahumada-Santos et al. 2014). This study aimed to determine the presence and distribution of V. parahaemolyticus, its toxigenic variants, and its relation to environmental variables in Caimanero Lagoon.
Materials and methods
Study areas and sampling sites
The study was conducted in Caimanero Lagoon, which is part of the Huizache-Caimanero lagoon system. This lagoon is located approximately 25 km southeast of the port of Mazatlán, Sinaloa (Mexico), between 22º50ʹ00ʺN and 106º01ʹ00ʺW. Water enters the lagoon by means of direct precipitation, drainage from surrounding streams, and inputs from a river through the marshes that connects the system to other rivers and the ocean (i.e., the Anonas Estuary with the Baluarte River and the Pacific Ocean, the Ostial Estuary with the Presidio River and the Pacific Ocean). Water level in the lagoon decreases through evaporation and because of the tidal flow through the Ostial Estuary. Caimanero Lagoon has a total surface area of 134 km2, with a maximum length of 19.6 km, ranging widths of 2.5-9.5 km, and ranging depths of 0.2-2.0 m. The largest dimensions are seen only during the rainy season (Ramsar 2007). Climate in the region is tropical, with mean temperature of 22 ºC and a marked rainy season from July to September that accounts for 80% of total rainfall (1,000 mm).
Five sampling sites were selected based on the ecological and hydrological characteristics of the lagoon and on the distance from the lagoon to shrimp farm discharges. Site 1 (22º50ʹ0.99ʺN, 106º1ʹ56.76ʺW) is an area where the Baluarte River drains and seawater enters from the Pacific Ocean. Site 2 (22º53ʹ5.59ʺN, 106º3ʹ40.36ʺW) was the primary access point to the lagoon and was located in an active fishing area (shrimp fishery cooperative). Site 3 (22º57ʹ6.67ʺN, 106º4ʹ21.33ʺW) and site 4 (22º57ʹ6.67ʺN, 106º4ʹ21.33ʺW) were both affected by aquaculture activities (i.e., shrimp farming and production of shrimp larvae). Site 5 (23º0ʹ40.86ʺN, 106º8ʹ59.91ʺW) corresponded to a zone influenced by the Huizache Lagoon and was the point farthest from the shrimp farming activities.
Sampling
Eight surveys were carried out from June 2014 to February 2016, and a total of 120 samples were collected (i.e., 40 water samples, 40 zooplankton samples, and 40 sediment samples). All samples were taken in duplicate. Water samples were collected 10 cm below the surface using a sterile plastic bag (200 mL). Zooplankton was collected from the surface runoff by towing a plankton net (202 µm mesh size, 0.30 m mouth diameter, 2 m length) provided by a flake collector (10 cm in diameter, 20 cm in length) with 4 circular windows (3 cm in diameter) for 5 min. Each collector was covered with the same mesh as the plankton net to allow water release and concentrate zooplankton (100 mL). Sediment samples were collected with a dredge (jaws 10 cm in diameter, 150 mL volume capacity) below the aqua-sediment interface in the first 5 cm depth. Sediment samples of approximately 100 g each were taken. All samples were placed in an ice chest and transported to the laboratory for analysis within 6 h.
Environmental variables were measured at each site. Temperature and pH were measured with a mercury thermometer and a field potentiometer (Orion). Salinity was measured using a refractometer (Fisher) (salinity range of 0 to 50) with ±0.5 precision. A YSI-50-B oximeter was used to measure dissolved oxygen. All equipment was calibrated prior to use.
Bacteriological analyses
Assessment of the presence or absence of V. parahaemolyticus in the different types of samples was done by PCR using primers for the thermolabile haemolysine (tlh) gene and the methodology described by Kaysner and DePaola (2004). The procedure was carried out as follows. A 100-mL water sample and a 50-mL zooplankton sample were separately filtered through a polyethersulfone membrane (45 mm diameter, 0.2 μm pore size; Supor-200 Pall Corporation) placed on a Millipore base. Each filter was then placed in 50 mL of Alkaline Peptone Water. For sediment analysis, 1 g of each sample was weighed, placed in a flask with 50 mL of Alkaline Peptone Water, and incubated at 35 ºC for 24 h. All samples were analyzed in duplicate. After incubation, 1 mL of the culture medium was extracted from flasks showing turbidity and placed in a 1.5-mL tube for DNA extraction. The tube was vigorously mixed with a vortex mixer (Genie 2) and incubated at 95 ºC for 5 min. The tube was mixed for a second time and placed on ice for 5 min. Finally, the tube was centrifuged at 1,400 rpm for 5 min, and the samples were stored at -20 ºC until use.
Identification of the tlh, tdh, trh, and orf8 genes by conventional PCR
The presence of the tlh gene (species-specific marker for V. parahaemolyticus) in samples with Alkaline Peptone Water was confirmed by PCR. The presence of the tlh gene was determined using a 12.5 µL reaction mix that was prepared with 6.19 µL of deionized water (18 Ω), 2.5 µL of Green-Go-Tag Flexi buffer with MgCl2 (Promega; Madison, WI, USA), 0.25 µL of dNTPs (10 µL) (Promega Corporation; Madison, WI, USA), 1.25 µL of forward primer (10 µL), 1.25 µL of reverse primer (10 µL), 0.06 µL of Taq polymerase (Axygen) (0.025 U·µL-1), and 1.0 µL of DNA from the sample that was to be analyzed. The samples that tested positive for the tlh gene were used for detection of toxigenic genes (tdh and trh), using the same component concentrations as the ones used for detection of the tlh gene. The primers used for the tlh, tdh, and trh genes were described by Bej et al. (1999). Amplification of the orf8 DNA segment was done with primers described by Myers et al. (2003). An Axygen MaxyGene thermocycler (Union City, CA, USA) was used to amplify the tlh gene and its toxigenic genes, with the following amplification conditions: denaturation cycle at 94 ºC for 10 min, 35 cycles at 94 ºC for 1 min, annealing at 58 ºC for 1 min, extension at 72 ºC for 2 min, and final extension at 72 ºC for 10 min. The annealing temperature for the orf8 gene primers was modified to 60 ºC. To visualize the obtained products, electrophoresis on a 1.2% agarose (w/v) gel was carried out with a TAE 1× buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.0) and 0.5 µL of GelRed (10,000X BIOTIUM) in an electrophoresis chamber (Enduro 10.10 horizontal Gel Box, 10 × 10 cm, Labnet) at 90 V for 40 min. Electrophoresis results were observed in a transilluminator with a UVP lamp. Molecular weight markers with size range of 100-3,000 bp (Axygen Biosciences, CA, USA) were used. Control strains were obtained from the Collection of Aquatic Important Microorganisms (CAIM), which was provided by the Research Center for Food and Development in Mazatlán, Sinaloa (Mexico). Positive controls were CAIM 320 for the tlh gene, CAIM 1772 for the tdh and trh genes, and CAIM 1400 for the orf8 gene. A reaction mixture with no DNA was used as a negative control. Only samples with duplicate sequence-specific amplifications were considered positive for V. parahaemolyticus.
Statistical analysis
A one-way analysis of variance (ANOVA) was used to determine significant differences in the presence of the tlh gene among sampling sites. One-way ANOVA was also used to evaluate significant differences in the monthly variation of environmental variables (i.e., temperature, salinity, pH, and dissolved oxygen) and the tlh gene. The analyses were implemented in SigmaPlot 11.0. Spearman’s correlation coefficient was used to determine the relationship between the frequency of the tlh gene indicative of V. parahaemolyticus and the environmental variables. The contribution of environmental parameters to the frequency of the tlh gene indicative of V. parahaemolyticus was analyzed using a principal component analysis, which was carried out with XLSTAT v2017.1. The level of significance was P < 0.05.
Results
Detection of the tlh gene and toxigenic genes
The tlh gene was isolated from all the samples. Frequency was highest during the dry season (June 2014, February 2015, May 2015, and February 2016), followed by the rainy season (September 2014 and 2015). The highest proportion was found in zooplankton samples (65%), compared to the water (57%) and sediment (50%) samples. With respect to sampling sites, the highest tlh gene frequency (37%) occurred at site 4, but it was not significantly different from frequencies at the other sites (P = 0.752) (Table 1).
Water | Zooplankton | Sediment | Total | ||||||||||||||
Sampling | Site | tlh | tdh | trh | orf8 | tlh | tdh | trh | orf8 | tlh | tdh | trh | orf8 | tlh | tdh | trh | orf8 |
June 2014 | 1 | + | - | - | + | + | - | - | - | + | + | - | - | 3 | 1 | 0 | 1 |
2 | 0 | + | - | - | - | + | - | - | + | 2 | 0 | 0 | 1 | ||||
3 | + | - | - | + | 0 | + | - | - | - | 2 | 0 | 0 | 1 | ||||
4 | + | - | - | - | + | - | - | - | 0 | 2 | 0 | 0 | 0 | ||||
5 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 | |
September 2014 | 1 | 0 | 0 | 0 | 0 | ||||||||||||
2 | 0 | 0 | 0 | 0 | |||||||||||||
3 | 0 | + | + | - | - | 0 | 1 | 1 | 0 | 0 | |||||||
4 | 0 | + | + | - | - | 0 | 1 | 1 | 0 | 0 | |||||||
5 | 0 | 0 | 0 | 0 | |||||||||||||
November 2014 | 1 | 0 | + | - | - | - | 0 | 1 | 0 | 0 | 0 | ||||||
2 | + | - | - | + | + | - | - | - | + | - | - | + | 3 | 0 | 0 | 2 | |
3 | 0 | + | - | - | - | 0 | 1 | 0 | 0 | 0 | |||||||
4 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 | |
5 | 0 | + | - | - | - | 0 | 1 | 0 | 0 | 0 | |||||||
February 2015 | 1 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 |
2 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 | |
3 | + | - | - | + | + | - | - | + | + | - | - | - | 3 | 0 | 0 | 2 | |
4 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 | |
5 | + | - | - | - | + | - | - | + | + | - | - | - | 3 | 0 | 0 | 1 | |
May 2015 | 1 | + | - | - | - | 0 | + | - | - | - | 2 | 0 | 0 | 0 | |||
2 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 | |
3 | + | - | - | - | + | - | - | - | 0 | 2 | 0 | 0 | 0 | ||||
4 | - | - | - | - | + | - | - | - | 0 | 1 | 0 | 0 | 0 | ||||
5 | + | - | - | - | + | - | - | - | + | - | - | - | 3 | 0 | 0 | 0 | |
September 2015 | 1 | 0 | 0 | 0 | 0 | ||||||||||||
2 | 0 | 0 | 0 | 0 | |||||||||||||
3 | 0 | 0 | + | - | - | - | 1 | 0 | 0 | 0 | |||||||
4 | 0 | 0 | 0 | 0 | |||||||||||||
5 | 0 | 0 | + | - | - | - | 1 | 0 | 0 | 0 | |||||||
November 2015 | 1 | 0 | + | - | - | - | 0 | 1 | 0 | 0 | 0 | ||||||
2 | + | - | - | - | 0 | 0 | 1 | 0 | 0 | 0 | |||||||
3 | + | - | - | - | + | - | - | - | 0 | 2 | 0 | 0 | 0 | ||||
4 | + | - | - | - | 0 | + | - | - | - | 2 | 0 | 0 | 0 | ||||
5 | 0 | 0 | + | - | - | - | 1 | 0 | 0 | 0 | |||||||
February 2016 | 1 | + | - | + | - | 0 | 0 | 1 | 0 | 1 | 0 | ||||||
2 | + | - | + | - | + | - | - | - | 0 | 2 | 0 | 1 | 0 | ||||
3 | + | - | + | - | + | + | - | - | 0 | 2 | 1 | 1 | 0 | ||||
4 | + | - | - | - | + | + | - | - | + | - | - | - | 3 | 1 | 0 | 0 | |
5 | + | - | - | - | + | + | + | - | + | - | - | - | 3 | 1 | 1 | 0 | |
Total | 23 | 0 | 3 | 4 | 26 | 5 | 1 | 2 | 20 | 1 | 0 | 2 | 69 | 6 | 4 | 8 |
The tdh and trh toxigenic genes were detected in zooplankton samples collected in September 2014 (sites 3 and 4) and February 2016 (sites 3-5). The trh gene was detected only in water samples collected in February 2016 (sites 1-3) (Table 1). The tdh gene was not detected in water, but the trh and orf8 genes were found in 13% (3/23) and 17% (4/23) of the positive samples, respectively. In zooplankton, tdh, trh, and orf8 were detected in 19% (5/26), 4% (1/26), and 8% (2/26) of the samples, respectively. In the sediment, tdh was detected in 5% (1/20) of the samples and orf8 in 10% (1/20) of the positive samples, but trh was not detected.
Environmental parameters and correlation with the tlh gene
Enviromental variables showed temporal variations in Caimanero Lagoon (Table 2). Seawater temperature varied between 24.7 and 31.1 ºC. Values for pH varied between 7.1 and 9.4, with no significant differences. Salinity was high in June 2014 (mean = 41.20), with significant differences (P < 0.05) between all months except for May 2015 and February 2016. Dissolved oxygen was variable, trending between 4.6 and 13.8 mg·L-1. In May 2015, dissolved oxygen concentration was high (mean = 10.10 mg·L-1) and significantly different (P < 0.05) from concentrations in September 2014 and February and September 2015.
Environmental parameters | 2014 | 2015 | 2016 | |||||||
June | September | November | February | May | September | November | February | |||
Temperature (ºC) | 31.1 ± 1.8ab | 31.8 ± 1.1b | 27.3 ± 1.3ab | 27.7 ± 1.1ab | 24.7 ± 2.4a | 30.5 ± 1.3ab | 24.8 ± 3.1a | 26.0 ± 4.8ab | ||
Salinity | 41.2 ± 18.8b | 10.0 ± 7.1ac | 11.4 ± 6.0ac | 22.2 ± 7.3ac | 31.2 ± 13.3b | 8.0 ± 5.7 a | 18.6 ± 6.8ac | 27.8 ± 6.3bc | ||
pH | 8.5 ± 0.3a | 7.7 ± 0.3a | 8.5 ± 0.7 a | 8.3 ± 0.3a | 8.0 ± 0.3a | 7.7 ± 0.4ª | 8.5 ± 0.4a | 8.0 ± 0.5a | ||
Dissolved Oxigen (mg·L-1) | 7.5 ± 1.1ab | 5.8 ± 0.6a | 6.9 ± 0.9 ab | 5.6 ± 0.2a | 10.1 ± 2.6b | 5.9 ± 0.7 a | 7.9 ± 1.2 ab | 8.1 ± 2.5 ab |
a, bMeans with different letters are significantly different (P < 0.05) between seasons.
A principal component analysis was used to analyze the contribution of environmental parameters to the presence of the tlh gene in all the samples from the lagoon. The relation between environmental parameters and the tlh gene explained 69.59% of total variance. Salinity accounted for 15.78% of the variability in the presence of the tlh gene, pH accounted for 31.45%, dissolved oxygen accounted for 37.55%, and temperature accounted for 15.21%. Salinity levels were significantly associated with the presence of the tlh gene (Fig. 1). The environmental parameters considered in this study to possibly affect the presence or absence of V. parahaemolyticus are listed in Table 3. The presence of the bacterium in the samples was significantly correlated with salinity, but no correlation was observed between the tlh gene and temperature (Table 3).
Site | Temperature (ºC) | Salinity | pH | Dissolved oxygen (mg·L-1) |
1 | 0.018 (P < 0.963) | 0.794 (P < 0.048) | 0.482 (P < 0.302) | -0.441 (P < 0.302) |
2 | -0.510 (P < 0.236) | 0.505 (P < 0.267) | 0.566 (P < 0.200) | 0.359 (P < 0.444) |
3 | -0.235 (P < 0.582) | 0.726 (P < 0.058) | 0.848 (P < 0.011) | 0.183 (P < 0.665) |
4 | -0.346 (P < 0.389) | 0.222(P < 0.619) | 0.321 (P < 0.462) | -0.012 (P < 0.977) |
5 | -0.350 (P < 0.389) | 0.760 (P < 0.037) | 0.100 (P < 0.840) | 0.250 (P < 0.536) |
Discussion
The distribution of the tlh gene indicative of V. parahaemolyticus and its relation to environmental parameters in the coastal lagoons in northwest Mexico are little known. The presence of the tlh gene in Caimanero Lagoon was detected throughout the survey. It was detected with less frequency at site 1 (water entry to the lagoon system), which was not affected by aquaculture activities or major salinity fluctuations owing to its geographic location. The lowest tlh gene frequency in Caimanero Lagoon was recorded in September, when heavy rainfall caused an abrupt decrease in salinity (salinity = 8). These results suggest that V. parahaemolyticus can be affected by low salinity.
Several studies have shown that when salinity decreases during the rainy season in a tropical zone, the concentration of V. parahaemolyticus increases (Reyes-Velázquez et al. 2010, Machado and Bordalo 2016). However, in this study the frequency of V. parahaemolyticus was higher during the dry season. Collin and Rehnstam-Holm (2011) obtained similar results, as they found V. parahaemolyticus in 73.2% of their samples at the end of the dry season. Flores-Primo et al. (2014) observed high densities of the tlh gene in oysters during the dry season. Deepanjali et al. (2005) observed that water temperature in the tropical coastal regions of India was always optimal and did not significantly affect V. parahaemolyticus. Esteves et al. (2015) indicated that, in a Medetirranean coastal lagoon, salinity was crucial for the development of Vibrio, compared to small changes in temperature. In the present study, temperature did not correlate with the presence of the tlh gene by site because there were little temperature variations in the lagoon. Similar results were reported by Turner et al. (2014), who showed that, for plankton, the tlh gene in V. parahaemolyticus did not correlate with temperature. Whitaker et al. (2010) reported that V. parahaemolyticus grows best when pH is neutral; however, Parveen et al. (2008) found low correlation between V. parahaemolyticus and this parameter (r2 = 0.3514, P < 0.085). In the present study, a positive correlation between pH and the presence of the tlh gene was found only for site 3. Variations in pH were normal in this study; therefore, pH could not atypically effect the tlh gene.
The highest tlh gene frequency was detected in zooplankton. Several studies have also found higher frequencies of the tlh gene in zooplankton than in water (Baffone et al. 2006, Turner et al. 2009, Caburlotto et al. 2010, Johnson et al. 2010, Martinez-Urtaza et al. 2012, Rehnstam-Holm et al. 2014). Vibrio parahaemolyticus can survive in sediments during the coldest months of the year, incorporating itself back into the water column when temperature rises in the summer (DePaola et al. 1994, Fukushima and Seki 2004, Böer et al. 2013). However, Caimanero Lagoon is in a tropical zone where temperature variations are small, and this is why V. parahaemolyticus was detected throughout the study period. The results of the present study are consistent with the findings by Johnson et al. (2010) and Vezzulli et al. (2013).
The tdh gene was detected at a higher frequency in zooplankton taken from sites affected by aquaculture activity. The presence of pathogenic genes in this study was low (i.e., in less than 10% of the samples), and previous studies in this area have reported similar results (Cabanillas-Beltrán et al. 2006, Velasco 2007, Sánchez 2016). However, Velazquez-Roman et al. (2012) and Hernández-Díaz et al. (2015) respectively reported 52.0% and 65.3% of pathogenic genes in strains that were isolated from environmental samples taken from coastal areas in Sinaloa. This increase in the percentage of pathogenic genes was probably due to the high number of samples analyzed and to the selective isolation of V. parahaemolyticus.
Nasu et al. (2000) indicated that serotypes O3:K6 produce tdh and enconde a single orf8 gene; however, we found samples that tested positive for the orf8 gene but negative for the tdh gene. Likewise, other studies have reported samples that tested positive for the tlh gene (species-specific), negative for the toxigenic genes (tdh, trh), and positive for the gene encoding the serotype O3:K6 (orf8) (Nair et al. 2007, Kam et al. 2008, Velazquez-Roman et al. 2012, Mala et al. 2016). Hara-Kudo et al. (2003) found tdh-negative and O3:K6-positive strains and suggested that these strains may have been variants that diverged from the ancestor of the present pandemic strains, which may have lost the tdh gene as they adapted to the environment and thus lost their virulence. On the other hand, Velazquez-Roman et al. (2014) mentioned that in some countries of the Americas there were reported cases of gastroenteritis due to pandemic strains of O3:K6 and its serovariants.
The years 2014 and 2015 were affected by an El Niño event, with atypical changes in temperature and precipitation in September 2014 (228.0 mm) and September 2015 (474.5 mm), indicating salinity variations at almost every site in the lagoon. Enviromental changes caused by climatic events such as El Niño have a direct impact on Vibrio populations because of the increase in temperature and changes in the ecology and hydrology of the systems (Ceccarelli and Colwell 2014). In the present study, water temperature in Caimanero Lagoon fluctuated slightly, but salinity showed the highest fluctuation and was the most important factor determining the presence of the tlh gene. These results clearly indicate that flood events can strongly affect V. parahaemolyticus abundance. This is the first time that an abrupt decrease in V. parahaemolyticus abundance following the rainy season and the concomitant decrease in salinity has been recorded in situ.
In conclusion, this study establishes the importance of environmental parameters affecting the distribution and presence of the tlh gene indicative of V. parahaemolyticus. Our results also indicate the optimal niches for the survival of this species and identify the effects of changes in salinity due to rainfall on the presence of the tlh gene, highlighting that a single environmental parameter alone should not be investigated to determine the presence and distribution of V. parahaemolyticus. The results confirm that the ecology of V. parahaemolyticus varies with respect to geographic locations. The presence of the pathogenic tdh and trh genes and the orf8 gene suggests that constant health surveillance is needed to prevent local public health problems.