Seasonal Abundance of Total and Pathogenic Vibrio ...

1069 Journal of Food Protection, Vol. 77, No. 7, 2014, Pages 1069–1077 doi:10.43l5/0362-028X.JFP-13-482 Copyright G, International Association for Food Protection

Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus Isolated from American Oysters Harvested in the Mandinga Lagoon System, Veracruz, Mexico: Implications for Food Safety ´ RRAGA-PARTIDA,2 ARGEL FLORES-PRIMO,1 VIOLETA PARDIÓ-SEDAS,1* LEONARDO LIZA 1 1 ´ ´ ´ NDEZ1 KARLA LOPEZ-HERNANDEZ, ROXANA USCANGA-SERRANO, AND REYNA FLORES-HERNA of Veterinary Medicine, Universidad Veracruzana, Avenida Miguel Ańgel de Quevedo s/n esquina Yań˜ez, Colonia Unidad Veracruzana, Veracruz, Veracruz, Me´xico 91710; and 2Centro de Investigacioń Cientı´fica y de Educacioń Superior de Ensenada, Biotecnologıá Marina, Carretera Tijuana-Ensenada 3918, Zona Playitas, Ensenada, Baja California, Me´xico 22860

1Faculty

MS 13-482: Received 10 November 2013/Accepted 2 March 2014

ABSTRACT The abundance of total and pathogenic Vibrio parahaemolyticus (Vp) strains in American oysters (Crassostrea virginica) harvested in two different harvest sites from the Mandinga lagoon System was evaluated monthly for 1 year (January through December 2012). Frequencies of species-specific genes and pathogenic genes exhibited a seasonal distribution. The annual occurrence of Vp with the species-specific tlh gene (tlhz) was significantly higher during the winter windy season (32.50%) and spring dry season (15.0%), with the highest densities observed during spring dry season at 283.50 most probable number (MPN)/ g (lagoon bank A, near human settlements), indicating the highest risk of infection during warmer months. Pathogenic Vp tlhz /tdhz frequency was significantly higher during the winter windy and the spring dry seasons at 22.50 and 10.00%, respectively, with highest densities of 16.22 and 41.05 MPN/g (bank A), respectively. The tlh/trh and tdh/trh gene combinations were also found in Vp isolates during the spring dry season at 1.25 and 1.3%, respectively, with densities of 1.79 and 0.4 MPN/g (bank A), respectively. The orf8 genes were detected during the winter windy season (1.25%) with highest densities of 5.96 MPN/g (bank A) and 3.21 MPN/g (bank B, near mangrove islands and a heron nesting area). Densities of Vp tdhz were correlated (R2 ~ 0.245, P , 0.015) with those of Vp orf8z. The seasonal dynamics of Vp harboring pathogenic genes varied with seasonal changes, with very high proportions of Vp tdhz and Vp orf8z isolates in the winter windy season at 46.2 and 17.0%, respectively, which suggests that environmental factors may differentially affect the abundance of pathogenic subpopulations. Although all densities of total Vp (Vp tlhz) were lower than 104 MPN/g, thus complying with Mexican regulations, the presence of pathogenic strains is a public health concern. Our results suggest that total Vp densities may not be appropriate for assessing oyster contamination and predicting the risk of infection. Evaluation of the presence of pathogenic strains would be a better approach to protecting public health.

Vibrio parahaemolyticus (Vp) is widely distributed in estuarine and coastal waters worldwide. This bacterium is gram negative, facultative anaerobic, and halophytic and has been recognized as the leading cause of acute gastroenteritis associated with the consumption of raw, undercooked, or contaminated shellfish, particularly oysters, throughout the world (15, 17, 50). In recent years, outbreaks of Vp infection have increased worldwide in regions with high seafood consumption. This organism is the leading cause of seafoodassociated bacterial gastroenteritis in the United States (12) and is one of most important foodborne pathogens in Asia, causing approximately half of the food poisoning outbreaks in Taiwan, Japan, and other Southeast Asian countries (31). The symptoms of the illness present 24 h after consumption * Author for correspondence. Tel: z52 229-9342075, Ext 24125; Fax: z52 229-9342075, Ext 24104; E-mail: [email protected], vpardio@ uv.mx.

and are characterized by inflammatory diarrhea, vomiting, abdominal cramps, and low-grade fever (18). The incidence and severity of the gastroenteritis are affected by the infectious dose and virulence of the bacterium (15, 29, 54). The infection can cause septicemia that may be life threatening to people with underlying medical conditions such as impaired hepatic and renal capacity, diabetes, or immune disorders (23, 45). However, septicemia and death rarely occur. The mechanism by which Vp infects humans has not been comprehensively determined, although thermostable direct hemolysin (TDH), a pore-forming protein that might contribute to the invasiveness of the bacterium (23), and TDH-related hemolysin have been recognized as primary virulence factors (18). The tlh (thermolabile hemolysin) gene is a species-specific marker for Vp, and the tdh and trh genes are pathogenicity markers for Vp (37). Based on studies conducted in different regions of the world, the tdh

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J. Food Prot., Vol. 77, No. 7

FIGURE 1. Mandinga Lagoon System. Oyster samples were collected from January to December 2012 at bank A (close to the settlement of Mandinga) and bank B (close to mangrove islands) in the Mandinga Grande Lagoon.

and/or trh genes have been found in 90 to 99.8% of clinical strains, whereas generally 0.2 to 10% of environmental Vp isolates are potentially pathogenic based on the presence of tdh and/or trh (31, 45, 48). Therefore, the presence of these genes should routinely be used for determining the pathogenicity of Vp strains. The frequency of Vp in the environment has been correlated with parameters such as temperature and salinity. This correlation could explain the seasonality of infections, which are more abundant in warmer months. Therefore, Vp is more likely to be detected in oysters harvested in spring and summer than in those harvested in winter (19). Because only a small proportion of the environmental strains are virulent, the presence of Vp strains with tdh and/or trh would have to be evaluated in the oysters to determine safety. Several other risk factors, such as the growing number of people consuming larger amounts of seafood, will affect Vp incidence (20). The rise in world bivalve consumption will increase the population at risk, amplifying disease transmission. The growing number of individuals with predisposed risk factors (e.g., age, diabetes, hypertension, liver disease, and immune disorders) also will increase the number of individuals susceptible to infections (2). According to the Mexican standard NOM-242-SSA1-2009 (44), Vp is not investigated routinely as part of epidemiologic surveillance and control of communicable diseases. Vp also is excluded from the microbiological surveillance system for infectious gastroenteritis, which is restricted to epidemiological surveillance in cases of a Vibrio cholerae O1 infection outbreak. Other authors have recommended evaluation and regulation of Vp occurrence in shellfish, including oysters, and inclusion of Vp in the epidemiologic surveillance system for gastrointestinal diseases (13, 18, 21, 29, 39, 55). Few reports on the presence of environmental Vp strains carrying the tdh gene (tdhz) in Mexico are available.

Vp tdhz incidence was reported in fresh oysters sold in Guadalajara, Mexico (46), and in oysters harvested from the Pueblo Viejo lagoon and coastal zone of Tamaulipas, Mexico (6, 8). The Mandinga Lagoon System (MLS) is one of the most important shellfish-producing estuarine lagoon systems on Mexico’s Gulf coast. Despite the importance of this intensive oyster harvesting area, the main problems in the MLS are anthropogenic pressures from nearby human settlements and urban developments and increasing tourist activity. Because of the limited information about the presence and abundance of pathogenic Vp in oysters from this lagoon system, Vp monitoring is crucial given the public health risk. The aim of this study was to investigate the seasonal distribution and densities of total and pathogenic Vp in the American oyster (Crassostrea virginica) harvested from the MLS in the state of Veracruz, Mexico, for a better assessment of the public health risk associated with oyster consumption. MATERIALS AND METHODS Reagents. Kits of tlh, tdh, trh, and orf8 (open reading frame 8) primers were provided by Sigma-Aldrich (Mexico City, Mexico). Deoxynucleoside triphosphates (dNTPs), GoTaq DNA polymerase, the 100-bp DNA ladder, and GoTaq Master Mix were acquired from Promega Corporation (Madison, WI). Area of study and sample collection. Oyster collection sites were on two banks of the Mandinga Grande Lagoon: one close to human settlement (bank A) and the second around mangrove islands with nesting herons (bank B) (Fig. 1). The MLS is in the southern state of Veracruz, Mexico, and flows parallel to the northwestern coastline of the Gulf of Mexico, between 19u029N and 96u069W in Alvarado, Veracruz. The MLS is formed by the confluence of the Jamapa River and effluents of the Huatusco, Cotaxtla, and Totolapan rivers, receives supplementary freshwater from the Arroyo Hondo River, and exits into the Gulf of Mexico near the city of Boca del Rio close to the city of Veracruz. This


V. PARAHAEMOLYTICUS IN OYSTERS FROM MANDINGA LAGOON, MEXICO

shallow (1 to 3 m depth) lagoon system is connected to the Gulf of Mexico by a long, narrow, and deeper channel through the Jamapa River. The MLS consists of four lagoons (Conchal, Larga, Chica, and Grande) and their associated flood zones and covers 3,250 ha. The dry season occurs from April to June, the rainy season occurs from July to October with tropical hurricane activity, and the windy season is from November to March with high velocity north winds (90 to 129 m/s) (10). The mean temperatures in these seasons are 24.6, 25.2, and 20.4uC, respectively. Oyster samples were collected from two permanent oyster harvesting areas on banks A and B from January to December 2012. A total of 80 legal-size live oysters (40 from each site) were collected monthly by divers and immediately transported to the laboratory according to Secretarıá de Salud approved method NOM-109-SSA1-1994 (43). Dead animals were discarded, and the remaining oysters were scrubbed and rinsed under cold running tap water to remove debris and attached algae and then analyzed for the presence of Vp within 2 h of collection following the Vibriomex Group methodology (30). Enumeration of Vp. Oysters were shucked under aseptic conditions, and 200 g of oyster sample (150 g of meat and 50 g of intervalve liquid) was blended for 120 s with 200 ml of phosphate-buffered saline to make a 1:1 dilution. The shellfish homogenate was added to alkaline peptone water (APW) in a three-tube most-probable-number (MPN) dilution series. Because the densities of Vp were unknown, dilutions of 1021, 1022, 1023, and 1024 were prepared in triplicate, each using 9 ml of APW, and inoculated in the MPN tubes according to the standard threetube MPN procedure (47). The tubes were incubated at 35uC for 24 h. After incubation, DNA was extracted and purified. DNA was extracted from each tube showing growth in APW. Bacterial genomic DNA was extracted by heating 200 ml of the enrichment broth from each positive tube in a thermal block (AccuBlock digital dry bath, Labnet, Edison, NJ) with a protocol of five cycles at 95uC for 5 min and 220uC for 5 min. The samples were then centrifuged (Prism R refrigerated microcentrifuge, Labnet) at 11,709 | g for 20 min, and the supernatant was recovered and treated with 600 ml of 100% ethanol and 20 ml of 3.0 M sodium acetate and held at 220uC for 20 min. Extracts were centrifuged again under the same conditions, the supernatant was discarded, 600 ml of 70% (vol/vol) ethanol was added, and the sample was centrifuged again under the same conditions. The supernatant was discarded. The pelleted DNA was recovered and resuspended in 100 ml of sterile biotechnology performance certified (BPC)–grade water (Sigma-Aldrich); 1 ml was used directly in the PCR for DNA amplification and the remainder was stored at 220uC. The populations of nonpathogenic, pathogenic, and pandemic Vp strains was calculated using the MPN tables corresponding 95% confidence limits (CLs), and the results were expressed as MPN per gram of oyster (47). In the second step, one loopful from the top 1 cm of each turbid broth tube from the MPN method categorized as positive for Vp tlhz based on the DNA amplification results was streaked onto CHROMagar Vibrio (CHROMagar Microbiology, Paris, France). Plates were incubated at 35uC for 24 h for the isolation of presumptive strains. At least 15 mauve well-grown colonies of presumptive Vp tlhz from each inoculated CHROMagar plate were selected and inoculated into APW tubes, incubated at 35uC for 18 to 24 h, and then subjected to DNA extraction and purification as described above and amplification as described below. Presumptive Vp strains confirmed as tlhz, tdhz, trhz, and orf8z with the direct PCR assay were scored as positive for the

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respective gene and stored in Trypticase soya agar (BIOXON, BD, Mexico City, Mexico) slants at 220uC. Identification of tdh, trh, and orf8 genes by conventional PCR amplification. Presumptive Vp isolates were confirmed by the presence of the species-specific tlh gene. The presence of tlh and the virulence genes tdh and trh were determined by multiplex PCR assay, and the presence of a segment of the orf8 DNA sequence was determined separately according to the procedure described by Bej et al. (3). Amplification of nonpathogenic tlh and the pathogenicity genes (tdh, trh, and orf8) was performed using a master mix including 6.19 ml of cold BPC-grade water, 2.5 ml of GoTaq Green buffer (1.5 mM), 0.25 ml of the dNTPs (0.2 mM), 1.25 ml of Fwd primer (1.0 mM), 1.25 ml of Rvs primer (1.0 mM), 0.06 ml of GoTaq polymerase (0.025 mM), and 1 ml of DNA sample (2.0 ng/ml) (3, 28, 42). The reactions for detection of the tlh gene were performed with an automated thermal cycler (MaxyGene Gradient, Axygen, Union City, CA) with the optimized cycling parameters: initial denaturation at 94uC for 10 min; 35 cycles of denaturation at 94uC for 1 min, primer annealing at 58uC for 1 min, and primer extension at 72uC for 2 min; and a final extension at 72uC for 10 min. The primer annealing temperature for the orf8 gene was changed to 60uC. Amplified products were separated by electrophoresis on 1.2% (wt/vol) agarose (Promega) in 1| TAE buffer (50| contained 242 g Tris and 0.5 M EDTA, pH 8.0 [Sigma-Aldrich] and 57.1 ml of glacial acetic acid [Mallinckrodt Baker, Xalostoc, Estado de Mexico, Mexico]) at a constant voltage of 90 V for 40 min and visualized by staining with 0.5 ml of GelRed under UV light on a benchtop UV transilluminator (MultiDoc-It Digital Imaging System, Ultra-Violet Products, Cambridge, UK). A 100-bp ladder (100 to 3,000 bp; Axygen) was used as a DNA size marker. DNA from Vp strain CAIM 1772 (3) was used as positive control for the nonpathogenic (tlh) and pathogenic (tdh and trh) genes, and DNA from Vp strain CAIM 1400 was used as positive control for the orf8 gene (33). Statistical analysis. MPN tables and formulas were used to identify each sample as previously described (47). The odds ratio (OR) was used to evaluate the differences in the presence of various genes corresponding to different months at a significance level of 0.05. Bacteriological data were log transformed for the analyses, and results for nonpathogenic and pathogenic Vp densities were analyzed for significant differences with an analysis of variance (P , 0.05) using the statistical software Minitab 16.0 (Minitab, Inc., State College, PA). Samples for which the genes were not detected were assigned a value of half of the 0.30 MPN/g limit of detection (0.15 MPN/g).

RESULTS AND DISCUSSION PCR assays targeting the tdh, trh, and orf8 genes in the Vp isolates revealed the presence of pathogenic strains. Representative PCR results are presented in Figure 2A and 2B. Among the 201 isolates, 36.3% (73 of 201) were identified as tlhz, 41.1% (30 of 73) were identified as tdhz, and 1.4% (1 of 73) was identified as trhz. Among Vp tdhz isolates, 23.3% (7 of 30) also carried the orf8 gene, representing 9.6% (7 of 73) of all Vp strains (Table 1). The prevalence of Vp tlhz in oyster samples from the Mandinga Grande Lagoon (36.3%) was lower than the 93.8% prevalence (46 of 49) found in oysters (Crassostrea madrasensis) from the southwest coast of India and the 94.8% prevalence in Pacific oysters (Crassostrea gigas)

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FIGURE 2. Agarose gel electrophoresis of the amplified DNA of V. parahaemolyticus isolates from American oysters (Crassostrea virginica) harvested in the Mandinga Lagoon System. (A) Pathogenic Vp tdhz and trhz strains. Lane 1, blank; lane 2, 100-bp DNA ladder; lanes 3 through 7, Vp isolates; lane 8, positive control (V. parahaemolyticus CAIM 1772); lane 9, negative control (V. vulnificus CAIM 610). Numbers on the right indicate the size of the amplification products corresponding to the 500- and 269-bp internal fragments of the trh and tdh genes, respectively. (B) Pathogenic Vp orf8 strains. Lane 1, blank; lanes 2 through 10, Vp isolates; lane 11, 100-bp DNA ladder; lanes 12 through 18, Vp isolates; lane 19, negative control (V. vulnificus CAIM 610); lane 20, positive control (V. parahaemolyticus CAIM 1400). Number on the right indicates the size of the amplification product corresponding to the 369-bp internal fragment of the orf8 gene.

collected from commercial growing areas in the North Island, New Zealand (14, 27), but similar to the prevalence detected in oysters from Mississippi (56%) and Alabama (44%) in the United States (55). In contrast, when compared with other studies in Mexico, the prevalence results in the present study are higher than the 26.1% prevalence of Vp tlhz reported in oysters harvested from the Pueblo Viejo Lagoon in Tamaulipas (6) and the 19.9% prevalence reported in oysters from three coastal districts (Victoria, Tampico, and Matamoros) in Tamaulipas State (8). In general, the Vp species-specific gene and the pathogenic genes more commonly associated with clinical Vp strains exhibited seasonal variation, based on the PCR assay results. Occurrence of Vp tlhz was significantly higher in January during the winter windy season (32.50%, OR95% ~ 8.53, CL95% ~ 4.91 to 14.80) and in April (15.00%, OR95% ~ 2.36, CL95% ~ 1.2 to 4.61) and May (13.75%, OR95% ~ 2.10, CL95% ~ 1.05 to 4.18) during the spring dry season, but Vp tlhz was not detected in August, September, and October (summer to autumn rainy season) (Table 1). The total Vp tlhz densities increased during the spring dry season with 283.5 MPN/g (bank A), and the highest density of 1,100.00 MPN/g (bank A) was detected in April during spring dry season (Table 2). Several authors (5, 6, 16, 49) have reported an increase of Vp densities during the dry season, corresponding to the warmer months of the year in Veracruz, Mexico, which are May and June with a mean temperature of 25.7 and 26.3uC, respectively.


Pathogenic Vp tlhz/tdhz frequency was significantly higher in January during the winter windy season (22.50%, OR95% ~ 21.00, CL95% ~ 9.67 to 45.56) and in April during the spring dry season (10.00%, OR95% ~ 4.33, CL95% ~ 1.86 to 10.08). The tlh/trh genes were also detected during the spring dry season in May with a frequency of 1.25% (OR95% ~ 122.40, CL95% ~ 0.18 to 79,620) (Table 1). Vp tlhz/tdhz was detected during all seasons, indicating that this pathogen is endemic to the Mandinga Grande Lagoon. Vp tlhz/tdhz was detected at high densities in the spring dry and winter windy seasons at 41.05 and 16.22 MPN/g (bank A), respectively, and 0.57 and 7.22 MPN/g (bank B), respectively; however, during the summer rainy season only 0.40 and 0.31 MPN/g (banks A and B, respectively) were found (Table 2). Vp tlhz/trhz was detected at levels of 1.79 and 0.59 MPN/g (bank A) during the spring dry and winter windy seasons, respectively. Pathogenic Vp strains have been detected in oysters in other studies of at least 1 year duration at prevalences of 3 to 70% for Vp tdhz and 17 to 60% for Vp trhz, depending on the methodology and the region (14, 16). Vp tdhz was detected in 13.2 and 14.1% of oysters harvested in winter and spring, respectively, along the Gulf of Mexico in Alabama, Louisiana, and Texas (11). The presence of Kanagawa hemolysin, characteristic of pathogenic Vp tdhz strains, was reported in oysters harvested in U.S. coastal waters along the Gulf of Mexico during warmer months and more recently in Mexican coastal lagoons on the Gulf of Mexico (6, 15). Another study in Alabama oysters revealed a high prevalence (12.8%) of Vp tdhz (16), which is similar to the prevalence found in our study (1.25 to 22.5%). In tropical countries, the seasonal cycle of the organism is correlated with the dry and rainy seasons; the lowest numbers are found in rainy months and the highest numbers are found in the dry season (36). Our observations suggest a similar trend. We identified 1.25% of natural occurring Vp tdhz/trhz isolates in June during the spring dry season in oysters harvested from both banks (Table 1) with a density of 1.20 MPN/g (Table 2), which represents the first isolation of this pathogenic form of Vp in Mexican seafood. A prevalence of 85% for Vp tdhz/trhz from oysters, water, and sediment samples along the Mississippi Gulf Coast has been reported (25). The orf8 genes appeared only in January (8.75%, OR95% ~ 854.80, CL95% ~ 1.66 to 441,100) during the winter windy season, with densities of 5.96 and 3.21 MPN/g at banks A and B, respectively. Only Vp tdhz isolates where positive for orf8 expression; isolates with the trh gene were negative for orf8. These results are in accordance with other reports of pandemic strains of Vp in environmental or clinical samples from several countries such as Bangladesh, Japan, Mexico, China, India, the United States, and France (1, 4, 5, 12, 22, 32, 38, 41, 49, 52). In several studies, orf8 genes have been found in foodborne Vp strains; thus, orf8 has been considered an additional virulence factor for Vp (7, 24, 35). Although more than 90% of clinical Vp isolates contain at least one pathogenicity marker, only ,5% of Vp strains isolated from food or environmental sources possess pathogenicity genes; however, these strains can cause

12

11

3

Spring: dry Apr.

May

June

0 0

a

3

3.75

0 2.50

0 0

6.25

3.75

13.75

15.00

8.75

5.00

0 0.29 (0.07–1.21) 0.45 (0.13–1.46)

0.80 (0.31–2.06) 0 0

2.36 (1.21–4.61) 2.10 (1.05–4.18) 0.45 (0.13–1.46)

8.53 (4.91–14.80) 0.61 (0.22–1.74) 1.18 (0.52–2.67)

OR (95% CL)

1

0 0

0 0

1

0

0

8

0

2

18

1.25

0 0

0 0

1.25

0

0

10.00

0

2.50

22.50

OR (95% CL)

0.37 (0.05–2.76)

0 0

0.37 (0.05–2.76) 0 0

0

4.33 (1.86–10.08) 0

21.00 (9.67–46.56) 0.78 (0.18–3.33) 0

tlh and tdh No. of Frequency isolates (%)

OR, odds ratio; 95% CL, 95% confidence limit.

Dec.

Autumn: rainy, windy Oct. 0 Nov. 2

Aug. Sep.

5

7

Mar.

Summer: rainy July

4

Feb.

32.50

No. of Frequency isolates (%)

Winter: windy Jan. 26

Month

Total Vp isolates (tlh gene)

0

0 0

0 0

0

0

1

0

0

0

0

0

0 0

0 0

0

0

1.25

0

0

0

0


0

0

0

0

OR (95% CL)

0

0 0

0 0

0

122.40 (0.18–79,620) 0

tlh and trh

0

0 0

0 0

0

1

0

0

0

0

0

0

0 0

0 0

0

1.25

0

0

0

0

0


0

0

0

0

0

OR (95% CL)

0

0 0

0 0

0

122.40 (0.18–79,620)

tdh and trh

Pathogenic Vp isolates (tdh, trh, and orf8 genes) orf8

0

0 0

0 0

0

0

0

0

0

0

7

0

0 0

0 0

0

0

0

0

0

0

8.75


TABLE 1. Vibrio parahaemolyticus strains isolated from American oysters (Crassostrea virginica) harvested from the Mandinga Grande Lagoon, Veracruz, Mexico a

0

0 0

0 0

0

0

0

0

0

854.80 (1.66–441,100) 0

OR (95% CL)

J. Food Prot., Vol. 77, No. 7 V. PARAHAEMOLYTICUS IN OYSTERS FROM MANDINGA LAGOON, MEXICO

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TABLE 2. Seasonal and monthly variations in densities of total (tlh) and pathogenic (tdh, trh, tdh/trh, and orf8) strains of Vibrio parahaemolyticus in American oyster (Crassostrea virginica) samples harvested from Mandinga Grande Lagoon during 1 year Mean (range) density (MPN/g) tlhz

Collection time

tlhz/tdhz

tlhz/trhz

Bank A Dry, spring (Apr.– June) 283.50 (3.60–1,100.00) 41.05 (,0.30–160.00) Rainy, summer– autumn (July–Oct.) 75.20 (,0.30–290.00) 0.40 (,0.30–0.74) Windy, winter (Nov.–Mar.) 35.10 (,0.30–120.00) 16.22 (,0.30–64.00) Bank B Dry, spring (Apr.– June) Rainy, summer– autumn (July–Oct.) Windy, winter (Nov.–Mar.)

1.79 (,0.30–3.60)

tdhz/trhz

tlhz/tdhz/orf8

0.4 (,0.30–1.20) 0.30 (,0.30–0.36)

,0.30

,0.30

,0.30

0.59 (,0.30–1.50)

,0.30

5.96 (,0.30–23.00)

3.20 (2.70–3.60)

0.57 (,0.30–1.40)

0.39 (,0.30–0.72)

,0.30

0.30 (,0.30–0.36)

6.10 (,0.30–21.00)

0.31 (,0.30–0.36)

,0.30

,0.30

0.30 (,0.30–0.36)

,0.30

,0.30

3.21 (,0.30–12.00)

59.90 (,0.30–210.00) 7.22 (,0.30–28.00)

a

Month Jan. Feb. Mar. Apr. May June July Aug. Sep. Oct. Nov. Dec. a

74.50 3.40 12.00 551.80 6.50 3.20 146.50 12.00

(120.00, 29.00) (6.40, 0.30) (21.00, 3.00) (1,100.00, 3.60) (9.40, 3.60) (3.60, 2.70) (290.00, 3.00) (3.00, 21.00) ,0.30 3.80 (7.4, ,0.30) ,0.30 111.80 (13.60, 210.00)

46.00 (64.00, 28.00) 0.30 (,0.30, 0.40) ,0.30 80.10 (160.00, ,0.30) ,0.30 2.50 (3.60, 1.40) ,0.30 0.30 (,0.30, 0.40) ,0.30 0.50 (0.70, ,0.30) ,0.30 ,0.30

,0.30 ,0.30 17.50 (23.00, 12.00) ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 1.60 (3.00, ,0.30) ,0.30 ,0.30 2.20 (3.60, 0.70) 1.20 (0.50, 0.70) 0.40 (0.40, 0.40) ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 0.30 (,0.30, 0.40) ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 ,0.30 0.89 (1.50, ,0.30) ,0.30 ,0.30

Monthly means (mean for bank A, mean for bank B).

gastroenteritis in humans (14, 34, 53). The presence of these pathogenic Vp strains (tdhz, trhz, or orf8z) raises important health issues and may be indicative of a risk of gastrointestinal illness in the usual consumers of oysters from the MLS during winter windy and spring dry seasons. Levels of pathogenic Vp in the present study were ,50 MPN/g, except for Vp tdhz detected in January (winter windy season, 64.0 MPN/g) and April (spring dry season, 160.0 MPN/g) in bank A (Table 2). The total densities of Vp in oysters observed in our study were lower than the limits set by the U.S. Food and Drug Administration (48) and Mexican (NOM-242-SSA1-2009) (44) regulations for Vp tlhz of 10,000 MPN/g. Densities lower than these limits also have been reported by other authors (13, 53, 55). However, marked differences in the ratio of nonpathogenic to pathogenic Vp density were identified at both collection sites in the present study. Pathogenic Vp tdhz strains represented 14.6, 0.5, and 46.2% of the total Vp isolates during the spring dry, summer rainy, and winter windy seasons, respectively, in bank A and 17.8, 5.1, and 12.1%, respectively, in bank B (Table 2). The Vp trhz strains represented 0.6 and 1.7% of the total isolates during the spring dry and winter windy seasons, respectively, in bank A and 12.2% of the total isolates during the spring dry season in bank B. The orf8 strains represented 17.0% of the

total isolates during the winter windy season in bank A and 9.4, 4.9, and 5.4% during the spring dry, summer rainy, and winter windy seasons in bank B, respectively. Vp tdhz and orf8z isolates were found in very high proportions in the winter windy season (46.2 and 17.0%, respectively) compared with results from other studies (5, 6, 34). This finding indicates that pathogenic isolates were unevenly distributed during the annual cycle. The monthly variation could indicate a rapid turnover in the population. Densities of total and pathogenic Vp strains have been reported as significantly higher (P , 0.01) in the environment in warmer months, and the pathogen is more likely to be detected in oysters harvested in spring and summer than in winter (16, 19). The high densities of pathogenic Vp tdhz and orf8z in the winter dry season suggests that these strains are more cold tolerant than are other Vp strains, although the mean temperature in this season is 20.4uC in the region. This finding suggests that environmental factors may differentially affect the abundance of pathogenic subpopulations. This finding is particularly relevant given previous observations that the highest percentage of total Vp tdhz was detected during warmer months (55). Other variables (e.g., chemical, biological, geological, and hydrological) may be responsible for differences in Vp densities at the two study sites and should be investigated. Even though



FIGURE 3. PCA projections of scores and loadings for the first two principal components for the analysis of densities of V. parahaemolyticus tlhz, tdhz, and orf8z. Score plot indicates densities of total and pathogenic strains, seasons, and sampling sites (banks A and B) of the loading plot variables.

total Vp densities in oysters comply with Mexican regulations, the presence of pathogenic strains is a public health concern, and these strains are not covered by current regulations. Hence, the use of total Vp density as an indicator of food contamination is inadequate, despite the fact that total Vp levels are lower than the permissible 10,000 MPN/g. Seasonal distribution. To evaluate the spatial and seasonal differences in density for total and pathogenic Vp strains, principal components analysis (PCA) was used with XLSTAT version 2013 software (Addinsoft, MultiON Consulting, Mexico City, Mexico). The seasons and sampling sites were the active variables. The first axis represents 48.52% of the explained variance, and the second axis represents 25.98% (Fig. 3). Relationships between seasons and sampling sites and between total and pathogenic strains explained 74.5% of the total variance. The mean densities of total Vp strains in oyster samples harvested each month during the annual cycle were positively correlated (R2 ~ 0.811, P , 0.0001) with densities of Vp tdhz strains during the spring dry and summer rainy seasons in bank A and during the winter windy season in bank B. The highest densities of total Vp (Table 2) were observed during April and July (means at both combined, 551.8.0 and 146.5 MPN/g, respectively), corresponding to the spring dry and summer rainy seasons, respectively, most associated with high densities in bank A (1,100.0 and 290.0 MPN/g, respectively), indicating that this area poses the highest risk of infection during warmer months. These results are in accordance with those found in Alabama (16), where Vp was more abundant in the environment during warmer months and thus pathogenic Vp was more likely to be detected in oysters harvested in spring

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and summer. In another study, pathogenic Vp was detected in oyster samples mainly in the summer months (July to September) at very low levels (3.0 MPN/g) (19). The density of total Vp was 111.8 MPN/g in December during the autumn rainy season, mostly associated with high density in bank B (210.0 MPN/g) (Table 2 and Figure 3). Densities of pathogenic Vp tdhz were correlated (R2 ~ 0.245, P , 0.015) with densities of Vp orf8z in both banks (23.0 and 12.0 MPN/g) in January (the winter windy season; January mean, 17.5 MPN/g). However, densities of pathogenic Vp trhz were not correlated with those of any other strain and were highest in the spring dry season (May and June, 1.6 and 2.2 MPN/g, respectively) associated with bank A (3.0 and 3.6 MPN/g, respectively) and in the autumn rainy season (December, 0.89 MPN/g) also associated with bank A (1.5 MPN/g). The highest densities of pathogenic Vp tdhz were observed in oysters harvested in January (winter windy season) and April (spring dry season) (monthly means of 46.0 and 80.1 MPN/g, respectively) from bank A (64.0 and 160.0 MPN/g, respectively), and the highest densities of pathogenic Vp tdhz/orf8z strains were found in January (17.5 MPN/g) associated with bank A (23.0 MPN/g) and bank B (12.0 MPN/g). Vp tdhz/trhz was detected only during the spring dry season in June (1.2 MPN/g) in oysters harvested from bank A (0.5 MPN/g) and bank B (0.7 MPN/g). This variability between sites suggests the impact of environmental factors. The MLS has been subjected to anthropogenic pressure, and changes in its environmental characteristics have resulting from human activities and modern real estate developments. Environmental interactions may enhance the pathogenicity in a subset of the Vp population. Environmental changes have been associated with the emergence of new pathogens, the reemergence of diseases almost eradicated, and changing patterns and distributions of numerous infectious agents. These natural phenomena may be further enhanced by human activity such as increased sewage input (26, 40, 51). Sea surface temperature and suspended particulate matter have been reported as strong predictors of total and potentially pathogenic Vp populations. Higher nutrient levels associated with highly turbid and polluted waters may have stimulated the growth of vibrios, and nutrients that were previously bound in sediments may become more available in the water column, resulting in more Vp growth (55). The Jamapa, Huatusco, Cotaxtla, and Totolapan river plumes contribute to MLS turbidity and southerly winds during the spring dry season and northerly winds in the winter windy season frequently resuspend sediment in the shallow waters of the Gulf of Mexico near the entrance of the MLS. The MLS is an important area economically for seafood production and consumption and recreation and is one of the most productive estuarine lagoon systems on Mexico’s Gulf Coast for year-round oyster harvesting, with oyster production of 338 to 1,075 tons per year. These oysters supply seafood restaurants and oyster outlets in nearby cities such as Veracruz and Boca del Rıó and are shipped to Cancun and Mexico City. With a national production of 50,715 tons in 2010, Mexico is the sixth largest

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oyster-producing country in the world. In Mexico, Veracruz State is the primary producer, with 26,328 tons representing 51% of the national production (9). To our knowledge, the present study provides the first evidence of the presence of pathogenic Vp (trhz, tdhz/trhz, and orf8z) in oysters harvested from a lagoon on Mexico’s Gulf Coast. Based on our findings, the seasonal dynamics of Vp harboring pathogenicity genes vary with seasonal changes in environmental conditions, mainly during the winter windy and spring dry seasons. The isolation of pathogenic Vp strains in oysters during the annual cycle should be considered a human health concern. Unlike other areas of the world, tropical zone water temperature variations are less pronounced throughout the seasons, which might explain the continual presence of Vp in oysters harvested from the Mandinga Grande Lagoon during the year. Additional research is needed to clarify the effects of the climate changes and water parameter variability on the occurrence of total and pathogenic Vp strains. Our results suggest uncertain outcomes when total Vp densities are used to assess oyster contamination and predict the risk of infection. Hence, the presence of pathogenic strains and the distribution of virulence factors should be evaluated. A year-round monitoring program for environmental parameters and densities of total and pathogenic Vp is mandatory (i) to predict and prevent climate-associated diseases, (ii) to generate information on conditions leading to an outbreak, (iii) to forecast warnings and prevent harvesting or consumption of contaminated shellfish, (iv) to detect the possibility of increased strain virulence, and (v) for risk assessment of postharvest pathogen growth. The detection of Vp tdhz, trhz, tdhz/trhz, and orf8z strains in oysters harvested in this lagoon reveals that the Mexican regulations concerning the microbiological quality of molluscan shellfish require revision to include routine monitoring of these pathogenic strains in harvesting areas and markets, which currently is not being applied for bivalves. Although no evidence of differences in densities of pathogenic Vp strains was found, the detection of Vp tdhz/orf8z strains must be noted and considered a public health concern; thus, both the nonpathogenic and pathogenic Vp strains should be monitored. Surveillance of this potential pathogen in shellfish is crucial to produce more reliable data for conducting risk assessments, to better understand changes in risk from environmental exposure, and to protect public health. ACKNOWLEDGMENTS This work was supported by research grant 114024 from the Mexican National Council of Science and Technology and the Ministry of Health (CONACyT-SSA) under the technical responsibility of Dr. Leonardo Liza´rraga-Partida. We thank the Vibriomex Group for supplying the Vp strains CAIM 1772 and CAIM 1400.


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