Abundance of Vibrio cholerae,V. vulnificus,and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh⁺ and/or trh⁺) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.     

Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)

Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g⁻¹. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g⁻¹). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g⁻¹) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g⁻¹ total V. parahaemolyticus or >10 g⁻¹ tdh- and/or trh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.     

Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g⁻¹. Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh⁺ V. parahaemolyticus than previously reported.     

Long-Term Study of Vibrio parahaemolyticus Prevalence and Distribution in New Zealand Shellfish

The food-borne pathogen Vibrio parahaemolyticus has been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence of V. parahaemolyticus in NZ oysters and Greenshell mussels and the prevalence of V. parahaemolyticus tdh and trh strains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. Total V. parahaemolyticus numbers and the presence of pathogenic genes tdh and trh were determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ, V. parahaemolyticus was detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers of V. parahaemolyticus tdh and trh strains were low, with just 3/215 Pacific oyster samples carrying the tdh gene. V. parahaemolyticus organisms carrying tdh and trh were not detected in South Island samples, and V. parahaemolyticus was detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers of V. parahaemolyticus organisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers of V. parahaemolyticus organisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the total V. parahaemolyticus numbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.     

Isolation of Pandemic Vibrio parahaemolyticus from UK Water and Shellfish Produce

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium found commonly in temperate and warm estuarine waters worldwide. V. parahaemolyticus is considered an emerging bacterial pathogen in Europe and has been responsible for several recent seafood-associated outbreaks. During ad hoc testing of raw shellfish produce in May 2012, pandemic group (O3:K6) V. parahaemolyticus was isolated from Pacific oysters (Crassostrea gigas), harvested in Southern England. Follow-on testing of water and shellfish, encompassing a small number geographically diverse sites, also retrieved pandemic group isolates. These strains are amongst the most northerly pandemic strains described to date and represent the first instance of pandemic V. parahaemolyticus isolated in the UK, highlighting the expanding geographical distribution of these foodborne pathogens in the environment.     

Experimental uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in two species of clams: Ruditapes decussatus and Ruditapes philippinarum

Aims: To describe uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in Ruditapes decussatus and Ruditapes philippinarum. Methods and Results: Ruditapes decussatus accumulated greater concentrations of pathogenic and nonpathogenic V. parahaemolyticus than R. philippinarum. These concentrations decreased earlier in R. decussatus. Nonpathogenic V. parahaemolyticus reached higher concentrations (approx. 1 log CFU g^?1 in tissues of R. decussatus and more than 1 log CFU g^?1 in R. philippinarum) than pathogenic V. parahaemolyticus at similar times. It also persisted longer in both species of clams (72 h in R. decussatus and 96 h in R. philippinarum), while pathogenic V. parahaemolyticus persisted 48 h in R. decussatus and 72 h in R. philippinarum. Conclusions: Ruditapes decussatus incorporated both isolates of V. parahaemolyticus faster than R. philippinarum and it eliminated both isolates earlier than R. philippinarum. Under same conditions, nonpathogenic V. parahaemolyticus might survive better than pathogenic V. parahaemolyticus within both species of clam. Significance and Impact of the Study: Pathogenic V. parahaemolyticus is an important cause of foodborne illnesses. This study shows it may be possible to use nonpathogenic V. parahaemolyticus to perform experimentation to evaluate bacterial evolution in clams, developing an in vivo model useful for risk analysis.     

Predatory Bacteria as Natural Modulators of Vibrio parahaemolyticus and Vibrio vulnificus in Seawater and Oysters

This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus and Vibrio vulnificus were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. The pathogens examined were V. vulnificus strain VV1003, V. parahaemolyticus O1:KUT (KUT stands for K untypeable), and V. parahaemolyticus O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, V. parahaemolyticus O3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns of V. parahaemolyticus O3:K6 host cells. Many VPB, including Bdellovibrio and like organisms (BALOs; Bdellovibrio bacteriovorus and Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. When V. parahaemolyticus O3:K6 was added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.     

Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization

Aims: The aim of this study was to explore a new PCR target gene for Vibrio parahaemolyticus, based on the histone‐like nucleoid structure (H‐NS) gene. Methods and Results: Primers for the H‐NS gene were designed for specificity to V. parahaemolyticus and incorporated into a PCR assay. The PCR assay was able to specifically detect all of the 82 V. parahaemolyticus strains tested, but did not result in amplification in the 47 other Vibrio spp. and nonVibrio spp. strains. The detection limit of the PCR assay was 0·14 pg purified genomic DNA and 1·8 × 10⁵ CFU g^?1 spiked oyster samples from V. parahaemolyticus RIMD2210633. Furthermore, a multiplex PCR assay targeting the hns, tdh and trh genes was successfully developed to detect virulent V. parahaemolyticus strains. Conclusions: The H‐NS‐based PCR assay developed in this study was sensitive and specific, with great potential for field detection of V. parahaemolyticus in seawater or seafood samples. Significance and Impact of the Study: The H‐NS gene was validated as a new specific marker gene in PCR assays for accurate detection and identification of V. parahaemolyticus, which has the potential to be applied in diagnostics and taxonomic studies.     

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh ⁺, ATCC33846-tdh ⁺, ATCC33847-tdh ⁺, ATCC17802-trh ⁺, and F13-environmental strain-tdh ⁺) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.     

Seasonal Variation in Abundance of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters along the Southwest Coast of India

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10⁴ organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.     

Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10⁴ CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh⁺ and trh⁺ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.     

Environmental Determinants of the Occurrence and Distribution of Vibrio parahaemolyticus in the Rias of Galicia, Spain

Infections associated with Vibrio parahaemolyticus on the coast of Galicia (in northwestern Spain) were reported to be linked to large outbreaks of illness during 1999 and 2000. Little information is available about the ecological factors that influence the emergence of V. parahaemolyticus infections in this temperate region. We carried out a 3-year study to investigate the occurrence and distribution of V. parahaemolyticus at 26 sites located in the four main rias of Galicia in association with environmental and oceanographic variables. V. parahaemolyticus was detected in all the areas investigated and throughout the complete period of study with an overall incidence of 12.5%. Salinity was the primary factor governing the temporal and spatial distribution of V. parahaemolyticus, whereas seawater temperature had a secondary effect and only modulated the abundance in periods and areas of reduced salinities. Higher occurrence of V. parahaemolyticus was observed during periods of lower salinity in autumn, with a total of 61 positive samples (18%) and a mean density of 1,234 most probable number/100 g. V. parahaemolyticus was primarily detected in areas of reduced salinity close to freshwater discharge points, where it was found in up to 45% of the samples. Characterization of the isolates obtained from the study resulted in the first identification of two pathogenic tdh-positive strains of V. parahaemolyticus recovered from the marine environment in Galicia. These isolates showed serotypes identical to and DNA profiles indistinguishable from those of the clinical clone of V. parahaemolyticus dominant in infections in Spain in the last 10 years.     

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10² to 10³ CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.     

Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10⁴ CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 10⁹ CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.     

Variability of Total and Pathogenic Vibrio parahaemolyticus Densities in Northern Gulf of Mexico Water and Oysters

Vibrio parahaemolyticus is indigenous to coastal environments and a frequent cause of seafood-borne gastroenteritis in the United States, primarily due to raw-oyster consumption. Previous seasonal-cycle studies of V. parahaemolyticus have identified water temperature as the strongest environmental predictor. Salinity has also been identified, although it is evident that its effect on annual variation is not as pronounced. The effects of other environmental factors, both with respect to the seasonal cycle and intraseasonal variation, are uncertain. This study investigated intraseasonal variations of densities of total and pathogenic V. parahaemolyticus organisms in oysters and overlying waters during the summer of 2004 at two sites in the northern Gulf of Mexico. Regression analyses indicated significant associations (P < 0.001) between total V. parahaemolyticus densities and salinity, as well as turbidity in water and in oysters at the Mississippi site but not at the Alabama site. Pathogenic V. parahaemolyticus organisms in Mississippi oyster and water samples were detected in 56% (9 out of 16) and 78% (43 out of 55) of samples, respectively. In contrast, 44% (7 out of 16) of oyster samples and 30% (14 out of 47) of water samples from Alabama were positive. At both sites, there was greater sample-to-sample variability in pathogenic V. parahaemolyticus densities than in total V. parahaemolyticus densities. These data suggest that, although total V. parahaemolyticus densities may be very informative, there is greater uncertainty when total V. parahaemolyticus densities are used to predict the risk of infection by pathogenic V. parahaemolyticus than previously recognized.     

Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India

The work was aimed to study the microbial quality of the seafood sold in the domestic markets and incidence of Vibrio parahaemolyticus. Samples comprising of shellfish, finfish, and cephalopods were collected from various fish markets in and around Cochin. Presumed V. parahaemolyticus were identified by standard biochemical tests, and further confirmed by polymerase chain reaction targeting species-specific tl gene (450 bp). About 81% of the samples were found to exceed the limits specified for total plate count while total presumptive V. parahaemolyticus count was above the limit in 71% of the samples ranging from 5.5 × 10⁵ to 9.7 × 10⁷ and 0.31 × 10² to 7.8 × 10⁶ cfu/g, respectively. Pathogenicity of the identified isolates was confirmed by Kanagawa phenomenon and urease activity. A total of 10% of the isolates exhibited weak haemolysis on Wagatsuma agar, and 1% of the isolates showed urease activity using Christensen’s urea agar. Random amplified polymorphic DNA analysis revealed two major clusters based on the species rather than seasonality. The gel pattern revealed 8–10 bands ranging from 0.45 to 3.0 kb. Antibiogram results revealed 85% of the strains sensitive to chloramphenicol and nitrofurantoin. Multiple antibiotic resistance index was found to be 0.4 thus suggesting the risk potential involved in consuming seafoods. The present study has clearly demonstrated the need to adopt seafood safety measures for the products meant for human consumption.     

The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters

The dynamics of Dinophysis acuminata and its associated diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX1) as well as pectenotoxins (PTXs), were investigated within plankton and shellfish in Northport Bay, NY, USA, over a four year period (2008–2011). Over the course of the study, Dinophysis bloom densities ranged from ~10⁴ to 10⁶ cells L⁻¹ and exceeded 10⁶ L⁻¹ in 2011 when levels of total OA, total DTX1, and PTX in the water column were 188, 86, and 2900 pg mL⁻¹, respectively, with the majority of the DSP toxins present as esters. These cell densities exceed – by two orders of magnitude – those previously reported within thousands of samples collected from NY waters from 1971 to 1986. The bloom species was positively identified as D. acuminata via scanning electron microscopy and genetic sequencing (cox1 gene). The cox1 gene sequence from the D. acuminata populations in Northport Bay was 100% identical to D. acuminata from Narragansett Bay, RI, USA and formed a strongly supported phylogenetic cluster (posterior probability = 1) that included D. acuminata and Dinophysis ovum from systems along the North Atlantic Ocean. Shellfish collected from Northport Bay during the 2011 bloom had DSP toxin levels (1245 ng g⁻¹ total OA congeners) far exceeding the USFDA action level (160 ng g⁻¹ total OA of shellfish tissue) representing the first such occurrence on the East Coast of the U.S. D. acuminata blooms co-occurred with paralytic shellfish poisoning (PSP) causing blooms of Alexandrium fundyense during late spring each year of the study. D. acuminata cell abundances were significantly correlated with levels of total phytoplankton biomass and Mesodinium spp., suggesting food web interactions may influence the dynamics of these blooms. Given that little is known regarding the combined effects of DSP and PSP toxins on human health and the concurrent accumulation and depuration of these toxins in shellfish, these blooms represent a novel managerial challenge.     

Variation analysis of pathogenic Vibrio spp. and Pseudomonas spp. in Changhai mollusc farming waters using real-time PCR assay during 2011–2014

Bacterial disease has caused high mortality of breeding molluscs from 2009 to 2011 in the Changhai area (Dalian, China). Vibrio spp. and Pseudomonas spp. have been detected as major pathogenic agents for aquatic animals in this area. In the present study, four virulence genes including vsm, toxR, aprX and carA were targeted to develop a real-time PCR assay for the quantitative detection of Vibrio splendidus, V. parahaemolyticus, Pseudomonas fluorescens and P. putida, respectively. The sensitivity and specificity of the assays were verified by experimental samples, and the variation tendencies of pathogenic V. splendidus, V. parahaemolyticus, P. fluorescens and P. putida strains from June to September were also detected in mollusc farming waters during 2011–2014. The concentration of V. splendidus increased from June to July, reduced in August, and then increased again in September. The highest count of V. parahaemolyticus appeared in July, and then dramatically decreased from August to September. Conversely, the counts of P. fluorescens and P. putida remained at lower levels from June to August, and then dramatically peaked in September. All four pathogenic bacteria displayed similar fluctuation tendencies of count variation in each year, and their concentrations were found to have a correlation with the average annual temperature. The variation tendency of pathogenic bacteria with temperature suggested that temperature was one of the most important factors to regulate the bacterial growth in a farming area, which could further provide information for the early warning of disease outbreak by using a convenient real-time PCR assay.     

Rapid Proliferation of Vibrio parahaemolyticus,Vibrio vulnificus,and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae of the non-O1/non-O139 serotype are present in coastal lagoons of southern France. In these Mediterranean regions, the rivers have long low-flow periods followed by short-duration or flash floods during and after heavy intense rainstorms, particularly at the end of the summer and in autumn. These floods bring large volumes of freshwater into the lagoons, reducing their salinity. Water temperatures recorded during sampling (15 to 24°C) were favorable for the presence and multiplication of vibrios. In autumn 2011, before heavy rainfalls and flash floods, salinities ranged from 31.4 to 36.1‰ and concentrations of V. parahaemolyticus, V. vulnificus, and V. cholerae varied from 0 to 1.5 × 10³ most probable number (MPN)/liter, 0.7 to 2.1 × 10³ MPN/liter, and 0 to 93 MPN/liter, respectively. Following heavy rainstorms that generated severe flash flooding and heavy discharge of freshwater, salinity decreased, reaching 2.2 to 16.4‰ within 15 days, depending on the site, with a concomitant increase in Vibrio concentration to ca. 10⁴ MPN/liter. The highest concentrations were reached with salinities between 10 and 20‰ for V. parahaemolyticus, 10 and 15‰ for V. vulnificus, and 5 and 12‰ for V. cholerae. Thus, an abrupt decrease in salinity caused by heavy rainfall and major flooding favored growth of human-pathogenic Vibrio spp. and their proliferation in the Languedocian lagoons. Based on these results, it is recommended that temperature and salinity monitoring be done to predict the presence of these Vibrio spp. in shellfish-harvesting areas of the lagoons.