Parahaemolyticus In Shellfish Research Articles

Trend of Pathogenic Vibrio parahaemolyticus Occurrences in Bivalve Molluscs Harvested in Sardinian Coastal Environments Between 2011 and 2018

The aim of the present study was to evaluate Vibrio parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2013 and 2015. The prevalence of pathogenic V. parahaemolyticus isolates is based on the detection of the two major virulence genes thermostable direct hemolysin (tdh) and thermolabile hemolysin (trh) To assess changes between 2011 and 2018 in the prevalence of V. parahaemolyticus in bivalve molluscs, we compared our results with those of previous investigations. In total, 2,933 samples were collected: 1,079 in 2013, 1,288 in 2014, and 566 in 2015. The mean prevalence of V. parahaemolyticus in shellfish was 3.5% in 2013, 1.7% in 2014, and 3.5% in 2015. The highest percentage of positive samples in 2013 and 2014 was observed in clams (3.5% and 2.7%, respectively), whereas in 2015, it was reported in oysters (15.1%). By comparing the sampling period of 2011–2014 with that of 2015–2018, an increase in the prevalence of V. parahaemolyticus was observed in shellfish (p < 0.05). In parallel, 208 potentially enteropathogenic V. parahaemolyticus strains were identified through the years 2011–2018 and, in particular, 10 trh+ and six tdh+ isolates. Our present study provides information regarding trends of V. parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2011 and 2018 suggesting that the prevalence varies depending on the sampling period and shellfish species.

Quantitative Risk Assessment of Vibrio parahaemolyticus in Shellfish from Retail to Consumption in Coastal Cities of Eastern China

Vibrio parahaemolyticus is the main foodborne pathogen worldwide that causes acute gastroenteritis. A quantitative microbiological risk assessment (QMRA) was conducted to evaluate the health risk associated with V. parahaemolyticus in shellfish in the coastal cities in the eastern part of the People's Republic of China. The QMRA framework was established from shellfish at retail to cooking at home to consumption. The prevalence and level of V. parahaemolyticus in shellfish, cooking methods, storage temperature, time after purchase, shellfish consumption frequency, and consumption amount were analyzed in the exposure assessment. The results of the exposure assessment were introduced into the beta-Poisson dose-response model, and Monte Carlo analysis was used to calculate the risk of gastroenteritis from shellfish consumption. The probability of illness caused by V. parahaemolyticus from shellfish consumption per person per year (Pill,yr) was 3.49E-05. Seasonal differences were noted in the Pill/meal; the maximum was 4.81E-06 in summer and the minimum was 2.27E-07 in winter. The sensitivity analysis revealed that the level of V. parahaemolyticus in shellfish and the amount of shellfish consumed per meal were main factors contributing to illness. This QMRA provided valuable information such as the probability of illness associated with the consumption of shellfish and reference points for prevention strategies and control standards of V. parahaemolyticus in shellfish.

Occurrence, Seasonal Distribution, and Molecular Characterization of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in Shellfish (Mytilus galloprovincialis and Ruditapes decussatus) Collected in Sardinia (Italy)

In this study, we investigated the occurrence, seasonal distribution, and molecular characterization of pathogenic vibrios in Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) from two harvesting areas of Sardinia (Italy). Samples collected before and after depuration were submitted for qualitative and quantitative determination of Vibrio spp. Vibrio spp. isolates were presumptively identified by means of biochemical methods. Identification and virulence profile of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus were performed by molecular methods. The prevalence of Vibrio spp. in M. galloprovincialis and R. decussatus was, respectively, 96 and 77%. The averaged enumeration (mean ± standard deviation) of Vibrio spp. in samples of M. galloprovincialis and R. decussatus collected at the harvesting time was 2.04 ± 0.45 and 2.51 ± 0.65 log CFU/g, respectively. The average contamination levels in samples collected after purification were 2.28 ± 0.58 log CFU/g (M. galloprovincialis) and 2.12 ± 0.67 log CFU/g (R. decussatus). Four potentially pathogenic V. parahaemolyticus isolates (tdh+ or trh+) were recovered from grooved carpet shells samples. No isolate was tdh+/trh+. The presence of potentially pathogenic vibrios in Sardinian waters strengthens the need for rational purification practices under controlled conditions to guarantee the protection of consumers.

MOLECULAR CHARACTERIZATION OF VIBRIO PARAHAEMOLYTICUS ISOLATED FROM SOME MOLLUSKS, SHELLFISH AND THEIR HARVESTING WATER FROM SUEZ CANAL AREA

The study included 410 samples of shellfish (164 clams, 86 mussels, and 160 shrimps) collected from Suez Canal area as the following: 156 from Port Said, 164 from Ismailia and 90 from Suez governorates. The harvesting water samples (48) were collected from the three sites. All samples were collected during the warmest season (June, July, and August) through two years. After extraction of muscle tissue and homogenization, enriched samples were identified by plating onto TCBS agar medium. Presumptive V. parahaemolyticus colonies were selected, purified, and further identified by API20 and PCR techniques targeting toxRgene. The pathogenicity of the isolates was examined by detection of Thermostable Direct Haemolysin (TDH) and related Haemolysin (TRH) genes. Results revealed that the overall prevalence of V. parahaemolyticus in shellfish was 38/410 (9.27%), where as in water was 12/48 (25%). Higher contamination rate was detected in shrimp (15%), and the highest prevalence was recorded in Ismailia governorate (12.2%). The detection rate of TDH and TRH genes was 21.05% and 5.26% consequently indicating its health hazards to the consumers. This study concluded that the examined shellfish may have the potential human health risk associated with the presence of pathogenic V. parahaemolyticus. The preventative measures should be considered.

Detection of Total and Pathogenic Vibrio parahaemolyticus in Shellfish Growing along the South Yellow Sea and the East China Sea

This study was conducted to monitor the densities of total and pathogenic Vibrio parahaemolyticus in 300 samples of nine shellfish species harvested from the coasts of the South Yellow Sea and the East China Sea (N 23° to 34°, E 116° to 124°), People's Republic of China, between May and October 2015. Total V. parahaemolyticus densities were measured, and V. parahaemolyticus isolates were biochemically identified with probes for the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh). We found that 202 of the 300 samples were positive for V. parahaemolyticus from all the sites: 58 of the 100 samples from the Fujian province, 71 of the 100 samples from the Zhejiang province, and 73 of the 100 samples from the Jiangsu province. In most (170) of the 300 samples, V. parahaemolyticus densities were 0.3 to 10 most probable number (MPN)/g; five lots exceeded 110 MPN/g, and two lots were estimated at 110 MPN/g. Among the 202 V. parahaemolyticus strains, only one was trh positive. Densities of V. parahaemolyticus in these shellfish were temperature dependent, with highest densities in June and July. Among the nine mollusk species, V. parahaemolyticus was most abundant in the agemaki clam (Sinonovacula constricta). The highest and lowest V. parahaemolyticus prevalences were found in oriental cyclina (Cyclina sinensis, 93.8%) and mussels (Mytilus edulis, 28.1%), respectively. Overall, although V. parahaemolyticus is widely distributed in marine environments, the density of V. parahaemolyticus was low and the prevalence of the main virulence factor was very low in shellfish along the coasts of the South Yellow Sea and East China Sea, which is important from a public health perspective. Data presented here will be useful for correlational research and can be utilized for developing risk management plans that establish food safety guidelines for V. parahaemolyticus in Chinese shellfish.

Different abundance and correlational patterns exist between total and presumed pathogenic Vibrio vulnificus and V. parahaemolyticus in shellfish and waters along the North Carolina coast.

Monitoring of Vibrio vulnificus and V. parahaemolyticus abundance is pertinent due to the ability of these species to cause disease in humans through aquatic vectors. Previously, we performed a multiyear investigation tracking Vibrio spp. levels in five sites along the southeastern North Carolina coast. From February 2013 to October 2015, total V. vulnificus and V. parahaemolyticus abundance was measured in water, oysters and clams. In the current study, pathogenic subpopulations were identified in these isolates using molecular markers, revealing that 5.3% of V. vulnificus isolates possessed the virulence-correlated gene (vcgC), and 1.9% of V. parahaemolyticus isolates harbored one or both of the virulence-associated hemolysin genes (tdh and trh). Total V. parahaemolyticus abundance was not sufficient to predict the abundance of pathogenic subpopulations. Specifically, pathogenic V. parahaemolyticus isolates were more often isolated in cooler waters and were sometimes isolated when no other V. parahaemolyticus strains were detectable. Vibrio vulnificus clinical (C-) genotypes correlated with total V. vulnificus; however, salinity, water depth and total suspended solids influenced C- and E-genotypes differently. Lastly, we documented individual oysters harboring significantly higher V. vulnificus levels for which there was no ecological explanation, a phenomenon that deserves closer attention due to the potentially elevated health hazard associated with these 'hot' shellfish.

Different abundance and correlational patterns exist between total and presumed pathogenic Vibrio vulnificus and V. parahaemolyticus in shellfish and waters along the North Carolina coast

Different abundance and correlational patterns exist between total and presumed pathogenic Vibrio vulnificus and V. parahaemolyticus in shellfish and waters along the North Carolina coast

Occurrence and Antibiotic Resistance of Vibrio parahaemolyticus from Shellfish in Selangor, Malaysia

High consumer demand for shellfish has led to the need for large-scale, reliable shellfish supply through aquaculture or shellfish farming. However, bacterial infections which can spread rapidly among shellfish poses a major threat to this industry. Shellfish farmers therefore often resort to extensive use of antibiotics, both prophylactically and therapeutically, in order to protect their stocks. The extensive use of antibiotics in aquaculture has been postulated to represent a major contributing factor in the rising incidence of antimicrobial resistant pathogenic bacteria in shellfish. This study aimed to investigate the incidence of pathogenic Vibrio parahaemolyticus and determine the antibiotic resistance profile as well as to perform plasmid curing in order to determine the antibiotic resistance mediation. Based on colony morphology, all 450 samples tested were positive for Vibrio sp; however, tox-R assay showed that only 44.4% (200/450) of these were V. parahaemolyticus. Out of these 200 samples, 6.5% (13/200) were trh-positive while none were tdh-positive. Antibiotic resistance was determined for all V. parahaemolyticus identified against 14 commonly used antibiotics and the multiple antibiotic resistance index (MAR) was calculated. The isolates demonstrated high resistance to several antibiotics tested- including second and third-line antibiotics- with 88% resistant to ampicillin, 81% to amikacin,70.5% to kanamycin, 73% to cefotaxime, and 51.5% to ceftazidime. The MAR index ranged from 0.00 to 0.79 with the majority of samples having an index of 0.36 (resistant to five antibiotics). Among the 13 trh-positive strains, almost 70% (9/13) demonstrated resistance to 4 or more antibiotics. Plasmid profiling for all V. parahaemolyticus isolates revealed that 86.5% (173/200) contained plasmids - ranging from 1 to 7 plasmids with DNA band sizes ranging from 1.2 kb to greater than 10 kb. 6/13 of the pathogenic V. pathogenic strains contained plasmid. After plasmid curing, the plasmid containing pathogenic strains isolated in our study have chromosomally mediated ampicillin resistance while the remaining resistance phenotypes are plasmid mediated. Overall, our results indicate that while the incidence of pathogenic V. parahaemolyticus in shellfish in Selangor still appears to be at relatively reassuring levels, antibiotic resistance is a real concern and warrants ongoing surveillance.

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses the greatest threat to human health worldwide. It has been the leading bacterial cause of seafood-borne illness. This study investigated the prevalence and drug resistance of V. parahaemolyticus isolated from retail shellfish in Shanghai. A total of 140 shellfish samples were collected from February 2014 to February 2015. The occurrence of V. parahaemolyticus in shellfish was 34.3%, which has increased compared to previous reports. In addition, discernible differences of total presumptive V. parahaemolyticus counts (TPVPC) were also observed in shellfish between market A and B. The results from PCR assays indicated that thermostable direct hemolysin (tdh) gene was positive in two isolates (2.1%), and the thermostable direct hemolysin-related hemolysin (trh) gene was not detected in all isolates. Antibiotic resistance profiles of those isolates were as follows: ampicillin (87.5%), cephazolin (31.3%), cephalothin (6.3%), amoxicillin/clavulanic acid (6.3%), piperacillin (6.3%), and amikacin (3.2%). Thirty-three out of 96 isolates were resistant to two or more antimicrobial agents. It is suggested that V. parahaemolyticus in retail shellfish could be a potential risk to consumers in Shanghai.

Temporal and Spatial Variation in the Abundance of Total and Pathogenic Vibrio parahaemolyticus in Shellfish in China.

We investigated the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish sampled from four provinces in China during May 2013 and March 2014 using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 67.7% of 496 samples. A total of 38.1% and 10.1% of samples exceeded 1,000 MPN g-1 and 10,000 MPN g-1, respectively. V. parahaemolyticus densities followed a seasonal and geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels that were 100-fold higher than those of the Liaoning and Shandong regions. Moreover, the levels of V. parahaemolyticus were at least 10-fold higher in the summer and autumn than in the cooler seasons. Pathogenic V. parahaemolyticus levels were generally lower than total V. parahaemolyticus levels by several log units and tended to be high in samples contaminated with high total V. parahaemolyticus levels. The aqua farms had a lower prevalence but higher abundance of total V. parahaemolyticus compared to retail markets. The catering markets showed the lowest levels of total V. parahaemolyticus, but 20.0% of samples exceeded 1,000 MPN g-1. The levels of both total and pathogenic V. parahaemolyticus in oysters were higher than in clams. The log-transformed abundance of V. parahaemolyticus was significantly correlated with both water temperature and air temperature but not water salinity. These results provide baseline contamination data of V. parahaemolyticus in shellfish in China, which can be applied to local risk assessments to prioritize risk control to key sectors and evaluate the effectiveness of future control measures.

Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay

Vibrio parahaemolyticus is a halophilic bacterium that commonly inhabits the marine and estuarine environments. This organism is also one of the leading causative pathogen of gastroenteritis often related to consumption of raw or undercooked seafood. In this study, molluscan shellfish (bloody clams and surf clams) and crustaceans (shrimps) were monitored in wet markets and hypermarkets. Two molecular methods were employed and compared to detect total and pathogenic V. parahaemolyticus in MPN enrichments: multiplex PCR and LAMP assay. The multiplex PCR was optimized to detect the total (toxR+), tdh+ and trh+ V. parahaemolyticus. On the other hand, the LAMP assay was employed to target the pathogenic strains only, the tdh+ and trh+, respectively. Out of 232 samples examined, 229 (98.7%) were positive for V. parahaemolyticus with counts ranging from 30 to >110, 000 MPN/g. Positive samples for tdh+ V. parahaemolyticus were obtained in 77 out of 232 (33.1%) samples ranging from 30 to >110, 000 MPN/g. Meanwhile, positive samples for trh+ were identified in 16 out of 232 (6.9%) samples examined ranging from 30 to 9600 MPN/g. Detection of samples with presence of tdh+ genes did not vary between methods, but a significant difference was observed when the LAMP assay was compared to PCR to detect trh+ V. parahaemolyticus. Therefore, on occasions where the density of the targeted genes is low, the LAMP assay serves as a better alternative. Nonetheless, this study constitutes an assessment of presence of total and potentially pathogenic V. parahaemolyticus in shellfishes for domestic consumption revealing the potential risk of contracting vibriosis if precautions and safety measures are not properly managed.

Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish

Vibrio parahaemolyticus is a marine microorganism, recognized as cause of gastroenteritis outbreaks associated with seafood consumption.In this study the development and the in-house validation of a colony hybridization method for the enumeration of total and potentially pathogenic V. parahaemolyticus is reported. The method included a set of three controls (process, hybridization and detection control) for the full monitoring of the analytical procedure. Four digoxigenin-labeled probes were designed for pathogenic strains enumeration (tdh1, tdh2, trh1 and trh2 probes) and one for total V. parahaemolyticus count (toxR probe). Probes were tested on a panel of 70 reference strains and 356 environmental, food and clinical isolates, determining the inclusivity (tdh: 96.7%, trh: 97.8%, toxR: 99.4%) and the exclusivity (100% for all probes). Accuracy and linearity of the enumeration were evaluated on pure and mixed cultures: slopes of the regression lines ranged from 0.957 to 1.058 depending on the target gene and R2 was greater than or equal to 0.989 for all reactions. Evaluation was also carried on using four experimentally contaminated seafood matrices (shellfish, finfish, crustaceans and cephalopods) and the slopes of the curves varied from 0.895 (finfish) to 0.987 (cephalopods) for the counts of potentially pathogenic V. parahaemolyticus (R2≥0.965) and from 0.965 to 1.073 for total V. parahaemolyticus enumeration (R2≥0.981). Validation was performed on 104 naturally contaminated shellfish samples, analyzed in parallel by colony hybridization, ISO/TS 21872-1 and MPN enumeration. Colony hybridization and ISO method showed a relative accuracy of 86.7%, and a statistically significant correlation was present between colony hybridization enumeration and MPN results (r=0.744, p<0.001).The proposed colony hybridization can be a suitable alternative method for the enumeration of total and potentially pathogenic V. parahaemolyticus in seafood.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7×103CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120CFU/g and most of the positive samples (n=17) showing values equal or below 20CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency.The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.

Isolation And Identification of Vibrio parahemolyticus Recovered From Some Shellfish Marketed In Zagazig City

The present investigation was undertaken to study the occurrence of V. parahaemolyticus in shellfish and to assess the genomic diversity within V. parahaemolyticus strains isolate using ERIC-PCR technique. A total of 130 samples of shellfish were collected from fish markets, Zagazig city included white shrimps (40); blue crabs (40) and mussels (50).V. parahaemolyticus were isolated and identified using standard bacteriological methods.ERIC-PCR was carried out for a total of 9 V. parahaemolyticus clones, representing white shrimp, blue crab and mussels (3 each).V. parahaemolyticus were isolated with the percentages of 17.5(7/40), 12.5(5/40) and 16.0(8/50), respectively. The overall percentage of the examined shellfish was 15.4(20/130).Overall, the ERIC-PCR results showed 6 separate clusters at similarity level 0.53. Isolates No 4, 2, 5, 3 grouped together (group I). Isolates No 1, 8, 9, 7 and 6 each clustered in their own groups (II, III, IV, V and VI, respectively). In addition, there is sub-clustering with group I at similarity level 0.66. The amplified bands were analyzed by dendrogram using Dice correlation coefficient. The phylogenetic analysis of tested strains exhibits high amount of heterogeneity.It could be concluded from the present study that V. parahaemolyticus are circulating among the marketed shellfish in Zagazig city. Variable V. parahaemolyticus clones indicate different sources of contamination and infections under the research conditions.

Total number, virulence genes, and heterogeneity of Vibrio parahaemolyticus in a single shellfish

Infection by Vibrio parahaemolyticus is established if seafood contaminated with pathogenic strains of this bacterium at a sufficiently high dose is consumed. We reported recently that V. parahaemolyticus isolates from 67% of patients constituted a homogeneous population of a pathogenic strain and a heterogeneous population of V. parahaemolyticus varying in the serotype and/or the virulence gene profile found in 33% of the patients. To assess the possibility that this result was due to the dominant presence of a homogeneous strain of V. parahaemolyticus in shellfish, we enumerated and characterized V. parahaemolyticus in shellfish samples including three kinds of molluscan bivalves. Using CHROMagar Vibrio isolation medium, presumed total Vibrio strains and presumed V. parahaemolyticus in each of 52 shellfish samples were enumerated. The highest numbers of presumed total Vibrio strains and presumed V. parahaemolyticus were detected in bloody clam and mussel, respectively. There was no correlation between the number of presumed total Vibrio strains and that of presumed V. parahaemolyticus. Five to ten strains of V. parahaemolyticus isolated from each shellfish sample were characterized. Virulence genes (tdh and trh) could be detected in none of the isolated strains. Diversities in the serotype and DNA fingerprints were confirmed among the isolated strains from a single shellfish sample representing six different shellfish groups. We therefore conclude that most of the single shellfish harbour a pathogenic strain-bearing heterogeneous population of V. parahaemolyticus and the concentrations of pathogenic strains are low.

STUDY ON DETECTION OF VIBRIO PARAHAEMOLYTICUS IN SHELLFISH BY USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) technology for rapidly detecting Vibrio parahaemolyticus in shellfish. In this study, the gyrB gene of V. parahaemolyticus was chosen to design six primers, the target DNA was dectected with white precipitate; specificity of the primers were identified with 14 V. parahaemolyticus strain, 15 non-parahaemolyticus Vibrio and 22 non-Vibrio strains; 84 shellfish sampled at random from market were tested by using this method and the results were evaluated with the Food and Drug Administration Bacteriological Analytical Manual most probable number method. The results indicated that this method is easy for operation, with a high level of specificity and the sensitivity of 19 cfu/mL for bacterial liquid and 87 cfu/g for shellfish samples. Detection of an initial inoculum of 1 cfu V. parahaemolyticus per gram of shellfish tissue homogenate was possible after enrichment of 2 h. These results indicate that the LAMP can provide a reliable method for the detection of V. parahaemolyticus in shellfish. PRACTICAL APPLICATIONS The detection of Vibrio parahaemolyticus involve traditional culture-based methods; such methods are labor-intensive and time-consuming. Molecular-based methods such as DNA probe hybridization and polymerase chain reaction (PCR) assays yielded rapid and specific results. However, the probe hybridization procedure and the gel electrophoresis technique are tedious and time-consuming; several real-time PCR assays require a dedicated real-time PCR machine, which is rather expensive and not yet widely available. Because of carrying out under isothermal conditions, the amplification efficiency of the loop-mediated isothermal amplification (LAMP) method is extremely high; LAMP is highly specific for the target sequence because of its recognition of the target sequence by six primers. Furthermore, because the LAMP assay synthesizes a large amount of DNA, the products can be detected by simple turbidity; thus, expensive equipment is unnecessary for providing a high level of precision. These features allow for a simple, rapid and cost-effective detection of V. parahaemolyticus.

Detection of Vibrio parahaemolyticus in mediterranean mussels (Mytilus galloprovincialis) in Slovenia

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Seča, Piran, Strunjan and Debeli Rtič) between 2006 and 2008. Samples were examined and analysed for the presence of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.

Distribution, serological and molecular characterization of Vibrio parahaemolyticus from shellfish in the eastern coast of China

To investigate the prevalence of Vibrio parahaemolyticus in shellfish, a total of 288 samples from retail markets in four coastal provinces of eastern China were collected and analyzed monthly from December 2008 to November 2009. Altogether 172 isolates were isolated and identified, of which 2 isolates were tdh + and 5 were trh +. The levels of V. parahaemolyticus were estimated by most probable number procedure and results suggested that the distribution of V. parahaemolyticus was disparity in season. Serotyping was performed among all isolates, 157 isolates were grouped into 9 O-groups and 42 isolates were determined by specific K-typing. Two tdh + isolates were identified as O3:K6 and O4:K68 serovars. Random amplified polymorphic DNA (RAPD) was performed to assess the genetic diversity of all isolates. The results showed that there were 73 different patterns, which were clustered into 18 groups except 6 miscellaneous patterns. The two tdh + isolates and two clinical isolates were grouped into the same cluster. This study demonstrated that V. parahaemolyticus from shellfishes were of high antigenic and genetic diversity. Comparison with serological method, RAPD might be a more efficient vehicle for epidemiology and risk assessment of V. parahaemolyticus.

Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus

Considering its widespread distribution in marine environments, its fast replication times and low infectious doses and the rapid spread of its strains in recent years, intensive and continuous monitoring of potentially pathogenic Vibrio parahaemolyticus is strongly recommended in order to assess the human health risk arising from shellfish consumption. The lack of epidemiological data points to the need to develop specific methods for detecting V. parahaemolyticus. In this note, the authors compare two plating media currently available for isolating V. parahaemolyticus in shellfish. Both approaches involve pre-enrichment of V. parahaemolyticus. One uses thiosulphate-citrate-bile salt sucrose (TCBS) as the isolation medium, while the other uses a chromogenic medium (CHROMagar Vibrio). Next, biochemical identification of isolates was performed with API 20E, followed by PCR assay aimed at the toxR gene to confirm the cultural and biochemical identification. Comparison of the two methods highlighted that CHROMagar Vibrio is more accurate and specific than TCBS. The analysis of data from 160 shellfish samples showed an accuracy and specificity of just 51% and 71% for TCBS compared with 88% and 95% for CAV.

Dynamics of Clinical and Environmental Vibrio parahaemolyticus Strains during Seafood-Related Summer Diarrhea Outbreaks in Southern Chile

Seafood consumption-related diarrhea became prevalent in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached a region in the south of Chile (Region de los Lagos) where approximately 80% of the country's seafood is produced. In spite of the large outbreaks of clinical infection, the load of V. parahaemolyticus in shellfish of this region is relatively low. The pandemic strain constitutes a small but relatively stable group of a diverse V. parahaemolyticus population, composed of at least 28 genetic groups. Outbreaks in Region de los Lagos began in 2004 and reached a peak in 2005 with 3,725 clinical cases, all associated with the pandemic strain. After 2005, reported cases steadily decreased to a total of 477 cases in 2007. At that time, 40% of the clinical cases were associated with a pandemic strain of a different serotype (O3:K59), and 27% were related to V. parahaemolyticus isolates unrelated to the pandemic strain. In the results published here, we report that in the summer of 2008, when reported cases unexpectedly increased from 477 to 1,143, 98% of the clinical cases were associated with the pandemic strain serotype O3:K6, a change from 2007. Nevertheless, in 2009, when clinical cases decreased to 441, only 64% were related to the pandemic strain; the remaining cases were related to a nonpandemic tdh- and trh-negative strain first identified in shellfish in 2006. Overall, our observations indicate that the pandemic strain has become a relatively stable subpopulation and that when the number of diarrhea cases related to the pandemic strain is low, previously undetected V. parahaemolyticus pathogenic strains become evident.