Parahaemolyticus In Shrimp Research Articles

Evaluation of outer membrane protein U (OmpU) as a novel capture target of Vibrio parahaemolyticus and rapid detection of acute hepatopancreatic necrosis disease (AHPND) using PCR combined with immunomagnetic separation

A polymerase chain reaction (PCR) assay combined with immunomagnetic separation (IMS) was established to detect a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND). The outer membrane protein OmpU, a conserved antigen among Vibrio species, was evaluated as a novel capture target for the immunomagnetic separation to capture and concentrate V. parahaemolyticus from environmental sources. A PCR assay targeting pirA- (284bp) and pirB- (392bp) like genes was used for screening AHPND V. parahaemolyticus. Immunomagnetic beads (IMBs), coated with anti-VpOmpU antibodies, presented high capture efficiency (>90%) for V. parahaemolyticus within 60min. When PCR assay was combined with IMS (IMS-PCR), the detection limit was 101CFU/mL in pure cultures and was unaffected by the presence of 105CFU/mL of competing microflora. When applied in artificially contaminated environmental samples, the IMS-PCR could also detect as few as 101CFU/mL AHPND V. parahaemolyticus within 4–5h. These results suggest that the IMS-PCR approach could be a rapid and effective method for the detection of AHPND V. parahaemolyticus in shrimp and water samples from environmental sources.

Pathogenicity of Vibrio parahaemolyticus in Different Food Matrices

The pathogenicity and virulence factors of Vibrio parahaemolyticus in four food matrices--shrimp, freshwater fish, pork, and egg-fried rice--were compared by measuring the thermostable direct hemolysin activity and total hemolytic titer. Significantly high thermostable direct hemolysin and also hemolytic titers (P < 0.05) were produced by V. parahaemolyticus in egg-fried rice > shrimp > freshwater fish > pork. Filtrates of V. parahaemolyticus in shrimp given intraperitoneally induced marked liver and kidney damage and were highly lethal to adult mice compared with filtrates of V. parahaemolyticus in freshwater fish > egg-fried rice > pork. From in vitro and in vivo pathogenicity tests, it seems the type of food matrix has a significant impact on the virulence of V. parahaemolyticus. These results suggest that hemolysin may not necessarily be the only virulence factor for pathogenicity of V. parahaemolyticus. This is the first report that shows that virulence factors produced by V. parahaemolyticus in seafood such as shrimp are more toxic in vivo than in nonseafood.

Quantitative Analysis of Pathogenic and Nonpathogenic V ibrio parahaemolyticus in Shrimp Derived from Industrial Processing

Vibrio parahaemolyticus is an important seafood-borne pathogen. V. parahaemolyticus carrying either tdh or trh or both genes is considered a pathogenic strain. The objectives of this study were to examine the prevalence and concentration of V. parahaemolyticus in shrimp processed in a factory using most probable number (MPN) combined with either polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) techniques. The pathogenic group (pathogenic Vp) is composed of strains carrying tdh (tdh+ Vp) and/or trh (trh+ Vp) and nonpathogenic group (total Vp) is consisted of both pathogenic and nonpathogenic strains. There was no difference in the total Vp detection by PCR and LAMP techniques. However, combined MPN with LAMP techniques, an increase in detection of pathogenic Vp in samples was identified and the concentrations of pathogenic Vp in shrimp from the first five processing steps were 3.2, 3.0, 3.1, 3.5 and 4.3 MPN/g and were below the detection level (<3 MPN/g) in shrimp from the last three steps. LAMP technique estimated significantly higher positive samples and MPN values for pathogenic V. parahaemolyticus than PCR technique. Practical Applications Loop-mediated isothermal amplification technique is more sensitive than polymerase chain reaction at determining low numbers of pathogenic Vibrio parahaemolyticus in seafood. Decreased temperature in the peeling step as well as frequent change in dipping water in the grazing step will reduce the total numbers of V. parahaemolyticus contaminated in shrimp.

QPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus

Abstract A quantitative polymerase chain reaction (qPCR) assay was developed, based on a TaqMan probe, to detect and quantify a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND). The assay uses a pair of PCR primers, which amplify a 135-bp DNA fragment, and a TaqMan probe selected from the plasmid pir A-like gene. This qPCR assay reacted with AHPND-pathogenic isolates of V. parahaemolyticus collected from Vietnam and Mexico, but not with non-pathogenic strains of Vibrio spp. For quantification, a plasmid (pVpPirA-1) containing the target pir A-like gene was constructed, purified and serially diluted to be used as a standard. With this standard, the qPCR assay was then used to quantify the virulence plasmid in shrimp samples collected from different farms. Up to 5.8 × 10 5 copy per mg tissue were detected in AHPND-affected shrimp collected from Vietnam. Lower quantities, up to 1.5 × 10 4 copies per mg of tissues were detected in affected shrimp collected from a Chinese farm. In the laboratory bioassays, similar plasmid quantities, 1.8 × 10 3 to 4.7 × 10 6 copies of plasmid per mg of tissues were found in the moribund/dead shrimp, 3.5 × 10 2 to 2.2 × 10 6 copies of plasmid per mL were detected in the water samples. This assay is specific with high sensitivity (10 copies of virulence plasmid) and can be used to detect AHPND-pathogenic V. parahaemolyticus in shrimp and water samples.

Phage therapy against Vibrio parahaemolyticus infection in the whiteleg shrimp (Litopenaeus vannamei) larvae

Vibrio parahaemolyticus is an important cause of disease, mortality, and economical losses in the shrimp aquaculture industry. Bacteriophages are natural bio-controlling agents, broadly recognized for their ability to reduce pathogen populations. Hence, in the present study, we evaluated the effectiveness of phage therapy in the prevention and control of vibriosis in Litopenaeus vannamei . Vibriosis was induced in shrimp larvae with 2 × 10 6 CFU mL − 1 of V. parahaemolyticus . The infected larvae were treated with different doses of selected phages and their efficacy was evaluated at different times after their application. Results revealed that selected lytic phages (A3S and Vpms1) are effective to reduce mortality caused by V. parahaemolyticus . In both cases, the early application (at 6 h post-infection) was effective to avoid mortality. Low multiplicity of infection (MOI) values (< 0.1) were enough to counteract V. parahaemolyticus infection. Delayed phage applications (> 6 h post-infection) hindered mortality and the progress of infection. This study provides the basis for the use of bacteriophages in the prevention and control of V. parahaemolyticus in shrimps. • We evaluated the effectiveness of phages to prevent and control vibriosis in shrimp. • Larvae infected with V. parahaemolyticus were treated with different doses of phages. • The phage therapy; effectively can prevent and control the mortality caused by Vibrio.

Prevalence, pathogenicity, and serotypes of Vibrio parahaemolyticus in shrimp from Chinese retail markets

Vibrio parahaemolyticus is one of the most common foodborne pathogens in Asian countries. V. parahaemolyticus contamination of retail shrimp in China has been reported previously, and such contaminated shrimp have been linked to outbreaks of foodborne diseases. However, to date, the prevalence of V. parahaemolyticus in retail shrimp in China has not been determined. This study aimed at identifying the prevalence of V. parahaemolyticus in shrimps in Chinese retail market. From May 2012 to April 2013, a total of 273 shrimp samples was obtained from retail markets in 16 provinces (19 cities) of China. V. parahaemolyticus was detected in 103 (37.7%) of 273 samples by the most probable number method. Bacterial densities were less than 100 MPN/g in 95.1% (98/103) of the samples. Five trh-positive isolates were identified from 247 isolates, and none of the isolates were tdh-positive. In multiplex PCR-based O-antigen serotyping of these 247 pathogenic isolates, all O-antigen serotypes, except O9, were detected, and serotype O2 was found to be the most prevalent (detected in 82 isolates). This is the first report on V. parahaemolyticus prevalence in retail shrimp in China, and the findings of this study can be used for microbiological risk assessment of shrimp in China.

Antibiotic Resistance of Vibrio parahaemolyticus Isolated from Cockles and Shrimp Sea Food Marketed in Selangor, Malaysia

Introduction: The main aim of this study is to determine the antibiotic profile of V. parahaemolyticus gastroenteritis associated with the consumption of contaminated shrimp and cockles marketed in Selangor Malaysia. V. parahaemolyticus is the leading cause of seafood-associated gastroenteritis in Asian Countries typically is associated with the consumption of raw shellfish and oysters specially shrimp and cockles. Rapid, sensitive and specific detection methods are needed to control V. parahaemolyticus infections. We describe a recognized the pathogenic V. parahaemolyticus in shrimp and cockles that will be the risk of gastroenteritis associated with the consumption of seafood marketed in Malaysia. Methods: This study was carried out between July 2011 and August 2013 at the Center of Excellence for Food Safety Research, Faculty of Food Science and Technology, Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, and Faculty of Biotechnology, Dep. of Cell and Molecular Biology, University Putra Malaysia and other centers as collaboration. The seafood samples were collected from different markets and more than 400 samples from shrimp and cockles were investigated for detection and isolation of V. parahaemolyticus. CHROMagar Vibrio and TCBS agar media were used for fast detection and isolation of V. parahaemolyticus isolates. PCR based methods targeted to toxR regulatory gene, tlh the species and family gene, tdh and trh the virulence genes were extensively used. The antibiotic susceptibility testing of 65 V. parahaemolyticus isolates recovered from retail shrimp and cockles seafood were determined with four types of E-test antibiotic strips. Results: All the 65 isolates were positive to toxR and tlh genes. Out of 65 isolates, only eight isolates (12.31%) were positive for tdh virulence gene isolated form cockles and shrimp (3 isolates from shrimp and 5 isolates from cockles), whereas twenty six (40%) isolates were positive for trh virulence gene isolated from shrimp and cockles (9 from shrimp and 17 from cockles). This result indicates high occurrence of tdh+ and trh+ isolates in shrimp and cockles marketed in Malaysia. None of the isolates tested possess both virulence genes. For the antibiotic E-test susceptibility test, overall, V. parahaemolyticus is remained susceptible to tetracycline (97%). A slight increase in the susceptibility of tetracycline is observed from 2011 to 2013. While reduced susceptibility was detected only in V. parahaemolyticus for ampicillin. The mean of MIC of the isolates toward ampicillin is increased from 64 μg/ml in 2011 to 128 μg/ml in year 2013. The current study demonstrates a high risk of pathogenic V. parahaemolyticus in the shrimp and cockles marketed in Selangor Malaysia. Conclusions: The potential risk of V. parahaemolyticus infection due to the consumption of contaminated seafood in Malaysia should not be neglected. The increased resistance of ampicilin from our studies in Malaysia since 2004 to 2013 could be in indication of antibiotic abuse in clinical and agricultural used of ampicilin in Malaysia.

Modeling the thermoultrasound inactivation of Vibrio parahaemolyticus in raw peeled shrimps.

Vibrio parahaemolyticus has been identified as a causative agent for seafoodborne diseases worldwide. The effect of thermoultrasound treatment on the survival of V. parahaemolyticus in raw peeled shrimps was investigated in this study as an alternative bacterial inactivation method in seafood as part of the postharvest washing process. Raw peeled shrimps inoculated with V. parahaemolyticus were treated with mild heat (47, 50, and 53°C) combined with ultrasound (0, 96, 150, and 204 W) based on a 3 × 4 full factorial design, and the bacterial survival curves were fitted with a Weibull model. Because of the high correlations of the shape parameter n and the scale parameter β in the Weibull model, an overall n was estimated from the whole set of bacterial inactivation data and β values were estimated for each set of inactivation curves accordingly. A response surface model was generated to describe the scale parameter as a function of temperature and ultrasonic power. The results indicated that the Weibull model with the overall n value could be used to describe the bacterial reduction with the time of exposure to the thermoultrasound treatments, which was well supported by the small root mean square errors (RMSE) and the high coefficients of determination (R(2)). The quadratic model was validated with independent experiments within the prediction range. Statistical indices (R(2) = 0.99; P < 0.0001; RMSE = 0.17) and validation parameters (bias factor = 0.97; accuracy factor = 1.03) indicated satisfactory performance of the quadratic model. The results indicated that the thermoultrasound treatment is effective, simple, and cost-effective for inactivation of V. parahaemolyticus in shrimps during the postharvest washing process.

A predictive model for assessment of decontamination effects of lactic acid and chitosan used in combination on Vibrio parahaemolyticus in shrimps

Vibrio parahaemolyticus is a major causative agent of human gastroenteritis in seafood products including shrimps. Lactic acid and chitosan are natural antimicrobials for food decontamination in the washing process of seafood. In this research, a 4-factor response surface model based on the Box–Behnken experimental design was developed to evaluate the effects of lactic acid (1%, 2%, and 3%, v/v), chitosan (0.4%, 1%, and 1.6%, w/v), rotational rate (90, 110, and 130rpm) and washing time (10, 20, and 30min) on reduction of V. parahaemolyticus inoculated in raw shrimps. These treatments achieved 2.2 to 4.3log10CFU/g reduction of V. parahaemolyticus in shrimps. Stepwise stratification led to a simplified model that has a satisfactory performance as evidenced by statistical indices (R2=0.92; p<0.0001; RMSE=0.196) and external validation parameters [bias factor (Bf)=1.01; accuracy factor (Af)=1.05]. The model generated an optimum treatment combination (3% lactic acid, 1.6% chitosan, and rotational rate at 110rpm) that could achieve greatest bacterial reduction of 4.5log10CFU/g. Among the four factors, lactic acid and chitosan were the major contributors for bacterial decontamination. Analysis of variances showed a significant interactive inactivation effect (p<0.05) from combined use of lactic acid and chitosan. The treatments did not have adverse effects on the quality attributes such as color and pH of the shrimps.

Modeling inhibitory activity of a novel antimicrobial peptide AMPNT-6 from Bacillus subtilis against Vibrio parahaemolyticus in shrimp under various environmental conditions

Vibrio parahaemolyticus is recognized as the leading cause of human gastroenteritis associated with the consumption of seafood. NT-6 antimicrobial peptide (AMPNT-6) that is a novel antimicrobial peptide secreted by Bacillus subtilis NT-6 isolated from Natto which is a Chinese traditional fermented food can be an effective method to reduce V. parahaemolyticus. This study aimed at investigating the factors that affected the application of AMPNT-6, such as temperature, salinity, pH value and sodium pyrosulfite concentration by response surface method and modeled the effect of AMPNT-6 on inhibition to V. parahaemolyticus in shrimp under various environmental conditions. Design-Expert software was chosen to perform regression fit and to establish the antibiotic mathematical model. The results indicated that all the above environmental conditions had no significant interactive effects on the antibacterial activity of AMPNT-6. The quadratic polynomial mathematical model was established and obtained the quadratic regression equation of the predictive value Y (bacterial colony number) and variables x1 (temperature), x2 (sodium chloride concentration), x3 (pH), x4 (sodium pyrosulfite concentration) and x5 (AMPNT-6 concentration) in a certain range: Y = 6.60 + 0.34x1 − 0.23x3 − 0.19x5 − 0.29x12 + 0.19x22 + 0.3x42 − 0.35x1x2 (R2 = 0.8963). The experimental values were shown to be in good agreement with predicted values. The established model had a high fitting degree and could predict the inhibition effect of AMPNT-6 toward V. parahaemolyticus growth in shrimp under various conditions.

MICROBIOLOGICAL RISK ASSESSMENT OF FISHERY PRODUCT IN INDONESIA: A PROPOSED MODEL FOR THE RISK OF Vibrio parahaemolyticus IN SHRIMP

Increasing fish consumption value should be supported by enhancing the safety and quality offish products. Microbiological level is most of importance since every food contains microorganismswhich could multiplicate due to temperature abuse and time delay during handling and processing.Risk assessment is a structurally and scientifically based approach aimed to protect consumerfrom risk (hazard) particularly microbiological hazard when consuming certain food. Microbiologicalrisk assessment of fishery products have not been structurally developed in Indonesia, eventhoughseveral initial data on hazard identification have been available. As an attempt to build an integratedrisk assessment, a model for microbiological risk of pathogenic Vibrio parahaemolyticus inshrimp will be proposed in this article. The available data could be used as a starting point whileother required data could be collected in collaboration with other related institutions.

Inhibitory activity of a novel antibacterial peptide AMPNT-6 from Bacillus subtilis against Vibrio parahaemolyticus in shrimp

Vibrio parahaemolyticus is a major foodborne pathogen in marine products. Effective methods for reducing V. parahaemolyticus in seafood would reduce the likelihood of foodborne outbreaks of Vibrio. AMPNT-6 is a novel antibacterial peptide secreted by Bacillus subtilis NT-6 isolated from Natto, a Chinese traditional fermented-soybean paste. The objective of this study is to investigate the antibacterial activity of AMPNT-6 against V. parahaemolyticus. The disc diffusion experiment indicated that AMPNT-6 posed an evident inhibitory activity against V. parahaemolyticus with a diameter of 15.5 mm and the MIC was measured at 1.25 mg/mL. Compared to the control, the significant bactericidal activity was observed through the changes of V. parahaemolyticus growth curve and inactivation kinetics when the final concentration of AMPNT-6 in bacterial suspension was added to 1 and 2 MIC. Furthermore the significant reduction of population size of V. parahaemolyticus in shrimp matrix was also found when the final concentration of AMPNT-6 reached to 2 MIC. These obtained results indicate that AMPNT-6 can potentially be used as a natural inhibitor to control V. parahaemolyticus borne on shrimp matrix.

Comparative evaluation of a chromogenic agar medium – PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples

Screening for pathogenic Vibrio parahaemolyticus has become routine in certain areas associated with food-borne outbreaks. This study is an evaluation of the CHROMagar Vibrio (CV) medium-PCR protocol and the conventional method (TCBS (thiosulfate-citrate-bile salts-sucrose) agar plus biochemical and Wagatsuma agar tests) for detection of V. parahaemolyticus in shrimp, water, sediment, and stool samples collected for biosurveillance in an endemic area of northwestern Mexico. A total of 131 environmental and clinical samples were evaluated. The CV medium-PCR protocol showed a significantly improved ability (P < 0.05) to isolate and detect V. parahaemolyticus, identifying isolates of this bacteria missed by the conventional method. Although some other bacteria, distinct from pathogenic V. parahaemolyticus, produced violet colonies similar to that of V. parahaemolyticus on CV medium, we were able to detect a superior number of samples of V. parahaemolyticus with the CV medium-PCR protocol than with the conventional method. The Kanagawa phenomenon is routinely determined on Wagatsuma agar for the diagnosis of V. parahaemolyticus (pathogenic) positive for thermostable direct hemolysin (TDH) in developing countries. In our results, Wagatsuma agar showed low sensitivity (65.4% at 24 h and 75.6% at 48 h) and specificity (52.4% at 48 h) for identifying V. parahaemolyticus positive for TDH. Overall, our data support the use of the CV medium-PCR protocol in place of the conventional method (TCBS-biochemical tests-Wagatsuma agar) for detection of pathogenic V. parahaemolyticus, both in terms of effectiveness and cost efficiency.

Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6 h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticus cells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R 2 > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR ( P = 0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.

Effect of the addition of four potential probiotic strains on the survival of pacific white shrimp ( Litopenaeus vannamei) following immersion challenge with Vibrio parahaemolyticus

Four bacterial strains isolated from the gastrointestinal tract of adult shrimp Litopenaeus vannamei, Vibrio alginolyticus UTM 102, Bacillus subtilis UTM 126, Roseobacter gallaeciensis SLV03, and Pseudomonas aestumarina SLV22, were evaluated for potential use as probiotics for shrimp. In vitro studies demonstrated antagonism against the shrimp-pathogenic bacterium, Vibrio parahaemolyticus PS-017. Feeding shrimp with diets containing the potential probiotics showed the best feed conversion ratio in comparison with the control groups. After feeding with the potential probiotics for 28 days, challenge by immersion indicated effectiveness at reducing disease caused by V. parahaemolyticus in shrimp.

Comparing the efficiency of chitosan with chlorine for reducing Vibrio parahaemolyticus in shrimp

Thailand is one of the leading exporters of frozen shrimp to many countries. Chlorine is the decontaminating agent most frequently used in the frozen shrimp industries to kill potential pathogens. However, long time contact to chlorine causes severe respiratory tract damage. In this study, chitosan was compared to chlorine for reducing Vibrio parahaemolyticus. In vitro investigation, chitosan could reduce more than 90% of V. parahaemolyticus, whereas chlorine completely eliminated this organism. In artificially inoculated shrimp, more than 90% reduction of V. parahaemolyticus was achieved by chitosan. A similar reduction was obtained by chlorine, however, at lower concentrations and less contact time. In naturally contaminated shrimp, neither agent completely eradicated V. parahaemolyticus, however, chitosan achieved a decrease of more than 60%. These results demonstrate the possibility of using chitosan to decontaminate pathogenic bacteria in the seafood factory, a change that would diminish health problems of the workers.

Effects of dietary vitamin C on the immune function of shrimps, Penaeus chinensis

Prepared in this experiment were six groups of diets, i.e. VC0, VC1, VC2, VC3, VC4 and VC5 with the contents of vitamin C (VCmg(100 g)−1 diet) of 0, 100, 200, 400, 800, and 1200 respectively. It was found that vitamin C increased the concentrations of immunoglobulin-like (IgG-like, IgA-like and IgM-like) substances in the serum of Penaeus chinensis after a feeding period of 3 weeks. The differences among groups were significant (P 0.05). Phenoloxidase (PO) activity in the serum of VC3 group shrimps was higher than that of VC0 and other groups, but no significant difference was observed between VC0 group and other groups. Furthermore, bactericidal activity of the serum to Vibrio parahaemolyticus in shrimps fed with the VC1 diet was higher than that in the other groups (P 0.05). It is, therefore, suggested that vitamin C (100–400 mg(100 g)−1 diets) could be used as an immunostimulant of P. chinensis.

Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp.

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. The gyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 other Vibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-microliter PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.

A study of the incidence of Vibrio parahaemolyticus in Malaysian shrimp undergoing processing for export.

The incidence of Vibrio parahaemolyticus in products of the Malaysian export shrimp processing industry was investigated through the stages from the catch to that of the cooked, peeled and frozen product. The organism was commonly found in freshly caught and landed shrimp, and could be detected by enrichment culture at all stages of processing. The number of V. parahaemolyticus in shrimp varied from nil to 4x10(4), and 19 of the 50 serotypes in the current antigenic scheme were found, O1-K38 and O1-K32 occurring most often. All the isolates were Kanagawa-negative; one strain was a sucrose-positive variant. The study indicated that specifications of 10(2) g-1 for V. parahaemolyticus in raw tropical shellfish are too stringent but that the Malaysian shrimp industry should be able to meet this requirement for cooked shrimp.