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~ We ship worldwide! We do not make money on freight charges. If you see a freight charge that is out of line for your country, please let us know. We will verify or correct it!The IP address used for your Internet connection is part of a subnet that has been blocked from access to PubMed Central. Addresses across the entire subnet were used to download content in bulk, in violation of the terms of the PMC Copyright Notice. Use of PMC is free, but must comply with the terms of the Copyright Notice on the PMC site. For additional information, or to request that your IP address be unblocked, For requests to be unblocked, you must include all of the information in the box above in your message.The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed.
An alkaline phosphatase (AP)-labelled probe was evaluated for specificity against 26 strains of V. parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non-vibrio species. Of the 124 isolates tested, the probe hybridized only with the 26 strains of V. parahaemolyticus, indicating species specificity. Two hundred and six suspect V. parahaemolyticus isolates from oysters were tested by this probe and API-20E diagnostic strips; there was 97% agreement between results. A digoxigenin (DIG)-labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP-labelled probe. When tested on 584 suspect V. parahaemolyticus isolates, results obtained with the AP- and DIG-labelled probes were in 98% agreement. These results suggest that the probes are equivalent for detection of the V. parahaemolyticus tlh gene.Vibrio parahaemolyticus is a halophilic bacterium often found within shellfish and in estuarine waters. Among a number of pathogenic vibrio species, it is commonly associated with oyster-borne gastroenteritis ( Rippey 1994).
The organism was first described after an outbreak of food poisoning in Osaka, Japan, in 1950 ( Fujino et al. 1951 ).Bacterial strainsEighty-four of the isolates used in this study were from environmental (water, sediment, seafood products) and clinical (patient) sources; 40 isolates were from unknown sources. Several of the strains were provided by E. Elliot and F. Khambaty, FDA, Center for Food Safety and Applied Nutrition (CFSAN, Washington, D.C., USA) and W. Landry, FDA, Dallas District Office (DDO, Dallas, TX, USA). The remainder were from the FDA, Gulf Coast Seafood Laboratory (GCSL, Dauphin Island, AL) and FDA, Seafood Products Research Center (SPRC, Bothell, WA) culture collections. A list of the isolates used in this study is given in Table 1. The isolates included 26 V. parahaemolyticus, 13 V. vulnificus, 26 V. cholerae non-O1; seven V. cholerae O1, 12 V. fluvialis, 13 V. mimicus, nine V. hollisae, two V. alginolyticus, three V. damsela, one V. furnissii (CDC1955–83), one V. metschnikovii (ATCC7708) and one V. anguillarum (ATCC19264).
Other isolates examined were four Aeromonas hydrophila, two Salmonella spp., two Escherichia coli and two Bacillus subtilis (ATCC strains 23857 and 33234). The B. subtilis strains were used because of their similarity to the V. parahaemolyticus tlh gene as determined by a FASTA search ( Pearson & Lipman 1988) in conjunction with the Genetics Computer Group software ver. 8 ( Devereux et al. 1984 ). Cultures were maintained at room temperature on T1N1 agar slants (1% tryptone (Difco), 1% NaCl and 2% agar) overlaid with sterile mineral oil, or were inoculated using sterile toothpicks into 96-well plates (Costar, Cambridge, MA, USA) containing 100 μl well−1 alkaline peptone water (APW, 1% peptone (Difco) and 1% NaCl, pH 8·5). Cultures in 96-well plates were incubated at 35 °C for 18 h, amended with 100 μl well−1 cryoprotectant (24% glycerol (Sigma) in tryptic soy broth (TSB, Difco)) and stored at −80 °C. Bacterial strains used in study Vibrio parahaemolyticus15Env 11ClinVibrio vulnificus13EnvVibrio cholerae non-O110Env 3Clin 13UnkVibrio cholerae O11Env 6ClinVibrio fluvialis4Env 8UnkVibrio mimicus8Env 5UnkVibrio hollisae4Clin 5UnkVibrio alginolyticus2EnvVibrio damsela3EnvVibrio furnissii (CDC1955-83)1UnkVibrio metschnikovii (ATCC7708)1UnkVibrio anguillarum (ATCC19264)1UnkAeromonas hydrophila4EnvSalmonella spp.2UnkEscherichia coli2UnkBacillus subtilis (ATCC23857, ATCC33234) 2UnkCulture preparationBacterial colonies were transferred from agar media using sterile toothpicks to 96-well plates containing 100 μl well−1 APW and incubated at 35 °C for 18 h. Growth in wells was transferred to T1N3 agar plates (1% tryptone
, 3% NaCl and 2% agar) using a flame-sterilized 48-prong inoculating block, with incubation at 35 °C, 18 h. Unless otherwise stated, all media and equipment were sterilized by autoclaving for 15 min at 121 °C.Analyses of oystersPacific oysters (Crassostrea gigas) were collected from representative growing areas and retail markets in Washington and Oregon states during an outbreak investigation from July to September 1997. Twelve live oysters or shucked oyster product were cooled in an ice chest with packaged ice, transported to the FDA District Laboratory in Bothell, WA, and analysed within 24 h of collection. Thirty-five samples, including four live samples from Oregon and four of shucked oyster product from Washington, were analysed by the FDA BAM method ( Elliot et al. 1995 ) using a three tube, multiple dilution MPN procedure with enrichment in APW and isolation on thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates. Two or three sucrose-negative (green) colonies were selected from each plate for screening, using the arginine reaction on arginine-glucose slants (AGS) and growth in tryptone with 3% NaCl.Gulf Coast oysters (Crassostrea virginica) were harvested by commercial means from the Mississippi Sound area of Alabama and held in tanks with flowing sea water (25 ppt, 27–29 °C) at the Gulf Coast Seafood Laboratory.