Evaluation of an alkaline phosphatase-labeled DNA probe for enumeration of Vibrio vulnificus in Gulf Coast oysters

Kaysner

Bowers

et al. 2000

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266

186

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.

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confidence: 99%

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

Kaysner

Bowers

et al. 2000

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266

186

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“…Under warm conditions, either direct plating method offers an alternative that is more rapid, economical, and less labor-intensive than the BAM-MPN procedure. Similar direct plating methods used for V. vulnificus have shown that direct plating methods are more precise than MPN analyses (9).…”

Section: Resultsmentioning

confidence: 99%

“…After 24 h, the remaining oysters were transferred to a refrigerator (3°C) and then analyzed 14 to 17 days later to simulate possible retail handling practices. The oysters were scrubbed, shucked, and mixed with an equal weight (1:1) of sterile phosphate-buffered saline (PBS) (7.65 g of NaCl per liter, 0.724 g of anhydrous Na 2 HPO 4 [Sigma] per liter, 0.21 g of KH 2 PO 4 [Sigma] per liter; pH 7.4) (11), and the mixture was blended for 90 s with a sterile Waring blender in preparation for analysis (9).…”

Section: Methodsmentioning

confidence: 99%

“…This oligonucleotide probe conjugated with alkaline phosphatase (tlh-AP) was purchased from DNA Technology A/S (Aarhus, Denmark). Briefly, oyster homogenate was serially diluted, inoculated into a series of MPN tubes containing alkaline peptone water (11) (9). Colony lifts were prepared and tested for hybridization with the tlh-AP probe as described by McCarthy et al (17) for confirmation of species identity.…”

Section: Methodsmentioning

confidence: 99%

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Evaluation of Two Direct Plating Methods Using Nonradioactive Probes for Enumeration of Vibrio parahaemolyticus in Oysters

Gooch

Kaysner

et al. 2001

Oysters (Crassostrea virginica) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine Vibrio parahaemolyticus densities at zero time and after 5, 10, and 24 h of postharvest storage at 26°C. After 24 h of storage at 26°C, oysters were transferred to a refrigerator at 3°C and then analyzed 14 to 17 days later. The V. parahaemolyticus numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (tlh), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and DirectVPDig techniques were highly correlated (r ‫؍‬ 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (r ‫؍‬ 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total V. parahaemolyticus, and they were more rapid and less labor-intensive.Vibrio parahaemolyticus is an enteric pathogen found in estuaries and various types of seafood throughout the world (1, 2, 13-15). V. parahaemolyticus infections can cause gastroenteritis in humans and are most frequently associated with consumption of raw or undercooked seafood and seafood recontaminated with the bacterium after cooking (19). Consumption of raw shellfish, primarily oysters, has been linked to four multistate V. parahaemolyticus illness outbreaks involving 650 reported cases in the United States since 1997 (Washington in 1997 and New York and Texas in 1998) (4-6). All patient isolates obtained from the 296 reported V. parahaemolyticus infections in Texas were serotype O3:K6, which commonly causes outbreaks in Asia but had not been identified previously in the United States (6). These outbreaks increased concern about V. parahaemolyticus densities in oysters and focused attention on the development of more efficient methods for environmental monitoring of this pathogen.The Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) most-probable-number (MPN) method (11) is most frequently used to enumerate V. parahaemolyticus in foods. The BAM-MPN method uses biochemical techniques to identify isolates and is time-consuming and labor-intensive. As an alternative, researchers recently described the use of nonradioactive DNA probes for identification of V. parahaemolyticus (17).In the present study we compared two direct plating methods using nonradioactive DNA probes (Direct-VPAP and Direct-VPDig) with a modification of the BAM-MPN method in which confirmation of the identities of V. parahaemolyticus isolates was accomplished with a DNA probe (MPN-VPAP) targeting the species-specific thermolabile hemolysin gene (tlh) (21). In the Direct-...

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“…541 paper as described by Kaysner and DePaola (25). Briefly, bacterial colonies were lysed, and the DNA was fixed to the filters by treatment with alkaline lysis buffer, ammonium acetate neutralization, and proteinase K. Alkaline phosphatase-labeled DNA probes (DNA Technology, Aarhus, Denmark) targeting the species-specific V. vulnificus hemolysin gene (vvhA) were used to confirm the identity of suspected isolates as V. vulnificus (14,25,39). The filters were hybridized with the probe at 55°C for 1 h, followed by washing and colorimetric development according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN).…”

Section: Methodsmentioning

confidence: 99%

Development and Validation of a Predictive Model for the Growth of Vibrio vulnificus in Postharvest Shellstock Oysters

DaSilva

Parveen

et al. 2012

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