Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test. Vibrio parahaemolyticus is an enteric pathogen transmitted to humans primarily through consumption of raw or mishandled seafood (2, 23). Like other members of the genus Vibrio, it is a gram-negative, halophilic bacterium that occurs naturally in estuarine environments (15). Its distribution is worldwide, but reported densities of V. parahaemolyticus in the environment and in seafood vary greatly according to season, location, sample type, fecal pollution, and analytical methodology (3, 5, 8, 16, 27). The public health significance of V. parahaemolyticus contamination in seafoods is debatable, since few environmental or seafood isolates produce the thermostable-direct hemolysin (TDH) or Kanagawa hemolysin associated with most (96.5%) pathogenic strains found among clinical isolates (12, 20, 21). However, a TDH-related hemolysin has been isolated from Kanagawa phenomenon-negative V. parahaemolyticus strains associated with gastroenteritis in Southeast Asia (13). In an evaluation (5) of four methods for the enumeration of V. parahaemolyticus in seawater and oysters, we found that the hydrophobic grid membrane filter method described by Entis and Boleszczuk (9) gave the highest density estimates. This method had been used to evaluate the effects of thermal stress on artificially contaminated seafoods but had not been used in environmental surveys. Past V. parahaemolyticus surveys were conducted regionally but did not compare distribution of the organism among the Pacific, Gulf, and Atlantic coasts. The present study reports the incidence of V. parahaemolyticus in U.S. oysters and seawater as determined by the hydrophobic grid membrane filter method. The National Shellfish Sanitation Program classification of shellfish-growing areas is based on fecal coliform densities in the overlying waters (14); however, a statistical evaluation is needed to correlate V. parahaemolyticus densities in shellfish with either fecal coliform or V. parahaemolyticus counts for the overlying waters. In shellfish, V. parahaemolyticus

The polymerase chain reaction was used to selectively amplify sequences within the cholera toxin operon from Vibrio cholerae 01. Oysters, crabmeat, shrimp, and lettuce were seeded with V. cholerae and then homogenized or washed with alkaline peptone water, followed by short-term (6to 8-h) enrichment. A detection limit of as few as 1 V. cholerae CFU per 10 g of food was obtained with amplification reactions from crude bacterial lysates. The method is extremely rapid and obviates the need for DNA isolation from a variety of complex food matrices.

A 500-base-pair DNA fragment of a presumptive jI-hemolysin gene of Listeria monocytogenes has been used to identify this organism by a modified colony hybridization technique. We have cloned this DNA fragment into M13 bacteriophage vectors and sequenced it by a dideoxynucleotide sequencing technique. From this sequencing information, several oligodeoxyribonucleotides were synthesized and used as synthetic probes to identify L. monocytogenes. The probes were specific for L. monocytogenes and did not react with any other Listeria strains in a colony hybridization assay. In particular, one of these probes (AD07) was used to detect L. monocytogenes in artificially contaminated raw-milk and soft-cheese samples.

A fragment of about 500 base pairs of the I-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe. Several recent outbreaks of listeriosis (3, 4) have underscored the lack of rapid methods to isolate and identify Listeria monocytogenes from suspected food sources (8; Joseph Lovett, personal communication). Current enrichment schemes may require up to 28 days to recover and characterize cultures of L. monocytogenes. Therefore, development of a quick, reliable method would be of great importance. Colony hybridization with radiolabeled DNA probes has been used to rapidly detect and enumerate specific microorganisms in food samples without the need for lengthy enrichment schemes (10). A similar approach should also be useful in identifying L. monocytogenes. L. monocytogenes frequently elaborates an extracellular

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