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PubMed1977

Occurrence of human-associated yeasts in bivalve shellfish from Long Island Sound.

Candida parapsilosis, C. tropicalis, and Torulopsis glabrata were the human-associated yeasts most frequently isolated from quahogs, oysters, and mussels collected from four estuarine areas along the northern shore of Long Island Sound. Some inconsistency and seasonal variation in the occurrence of these and other yeast species were noted. In particular, C. albicans densities were greatest during colder months in the more heavily polluted waters. A total of 347 yeasts were isolated and cultured at 37 degrees C and, of these, 219 of 62% were human-associated forms. Generally, these yeasts in the animals sampled reflected the overall pollution status of the estuary from which they were taken. This study represents a clear demonstration of potentially pathogenic yeasts in a valuable marine resource.

Animals↗
PubMed1999

Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA.

To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.

Animals↗
PubMed2006

Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes.

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10(4) CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3x10(9) CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

Animals↗