IAFP 2009 Program Book and Abstract Book - AB72

Purpose: This study examined the effect of feeding habits and environment on the abundance of pathogenic vibrios in the intestinal contents of common finfish species in the Gulf of Mexico. Methods: Ten common finfish species from inshore (Mobile Bay) and offshore (Gulf of Mexico) were collected in fall of 2008. Their intestinal contents were examined by a variety of molecular methods in a most probable number (MPN) format to determine levels of V. parahaemolyticus, V. vulnificus and V. cholerae. The Qualicon Bax Q7 real-time PCR system was used to screen overnight alkaline peptone water enrichments for genes specific to each of the Vibrio spp. These enrichments were further examined for a variety of pathogenicity markers, using real-time PCR and loop mediated amplification assays. Results: Levels of V. parahaemolyticus and V. vulnificus often exceeded 10,000,000 MPN/g and appeared to be influenced by both feeding habits and environment. Highest levels were found in bottom dwelling fish from Mobile Bay that typically feed on invertebrates. Piscivores (mackerel) collected offshore harbored few vibrios (< 3 MPN/g). Salinity appeared to be influential in the distribution of these vibrios, with V. cholerae detection (< 10 MPN/g) being limited to low salinities (< 15ppt). V. vulnificus was prevalent at all inshore sites. V. parahaemolyticus was most widely distributed and more abundant than other Vibrio spp. in fish collected offshore in the Gulf (maximum 9300 MPN/g). Significance: These data indicate that extremely high levels of vibrios can occur in the intestines of finfish, depending on harvest environment and feeding habits. Preventing contamination of edible flesh during evisceration and filleting may greatly reduce risk. P1-30 Evaluation of Immunomagnetic Separation (IMS) Coupled with Real-time PCR for Enumeration of Vibrio DSC parahaemolyticus in Spiked Oyster Homogenates SHREYA DATTA, Janet G. Simonson and Marlene Janes LSU, Food Science, Baton Rouge, LA, USA Introduction: Vibrio parahaemolyticus is a naturally occurring marine bacterium that causes a majority of the seafoodassociated gastroenteritis cases in the United States. There is a need for an approved and standardized method that is sensitive, reliable and capable of accurate identification of V. parahaemolyticus. Purpose: The purpose of this study is to develop an antibody based method coupled with real-time PCR (q-PCR) for enumeration of V. parahaemolyticus in spiked oyster homogenates. Methods: The monoclonal antibody (MnAb) produced against the flagellar cores of V. parahaemolyticus was used in an immuno-magnetic separation (IMS) protocol. Live oysters were shucked, weighed and mixed in a ratio of 1:1(wt/wt) with sterile PBS and stomached for 2 min. Oyster homogenate was filtered and spiked with a serially diluted overnight grown culture of V. parahaemolyticus ATCC 33847. One ml of spiked homogenate dilution was subjected to the IMS protocol. The magnetic beads were collected, using a magnetic concentrator and the supernatant fluid was spread on VVA agar plates. For q-PCR, the magnetic beads were collected from the selected dilution, washed three times with PBS and suspended in 100 µl sterile water. The sample was then heated at 100°C for 15 min, and centrifuged at 16000 times g for 10 min; the supernatant was used as the DNA template in q-PCR. Results: An average binding of 69% was exhibited in the binding study using the IMS protocol. When IMS was coupled with q-PCR, after successive washing steps, the binding capacity decreased to 40% after the first wash, 17 % after the second and 10% after the third. Significance: IMS coupled with q-PCR method could be used to concentrate and detect V. parahaemolyticus from mixed cultures, seawater and shellfish homogenates. P1-31 Validation of a PCR Assay for Screening Vibrio in Foods MORGAN WALLACE, Anita C. Wright, Bridget Andaloro, George Tice and Timothy R. Dambaugh DuPont Qualicon, Wilmington, DE, USA Introduction: The species of Vibrio that cause the majority of human illness are cholera (Vc), parahaemolyticus (Vp), and vulnificus (Vv). Since culture-based screening methods can be difficult and time-consuming, well validated, rapid methods for the detection of these pathogens are needed. Purpose: This study evaluated the inclusivity, exclusivity, and effectiveness of a real-time multiplex PCR assay for detecting Vibrio. Methods: Inclusivity testing (n = 50 strains) was performed at ~105 CFU/ml, while exclusivity testing (n = 50 strains) was performed at ~108 CFU/ml from broth cultures. For spiked foods, Vibrio strains were inoculated to yield fractional positive results for plus/minus screening, or at levels informative of method performance for MPN-based approaches. Samples were tested with the FDA-BAM culture-based method and by PCR using the BAX® system. Ahi tuna was spiked at three levels with Vc and tested for presence or absence of target in sets of twenty 25 g sub-samples with five unspiked sub-samples, with PCR testing from the BAM enrichments. Similarly, five replicates of scallops were tested, using the MPN approach of the BAM spiked with Vv at a level giving fractional results for the fifteen 1 g samples, and each MPN tube was tested by PCR, as were five 25 g samples enriched in a comparable manner. Naturally occurring fractionally positive, lowlevel Vc in raw shrimp was also tested, using twenty 25 g samples with both the BAM method and PCR testing from the same enrichments. Results: All inclusivity/exclusivity testing demonstrated expected results. For effectiveness testing, no statistically significant differences were found when comparing test and reference methods. In fact, comparing PCR and culture, results for the spiked ahi tuna (36 positive of 65 samples tested) and shrimp (5 positive of 20 samples tested) were identical, with no false negative or false positive results by PCR. Scallop data gave identical MPN results for test and reference methods, and 25g enrichments were all positive by PCR. Significance: This data indicates that this PCR method for the detection of Vibrio is as effective as culture-based methods while providing significant time and labor savings. 72 — Abstract Book Poster

IAFP 2009 Program Book and Abstract Book

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