IAFP 2009 Program Book and Abstract Book - AB93

Methods: One ml of 2 × 106 PFU/ml Murine norovirus-1 (MNV) was added to 2 g sterile solid dairy manure (DM), dried for 15 min and stored in a heat-sealed membrane bag (0.22 µm pore size). Composting was set up in a pile 0.3 m high and 1.2 m in diameter in an open field, using raw solid DM as material. The virus sample was placed in the center layer of the compost. Samples were collected at particular time points, at which times the temperature and water activity of the center layer of the compost were also measured. The sample was diluted with phosphate buffer and MNV infectivity was analyzed using the plaque assay. Results: Compost temperature reached 61.7°C at day 1 and 65.2°C at day 2, after which it decreased to 47.8°C at day 5 and remained at ~45°C until day 15. The temperature then decreased to ~10°C at day 25 and was maintained at ~5°C at day 30. MNV was stable during the composting process. There were ~6.1 and 5.9 log PFU/ml infectious MNV at day 1 and day 2, respectively, and after 30 days, 5.3 log MNV were still detected. In contrast, Aichi virus had > 4 log loss of infectivity at day 1 and no viral RNA was detected at day 2. Significance: The stability of MNV during the composting process results in a potential danger of eating fresh produce grown on land to which manure has been applied. P1-85 Comparison of Methods for Recovery of Hepatitis A Virus (HAV) from Fresh Produce and Detection of HAV by DSC Real-time RT-PCR and Cell Culture JI-YEON HYEON, Jae-Hoon Lee, Jeong-Hwan Cheon, Joong-Bok Lee, In-Gyun Hwang, Hyo-Sun Kwak, Yong-Choon Park, Jeong-Su Lee and Kun-Ho Seo Konkuk University, College of Veterinary Medicine, Dept. of Public Health, Seoul, Korea, South Introduction: Hepatitis A virus (HAV) outbreaks associated with consumption of fresh fruits and raw vegetables have been reported in recent years in Korea. However, limited methods have been developed for detection of the virus in foods, and a method that isolates and detects HAV from fresh produces has been not standardized. Purpose: The purpose of this study was to compare methods for elution and concentration of HAV on the surface of leafy vegetables and to develop a rapid, sensitive, and reliable detection method using real-time RT-PCR and cell culture. Methods: Leafy vegetable samples (lettuce, Chinese cabbage) were artificially inoculated with HAV and processed by the sequential steps of homogenization, elution, concentration, RNA extraction, and real-time RT-PCR or cell culture, using FRhk-4 cell. Viruses were eluted from the food surface by two different elution buffers, buffer A [100 mM Tris-HCl, 50mM glycine, 3% beef extract pH 9.5] and buffer B [250 mM Threonine, 300 mM NaCl, pH 9.5]. In addition, an ultrafiltration method was evaluated by comparing it with a widely used viral concentration method, PEG precipitation. The FRhk-4 cells were infected with viruses recovered from the samples to determine infectivity of the viruses, and integrated cell culture real-time RT-PCR was performed at 72, 120,168, 216, 264, and 288 h post infection. Results: The sequential step of elution using buffer A and ultrafiltration were evaluated as a primary virus concentration step for vegetable samples, and detection limit of this method was found to be 1.9 × 102 RNA copies/g. Cytopathic effect and rapid decrease of Ct value were observed at 14 days post infection in the FRhk-4 cell infected with the viruses extracted and concentrated by buffer A and ultrafiltration. Significance: The optimized detection method evaluated in this study was successful in detecting HAV contamination from a wide range of fresh produce, by molecular and cell culture methods. This method could be used for studies and routine surveillance of viral quality of vegetables. P1-86 Prevalence of Vibrio cholerae, V. parahaemolyticus and V. vulnificus in Retail Frozen Shrimp Determined with a Real-time PCR Assay TIMOTHY R. DAMBAUGH, M. Morgan Wallace, Thilan Wijesekera, Daniel F. Delduco, Bridget W. Andaloro and George Tice DuPont Qualicon, Wilmington, DE, USA Introduction: Vibrio species are a frequent cause of bacterial seafoodborne disease. The prevalence of Vibrio in coastal waters and in aquaculture operations, combined with globalization of food production, may place consumers at risk of seafoodborne diseases. Purpose: The study was initiated to assess the prevalence and levels of Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) in retail frozen raw and cooked shrimp, using a multiplex real-time PCR assay specific for these Vibrio species. Methods: Twenty-five grams of thawed whole shrimp were homogenized 1:10 in alkaline peptone water. After 18 h incubation at 35°C, enrichments were streaked on TCBS, mCPC and Vibrio CHROMagar™ plates, and samples were processed for the BAX® system Vibrio assay. Presumptive colonies were propagated for cultural confirmation. Initial contamination levels were determined by the FDA-BAM MPN method for Vp and Vv. Results: PCR screening of imported frozen raw and cooked shrimp demonstrated 64% (n = 14) of retail batches contaminated with culturable Vp. Of these, 50% of the batches of raw shrimp were also contaminated with viable Vc or Vv. Cycle threshold (Ct) values indicated Vp levels were 2 logs higher than Vc or Vv after enrichment. MPN analysis revealed an initial 9300 MPN/gm for Vp and 6.5 MPN/gm for Vc for one retail batch of raw shrimp. For Vp positive raw shrimp batches, 100% of individual shrimp tested at 1:10 enrichment were positive for Vp. In contrast, sporadic Vc contamination (6 confirmed positive out of seventy 25 g samples tested) was observed in one retail batch. Morphologic and molecular characterization by ribotyping revealed the presence of multiple strains of Vibrio in dually contaminated samples. Significance: The prevalence of Vp and other Vibrio species in retail shrimp revealed in this study reinforces the need for both safe seafood preparation practices and increased microbial quality standards. Abstract Book — 93 Poster

IAFP 2009 Program Book and Abstract Book

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