Food Protection Trends - September 2009 - 566

FIGURE 1. Average percent recovery of virus. The comparison of average recovery by cationic beads in salsa from both the supernatant () and pellet () in virus elution buffer (pH 9.6) (A) and 0.1N HCl (pH 2.0) (B) for HAV and AiV determined by virus cell culture infectivity by TCID50. To determine if viruses bound to beads are able to infect cell culture, salsa samples were inoculated with virus (HAV, AiV, FCV and RCN) and run on Pathatrix™ as previously described. After washing, beads were recovered in HCl (0.1N) or virus elution buffer (VEB) comprised of 100 mM Tris–HCl, 0.05M glycine, and 3% beef extract at pH 9.6 (Dubois et al., 2002). The eluted beads and solution were rocked for 24 h at room temperature. The beads were then pelleted using magnetic forces, the supernatant (1 ml) was collected, and the bead pellet was resuspended in 1 ml sterile water. The pH of both the supernatant and pellet samples were adjusted to 7.4 with 0.1 N HCl or 0.1 N NaOH. Infectivity was determined by TCID50 and calculated by the Reed Meunch method. Different virus elution solutions for virus recovery The effect of different recovery media of varying pH on viral recovery was determined. Salsa and milk samples were inoculated with HAV and AiV (107 log TCID50/g or ml) and run on Pathatrix™ as previously described. After the beads had been washed with sterile water, 1 ml of either HBSS, distilled deionized water (ddH2O), virus elution buffer (100 mM Tris–HCl, 0.05 M glycine, 3% beef extract, pH 9.6) or HCl (0.1N) was added to the cationic beads. Samples were rocked for 24 h at room temperature, pH was adjusted to 7.4, and infectivity of bead samples was determined by TCID50 and calculated by the Reed Meunch method. Viral recovery (%) was calculated, using the initial TCID50 value of the inoculated virus as 100%. medium containing 2% FBS was added to the plates (7). Plates were incubated at 37°C for specific times according to virus type, and cytopathic effects were observed microscopically and virus titers calculated. HAV was read for cytopathic effect 14 days post-inoculation (dpi), and FCV, AiV, and RCN were read 3–5 dpi. from local grocery stores in Newark, DE. Milk (250 ml) was inoculated with HAV, AiV and FCV virus (107–108 TCID50/ml). Samples were placed in sterile stomacher bags (Fisher Scientific, Pittsburgh, PA) and placed in one of the sample pots of the Pathatrix™ machine according to manufacturer’s directions. Viral loading capacity of the cationic beads The viral loading capacity of the beads was investigated in inoculated milk samples (25 ml of UHT milk plus 225 ml of dH2O) with >107, 106, 105, 104, and 103 TCID50/ml of HAV and AiV. Samples were run on the Pathatrix™ and infectivity was determined as described previously. Food sample preparation Fresh salsa (containing tomatoes, green onions, green chilies, spices) was purchased at a local grocery store in Newark, DE. A 25-g sample of salsa was added to 225 ml of distilled deionized water according to the manufacturer’s instructions for the regular-size sample cups used in the machine. Samples were inoculated with one virus type (107–108 TCID50/g) including HAV, AiV, FCV and RCN. Milk (UHT, 1% low-fat) was purchased Magnetic capture of foodborne viruses Samples were run on Pathatrix™ for 60 min at room temperature (25°C), using 50 µl of positively charged cationic beads (ZCCB-CAT, Matrix MicroScience) according to manufacturer’s instructions. After recirculation, the beads were washed with sterile water and recovered on a magnetic rack. Infectivity was determined by use of a tissue culture infectious dose 50% (TCID50) assay and Reed Meunch calculations (3). Statistical analysis Experiments were performed in triplicate on different days and are recorded as the means and standard deviations of these results. Difference of means t-tests were performed using Microsoft Excel 2007, and P values ≤ 0.05 were considered significant. 566 FOOD PROTECTION TRENDS | SEPTEMBER 2009

Food Protection Trends - September 2009

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