IAFP 2009 Program Book and Abstract Book - AB81

Purpose: The objectives of this study were to determine the effects of growth and recovery temperatures on apparent pressure-resistance of L. monocytogenes in milk. Methods: (1) L. monocytogenes was grown at various temperatures (10, 15, 20, 25, 30, 35, 40, 43°C) to early stationary phase, suspended in UHT-processed whole milk, pressure treated (400 MPa, 2 min, 21°C) and recovered on TSAYE at 35°C. (2) L. monocytogenes was grown at two temperatures (35 and 43°C), pressure treated in milk (400 and 500 MPa, respectively), and recovered on TSAYE at various temperatures (4, 10, 15, 20, 25, 30, 35, and 40°C). (3) L. monocytogenes was grown at 35°C, pressure treated in milk (400 and 450 MPa, 2 min, 21°C), and recovered on TSAYE at 10°C for various intervals (1, 2, 3, 6, 9, and 12 days) then at 35°C for 5 days. All studies were conducted in triplicate. Results: (1) There was no significant difference (P > 0.05) in pressure resistance of L. monocytogenes grown at 10 to 25°C with approximately 6.5 log CFU/ml population reductions. Pressure resistance increased directly with higher growth temperatures; less than 1 log CFU/ml reduction was observed at 43°C. (2) Regardless of growth temperature and pressure treatment, the greatest recovery of L. monocytogenes was within 4 to 20°C by as much as 2 log CFU/ml greater than at other recovery temperatures; recovery at 10°C required 24 days. (3) The time for full recovery could be reduced by incubating at 10°C for 2 days then 35°C for 5 days. Significance: Growth and recovery temperatures affect the apparent pressure resistance of L. monocytogenes and should be factored into the determination of adequate inactivation treatments. P1-54 Impact of Affinity Purification on the Performance of Antibodies Specific for Listeria spp. and Their Use in a Multiplex Luminex Bead Array for Food Testing Katelin Mao, Michael Federman, Christopher Baun, George Anderson and JOSHUA LEVIN KPL, Inc., Gaithersburg, MD, USA Introduction: Listeria monocytogenes is a major foodborne pathogen that causes the human disease listeriosis, which is associated with consumption of contaminated food products. Listeriosis results in a high mortality rate among immunosuppressed populations and a high rate of miscarriage in pregnant women. Immunoassay systems are efficient methods for early detection of Listeria spp. that fulfill most regulatory needs. However, rapid detection of Listeria using immunoassays is highly dependent on antibodies that have a high degree of sensitivity and specificity for the organism. Purpose: The goal of this work was to investigate the importance of affinity purification in the sensitivity and specificity of anti-Listeria antibodies, using multiple immunoassay detection systems, particularly in a multiplexed assay system such as Luminex. Methods: We purified goat anti-Listeria antisera both by standard Protein G chromatography and by affinity chromatography. The purified antibodies were tested in both ELISA and Western blot. Protein G-purified and affinitypurified antibodies were also both tested in a Luminex bead-array that was developed for the detection of five target organisms simultaneously. Results: The affinity-purified antibody had a higher level of sensitivity and specificity than the Protein G purified antibody in both ELISA and Western blot. The two antibodies had similar performance in avidity assays. In the Luminex bead-array assay, both antibodies effectively detect Listeria among other organisms, with good specificity in a variety of food samples. Under these assay conditions, the affinity-purified anti-Listeria antibody outperformed the Protein G-purified antibody, but only when used in both capture and detection. Significance: The results demonstrate the importance of using an affinity-purified antibody for Listeria detection in immunological tests. Furthermore, we have demonstrated the application of using this antibody in a multiplexed Luminex bead-array assay for the simultaneous detection of multiple food pathogens. P1-55 Virulence for Mice, Resistance to Synthetic Gastric Fluid and Biofilm Formation of a Strain of Listeria monocytogenes Serotype 4b Isolated from a Listeriosis Outbreak Associated with Hot Dogs Nancy G. Faith, Jae-Won Kim, Sophia Kathariou, Robert Sahagian, John Luchansky and CHARLES J. CZUPRYNSKI University of Wisconsin-Madison, Food Research Institute, Madison, WI, USA Introduction: One of the most severe listeriosis outbreaks occurred in 1998 to 1999 as a result of contamination of hot dogs with serotype 4b Listeria monocytogenes. There has been little characterization of virulence attributes of strains isolated from this outbreak. Purpose: The purpose of this study was to compare several characteristics of strain H7550 isolated from vacuumpackaged frankfurters in the 1998 to 1999 outbreak of listeriosis, a plasmid-free derivative (H7550cds) of that strain lacking the cadmium resistance plasmid pLM80, and selected transposon mutant derivatives. Methods: Virulence was assessed following intragastric (i.g.) inoculation of anesthetized A/J mice with approximately 106 CFU of the individual strains. We also compared the strains’ resistance to inactivation in synthetic gastric fluid (pH 4.5), and ability to form biofilms on a plastic surface in vitro. A transposon mutant of strain H7550 that lacks adenylosuccinate lyase activity (strain J22F), and a non-hemolytic (LLO-) transposon mutant of strain H7550cds (J29H), were also included in our experiments. Results: Comparable numbers of CFU were recovered from the spleen, liver, blood gallbladder, and ceca of mice inoculated with either strain H7550 or H7550cds. Mutants J22H and J29H were avirulent in our mouse model; we did not recover viable cells from any of the internal organs. We observed no significant difference in the resistance of stationary phase cells of the plasmid-harboring versus plasmid-free strains to synthetic gastric fluid at pH 4.5. Mid log phase cells of mutant strain J22H appeared to be more resistant to inactivation at pH 4.5 than mid log phase cells of the other strains examined. Strains H7550 and H7550cds both formed biofilms on plastic surfaces within 24 h in vitro. Strain J22F was better able to form biofilms in vitro than its parent strain H7550, whereas the LLO-negative mutant strain J29H was not significantly different from its parent strain H7550cds in biofilm formation. Abstract Book — 81 Poster

IAFP 2009 Program Book and Abstract Book

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