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The aims of this project are to (i) develop rapid genetic and phenotypic assays to detect L. monocytogenes with a reduced invasion phenotype and define the distribution of these strains among isolates from human clinical cases, foods, and other sources, (ii) quantify the relative virulence attenuation of L. monocytogenes with premature inlA stop codons, and (iii) develop revised dose response models and risk assessments incorporating knowledge on strain-specific virulence characteristics. <P>
The specific project objectives are: <OL> <LI> Develop and implement a rapid cell culture assay to screen L. monocytogenes isolates for reduced invasion efficiency for human intestinal epithelial cells. Objective <LI> Develop and implement a TaqMan genotyping assay to determine the prevalence of L. monocytogenes with premature inlA stop codons among isolates from different sources along the food chain. <LI> Utilize tissue culture and guinea pig models to quantify the virulence and pathogenic potential of L. monocytogenes with and without premature inlA stop codons. <LI> Develop dose response models for L. monocytogenes subtypes with and without premature inlA stop codons, and develop a revised quantitative risk assessment to describe the relative human health risk attributable to these subtypes.
NON-TECHNICAL SUMMARY: Members of the bacterial species Listeria monocytogenes cause an estimated 500 foodborne deaths annually. This project will characterize Listeria monocytogenes isolated from humans, foods, animal, and various environments to determine whether and how different subtypes within this bacterial species differ in their ability to cause human disease. This information will help us to better identify the specific bacterial subtypes that cause human disease allowing mare targeted control and intervention strategies.
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APPROACH: <BR> 1. Develop a rapid high-throughput cell culture assay to screen a large number of Listeria monocytogenes isolates using human intestinal cell line Caco-2. Screen approximately 300 Listeria monocytogenes isolates representing 90 unique ribotypes using the Caco-2 cell invasion assay. <BR> 2. Perform DNA sequencing of inlA to identify mutations associated with reduced invasion of Caco-2 cells. Design TaqMan SNP typing primer and probe sets using the Primer Express software to detect mutations associated with reduced invasion of Caco-2 cells and validate primers and probes using Listeria monocytogenes isolates with known inlA sequences. Screen approximately 300 Listeria monocytogenes isolates with subtypes associated with inlA mutations associated with reduced invasion of Caco-2 cells. <BR> 3. Construct isogenic mutant pairs representing the three most common inlA premature stop codon mutations to directly assess the effect of each mutation on virulence. Characterize mutants using tissue culture and guinea pig models of infection. <BR>4. Develop dose response models for Listeria monocytogenes subtypes with and without premature inlA stop codons. Develop a revised quantitative risk assessment to describe the relative human health risk attributable to these subtypes considering both virulence differences and differences in growth potential. This risk assessment will be based on previously collected Listeria monocytogenes food contamination data for more than 30,000 food samples.