# R/SHH-7

Status: Ended

Started: 2/1/1998

Ended: 1/31/2000

1. To determine the incidence of L. monocytogenes lineage III strains and other possibly avirulent strains in salmon and in salmon processing plants and to complete the characterization of potentially avirulent stains to achieve their reclassification as a new Listeria species, subspecies or subset non-pathogenic for humans.
2. To format a polymerase chain reaction (PCR)-based assay for the specific detection of pathogenic L. monocytogenes which can be used for verification of the efficacy of HACCP programs in the smoked fish industry. This assay will be formatted for screening both raw materials and environmental samples.
3. To apply a fingerprinting system based on automated ribotyping to track the origins and spread of pathogenic L. monocytogenes throughout the processing chain in the smoked fish industry.
4. To utilize the information generated under objectives 1-3 to develop specific recommendations for control of pathogenic L. monocytogenes as part of a comprehensive HACCP program for the smoked fish industry.

1. Characterize 100 L. monocytogenes isolates from salmon and environmental samples genetically, phenotypically and for cytopathogenic potential.
2. Develop and optimize primers, detection reagents and PCR assays for the specific and sensitive detection of L. monocytogenes from salmon and environmental samples. Optimize minimal enrichment procedures and DNA preparation methods for use with the new PCR assay(s).
3. Isolate and ribotype L. monocytogenes from raw material, environmental samples, equipment and finished products in three salmon smoking plants. Determine contamination sources and profiles.
4. Prepare protocols for PCR assays and molecular strain typing strategies for use in verifying the efficacy of critical control points (CCPs) identified in objectives 1-3. Set up a WWW page containing this HACCP information to assure accessibility to any interested parties.

Compliance with zero-tolerance for L. monocytogenes in ready to eat seafoods represents a significant challenge for the smoked fish industry. Our proposed project will determine the relative frequency and characteristics of L. monocytogenes with attenuated virulence or avirulence in cold-smoked salmon products. These data will provide the basis for proposing a new non-pathogenic or less pathogenic Listeria subset to the FDA. Regulatory recognition of avirulent Listeria spp., coupled with application of our proposed new technology for their rapid detection, may reduce the incidence of costly product recalls. We also propose development, validation and application of a molecular detection and tracking system for L. monocytogenes, followed by incorporation of this strategy into HACCP plans. This project will be conducted in close collaboration with the New York Sea Grant extension program and with New York State salmon processors.

Listeria monocytogenes is a foodborne pathogen that causes a rare, but severe foodborne disease in humans. While L. monocytogenes is common in many environments and found in many ready-to-eat foods (including deli meats, hot dogs, but also smoked fish), current regulations specify a zero-tolerance level for this organism in ready-to-eat foods. Control of L. monocytogenes is a specific challenge for the cold smoked fish industry as there is no kill step for L. monocytogenes in this process. This research team applied molecular approaches, including PCR based detection strategies and DNA fingerprinting methods, to study the ecology of L. monocytogenes in the cold-smoked fish industry. A total of 531 samples, including raw materials, finished goods, and environmental samples were collected from three facilities over five collection periods. Through ribotyping, we discovered each facility has its own contamination "fingerprint" of Listeria contamination persisting in the environment. The data suggests that while both raw materials and the environment contribute to the contamination of finished products, L. monocytogenes which persist in the smoked fish processing environment are the most common source of finished product contamination. By comparing the frequency of occurrence of different molecular subtypes between fish industry isolates (environmental and finished product isolates) and human clinical isolates, we have found a statistically significant difference in the populations of L. monocytogenes that cause human disease and those we found in the fish industry. Tissue culture studies further confirmed that at least some of the L. monocytogenes subtypes found in smoked fish and the processing environment are virulence attenuated. These data suggest that science-based control measures and action limits should consider not only the presence of L. monocytogenes in food and the environment but should ultimately focus on the specific subtypes associated with human disease. Furthermore, these results suggest that L. monocytogenes in the cold smoked fish industry does not lend itself to a HACCP type control strategy as there is no specific critical control point or kill step. It is recommended that HACCP prerequisite programs and specifically sanitation standard operating procedures (SSOPs) may provide a suitable approach to Listeria control in this industry. Specifically, strict and thorough sanitation procedure to reduce Listeria levels in the environment and barriers to minimize its movement to post-processing areas and control of contamination in raw materials (fish) should significantly reduce contamination of finished products.