We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n ‫؍‬ 206), 8 (7.8%) raw material samples (n ‫؍‬ 102), 23 (18.1%) samples from fish in various stages of processing(n ‫؍‬ 127), and 7 (7.3%) finished product samples (n ‫؍‬ 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P < 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important foodborne pathogen in the food industry.The advent of molecular methodology has revolutionized our ability to investigate and understand microbial ecology, offering new and unique opportunities to explore the ecology of food-borne pathogens, including Listeria monocytogenes, throughout the food chain and in the food processing environment. Highly discriminatory molecular typing methods, including multilocus enzyme electrophoresis, pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA, ribotyping, and phage typing, have been successfully applied to investigations of contamination patterns in foods and in the food processing environment and are increasingly used for surveillance of human disease cases and for tracking of outbreak sources (2-4, 7, 12, 26, 34, 36, 37, 39). While each method provides discriminatory differentiation of L. monocytogenes subtypes, highly automated and standardized methods provide a simplified approach to molecular subtyping and data analysis. The RiboPrinter Microbial Characterization System (Qualicon, Inc., Wilmington, Del.) is one example of such an approach. This system is based on ribotyping, a subtyping method based upon scoring restriction poly...

The effects of baking, broiling, deep frying and cooking in a microwave oven on the proximate, mineral and fatty acid composition of grouper (Epinephelus morio), red snapper (LuGanus campechanus), Florida pompano (Trachinotus carolinus) and Spanish mackerel (Scomberomorus macuhtus) were determined. The lipid content of low fat species was not significantly changed by cooking, but lipid was lost from fatty fillets during cooking. The fatty acid composition of all fillets was not significantly changed by baking, broiling or microwave cooking. Deep fried fiets absorbed the major fatty acids in the cooking medium, and as the filet lipid content increased the extent of absorption of fatty acids from the cooking medium decreased. Sodium, potassium and magnesium levels were decreased when low fat species were cooked, but these minerals were not lost when raw fillets containing higher lipid levels were cooked. Cooking did not significantly affect the concentration of the microelements, zinc, copper, iron and manganese.

Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments. Each of the four plants was sampled monthly for approximately 1 year. At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots. In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources. A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested. Presumptive Listeria spp. were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples. L. monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%). Among the environmental samples, L. monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces. Listeria spp. were isolated from environmental samples in each of the four plants, whereas L. monocytogenes was not found in any of the environmental samples from one plant. Overall, the L. monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples. Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L. monocytogenes isolates. For each of the three plants with L. monocytogenes-positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period. In one plant, a specific L. monocytogenes ribotype represented 44% of the L. monocytogenes-positive environmental samples and was also responsible for one of the two finished product positives found in this plant. In another plant, a specific L. monocytogenes ribotype persisted in the raw fish handling area. However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas. Molecular subtyping methods can help identify plant-specific L. monocytogenes contamination routes and thus provide the knowledge needed to implement improved L. monocytogenes control strategies.

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Four ready-to-eat smoked fish plants were monitored for 2 years to study Listeria contamination patterns and the impact of plant-specific Listeria control strategies, including employee training and targeted sanitation procedures, on Listeria contamination patterns. Samples from the processing plant environment and from raw and finished product were collected monthly and tested for Listeria spp. and Listeria monocytogenes. Before implementation of intervention strategies, 19.2% of raw product samples (n = 276), 8.7% of finished product samples (n = 275), and 26.1% of environmental samples (n = 617) tested positive for Listeria spp. During and after implementation of Listeria control strategies, 19.0% of raw product samples (n = 242), 7.0% of finished product samples (n = 244), and 19.5% of environmental samples (n = 527) were positive for Listeria spp. In one of the four fish plants (plant 4), no environmental samples were positive for L. monocytogenes, and this plant was thus excluded from statistical analyses. Based on data pooled from plants 1, 2, and 3, environmental Listeria spp. prevalence was significantly lower (P < 0.05) for nonfood contact surfaces and the finished product area and for the overall core environmental samples after implementation of control strategies. Listeria prevalence for floor drains was similar before and after implementation of controls (49.6 and 54.2%, respectively). Regression analysis revealed a significant positive relationship (P < 0.05) between L. monocytogenes prevalence in the environment and in finished products before implementation of control strategies; however, this relationship was absolved by implementation of Listeria control strategies. Molecular subtyping (EcoRI ribotyping) revealed that specific L. monocytogenes ribotypes persisted in three processing plants over time. These persistent ribotypes were responsible for all six finished product contamination events detected in plant 1. Ribotype data also indicated that incoming raw material is only rarely a direct source of finished product contamination. While these data indicate that plant-specific Listeria control strategies can reduce cross-contamination and prevalence of Listeria spp. and L. monocytogenes in the plant environment, elimination of persistent L. monocytogenes strains remains a considerable challenge.

Reliable data on the sources of Listeria monocytogenes contamination in cold-smoked fish processing are crucial in designing effective intervention strategies. Environmental samples (n = 512) and raw fish samples (n = 315) from two smoked fish processing facilities were screened for L. monocytogenes, and all isolates were subtyped by automated ribotyping to examine the relationship between L. monocytogenes contamination from raw materials and that from environmental sites. Samples were collected over two 8-week periods in early spring and summer. The five types of raw fish tested included lake whitefish, sablefish, farm-raised Norwegian salmon, farm-raised Chilean salmon, and feral (wild-caught) salmon from the U.S. West Coast. One hundred fifteen environmental samples and 46 raw fish samples tested positive for L. monocytogenes. Prevalence values for environmental samples varied significantly (P < 0.0001) between the two plants; plant A had a prevalence value of 43.8% (112 of 256 samples), and plant B had a value of 1.2% (3 of 256 samples). For plant A, 62.5% of drain samples tested positive for L. monocytogenes, compared with 32.3% of samples collected from other environmental sites and 3.1% of samples collected from food contact surfaces. Ribotyping identified 11 subtypes present in the plant environments. Multiple subtypes, including four subtypes not found on any raw fish, were found to persist in plant A throughout the study. Contamination prevalence values for raw fish varied from 3.6% (sablefish) to 29.5% (U.S. West Coast salmon), with an average overall prevalence of 14.6%. Sixteen separate L. monocytogenes subtypes were present on raw fish, including nine that were not found in the plant environment. Our results indicate a disparity between the subtypes found on raw fish and those found in the processing environment. We thus conclude that environmental contamination is largely separate from that of incoming raw materials and includes strains persisting, possibly for years, within the plant. Operational and sanitation procedures appear to have a significant impact on environmental contamination, with both plants having similar prevalence values for raw materials but disparate contamination prevalence values for the environmental sites. We also conclude that regular L. monocyrogenes testing of drains, combined with molecular subtyping of the isolates obtained, allows for efficient monitoring of persistent L. monocytogenes contamination in a processing plant.

Cryopreserved aortic valve allografts elicit a substantial allogeneic response in recipients. This alloreactivity may contribute to the observed morphologic changes in aortic valve allografts and eventual long-term deterioration of allograft function.

Aortic valve replacement with or without concomitant procedures was performed using an allograft aortic valve in 534 patients. From December 1969 to May 1975 (group I), a 4°C stored valve was used (124 patients) and from June 1975 to July 1990 (group II), a cryopreserved valve (410 patients) was used. The 30‐day mortality was 8.9% (confidence limits [CL] 6.2%–12.3%) for group I and 2.7% (CL 1.9%‐3.8%) for group II. Actuarial patient survival including early hospital mortality at 14 years was 57% for group I and 71% for group II (p = 0.014). Actuarial freedom from thromboembolism for all patients (n = 534) was 94% at 14 years, and for patients who underwent isolated aortic valve replacement with or without coronary artery bypass graft (n = 457) was 97% at 14 years (p = 0.017). Actuarial freedom from allograft valve endocarditls at 14 years was 92% in group I and 94% in group II (p = 0.36). The actuarial freedom from moderate or severe allograft valve incompetence at 14 years was 50% (group I) and 78% (group II) (p = 0.27). Reoperation was undertaken for endocarditis, leaflet structural deterioration (SD), or technical reasons. The actuarial freedom from reoperation (all causes) at 14 years was 63% (group I) and 86% (group II) (p = 0.39). Reoperation for SD occurred in 34 patients in group I and three patients in group II. The actuarial freedom from reoperation for SD at 14 years was 67% (group I) and 95% (group II) (p = 0.001). To reflect a more accurate depiction of the prevalence of SD, patients were analyzed according to the development of “assumed structural deterioration” (at reoperation, at death with moderate or severe allograft valve incompetence and macroscopic valve deterioration on autopsy, and in the presence of moderate or severe allograft valve incompetence in patients not undergoing reoperation). The actuarial freedom from “assumed structural deterioration” at 14 years was 51% (group I) and 85% (group II) (p = 0.000003). The long‐term results confirm the low incidence of thromboembolism and endocarditis regardless of the method of preservation and demonstrate the overall acceptable performance of the viable cryopreserved allograft valve and its superiority over the 4°C stored valve.