Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10 2 cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10 ؊2 spore/g for types A, B, and F to 10 ؊1 spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing L. monocytogenes was low. During processing, the frequency of fish contaminated with L. monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating L. monocytogenespulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an L. monocytogenes eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminatingL. monocytogenes. None of the control samples taken in the 5 months after the eradication program was implemented containedL. monocytogenes.

The prevalence of Clostridium botulinum type E gene in fish and fishery products of commercial importance in Finland was determined using a quantitative PCR analysis. The contamination level in 438 raw fish samples from intestines, surface and whole fish and 208 fish roe samples varied from 10-40% and from 4-14% respectively, depending on the fish species studied. The presence of C. botulinum in European wild freshwater fish and roe was demonstrated for the first time by isolation of the organism from PCR-positive samples. Five percent of 214 vacuum-packed and 3% of 123 air-packed fishery product samples examined at retail level were positive for the botulinum neurotoxin type E gene. A contamination level of 10% in vacuum-packed hot-smoked whitefish was detected. The results demonstrate that C. botulinum type E poses a serious health risk for those consuming fishery products from the Baltic Sea area.

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Secl-related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Secl-related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc-l8-2/Munc18b/muSecl protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associates in vitro with syntaxins l A , 2, and 3. In situ hybridization analysis of embryonic mouse tissues revealed prominent expression of the munc-I 8-2 mRNA in the epithelia of several tissues. Cell-fractionation studies demonstrated that the majority of Munc-18-2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc-18-2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS-1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin l A , it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc-18-2 as a predominantly epithelial vesicle-transport protein with a polarized distribution and provided novel in vivo evidence for the association of Secl -related proteins with members of the syntaxin family.Keywords: vesicle transport; Secl-related protein ; Munc-1 8-2; Madin-Darby canine kidney cell ; epithelial cell.The soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein (SNAP)-receptor (SNARE) hypothesis of vesicle transport postulates that membrane proteins related to the synaptic-vesicle-associated membrane protein synaptobrevin, the presynaptic-plasma-membrane protein syntaxin and synaptosonial-associated protein of 25 kDa (SNAP-25) participate in the creation of the specificity of transport-vesicle-fusion events [ l , 21. Although increasing experimental evidence supports this hypothesis, the molecular interactions that promote the assembly of SNARE complexes and the precise events that lead to fusion o f membrane bilayers are largely unknown. Two families o f proteins suggested to regulate SNARE-complex formation are the small Ypt/Rab GTPases [3] and the Secl-related proteins.In Sacchuromyces cerevisiae, four proteins belonging to the Secl protein family are known. Seclp is required for vesicle transport from the Golgi complex to the plasma membrane 14,

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The distribution of Clostridium botulinum serotypes A, B, E and F in aquatic environments of the Baltic Sea and Finnish mainland was examined. A total of 110 samples were tested with a neurotoxin-specific PCR assay. Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found. Spore counts were quantified by the most probable number method, Cl. botulinum type E kg(-1) averaging 940 in sea and 370 in freshwater samples. The overall prevalence and spore counts of Cl. botulinum type E in aquatic sediments correlated significantly with offshore bottom oxygen content, depth, and bioturbation activity, whereas there was no correlation with bottom water temperature. These findings indicate the possibility of Cl. botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments.