ABSTRACT

The prevalence of Clostridium botulinum type E in five commercially important raw fish species and seven various types of fishery products was determined by using quantitative PCR-based detection method. The applicability of randomly amplified polymorphic DNA (RAPD) analysis and repetitive element sequence-based PCR (rep-PCR) in subtyping C. botulinum strains was evaluated. Additionally, 92 type E isolates from different types of fish and seafood samples were characterized by pulsed-field gel electrophoresis (PFGE) and RAPD to determine the genetic biodiversity of type E strains. Challenge tests and predictive models were used to evaluate the safety of certain types of vacuum-packaged fishery products that were found positive for type E spores in the prevalence study. Additionally, the ability of sodium nitrite (NaNO₂) and potassium nitrate (KNO₃) to prevent the outgrowth and toxigenesis by nonproteolytic C. botulinum was assessed.

Both farmed and wild fish species in Finland were found to be contaminated by C. botulinum type E, with prevalence levels of 17% and 20%, respectively. Eight percent of fish roe samples, 5% of vacuum-packaged and 3% of air-packaged fishery product samples were positive for type E.

RAPD analysis was found to be a discriminating and reproducible method for the genotyping of type E strains. The highest discrimination was achieved by combining RAPD analysis with PFGE. The distinct advantages of RAPD as compared to PFGE were the 100% typeability and rapid performance. Rep-PCR lacked the discrimination to be a useful tool in C. botulinum typing.

High genetic biodiversity was observed among type E isolates regardless of the isolation source or geographical origin. Extensive genetic variation was observed between strains isolated from different fish species as well as among isolates from one fish species or even from one individual fish. Processing did not appear to favor survival of any particular genotype. The wide genetic biodiversity observed among C. botulinum type E strains was concluded to facilitate the use of DNA-based typing methods as a tool in contamination studies in food industry and in investigations of botulism outbreaks.

Using inoculums corresponding to the natural type E contamination level of fish, vacuum-packaged unprocessed and cold-smoked rainbow trout stored at 8^oC became toxic in two and three weeks, respectively. Even at 4^oC, the cold-smoked product turned toxic after four weeks. Vacuum-packaged raw pickled (gravad) rainbow trout stored at 6^oC was evaluated to be safe with respect to type E, provided that the declared shelf life was within reasonable limits. The results strongly indicated that unless the constant maintenance of a chill chain temperature below 3^oC cannot be guaranteed, the shelf life of vacuum-packaged cold-smoked rainbow trout in its current formulation should be limited to ten days. However, the use of nitrite or nitrate in vacuum-packaged cold-smoked rainbow trout was found to reduce the hazard arising from C. botulinum type E at slightly abusive storage temperatures.

The applicability of currently available predictive microbiological models for safety evaluation of fishery products with respect to C. botulinum type E was found to be highly questionable. The models tested were hampered by the limitations of the controlling environmental factors set by the programs which had an adverse effect on the reliability of predictions.