CONTENTS

TITLE PAGE

ACKNOWLEDGEMENTS

ABSTRACT

LIST OF ORIGINAL PUBLICATIONS

1. INTRODUCTION

2. REVIEW OF LITERATURE

2.1 Foodborne botulism

2.1.1 Pathogenesis and clinical symptoms

2.1.2 Epidemiology

2.1.3 Diagnostics

2.2 DNA-based typing methods

2.2.1 Pulsed-field gel electrophoresis (PFGE)

2.2.2 PCR-based typing

2.2.3 Typing of Clostridium botulinum by DNA-based methods

2.3 Clostridium botulinum type E

2.3.1 Phenotypic characteristics

2.3.2 Prevalence in environment

2.3.3 Prevalence in raw fish

2.3.4 Prevalence in fishery products

2.3.5 Fish-related botulism outbreaks caused by type E

2.4 Methods to control Clostridium botulinum type E in foods

2.4.1 Physical treatments

2.4.2 Chemical treatments

2.4.3 Combination of preservative factors (hurdles concept) and predictive microbiology

2.4.4 Recommendations to ensure the safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum

2.5 Risk factors predisposing fishery products to growth and toxigenesis by Clostridium botulinum type E

3. OBJECTIVES OF THE PRESENT STUDY

4. MATERIALS AND METHODS

4.1 Sampling in prevalence study (I)

4.2 Clostridium botulinum strains genotyped (II, III)

4.3 Detection, quantification and isolation of Clostridium botulinum (I-V)

4.4 Toxin analysis (I, IV, V)

4.5 Cultivation of Clostridium botulinum strains and isolation of chromosomal DNA (II, III)

4.6 PCR-based typing methods (II, III)

4.7 Macrorestriction analysis by PFGE and cluster analysis (III)

4.8 Challenge tests (IV, V)

4.8.1 Fish (IV, V)

4.8.2 Brining (IV, V)

4.8.3 Sample inoculation (IV, V)

4.8.4 Processing and storage conditions (IV, V)

4.8.5 Sampling procedures (IV, V)

4.8.6 Microbiological analyses and sensory evaluation (V)

4.8.7 Physical and chemical determinations (IV, V)

4.8.8 Predictive models (IV)

4.8.9 Statistical analyses (V)

5. RESULTS

5.1 Prevalence of Clostridium botulinum type E in Finnish raw fish and fishery products (I)

5.2 Application of RAPD and rep-PCR typing techniques for characterization of group I and II Clostridium botulinum (II)

5.2.1 RAPD

5.2.2 Rep-PCR

5.3 Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products (III)

5.3.1 Macrorestriction digests and cluster analysis

5.3.2 RAPD analysis

5.4 Predicted and observed growth and toxigenesis by Clostridium botulinum type E in vacuum-packaged fishery product challenge tests (IV)

5.4.1 Vacuum-packaged unprocessed rainbow trout

5.4.2 Vacuum-packaged raw-pickled (gravad) rainbow trout

5.4.3 Vacuum-packaged cold-smoked rainbow trout

5.5 Application of sodium nitrite (NaNO₂) and potassium nitrate (KNO₃) to control outgrowth and toxigenesis by nonproteolytic Clostridium botulinum in vacuum-packaged cold-smoked rainbow trout (V)

5.5.1 Effect of nitrite and nitrate on the Clostridium botulinum growth and toxigenesis

5.5.2 Effect of nitrite and nitrate on the microbiological and sensorial quality of the product

5.5.3 Nitrite and nitrate depletion in the product

6. DISCUSSION

6.1 Prevalence of Clostridium botulinum type E in Finnish raw fish and fishery products (I)

6.2 DNA-based characterization of isolated strains (II, III)

6.2.1 Application of different DNA-based typing techniques for characterization of group I and II Clostridium botulinum

6.2.2 Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products

6.3 Safety evaluation of vacuum-packaged fishery products using challenge tests (IV, V)

6.3.1 Growth and toxin production by Clostridium botulinum type E in vacuum-packaged fishery products

6.3.2 Use of predictive models in safety evaluation

6.3.3 Use of nitrite and nitrate in vacuum-packaged cold-smoked rainbow trout

7. CONCLUSIONS

8. REFERENCES