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Fish products under vacuum (VAC) and/or modified atmosphere packaging (MAP) conditions can have a significantly extended shelf life. Prevention of toxin production by Clostridium botulinum is essential for processors of VAC and MAP refrigerated fishery products. The objective of this study was to determine if C. botulinum toxin development precedes microbiological spoilage and sensory rejection in fully cooked breaded and battered Alaskan Pollock or raw aquacultured flounder fillets. Aquacultured summer flounder (Paralichthys dentatus) fillets and fully cooked breaded and battered Alaskan pollock (Theragra chalcogramma) were either aerobically packed (Oxygen Transmission Rate (OTR) of 3,000 cc/m2/24h@70°F for flounder and 6,000 cc/m2/24h@70°F for Pollock), vacuum packed or MAP packaged in a 100% CO₂ atmosphere (OTR of 7.3 cc/m2/24h@70°F). Flounder fillets were stored at either 4 or 10°C while pollock portions were stored at 8 and 12°C. Based on the time to spoilage (counts >107 CFU/g), additional samples were inoculated with five strains of nonproteolytic C. botulinum and analyzed qualitatively for botulinum toxin using a mouse bioassay. For flounder at 4°C, toxin formation did not occur after 35 days in aerobically packed fillets. Vacuum packed and 100% CO2 fillets produced toxin before spoilage at days 20 and 25, respectively. In the aerobic packages at 10°C, toxin production occurred after spoilage at day 8, but before spoilage in the vacuum and 100% CO₂ packages at day 9. Sensory evaluation of toxic vacuum and 100% CO₂ packages at 4°C revealed toxin production proceeded spoilage and absolute sensory rejection. However, at 10°C toxin production was evident only after absolute sensory rejection and microbiological spoilage for aerobically packed fillets. Vacuum packages and 100% CO₂ packages were toxic during spoilage but before absolute sensory rejection. Toxin was not present in the aerobically and 100% CO₂ packed pollock samples at 8°C and the 100% CO2 packed samples at 12°C after 35 days. Aerobically packed portions stored at 12°C first produced toxin at day 25; toxicity occurred after absolute sensory rejection and before spoilage. The vacuum packed portions first formed toxin at day 25 for 8 and 12°C storage before spoilage and absolute sensory rejection. / Ph. D.

Le botulisme est une maladie nerveuse, commune à l’homme et aux animaux, due à l’action de la toxine botulique produite par Clostridium botulinum. Il existe 8 types de toxines dénommées A à H. Les bactéries capables de produire cette toxine se différencient en six groupe sur la base de leurs caractéristiques phénotypiques et biologiques. Les souches de C. botulinum responsables du botulisme humain appartiennent aux groupes I et II selon qu’elles soient protéolytiques ou non. Elles produisent les toxines A, B, E et F, ainsi que le nouveau type H récemment découvert. C. butyricum et C. baratii sont également capables de produire les toxines botuliques de type F et E et appartiennent au groupe V et VI. C. argentinense appartient au groupe IV et est capable de synthétiser la toxine de type G. Elle a été soupçonnée d’être impliquée dans des cas de botulisme infantile en Argentine. Les souches de C. botulinum responsables du botulisme animal appartiennent au groupe III (C. novyi sensu lato) et produisent les toxines C, D et leurs formes mosaïques C/D et D/C. La toxine botulique est le poison le plus puissant connu à ce jour. La dose létale nécessaire pour tuer une personne en bonne santé par intoxication alimentaire est de 70 µg seulement. C’est pourquoi cette toxine a fait l’objet d’études particulièrement approfondies, notamment celles impliquées dans des cas de botulisme humain. Elle peut également être utilisée pour le traitement de certaine pathologie ou la chirurgie esthétique (Botox). Malheureusement, elle peut également être utilisée à mauvais escient, en tant qu’arme de guerre ou à des fins de bioterrorisme. C’est pourquoi l’emploi de la toxine botulique ou de sa bactérie productrice fait l’objet d’une législation particulièrement stricte. Mon projet de doctorat s’est organisé autour de plusieurs projets de recherche visant à développer des méthodes de détection et de typage de du germe et de sa toxine (projets Européens BIOTRACER et AniBioThreat ; projets NRBC-bio ; LNR botulisme aviaire en France). Lors de mes recherches j’ai concentré mon travail sur le développement de méthodes capable de suivre et remonter à la source d’une contamination, qu’elle soit délibérée, accidentelle ou naturelle. Afin d’y parvenir j’ai investigué les gènes des flagellines de C. botulinum groupe I à III, responsables du botulisme humain et animal. L’analyse des gènes flaA et flaB a mis en évidence 5 groupes majeurs et 15 sous-groupes, certain étant spécifiques de régions géographiques. FlaB s’est montré spécifique de C. botulinum type E. Les gènes flagellines fliC, spécifiques à C. botulinum du groupe III, se divisent 5 groupes, avec fliC-I et fliC-IV associés aux types mosaïques C/D et D/C. J’ai étudié la prévalence des souches productrices de toxine de type mosaïques chez les volailles et les bovins. Les résultats montrent que les types C/D et D/C sont majoritaires en Europe. Enfin, j’ai séquencé 17 génomes provenant de souches responsables de botulisme animal en France (14 types C/D et 3 types D/C). Leur analyse montre que ces souches sont très proche génétiquement, entre elles et avec les souches Européennes. Grâce à ces données j’ai mis en évidence un large contenu extra chromosomique dans les souches C/D, qui peut être utilisé pour créer une carte d’identité génétique. D’autre part, l’étude des séquences Crisps à des fins de typage ne s’est pas avérée suffisamment résolutive, du fait de système Crispr-Cas déficient chez les souches C/D. Enfin, un très haut degré de discrimination a été atteint par typage SNP, qui a permis de distinguer jusqu’à l’origine de chaque souche. L’ensemble de ces résultats est développé dans le présent manuscrit / Clostridium botulinum is the etiologic agent of botulism, a deadly paralytic disease that can affects both human and animals. Different bacteria, producing neurotoxins type A to H, are responsible for the disease. They are separated into different groups (I to VI) on the basis of their phenotypical and biological characteristics. Human botulism is mainly due to Groups I and II producing neurotoxins A, B, E and F, with type H recently discovered. Also C. butyricum and C. baratii species (Groups V and VI), producing toxins type F and E respectively, are scarcely reported. C. argentinense Group IV, producing toxin type G, which has been suspected to be associated with infant botulism in Argentina. Animal botulism is mainly due to Group III, which is constituted by C. novyi sensu lato species. They produce toxin types C, D and their mosaic variants. Botulinum neurotoxins are the most powerful toxin known to date with as little as 70 µg enough to kill a person by food poisoning. Therefore, it received a great deal of attention. Botulinum neurotoxins have been deeply studied, especially human related toxins compared to animal. The toxins found to be useful for medical or cosmetic (Botox) treatments, but it was also used as a biological warfare agent, and for bioterrorism. Its extreme potency is equal to its dangerousness. Therefore, governments show concerns of its potential misuse as a bioterrorism weapon; research programs are funded to study and raise awareness about both the toxins and the producing organisms. My PhD work was structured by the different projects I was involved in, which were related to C. botulinum detection and typing, like BIOTRACER and AniBioThreat European projects, the French national CBRN program, or the NRL for avian botulism. The main transversal objective I followed lead me to develop new methods to trace back the origin of C. botulinum contamination, in case of a deliberate, accidental or naturally occurring botulism outbreak. I investigated flagellin genes as potential genetic targets for typing C. botulinum Group I-II and III, responsible for human and animal botulism respectively. Flagellin genes flaA and flaB showed the investigated C. botulinum Group I and II strains to cluster into 5 major groups and up to 15 subgroups, some being specific for certain geographical areas, and flaB being specific to C. botulinum type E. Flagellin fliC gene investigated in C. botulinum Group III showed to cluster into five groups, with fliC-I and fliC-IV associated to type C/D and D/C respectively, being not discriminative enough to differentiate highly genetically related strains. I also studied the prevalence of mosaic toxin genes in C. botulinum Group III in animal botulism, mainly in poultry and bovine. The results brought out the mosaic toxin types C/D and D/C to be predominant in the samples investigated throughout Europe. Finally, I explored the full genome sequences of 14 types C/D and 3 types D/C C. botulinum Group III strains, mainly originating from French avian and bovine botulism outbreaks. Analyses of their genome sequences showed them to be closely related to other European strains from Group III. While studying their genetic content, I was able to point out that the extrachromosomal elements of strains type C/D could be used to generate a genetic ID card. Investigation of Crispr typing method showed to be irrelevant for type C/D, due to a deficient Crispr-Cas mechanism, but deserve more investigation for type D/C. The highest level of discrimination was achieved while using SNP core phylogeny, which allowed distinguishing up to the strain level. Here are the results I’m going to develop in this manuscript

Extended shelf-life cook-chill meals can pose a potential risk of botulism if they are subjected to a temperature abuse. Spores of group II non-proteolytic Clostridium botulinum can survive the mild heat treatment typically given to these products and can grow at refrigeration temperatures. To circumvent this safety issue, existing preservation methods can either affect the sensory properties of these foods or damage their image. Therefore, additional natural preservation hurdles are needed. Thus, the aim of this study was to develop a novel bio-preservation method based on the principle of antibiosis between protective cultures (PCs) and C. botulinum. Consequently, the objectives were to select effective anti-botulinal cultures and study their inhibition pattern in microbiological media and foods, identify the conditions for effective inhibition and the nature of the antibiosis. This research demonstrates for the first time that the bacteriocinogenic protective cultures inoculated at high levels had an anti-botulinal effect in a range of commercial cook-chill products, which supported active growth of non-proteolytic C. botulinum. The protocol for commercial application of the protective cultures was developed. / Doctor of Philosophy (PhD)

The application of infrared imaging employing focal plane array Fourier transform infrared (FPA-FTIR) instrumentation for the identification of bacteria was investigated. FPA-FTIR spectroscopy was shown to provide new opportunities for bacteria identification with unprecedented reliability and throughput by allowing 102--103 FTIR spectra to be acquired simultaneously from surface areas of 90 x 90 to 200 x 200 mum with a spatial resolution of ∼6 mum. The combination of data redundancy and spatial resolution afforded by infrared imaging made it possible to acquire highly reproducible spectra from bacterial films. A protocol for enhancing the reliability of bacteria identification by transmission-mode FPA-FTIR spectroscopy was developed by optimizing spectral acquisition parameters, spectral processing and data analysis; using the differentiation of two Campylobacter species as a test case. The results for this test case were compared with those obtained from three alternate FTIR spectral acquisition modes. The optimized protocol was employed for the generation of a spectral database of foodborne bacteria, containing over 1,000,000 spectra acquired by infrared imaging of 36 species from 19 genera. The development of a modular hierarchical clustering (MHC) model, in combination with the use of a region selection algorithm, allowed all species in the database to be differentiated from each other down to the species level based on differences in their infrared absorption profiles. A validation study involving the identification of well-characterized isolates by comparison of their spectra to those in the database demonstrated the robustness of the MHC model. In a further study employing 44 strains of Clostridium botulinum, the discriminatory power of FPA-FTIR spectroscopy was compared with that of pulsed-field gel electrophoresis, and the region selection algorithm was applied to identify growth medium-independent spectral regions that allowed for the differentiation of Group I and Group II C. botulinum strains in two blind validation studies. The research carried out also demonstrated the high-throughput potential of bacteria identification by infrared imaging when combined with the use of a microarray system for sample deposition. Overall, the novel FPA-FTIR spectroscopy-based bacteria identification protocol developed in this work provides a rapid-response and reagent-free technique suitable for routine use in both food and clinical microbiology laboratories.

Initial challenge studies with spores of proteolytic Clostridium botulinum types A and B (~104 spores/g) showed that while air- and gas-packaged English-style crumpets (aw 0.990) and pizza crust (aw 0.960) were toxic after 42-days storage at ambient temperature (25°C), no neurotoxin was detected in bagels (a w 0.944). Further challenge studies with similarly packaged crumpets inoculated with C. botulinum (~102 spores/g), pre- or post-baking, demonstrated that all crumpets were toxic within 4 to 6 days at 25°C and that toxigenesis preceded spoilage. Furthermore, reformulating crumpets to pH 8.3 and packaging in 100% CO2 had little effect in delaying the growth of C. botulinum compared to crumpets formulated to pH 6.5 and packaged in 60% CO2. / Subsequent studies were directed at determining the levels of additional barriers that could be used to ensure the safety of high-moisture MAP crumpets. While ethanol vapour proved to be an effective additional barrier in crumpets (100-g, [aw 0.990, pH 6.5]) challenged with ~102 spores/g of C. botulinum, spoilage preceded toxigenesis due to absorption of ethanol from the package headspace by crumpets. Modelling studies in Trypticase Peptone Glucose Yeast (TPGY) broth confirmed the anti-botulinal nature of ethanol and showed that a level of ~4% (vol/vol) could be used for complete inhibition of this pathogen, depending on the aw and pH of the growth medium. However, while ethanol vapour could be used to inhibit the growth of C. botulinum in high-moisture crumpets, its anti-botulinal efficacy was influenced by the method of crumpet leavening (yeast v chemical). / Preliminary studies were also done to assess the potential of mastic oil, a novel inhibitor, against C. botulinum. While direct and indirect application of ethanolic extracts of mastic oil inhibited the growth of C. botulinum in vivo, they failed to do so in crumpets.

Ziel der vorliegenden Arbeit war es, einen Beitrag zur Aufklärung der Ätiologie der Graskrankheit mit Hilfe der DGGE, besonders im Hinblick auf in vitro unkultivierbare Bakterien der Darmflora zu leisten. Es sollte ebenfalls geprüft werden, ob die DGGE die Diagnose der Graskrankheit erleichtern kann. Weiterhin sollte der Einfluß von C. botulinum auf die Erkrankung durch den Nachweis von Toxin, Bakterien und Antikörpern untersucht werden. Es standen zur Untersuchung Proben des Colons, Caecums und Kotes von erkrankten Pferden und Kontrolltieren, Kotproben von klinisch gesunden Pferden, die aus denselben Beständen wie die erkrankten Tiere stammen sowie Serum aller drei Gruppen zur Verfügung. Wegen der hohen individuellen Variabilität der Darmflora war kein eindeutiges Merkmal der Graskrankheit im Profil der mikrobiellen Gemeinschaft des Darmes oder Kotes nachweisbar. Allerdings ließ sich anhand der Clusteranalyse ein Abgrenzung der Flora des Caecums und besonders des Colons der erkrankten und gesunden Tiere erkennen. Für eine Diagnose der Graskrankheit am lebenden Tier anhand der Kotflora ist die DGGE jedoch wegen ihrer geringen Aussagekraft und methodischen Probleme nicht geeignet. Der Verdacht, dass C. botulinum an der Ätiologie der Graskrankheit beteiligt ist, konnte durch die Ergebnisse im Tierversuch und ELISA weiter untermauert werden.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Includes bibliographical references.

The neuroparalytic syndrome called botulism is caused by the neurotoxins produced by bacteria in the genus Clostridium. There are seven toxigenic types of C. botulinum (A to G) based on antigenically distinct toxins produced by different strains of the organism. Animal botulism is caused by C. botulinum type C and D neurotoxins and has a severe economic impact on cattle farming in South Africa and neighbouring countries. Current treatment regimes include the use of acetylcholine for symptomatic treatment, but this is unfortunately very seldom successful. All indications are that there is no cure for this disease and that effective control can only be achieved through development of efficacious vaccines. The botulinum vaccine currently in use in South Africa contains an adjuvanted toxoid form of the type C and o neurotoxins. However, this bivalent vaccine relies on problematic anaerobic cultivation of the Clostridium bacterium followed by isolation, purification and inactivation of the toxin by treatment with formalin. Apart from the fastidious growth requirements of this organism, it has been reported that the production of toxin by these cells declines rapidly and eventually ceases, following laboratory passaging of the bacterial cultures. In addition, improper inactivation of the toxins may also lead to the demise of animals following vaccination. Thus, there exists a great need for a safe, effective and inexpensive vaccine against botulism. To investigate the potential of types C and D botulinum neurotoxins as efficacious recombinant vaccine candidates against botulism, full-length copies of the genes were obtained by polymerase chain reaction (PCR) amplification from bacteriophage DNA isolated from Clostridium botulinum type C (Stockholm) and D (South Africa) cultures. The full-length genes were cloned and subsequently sequenced to verify their integrity. By making use of PCR-based site-directed mutagenesis procedures, three amino acid mutations were introduced in the zinc-binding motif of the respective neurotoxins. Mutation of this domain has previously been reported to successfully detoxify type C neurotoxin. The wild-type and mutant genes were subsequently expressed in insect cells using the BAC-to-BAC™ baculovirus system. Although, unique protein bands corresponding to the size of the neurotoxins could not be seen in Coomassie brilliant blue-stained gels, Western blot analysis indicated immunoreactive material for wild-type and mutant type C corresponding to the size of the type C toxin light chain. However, there was no conclusive evidence to support the successful expression of the full-length wild-type and mutant type D genes. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

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