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Shelf-stable canned butter is currently available in retail stores, and many home-preservationists promote home-canning of butter. Non-cultured butter is a low-acid canned food, which would presumably require thermal processing. The lack of a thermal process step in canned butter products raises questions of potential safety, because they are hermetically sealed and generally exhibit anaerobic growth conditions, which are optimal for Clostridium botulinum growth. Without thermal processing, low-acid canned foods (LACF) must have inhibitory factors present to prevent C. botulinum growth. Some potential intrinsic inhibitory factors, or "hurdles", within butter include: reduced water activity (aw), acidity (pH) in cultured products, elevated salt content, and the micro-droplet nature of the aqueous phase in the butter emulsion. It was hypothesized that a normal intact butter emulsion would have sufficient "hurdles" to prevent C. botulinum growth, while a broken butter emulsion would result in a larger aqueous phase that would allow for growth. Butter was prepared using a batch churn method with either inoculated or uninoculated cream. Butter samples with four different salt amounts (0, 0.8, 1.6, & 2.4% added NaCl) were prepared and placed in coated aluminum cans for storage. Samples were stored for 1 or 2 week periods at either 22°C or 41°C and then plated for C. sporogenes growth. Samples stored at 41°C showed a significant increase over those stored at 22°C. This growth increase occurred due to incubation near the optimal growth temperature for C. sporogenes and damage to emulsion structure. Furthermore, sodium chloride (NaCl) addition was found to have a significant effect on C. sporogenes growth, with 0.8 % NaCl promoting more growth than 0%, but with decreases in growth beyond 0.8%. Uninoculated control plates were also found to have bacterial growth. This growth was attributed to other anaerobic bacteria present within the cream. It was concluded that removal of the butter structure "hurdle" could result in C. botulinum growth even at elevated salt levels and therefore home preparation of canned butter is not advisable. It is also possible that commercially canned butter, if heat abused, could potentially allow for C. botulinum growth and therefore consumption is not recommended.

The risk of toxin production by Clostridium botulinum type E in pasteurized oysters was evaluated. Thermal death time studies for type E spores in a oyster homogenate showed that the spores survived pasteurization at 550 C; D-values ranged from 65 to 100 min at 700 C and 880 to 1300 min at 550 C. When tubes of oyster homogenate were inoculated with 1x103 type E spores/ml and stored at 3, 6, 12, and 30oC, no growth was observed; however, when the inocculum was increased to 1x104 spores/ml, toxin was produced during storage at 30oC, but not at lower temperatures. For containers of whole oysters inoculated with a low spore level, toxin production occurred during incubation at 6, 12, and 30oC. / Master of Science

A procedure to rapidly screen Clostridium botulinum type A and Clostridium sporogenes for plasmids was developed. Plasmid profiles of five C. botulinum type A strains and seven C. sporogenes strains were determined and a possible relationship of plasmids to toxin production was examined. The differentiation of these organisms by plasmid fingerprinting was also studied. The plasmid isolation procedure was a modified cleared lysate technique, including lysis under alkaline conditions. Samples were subject to agarose gel electrophoresis to detect plasmid DNA. Culture age affected plasmid detection due to changes in the cell density and lysing efficiency. Middle to late log cultures were used throughout the study because they provided optimum plasmid detection. Four out of five C. botulinum type A strains and three out of seven C. sporogenes strains contained extrachromosomal DNA. For those C. botulinum type A strains which contained plasmids, there were always two, one 15 to 15.6 Mdal and the other 6.2 Mdal. C. sporogenes showed less consistency in plasmid size and number and their plasmids were generally of a greater molecular weight than those in C. botulinum type A. One C. sporogenes strain contained two plasmids and two strains contained one plasmid. Toxin production may be plasmid-mediated in the plasmid containing strains, but there was no apparent general relationship, because one of the toxic strains did not show the presence of plasmids. Plasmid screening may be useful in the differentiation of these closely related organisms without toxin testing. / Master of Science

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Les toxines botuliques (BoNTs) sont les substances les plus toxiques connues. Elles sont responsables du botulisme, une maladie rare mais le plus souvent mortelle sans prise en charge médicale. Cependant, les applications médicales des BoNTs sont de plus en plus nombreuses du fait de leurs propriétés paralysantes. Leur toxicité par voie inhalée en fait un des 6 principaux agents du risque intentionnel. Les BoNTs, produites par Clostridium botulinum, se répartissent en 7 types sérologiques qui se déclinent en sous-types. Cette biodiversité rend difficile leur identification par les méthodes classiques utilisées pour les toxines protéiques (approches immunologiques). Jusqu’à présent, seule l’analyse génétique permettait de distinguer les différents sous-types entre eux. Dans ce travail a été développée une méthode d’analyse en LC-QqQ-MS/MS en mode MRM pour identifier les différents sous-types de la BoNT/A dans des matrices complexes à partir de peptides communs et spécifiques à ces sous-types. Un traitement d’échantillon par immunocapture sur billes magnétiques couplées à des anticorps anti-peptides a été développé pour isoler la toxine de l’échantillon avant analyse. Des surnageants de culture des sous-types A1 à A3, A5, A7 à A8 ont été utilisés pour valider la méthode. La limite de détection de la méthode est compatible avec les taux de toxine retrouvés habituellement dans les échantillons naturellement contaminés. Cette méthode de spectrométrie de masse a ensuite été utilisée pour quantifier les différents sous-types de la BoNT/A dans une matrice complexe (surnageants de culture de C. botulinum). Une technique de quantification, utilisant un isotope stable de la chaine légère de type A1, ([13C6]K et [13C6]R), a été retenu comme étalon interne. Les différents sous-types de BoNT/A ont été quantifiés dans les surnageants et la quantité de BoNT correspondante à une dose létale minimale de 100% a été déterminée pour chaque sous-type. / Botulinum neurotoxins (BoNTs) are the most poisonous substances known. They are responsible for human botulism, a rare but potentially fatal disease if not quickly treated. However, BoNTs were approved for the treatment of numerous medical applications due to their temporary paralysis effects. BoNTs are among the six agents with the highest risk of potential use as bio-weapons because of their high toxicity in aerosol form. BoNTs, produced by Clostridium botulinum, are divised into seven toxinotypes and each toxinotype contains several subtypes. This biodiversity makes more difficult their identification with classical methods by immunological ways. Until now, only molecular genetical methods could differenciate subtypes among them. The aim of this work was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) in MRM mode to efficiently discrimate the distinct subtypes from specific and common peptides. Immunocapture sample preparation with antipeptides antibodies was used and allowed the isolation of the toxin from the sample. Subtyping was performed with crude supernatants (BoNT/A1 to /A3, /A5, /A7 and /A8) in order to validate the method. Limit of detection (LOD) of the proposed method is in the range of minimal toxin concentration found in naturally contamined samples. In a second part of this work, this mass spectrometry method was used to quantify the neurotoxin in complex matrices (supernatants of Clostridium botulinum cultures). Isotope labeled light chain (13C6]K et [13C6]R) from botulinum A1 neurotoxin was produced and used as internal standart. Subtypes were quantified in supernatants and the quantity of neurotoxin for one minimal lethal dose 100% was determined for each subtype

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flasque, est produite par des bactéries anaérobies du genre Clostridium. Les BoNTs, classifiées en 7 types (A à G), forment divers complexes avec des protéines non toxiques, dont le composant non toxique non hémagglutinant (NTNH) et les hémagglutinines (HAs). Les gènes sont organisés, au sein du locus botulique, selon deux opérons divergents, ntnhbont/ A et ha34-ha17-ha70 pour le type A, dont l'expression est positivement régulée par le facteur sigma alternatif BotR. Un pic d'expression synchrone de tous les gènes du locus de type A est mesuré par RT-PCR en temps réel lors de la transition entre les phases exponentielle et stationnaire de croissance, en parallèle avec l'augmentation du titre en toxine du surnageant de culture. Dans un modèle d'épithélium intestinal, BoNT/A purifiée est transcytosée après liaison via le domaine Hc/A à des récepteurs apicaux comprenant des gangliosides et des protéines potentiellement apparentées à SV2. L'intensité de liaison et l'efficacité de transport de la toxine sont supérieures dans les cellules intestinales de type crypte plutôt qu'entérocyte. Injectés dans la lumière d'une anse iléale ligaturée, BoNT/A inhibe les contractions des muscles lisses et le domaine Hc/A fluorescent progresse de la muqueuse, via certaines cellules des cryptes, vers la sous-muqueuse et la musculeuse où il cible certaines terminaisons nerveuses, majoritairement cholinergiques. Hc/A entre par des voies distinctes dans les cellules neuronales (voie clathrine dépendante de la dynamine) et les cellules intestinales (voie non-clathrine, dépendante de Cdc42 et de la dynamine).

Although nitrite is a known inhibitor of Clostridium botulinum in cured meats, the mechanism of inhibition is not understood. The observation has been made that iron is required for growth of C. botulinum and that the role of nitrite may be to alter the pathway of iron uptake by these organisms. Since the color change in cured meats is due to the binding of nitrite to the heme group of meat pigments, it was hypothesized that nitrite may also be tying up an essential iron source, heme. This experiment was an investigation of the possibility that myoglobin added to a meat system would stimulate growth and toxin production by C. botulinum much more than myoglobin that had been nitrosylated before inclusion in the product. Treatments were included to compare the effects of a heme iron source, myoglobin, with that of an ionic source, ferric chloride. To help understand the role of free iron in botulinal growth, several treatments contained a metal ion chlator, ethylenediaminetetraacetic acid (EDTA). Nitrite caused a definite delay of growth, as evidenced by gas bubbles, when compared with a non-nitrite system. Addition of ferric chloride resulted in an increase in the rate of of appearance of swollen samples, although growth was enhanced even more when myoglobin was added. When nitrosylated myoglobin was included, growth was inhibited more than in the treatment with nitrite alone. EDTA inhibited growth of C. botulinum but a conclusion should not be made with respect to the chelation of iron since EDTA chelates many other metals. Residual nitrite levels had declined to below 10 ppm by the time swelling occurred. Although swelling did not occur until nitrite had declined in the products, the absence of nitrite alone did not allow growth and toxin production. Since nitrosylated myoglobin and EDTA inhibited botulinal growth even after residual nitrite had declined, it is possible that the inhibitory action of nitrite is creating a nutritional deficiency for C. botulinum.

The effect of added iron on the growth of Clostridium botulinum type A in a chemically defined medium was studied. Growth of C. botulinum was supported by an iron level of 0.05 ug/ml with maximum growth observed at a level of 3 ug iron/ml. Electron paramagnetic resonance (EPR) studies were conducted to detect the presence of iron-sulfur centers and iron-nitric oxide complexes in untreated and nitrite treated cell-free extracts of C. botulinum type A. Untreated extracts of C. botulinum exhibited EPR signals in the oxidized and reduced states characteristic of a "HiPiP-type" iron-sulfur center (g=2.02) in the oxidized state and a reduced signal at g=l.94, characteristic of a reduced iron-sulfur center. Extracts of C. botulinum treated with nitrite exhibited an EPR signal at g=2.035, characteristic of iron-nitrosyl complexes, with the simultaneous disappearance of the the signal at g=l.94. This indicates that nitrite reacts with the iron-sulfur centers in botulinal cells to form iron-nitrosyl complexes. Addition of ascorbate with nitrite intensified the EPR signal at g=2.035, probably by enhancing the reduction of nitrite to nitric oxide. A cytochrome c reduction method was used for the determination of ferredoxin activity in untreated and nitrite treated cells of C. botulinum type A from which ferredoxin had been partially purified. Untreated extracts of C. botulinum reduced cytochrome c which demonstrates ferredoxin activity within the cells. Treatment of the cells with nitrite at a level of 1000 ppm for 45 min was found to inhibit ferredoxin activity by 90%. Boiling the partially purified ferredoxin from the untreated cells for 5 min inactivated the protein. Pyruvate-ferredoxin oxidoreductase activity in partially purified extracts of nitrite treated and untreated cells of C. botulinum was determined by assaying for FAD reduction and acylhydroxamate formation. Nitrite treated cells exhibited an inhibition of 70% of FAD reducing activity and 80% inhibition of acylhydroxamate formation when compared to the untreated cells. Boiling inhibited the activity of partially purified oxidoreductase activity by more than 90% in both the assays.