Effects of purification on the bioavailability of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.

Evaluation of the Botulism Hazard from Vegetables in Modified Atmosphere Packaging.

The ability of Clostridium botulinum types A, B, and E spores to grow and produce botulinal toxin was investigated in five vegetables (lettuce, cabbage, broccoli, carrots, and green beans) packaged under vacuum or in air. The vegetables were packaged in two types of polyethylene-based bags differing in oxygen and carbon dioxide transmission rates. Vegetables were inoculated with a heat-shocked spore mixture of seven proteolytic and three nonproteolytic strains of C. botulinum types A, B, and E, Inoculated samples and uninoculated controls were incubated at 4, 12, or 21°C. Vegetables were tested for botulinal toxin prior to spoilage, at the onset of spoilage, and after gross spoilage had occurred as evaluated by odor, sliminess and loss of turgidity, gas production, and liquid accumulation in the packages. In nearly all vegetables during incubation the pH decreased, the number of microorganisms increased, the O2 level decreased, and the CO2 level increased. Botulinal toxin was detected in all grossly spoiled broccoli samples incubated at 21°C, in half of the inoculated grossly spoiled broccoli samples incubated at 12°C, and in one-third of the inoculated grossly spoiled lettuce samples incubated at 21°C. The pH of the toxic samples of lettuce and broccoli were 4.48 to 4.88 and 5.72 to 6.62, respectively, and the percent O2 ranged from nondetectable in lettuce to 0.40 to 1.76% in broccoli. The toxic vegetables were considered inedible. No botulinal toxin was detected in any cabbage, carrot, or green bean samples, or in any uninoculated control samples. The data suggest that the probability of botulinal toxin being produced prior to spoilage is less than 1 in 105 in the foods examined.