An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production.

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2 µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8 mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100 l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45 + 0.2 µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.

Tetanus toxin production is triggered by the transition from amino acid consumption to peptides.

Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings.

Fed-batch production of tetanus toxin by Clostridium tetani.

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L(f)/mL) for NZ(0) > or = 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield ( approximately 60 L(f)/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration ( approximately 45 L(f)/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification.

Effect of medium composition on the production of tetanus toxin by Clostridium tetani.

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N-Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 Lf/mL), achieved at G0= 9.7 g/L and NZ0= 43.5 g/L, was 80% higher than that obtained with standard cultivations (G0= 8.0 g/L and NZ0= 25.0 g/L).

Estudo clínico do tétano: revisäo; 1994 / Clinical aspects of tetanus: review

O autor fez uma revisäo sobre o tétano abordando os principais estudos referentes a etiologia, epidemiologia, patologia, diagnóstico, quadro clínico, diagnósticos diferenciais, exames laboratoriais subsidiários, tratamento, complicaçöes e profilaxias

Botulism and tetanus: selected epidemiologic and microbiologic aspects.

Botulism is rare in both developing and developed countries. During 1980 only 89 cases (18 food borne, 68 infant, 2 wound, 1 unspecified) were reported in the United States. Coproexamination is essential for laboratory confirmation of infant botulism. Botulinal antitoxins of equine origin are used for treating food-borne and wound botulism but are usually not recommended for infant cases. Tetanus is much more common in some developing countries than in developed countries. During 1980 only 95 cases of tetanus were reported in the United States; in 68 (72%) of these cases, the patient was 50 years or older, and in only two (2.1%) cases was the patient younger than one year. Tetanus neonatorum is a major problem in some developing countries. Diagnosis of tetanus is based primarily on clinical findings, but laboratory studies can be helpful, especially in epidemiologic investigations. Human hyperimmune immunoglobulin is now used in the treatment of tetanus.