[Studies on the Seemueller phenomenon. V. The importance of diphthin for the origin of the phenomenon. Summary observation].
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Using the "tetanus-mouse" model the differences in immunity development between animals exposed to irradiation (300 R and 400 R) and animals not exposed to irradiation after nasal and subcutaneous vaccination was investigated. Immunization was carried out 1, 3, 6 and 10 days before and after exposure to irradiation. Efficacy of immunization was tested by challenge with 10 X LD50 tetanus-toxin and by antitoxin determination with L+-method. The degree of the immune response was dependent on 1. the irradiation dose, 2. the interval between active immunization and the ensuing or preceding irradiation, 3. the mode of vaccination (local or parenteral) and 4. the vaccination dose.
An enzyme-linked immunosorbent assay for determining the toxigenicity of Corynebacterium diphtheriae is presented. The assay uses hyperimmune horse diphtheria antitoxin as a capture antibody and mouse monoclonal diphtheria antitoxin as a detecting antibody. Growth of bacteria and capture of diphtheria toxin by antitoxin are carried out in one step. Toxin produced by as little as 100 toxin-producing corynebacteria is detectable, corresponding to a sensitivity of 10 ng of diphtheria toxin per ml. Demonstration of toxin after incubation of the bacteria for 4.75 h, as well as after 18 h, was in accordance with the modified Elek gel diffusion method and the guinea pig inoculation test. However, heavy inocula incubated overnight produced significantly lower optical density than did diluted inocula; thus, the higher optical density was used as an indicator of toxin production. A decrease in optical density was also seen by shortening the incubation time. For laboratory safety, ethanol was added to the microtiter plate wells before washing out of the bacteria. This resulted in a further decrease in optical density. Using 4.75-h incubation time gave a single false-negative result. No false-positive results were ever seen. Incubation for 18 h is suitable for large-scale screening, and 4.75 h of incubation is suitable for rapid identification of toxin-producing C. diphtheriae.
The cecal contents of guinea pigs with clindamycin-associated colitis contained a heat-labile toxin. This toxin was lethal for guinea pigs and mice, produced vascular permeability in the skin of rabbits, and was cytotoxic in tissue culture. The lethality in mice, vascular permeability in rabbit skin, and cytotoxicity in tissue culture monolayers were neutralized by Clostridium sordellii antitoxin.
Fiock, Mary A. (Fort Detrick, Frederick, Md.), Allen Yarinsky, and James T. Duff. Studies on immunity to toxins of Clostridium botulinum. VII. Purification and detoxification of trypsin-activated type E toxin. J. Bacteriol. 82:66-71. 1961.-A procedure was described for production, purification, and conversion to toxoid of the Clostridium botulinum type E trypsin-treated toxin. A medium containing 1.5% trypticase, 0.5% yeast extract, 0.075% cysteine hydrochloride, and 1.0% glucose consistently yielded activated culture toxicities of 300,000 mouse intraperitoneal ld(50)/ml. Purification of the toxin was accomplished by precipitation with ammonium sulfate, extraction with calcium chloride, and reprecipitation with ethanol in the cold. The resulting partially purified toxin had a specific activity of 45,000,000 ld(50)/mg nitrogen. Purified toxins were converted to toxoid by incubation with formalin and adsorbed on aluminum phosphate. Good immune responses were obtained to the toxoids in mice, guinea pigs, and rabbits.
Gerwing, Julia (The University of British Columbia, Vancouver, Canada), Claude E. Dolman, and David A. Arnott. Activation phenomenon of Clostridium botulinum type E toxin. J. Bacteriol. 84:302-306. 1962-Highly purified preparations of both nonactivated and trypsin-activated type E botulinus toxins have been analyzed in an ultracentrifuge. Untreated botulinus type E toxin was found to have a sedimentation constant (S(20,w)) of 5.6 Svedberg units, whereas trypsin-activated toxins would not form a boundary under identical conditions. These and other considerations indicate that the mechanism of tryptic activation involves a fragmentation process whereby more toxic sites become exposed.
Clostridium novyi alpha-toxin and C. difficile toxins A and B (all 200 to 300 kDa) and C. botulinum C2-I toxin (50 kDa) caused a delayed and persistent retraction and rounding of microinjected cells. Microinjected phalloidin acted fast and reversibly. Unlike C2-I toxin, phalloidin passed through the intercellular junctions. Specific antitoxin applied to the medium did not prevent the action of microinjected C. novyi or C. difficile toxin B. Microinjected antitoxin protected against the toxins applied with the medium or injected into the same cells.
Electrical stimulation of multiple peripheral nerves, elicited "H" reflexes in a patient, 61 years old, with botulism. These reflexes are extremely suggestive of some central release or failure of inhibitory control of a monosynaptic or polysynaptic spinal reflex arc. This "central" action of botulinus toxin is similar to that suggested for tetanus toxin.
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The actions of tetanus toxin, botulinum A toxin, and black widow spider venom on the release of methionine-enkephalin-like immunoreactivity have been studied; a particulate fraction prepared from rat striata was used. Depending on the duration of preincubation, tetanus toxin diminished the release evoked by veratridine (50 microM final concentration), and abolished it at final concentrations between 0.1 and 1 micrograms/ml. Botulinum A toxin was about 10 to 20 times less potent. Heating or pretreatment with antitoxin inactivated the clostridial toxins. The particulate fraction pretreated with V. cholerae neuraminidase retained its toxin sensitivity. Tetanus toxin also depressed the release due to sea anemone toxin II and high K+. Spider venom stimulated the release in a concentration-dependent manner and required the presence of Ca2+; its effects were depressed by tetanus toxin. These results support the view that both clostridial toxins and spider venom act as broad-range presynaptic neurotoxins on peptidergic transmitter systems.
BOTULISM AND BIOWARFARE: Botulism is a severe neuro-paralysing infection due to a toxin produced by Clostridium botulinum. The use of the botulinum toxin for terrorist aims in the form of aerosols is a perfectly credible eventuality. The botulinum toxin is the most potent toxin known; it is easy to produce and can lead to massive destruction. DEPENDING ON THE CONTAMINATION: The clinical forms of botulism depend on the mode of contamination. Botulism through inhalation can only be the result of a deliberate act using an aerosol. The clinical symptomatology is identical to that of the other forms. PREVENTION: In the case of a bio-terrorist attack with an aerosol of botulinum toxin, the subjects exposed should be vaccinated as a prophylactic measure with trivalent antitoxin vaccine (types A, B and E). This vaccine must be administered as rapidly as possible in symptomatic patients. A single case of botulism acquired by inhalation corresponds to an act of terrorism.
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