This toxin is made by Clostridium botulinum and sometimes Clostridium butyricum and Clostridium baratii bacteria. These bacteria can produce the toxin in food, wounds, and the intestines of infants. These bacteria make spores, which act like protective coatings. Spores help the bacteria survive in the environment, even in extreme conditions. But under certain conditions, these spores can grow and make one of the most lethal toxins known. The conditions in which the spores can grow and make toxin are:.
For example, improperly home-canned, preserved, or fermented foods can provide the right conditions for spores to grow and make botulinum toxin. Skip directly to site content Skip directly to page options Skip directly to A-Z link. Section Navigation. Dilute a portion of untreated sample fluid or culture to , , and in gel-phosphate buffer. Make the same dilutions of each trypsinized sample fluid or culture. Inject each of separate pairs of mice intraperitoneally i. Repeat this procedure with trypsin-treated duplicate samples.
Heat 1. Cool heated sample and inject each of a pair of mice with 0. These mice should not die, because botulinal toxin, if present, will be inactivated by heating.
Observe all mice periodically for 48 h for symptoms of botulism. Record symptoms and deaths. Typical botulism signs in mice begin usually in the first 24 h with ruffling of fur, followed in sequence by labored breathing, weakness of limbs, and finally total paralysis with gasping for breath, followed by death due to respiratory failure.
Death of mice without clinical symptoms of botulism is not sufficient evidence that injected material contained botulinal toxin. On occasion, death occurs from other chemicals present in injected fluid, or from trauma. If after 48 h of observation, all mice except those receiving the heated preparation have died, repeat the toxicity test, using higher dilutions of supernatant fluids or cultures.
It is necessary to have dilutions that kill and dilutions that do not kill in order to establish an endpoint or the minimum lethal dose MLD as an estimate of the amount of toxin present. The MLD is contained in the highest dilution killing both mice or all mice inoculated. Typing of toxin. Rehydrate antitoxins with sterile physiological saline. Do not use glycerin water. Dilute monovalent antitoxins to types A, B, E, and F in physiological saline to contain 1 international unit IU per 0.
Prepare enough of these antitoxin solutions to inject 0. Use the toxic preparation that gave the higher MLD, either untreated or trypsinized. Prepare dilutions of the toxic sample to cover at least 10, , and MLD below the previously determined endpoint of toxicity if possible see 2, above. The untreated toxic preparation can be the same as that used for testing toxicity.
If a trypsinized preparation was the most lethal, it will be necessary to prepare a freshly trypsinized fluid. The continued action of trypsin may destroy the toxin. Inject the mice with the monovalent antitoxins, as described above, 30 min to 1 h before challenging them with i.
Inject pairs of mice protected by specific monovalent antitoxin injection i. Also inject a pair of unprotected mice no injection of antitoxin with each toxic dilution as a control. The use of 4 monovalent antitoxins types A, B, E, and F for the unknown toxic sample prepared at 3 dilutions requires a total of 30 mice — 6 mice for each antitoxin 24 mice plus 2 unprotected mice for each of the 3 dilutions 6 mice as controls.
Observe mice for 48 h for symptoms of botulism and record deaths. If test results indicate that toxin was not neutralized, repeat test, using monovalent antitoxins to types C and D, plus polyvalent antitoxin pool of types A through F.
Place each smoked fish subsample which may consist of 1 or more fish, depending on size, and may be either vacuum-packed or bulk-smoked fish in a strong water-tight plastic bag. Add freshly steamed and cooled TPGY broth to subsample. Squeeze bag to expel as much air as possible and seal it with hot-iron bag sealer or other air-tight closure device.
Precautions should be taken during incubation period since bag may swell and split from gas formation. Use refrigerated centrifuge. If above 6. Refrigerate for overnight storage. Remove culture and let cool to room temperature before injecting mice. Trypsinized extract cannot be stored overnight. Toxicity screening.
Dilute trypsinized and nontrypsinized broth cultures to , , and in gel-phosphate diluent. NOTE : Do not store trypsinized material overnight.
Inject mice i. Inject 2 mice per dilution, i. Observe mice for botulism symptoms and record condition of mice at frequent intervals for 48 h. If no deaths occur, no further tests are indicated. Deaths are presumptive evidence of toxin and should be confirmed. Confirmation with protected mice.
Dilute new portion of nontrypsinized or trypsinized culture whichever showed the highest titer to , , and in gel-phosphate diluent. Do not store trypsinized material overnight. Inject 6 mice i.
These will be compared to 6 mice without this protection controls. After 30 min, inject 0. Record their condition at intervals up to 48 h.
If unprotected mice die and protected mice live, the presence of type E toxin is indicated. If all protected mice die, repeat confirmation with higher dilutions of toxic culture in type E-protected mice and with mice protected against C.
Isolate and identify cultures from samples containing toxin of type E, if possible. Obtain C. Reconstitute lyophilized antisera with sterile saline. Dilute sera with sterile saline for mouse injection. Telephone Retesting at higher dilutions of toxic fluids is required, and mixtures of antitoxins must be used in place of monovalent antiserum.
Some other toxic material, which is not heat-labile, could be responsible if both heated and unheated fluids cause death. The heat-stable toxic substance could possibly mask botulinal toxin. Toxic cultures may be more antigenic than purified toxins and the level of detection using the ELISA may be more sensitive than the mouse bioassay.
Prepare the sample and control dilutions while the plate is being blocked. The plate should be taken to the plate reader immediately after addition of the amplifier reagent and be ready to read the reactions.
Read absorbance at nm with nm subtraction reference filter to account for plate absorbance. The digoxigenin label substitutes for the biotin label in the amplified ELISA and is detected using an anti-digoxigenin horse radish peroxidase conjugate and TMB substrate.
Contact J. Ferreira FDA , S. Sharma FDA Maslanka CDC , or J. Andreadis CDC for questions regarding this method. Toxic cultures may be more antigenic than purified toxins and the level of detection using the DIG-ELISA may be more sensitive than the mouse bioassay. Both TPGY and CMM are tested since more toxin may be generated in one medium compared to the other and the confirmatory mouse bioassay also utilizes these media.
Generally, a fold dilution will show that the true toxin type will have a very high absorbance and the crossing type will have a negative absorbance. In either case the toxic sample must be confirmed using the mouse bioassay. Casein buffer control is used as a system control.
Duplicate wells are tested for each toxin type. Results are compared to the positive control that consists of toxin spiked into casein to demonstrate if the product inhibits the ELISA.
The product may be diluted further to remove inhibitory substances but will lower the sensitivity of the test. Positive sample wells will begin to turn a blue-green color. High toxin samples will develop color within a few minutes. The plate should be taken to the plate reader immediately after addition of the stop solution. Measure absorbance at nm on microplate reader. Craven or Joseph L. Telephone: ; FAX: Clostridium botulinum organisms generally produce one of four neurotoxin types A, B, E, and F associated with human illness.
Neurotoxin type determination is important in determining the identification of the bacterium. A PCR method was developed to identify 24 hour botulinal cultures as potential type A, B, E and F neurotoxin producers as well as culture of other clostridial species which also produce botulinal neurotoxins. Components of the PCR and amplification conditions were adjusted for optimal amplification of toxin gene target regions enabling the simultaneous testing for types A, B, E, and F in a single thermal cycler.
Each primer set was specific for its corresponding toxin type. Additionally, a DNA extraction procedure was included to remove inhibitory substances that may affect amplification.
This procedure is rapid, sensitive, and specific for the identification of toxigenic C. Because of the severity of neuroparalytic illness caused by botulinal neurotoxin, a rapid diagnosis for the specific toxin type is necessary during illness outbreaks suspected of being foodborne. The PCR technique has also been used to detect multiple botulinal toxin-producing types within a single PCR assay 4,6. The PCR assay for the toxin gene type is determined after a hour anaerobic culture to obtain vegetative cells.
ELISA procedures may require up to five days of culture growth before toxin is detected 5,9. The PCR method may also be used in conjunction with the mouse bioassay to determine toxin type. Procedure for amplification of C. Cell lysis by boiling can also be performed to simplify the procedure. Remove a 1. Remove the supernatants and place into a sterile microcentrifuge tube.
Manufacturers' protocol supplied with kits are followed. Unless DNA concentrations are determined before PCR analysis, it may be necessary to test dilutions of the DNA sample to avoid false negative results caused by too little or too much DNA when using commercially available kits.
Note: DNA purification before amplification is recommended to reduce the possibility of inhibitory substances in cultures from affecting the PCR and to increase the concentration of target DNA. The use of the described extraction procedure that incorporates Proteinase K and lysozyme consistently lysed C. The amount of isolated DNA yielding positive results using this amplification method ranged from approximately 0.
Using DNA concentrations outside this range may result in false negative results. This method is rapid and reliable for the identification of type A, B, E and F toxin-producing clostridial strains. PCR results for typing clostridial toxin genes were obtained in approximately 4 hours following a hour incubation of the culture. This method is not limited by culture production of the neurotoxin which requires up to five days incubation prior to analysis by ELISA or the mouse bioassay 3,5.
The PCR products also can be toxin gene typed or confirmed by using type-specific oligonucleotide or polynucleotide DNA probes. Oligonucleotide Primers. Desalted oligonucleotide primers are obtained from commerical suppliers.
Primers were derived from published DNA sequences for C. PCR reaction preparation. Primer sets for each of the types are used in separate PCR reactions. If necessary add approx. Thermal cyclers equipped with heated covers will not require the addition of a mineral oil overlay. Positive and negative controls should be included in each analysis. Negative controls containing all of the reagents but lacking template DNA processed as described above are used to monitor for contamination with C.
Temperature cycling. These four primer pairs can not be used together in one multiplex reaction because the primers are incompatible. Agarose gel analysis of PCR products. Prepare a 1. Agarose may be melted in 0.
Cast gel and allow to solidify. An appropriate molecular weight marker must be included on each gel in order to determine the approximate molecular weight of PCR products.
Molecular weight markers should contain fragments which bracket the target sequence size. A short-wave UV light is used to visualize bands relative to the molecular weight marker. Photographs of the gels are used to document the results using either a polaroid camera or a comparable gel documentation system. Chapter Solomon and Timothy Lilly, Jr. For additional information, contact Shashi Sharma Clostridium botulinum is an anaerobic, rod-shaped sporeforming bacterium that produces a protein with characteristic neurotoxicity.
Millipore filters: 0. Opening of canned foods see Chapter Detection of viable C. Selection of typical C. Detection and identification of botulinal toxin Preparation of food sample. Culture one portion of sample for detection of viable C. Centrifuge samples containing suspended solids under refrigeration and use supernatant fluid for toxin assay. Extract solid foods with equal volume of gel-phosphate buffer, pH 6.
Centrifuge macerated sample under refrigeration and use supernatant fluid for toxin assay. Rinse empty containers suspected of having held toxic foods with a few milliliters of gel-phosphate buffer.
Use as little buffer as possible to avoid diluting toxin beyond detection. To avoid or minimize nonspecific death of mice, filter supernatant fluid through a millipore filter before injecting mice. For non-proteolytic samples or cultures, trypsinize after filtration. Determination of toxicity in food samples or cultures Trypsin treatment.
General Hints Regarding C. Typical symptoms of botulism and death may occur within 4 to 6 hours. If deaths occur after 24 hours, be very suspicious, unless typical botulism symptoms are clearly evident.
If deaths occur in mice injected with the or dilution but not with any higher dilution, be very suspicious. Deaths may have been from nonspecific causes. Mice can be marked on tails with dye to represent various dilutions. Dye does not come off easily. Mice injected with botulinal toxin may become hyperactive before symptoms occur. Food and water may be given to the mice right away; it will not interfere with the test. Rehydrated antitoxin may be kept up to 6 months under refrigeration, and may be frozen indefinitely.
TPGY medium is relatively stable and can be kept weeks under refrigeration. With cooked meat medium, vortex tubes completely; toxin may adhere to meat particles. Trypsin is not filtered. Use 0. It can be kept up to 1 week under refrigeration.
Viable C. The presence of toxin in food is required for an outbreak of botulism to occur. Ingested organisms may be found in the alimentary tract, but are considered to be unable to multiply and produce toxin in vivo, except in infants. Swollen cans are more likely than flat cans to contain botulinal toxin since the organism produces gas during growth. Presence of toxin in a flat can may imply that the seams were loose enough to allow gas to escape. Botulinal toxin in canned foods is usually of a type A or a proteolytic type B strain, since spores of the proteolytics can be among the more heat-resistant.
Spores of nonproteolytics, types B, E, and F, generally are of low heat resistance and would not normally survive even mild heat treatment. The protection of mice from botulism and death with one of the monovalent botulinal antitoxins confirms the presence of botulinal toxin and determines the serological type of toxin in a sample. The following reasons may explain why deaths occur in mice that are protected by one of the monovalent antitoxins: There may be too much toxin in the sample.
More than one kind of toxin may be present. Deaths may be due to some other cause. Safety Precautions for the Clostridium botulinum Laboratory Place biohazard signs on doors to restrict entrance and keep the number of people in the laboratory to a minimum. All workers in the laboratory should wear laboratory coats and safety glasses. Use a biohazard hood for transfer of toxic material, if possible. Centrifuge toxic materials in a hermetically closed centrifuge with safety cups.
Personally take all toxic material to the autoclave and see that it is sterilized immediately. Do not work alone in the laboratory or animal rooms after hours or on weekends. Have an eye wash fountain and foot-pedaled faucet available for hand washing.
No eating and drinking in the laboratory when someone works with toxins. In a very visible location, list phone numbers where therapeutic antitoxin can be obtained in case of emergency. Reduce clutter in the laboratory to a minimum and place equipment and other materials in their proper place after use.
References Arnon, S. Infant botulism, pp. In : Pediatrics, 18th ed. Rudolph and J. Hoffman eds. Centers for Disease Control. Botulism in the United States, Handbook for epidemiologists, clinicians, and laboratory workers.
DHEW Publ. Hauschild, A. Hilsheimer, K. Weiss, and R. Clostridium botulinum in honey, syrups, and dry infant cereals. Food Prot. Contact Joseph L. Ferreira for questions about method. Cooked meat medium CMM. Check pH and adjust to 7. Goat type A or E, rabbit type B, or horse F antitoxin.
Botulinal complex toxin standards A, B, E, and F.
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