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. 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:. A specific toxicity of 1. Botulinum neurotoxins BoNT , which are produced by Clostridium botulinum , an anaerobic spore former, belong to the most toxic biological substances. For more than a century, research focused on the structure and pathogenesis of the seven distinct neurotoxins A—G and the associated diseases.
Types A, B and E are generally considered as being pathogenic for humans, types C and D are often isolated in cases of animal botulism. One of the first reports on botulism was written by Justinus Kerner, a German physician and poet. But he did not only increase our basic knowledge on the disease and the causative agent. However, it lasted years until the benefits of BoNT were discovered and investigated again [2]. Since then, BoNT has become an important agent in treating strabism, blepharospasm, dystonia and other, similar diseases, caused by neuro-muscular disorders [3].
This, on the other hand, increased the demand for purified BoNT, which is also needed in basic neurologic research, e. Previously published purification procedures for BoNT usually include an initial precipitation [5 , 6] or at least a centrifugation step [7]. Based on previous experiences with hollow-fiber cross flow filtration [8] , we have attempted to develop an initial, self-contained two-step filtration, which separates the metabolites including the toxin from the bacteria and the low molecular weight substances, e.
Kozaki, Osaka Prefecture University, Japan were used in this study. The separation was run at a constant voltage of V. To reduce the number of animals needed in this study, 0. In addition samples of the fermentor, harvests and the purified toxins were diluted 2-fold in between the positive and negative fold dilutions. The minimal lethal dose MLD describes the reciprocal value of the highest dilution at which both mice died.
The hemagglutinating activity of harvest and chromatographic fractions was quantified in a microtiter plate assay. Plates were incubated at room temperature overnight.
The hemagglutination titer corresponds to the reciprocal value of the highest dilution at which agglutination of the cells could be observed. Inoculum of both strains was prepared in ml flasks. The flasks were placed in an anaerobic jar. Initially, the pH was adjusted to 6. For the two following batches of each strain, no inoculum was prepared. In general, type C and D toxin were purified according to the same procedure.
The filtration and further purification steps of the toxins are summarized in Fig. Column hardware and resins were supplied by Amersham Pharmacia if nothing else is stated. If a presumptive toxin band could be detected electrophoretically within a peak, the fraction was checked for the presence of toxin in the mouse bioassay, but only the purified toxin was quantified.
For hydrophobic interaction chromatography, the concentrated supernatant was applied to a phenylsepharose HP column, equilibrated with 50 mM Tris-HCl, pH 8. The toxin was eluted with a linear gradient from 1. The dialyzed fractions were further purified on an anion exchange column, which was packed with Source 30 Q. Again, the fractions were checked for the presence of toxin and the positive fractions pooled and dialyzed against 50 mM Bis-Tris, pH 6.
The next step was the application of the pool to a Source 15 Q column, pre-equilibrated with the dialyzing buffer. Elution was run with a linear gradient of 0. For the analytical anion exchange chromatography on a MonoQ column, 1 ml of the toxin solution was diluted fold to decrease the NaCl concentration.
The same buffers with pH 8. Protein concentrations of the fermentor harvest, the filtrated cultures and the purified toxin were measured with a commercially available assay BCA Protein Assay, Pierce. During the three consecutive batches, an average amount of 4. This corresponds to 4. The data given represent the average values of the three consecutive batches run with each strain. During the hollow-fiber filtration, the cultures could be concentrated more than fold.
In hydrophobic interaction chromatography, no toxin was detected in the fractions containing the unbound proteins. The toxin started to elute at an ammonium sulfate concentration of mM. However, separation of the hemagglutinin could only partially be achieved, although alkaline conditions pH 8.
In the following anion exchange chromatography at pH 8. At pH 6. No hemagglutinin activity could be detected within any fraction. During final polishing on Superdex , only one homogeneous peak eluted, containing the neurotoxin. Chromatograms of the purification steps are shown in chronological order. According to the mouse bioassay, an average overall toxicity yield of A high purity of the toxins was proven with the use of the MonoQ column.
All the protein present in the sample bound to the column and eluted in one distinct peak. No impurities could be detected electrophoretically.
The purification usually starts with an ammonium sulfate [5] or an acid precipitation [6].
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