Rapid Detection of Clostridium botulinum Toxin by Capillary Tube Diffusion

A micro capillary agar-gel diffusion system for the detection of botulinal toxin in foods and cultures was developed and evaluated. Toxins types A, B, and E, produced in culture broth with and without added trypsin, and type E toxin, produced in inoculated canned clams, were tested with this system and with the mouse bioassay procedure. With nontrypsinized toxin, the capillary diffusion system detected as little as 100 minimal lethal doses (MLD) per ml but was effective only at higher levels, 10(6) to 1.5 x 10(7) MLD/ml, when used with trypsinized toxin. The inability to detect lower levels of trypsinized toxin was due to thioglycolate present in the medium used to produce toxin. Evidently, trypsinization of toxin produces polypeptides still held together by disulfide bonds. Cleavage of these bonds by reduction with thioglycolate reduces the sensitivity of the capillary method. Trypsinized toxin produced in broth without thioglycolate was detected as readily as nontrypsinized toxin. Toxin was detected in canned clams containing as low as 100 MLD/ml. No cross-reactions were observed with type E toxin and types A and B antitoxins. Extensive studies using the capillary method for detecting types A and B toxins were not performed; however, a suspected sample of commercially canned mushrooms gave a positive type B reaction but not a type A reaction. This typing was confirmed later by the mouse bioassay. Toxin was present at a level of 100 MLD/ml. The procedure developed may prove useful as a rapid screening method for the detection of botulinal toxin in foods, with final identification made by using the mouse bioassay.     

Differentiation of Clostridium difficile Toxin from Clostridium botulinum Toxin by the Mouse Lethality Test

The mouse lethality test is the most sensitive method for confirming the diagnosis of infant botulism. Both Clostridium difficile and Clostridium botulinum produce heat-labile toxins which are lethal for mice and can be found in the feces of infants. These two toxins can be distinguished from one another in this assay when both are present in the same fecal specimen because they appear to be immunologically distinct toxins.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Molecular Construction of Clostridium botulinum Progenitor Toxin

Molecular dissociation of purified type F progenitor toxin with an S20,W of 10.3 and a molecular weight of 235,000 into two components, toxic and atoxic, was demonstrated by ultracentrifugation, gel filtration, and diethylaminoethyl-Sephadex chromatography at pH 7.5. The ultracentrifugal analysis indicated that type F progenitor toxin dissociates into components of the same molecular size of 5.9S. The toxic component contained a toxicity of 2.5 times 10-8 50% lethal doses per mg of N. Much higher stability of progenitor toxin than that of derivative toxin, particularly at pH below 5, suggests that only progenitor toxin can act as an oral toxin.     

Toxin Production in Clostridium botulinum as Demonstrated by Electron Microscopy

Spheroplasts of Clostridium botulinum 62A were prepared with the use of lysozyme. These spheroplasts were then exposed to ferritin-labeled type A antitoxin. Ultrathin sections of these specimens revealed the ferritin-labeled antibody symmetrically arranged around the outer spore coats but not within the spore cortex. The ferritin-labeled antibody was also observed in the bacterial cytoplasm. Here it was arranged in aggregates and strands, although it was not associated with any identifiable cell structure. Controls included sections of C. botulinum spheroplasts treated with a 1.5% solution of ferritin as well as spheroplasts of C. roseum and Bacillus subtilis treated with conjugated type A antitoxin or a 1.5% solution of ferritin. No intracellular or extracellular ferritin was demonstrable in these specimens.     

Purification of Clostridium botulinum Type F Progenitor Toxin

Clostridium botulinum type F progenitor toxin was purified to a homogeneous state as judged by gel filtration on Sephadex G-200, ultracentrifugation, and disc electrophoresis. The sedimentation constant, corrected to water at 20 C, of type F progenitor toxin was determined to be 10.3 and the molecular weight to be 235,000 by ultracentrifugation at pH 6.0. The purified toxin contained a toxicity of 1.2 x 10(8) 50% lethal doses/mg of N. In agar gel double diffusion, it formed two precipitin lines at pH 6.0. The progenitor toxin of type F differs from that of type A in that it contains no hemagglutinin and from that of type E in that it is not activable.     

Radioimmunoassay for Type A Toxin of Clostridium botulinum

A preparation of pure type A toxin of Clostridium botulinum was labeled with (131)I in the presence of chloramine-T and carrier-free isotope. The radioactive toxin ((131)I Tox) was used in a radioimmunoassay procedure similar to that of Berson and Yalow. Dilutions of antibody to the toxin, capable of binding 50% of the added (131)I Tox, were mixed with a sample of the labeled toxin and various concentrations of unlabeled toxin ('Cold' Tox). When concentration of Cold Tox were plotted against the ratio (131)I bound/(131)I not bound, a standard curve was established that could be used to estimate the concentration of Cold Tox in a test mixture. This assay was sensitive to as little as 100 mouse minimum lethal dose and was highly specific for the serological type of the toxin used.     

Activation of Clostridium botulinum Type B Toxin by an Endogenous Enzyme

It was previously postulated, based on indirect evidence, that Clostridium botulinum type B produces neurotoxin which is initially of low toxicity but which then becomes activated to highly toxic form by the action of an endogenous enzyme(s). The first direct in vitro experimental evidence in support of this hypothesis is presented here. The mildly active toxin (progenitor toxin) produced by C. botulinum type B (Lamanna) was isolated from the filtrate of a 24-hr culture and partially purified chromatographically. An enzyme that activates the progenitor toxin was also isolated from the filtrate of a 96-hr culture and purified 200-fold. The enzyme hydrolyzes synthetic substrates of trypsin but not of chymotrypsin.     

Purification and Properties of Clostridium botulinum Type F Toxin

Clostridium botulinum type F toxin of proteolytic Langeland strain was purified. Toxin in whole cultures was precipitated with (NH₄)₂SO₄. Extract of the precipitate was successively chromatographed on diethylaminoethyl-cellulose at pH 6.0, O-(carboxymethyl) cellulose at pH 4.9, Sephadex G-200 at pH 8.1, quaternary aminoethyl-Sephadex at pH 4.9, and finally diethylaminoethyl-cellulose at pH 8.1. The procedure recovered 14% of the toxin assayed in the starting culture. The toxin was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double gel diffusion serology, and isoelectric focusing. Purified toxin had a molecular weight of 150,000 by gel filtration and 155,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific toxicity was 9.6 × 10⁶ mean lethal doses per absorbancy (278 nm) unit. Sub-units of 105,000 and 56,000 molecular weight are found when purified toxin is treated with a disulfide reducing agent and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Reciprocal cross neutralizations were demonstrated when purified type F and E toxins were reacted with antitoxins which were obtained with immunizing toxoids prepared with purified toxins.     

Effect of Sodium Nitrite on Toxin Production by Clostridium botulinum in bacon

Pork bellies were formulated to 0, 30, 60, 120, 170, or 340 μg of nitrite per g of meat and inoculated with Clostridium botulinum via pickle or after processing and slicing. Processed bacon was stored at 7 or 27 C and assayed for nitrite, nitrate, and botulinal toxin at different intervals. Nitrite levels declined during processing and storage. The rate of decrease was more rapid at 27 than at 7 C. Although not added to the system, nitrate was detected in samples during processing and storage at 7 and 27 C. The amount of nitrate found was related to formulated nitrite levels. No toxin was found in samples incubated at 7 C throughout the 84-day test period. At 27 C, via pickle, inoculated samples with low inoculum (210 C. botulinum per g before processing and 52 per g after processing) became toxic if formulated with 120 μg of nitrite per g of meat or less. Toxin was not detected in bacon formulated with 170 or 340 μg of nitrite per g of meat under these same conditions. Toxin was detected at all formulated nitrite levels in bacon inoculated via the pickle with 19,000 C. botulinum per g (4,300 per g after processing) and in samples inoculated after slicing. However, increased levels of formulated nitrite decreased the probability of botulinal toxin formation in bacon inoculated by both methods.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Significance of 12S Toxin of Clostridium botulinum Type E

The pathogenesis of type E botulism is discussed as an aspect of the physicochemical and biological properties of 12S toxins (prototoxin and trypsin-activated 12S toxin) and the Ealpha and Ebeta components of each 12S toxin. A molecular weight of 350,000 was determined for each 12S toxin and 150,000 for Ealpha and Ebeta. Owing to the structure comprising the subunits Ealpha and Ebeta, 12S toxins are much more stable than Ealpha at low pH values and high temperatures. Such was also the case with type A 19S toxin and its alpha component. The Ealpha component alone accounts for the total toxicity of type E toxin. The toxic substance detected in the blood of the animals administered 12S toxins orally or parenterally was identified as Ealpha from the molecular size and the chromatographic pattern. Prototoxin escaping from detoxification in the stomach owing to the subunit structure may undergo dissociation in the intestine to release the Ealpha component. After absorption, the activated Ealpha appeared in the circulating blood without any further signs of dissociation or enzymatic digestion.     

Kinetics of the Thermal Inactivation of Type E Clostridium botulinum Toxin

Rate of inactivation curves for the "free" toxin, prototoxin, or activated toxin in crude filtrates of Clostridium botulinum type E were nonlinear, consisting of a fast inactivating rate followed by a slow inactivating rate. Thermodynamic parameters were calculated over a temperature range of 125 to 145 F (51.7 to 62.8 C) for the two different inactivation rates. Energy of activation was low at the lower temperature and high at the higher temperature. The thermal requirement for inactivating similar concentrations of the "free" toxin, prototoxin, or activated toxin was considered to be the same.     

Homogeneity and Molecular Weight of Toxin of Clostridium botulinum Type B

Gerwing et al. described the isolation and purification from culture filtrates of the toxin of Clostridium botulinum type B and characterized it as a homogeneous protein of less than 10,000 molecular weight. Analysis by various methods of samples of this toxin obtained from Gerwing et al., and preparations produced by their methods in our laboratories, furnished convincing evidence that neither her preparation nor ours was homogeneous. The molecular weight of the toxic component isolated from either of the preparations was 100,000 or greater and resembled, in a number of respects, the alpha component isolated by us from the crystalline toxin of C. botulinum type A.     

Storage Stability of Clostridium botulinum Toxin and Spores in Processed Cheese

Growth initiated from detoxified spores of Clostridium botulinum 62A resulted in toxin production of 50 to 10,000 mouse lethal doses (MLD) per gram of processed soft surface-ripened cheese. Regular assays during subsequent storage of toxic samples at 2 to 4 C revealed a characteristic two- to fivefold increase in toxin titer during the initial 1 week to 12 months of storage. Thereafter, the toxin titer remained constant for 2 to 4 years, after which the toxicity declined rapidly. At the end of 6 years of storage at 2 to 4 C, the samples still contained 20 to 5,000 MLD of toxin per gram, with the usual toxin level at 200 to 500 MLD. Toxic culture filtrates of C. botulinum incorporated into cheese and stored at 30 C for 60 days showed no decline in toxin in processed type I cheese, but toxin decreased slightly in processed type II and type III cheese. The surface flora of these cheeses did not attack but, on the contrary, protected C. botulinum toxin during storage at 30 C. Initial difficulties in recovering C. botulinum organisms from type I cheese were traced to growth inhibitory activity which could be removed by washing with distilled water in a centrifuge. Viable spores or vegetative cells could be recovered from all samples after 4 to 5 years of storage at 2 to 4 C. After 6 years, organisms were recovered from all except three samples of type I cheese. Two other samples showed a large decrease in viable organisms. In type III cheese, spores remained remarkably stable for 6 years at the level of the initial inoculum, i.e., approximately 10(5) spores per gram.