Botulism neurotoxins

Schiavo, G., Rosetto, O., Tonello, F., Montecucco, C. (1995). Intracellular targets and metalloprotease activity of tetanus and botulism neurotoxins. Curr. Top. Microbiol. Immunol. 195 257-74. 

Botulism neurotoxins bind with synaptic vesicular proteins and block the release of acetylcholine from the presynaptic membrane (Osborne et al, 2007). Clinical signs of botulism are weakness, tremors, recumbency, laryngeal paresis, and other signs of nervous system dysfunction (Braun et al, 2005). Botulism toxins do not appear to be excreted in milk (Galey et al, 2000). 

Adler, M., Oyler, G., Apland, J.P. et al. (2008). Mechanism of action of botulism neurotoxin and overview of medical countermeasures for intoxication In Chemical Warfare Agents Chemistry, Pharmacology, Toxicology, and Therapeutics (Romano, J.A., Jr., Lukey, B.J., Salem, H., eds), pp. 389-422. CRC Press, Boca Raton, FL. 

Sanchez-Prieto J, Shira TS, Evans D, Ashton A, Dolly JO, Nicholls DG (1987) Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes. In Eur. J. Biochem. 165 675-81 Schiavo G, Montecucco C (1995) Tetanus and botulism neurotoxins isolation and assay. Methods Enzymol. 248 643-52... 

Montecucco C, Schiavo G (1993) Tetanus and botulism neurotoxins a new group of zinc proteases. Trends Biochem. Sci. 18 324-7. 

Arndt, J.W., Chai, Q, Christian, T., and Stevens, R.C. 2006. Structure of botulism neurotoxin type D light chain at 1.65 A resolution repercussions for VAMP-2 substrate specihcity. Biochemistry 45 3255-3262. 

Detection and identification of the botulism neurotoxin have been essential for diagnosis of the illness and for identifying the causative food. When van Ermengem showed the lethality for animals of the ham that caused the Ellezelles botulism outbreak in 1895 (73), the bioassay naturally became the standard test for botulism neurotoxin. The mouse is very sensitive, and the mouse LD50 determined by i.p. injection became the quantitative unit. 

Table 1. Clostridia that produce botulism neurotoxin...

Tests for detection and quantitation of botulism neurotoxins are used for the following purposes ... 

Table 2. Methods for detection of botulism neurotoxin and determining neurotoxigenicity of bacterial strains...

The small size of the mouse and its high sensitivity to all types of botulism neurotoxin have made it ideal for assaying biologic activity. The usual test is performed by intraperi-toneal injection (31,39,63). The ability to observe the classic symptoms of the illness when working with unknown specimens enhances its specificity (Figure 1). 

A stable reference toxin derived from a preparation of crystalline type A neurotoxin was proposed for standardizing botulism toxin bioassays performed in different laboratories under diverse conditions (63). Standardized procedures for using the bioassay for detecting botulism neurotoxin in foods and identifying toxigenicity and toxin type of the organisms were established on the basis of the results of a collaborative study (40). 

Intramuscular injection of 0.1 ml of toxin into the gastrocnemius muscle of mice with the observation of local paralysis as the endpoint has been studied as an alternative method by Sugiyama et al. (68). The 50% paralytic endpoint dose (ED50) was between 0.06 and 0.09 i.p. LD50 for all toxin types A-F. Pearce et al. (54) used both the lethality and muscle paralysis assays to compare the potency of two commercial preparations of type A botulism neurotoxin marketed for clinical use in treatment of neuromuscular disorders. They found disparities between the relationship between the activities for each preparation 0.2 lethal units of the... 

Many of the early ELISA methods devised for botulism neurotoxin detection, like most of the in vitro tests,suffered from a lack of specificity, due to impurities in the antigen preparation used to produce the antitoxins. More purified toxins are now available for the production of better quality antitoxins. The most sensitive ELISA protocols use an indirect assay sometimes referred to as the sandwich assay. In the basic procedure, a specific antitoxin is first adsorbed to the surface of the wells of a plastic plate. The toxin added to the wells is then bound by these antibodies and detected with a second antitoxin which is conjugated to an enzyme or other labeling molecule. The amount of label is measured by supplying the enzyme substrate, which is converted to a colored product that is measured colorimetrically. Some ELISA protocols use a polyclonal antitoxin on one side of the sandwich and a monoclonal on the other side. Other assays use the same antibody for both sides but label the antibody the second time it is used. A modification of the sandwich assay is the double sandwich ELISA, which employs a third antibody that is conjugated to an enzyme and is directed against the second antitoxin it is an anti-antibody such as rabbit anti-horse IgG. The steps in a typical application of this assay for botulism toxin are shown in Figure 2. 

Figure 2. Double sandwich ELISA. Scheme for a typical adaptation for detecting botulism neurotoxin.

Figure 6. Results of type A, B, and E botulism neurotoxin assays using the ELCA sensitivity and specificity for each type can be seen. Photograph from reference 10, used with permission of the authors and the publisher.

The biosensor-fiberoptic approach to detection of botulism neurotoxin promises the most rapid results, but at this early stage in its development, its sensitivity is much less than that of the bioassay and the microtiter assays. 

Franciosa, G., Ferreira, J.L., and Hatheway, C.L., 1994, Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR evidence of unexpressed type B toxin genes in type A toxigenic organisms, J. Clin. Microbiol. 32 1911-1917. 

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