Botulism

Bacteriology at UW-Madison

The Microbial World

Lectures in Microbiology by Kenneth Todar PhD University of Wisconsin-Madison Department of Bacteriology

Botulism

Clostridium botulinum

Clostridium botulinum is a large anaerobic Gram-positive bacillus that forms subterminal endospores. It is widely distributed in soil, sediments of lakes and ponds, and decaying vegetation. Hence, the intestinal tracts of birds, mammals and fish may occasionally contain the organism as a transient.

Clostridium botulinum. CDC.

Seven toxigenic types of the organism exist, each producing an immunologically distinct form of botulinum toxin. The toxins are designated A, B, C1, D, E, F, and G). In the U.S. type A is the most significant cause of botulism, involved in 62% of the cases. Not all strains of C. botulinum produce the botulinum toxin. Lysogenic phages encode toxin serotypes C and D, and non lysogenized bacteria (which exist in nature) do not produce the toxin. Type G toxin is thought to be plasmid encoded.

Foodborne botulism in the United States 1990-2000. CDC

Pathogenesis of Botulism

Food-borne Botulism

In food-borne botulism the botulinum toxin is ingested with food in which spores have germinated and the organism has grown. The toxin is absorbed by the upper part of the GI tract in the duodenum and jejunum, and passes into the blood stream by which it reaches the peripheral neuromuscular synapses. The toxin binds to the presynaptic stimulatory terminals and blocks the release of the neurotransmitter acetylcholine which is required for a nerve to simulate the muscle.

Food-borne botulism is not an infection but an intoxication, since it results from the ingestion of foods that contain the preformed clostridial toxin. In this respect, it resembles staphylococcal food poisoning. Botulism results from eating uncooked foods in which contaminating spores have germinated and produced the toxin. C. botulinum spores are relatively heat resistant and may survive the sterilizing process of improper canning procedures. The anaerobic environment produced by the canning process may further encourage the outgrowth of spores. The organisms grow best in neutral or "low acid" vegetables (>pH4.5).

Clinical symptoms of botulism begin 18-36 hours after toxin ingestion with weakness, dizziness and dryness of the mouth. Nausea and vomiting may occur. Neurologic features soon develop: blurred vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles and respiratory paralysis.

Botulinum toxin may be transported within nerves in a manner analogous to tetanospasmin (tetanus toxin), and can thereby gain access to the CNS. However, symptomatic CNS involvement is rare.

Infant Botulism

Infant botulism is due to infection caused by C. botulinum. The disease occurs in infants 5 - 20 weeks of age that have been exposed to solid foods, presumably the source of infection (spores). It is characterized by constipation and weak sucking ability and generalized weakness. C. botulinum can apparently establish itself in the bowel of infants at a critical age before the establishment of competing intestinal bacteria (normal flora). Production of toxin by bacteria in the GI tract induces symptoms. This "infection-intoxication" is restricted to infants. C. botulinum organisms, as well as toxin can be found in the feces of infected infants. Almost all known cases of the disease have recovered. The possible role of infant botulism in "sudden infant death syndrome-SIDS" has been suggested and is under investigation. C. botulinum, its toxin, or both have been found in the bowel contents of several infants who have died suddenly and unexpectedly.

The Botulinum Toxins

The botulinum toxins are very similar in structure and function to the tetanus toxin, but differ dramatically in their clinical effects because they target different cells in the nervous system. Botulinum neurotoxins predominantly affect the peripheral nervous system reflecting a preference of the toxin for stimulatory motor neurons at a neuromuscular junction. The primary symptom is weakness or flaccid paralysis. Tetanus toxin can affect the same system, but tetanospasmin shows a tropism for inhibitory motor neurons of the central nervous system, and its effects are primarily rigidity and spastic paralysis.

Botulinum toxin is synthesized as a single polypeptide chain with a molecular weight around 150 kDa. In this form the toxin has a relatively low potency. The toxin is nicked by a bacterial protease (or possibly by gastric proteases) to produce two chains: a light chain (the A fragment) with a molecular weight of 50 kDa; and a heavy chain (the B fragment), with a mw of 100kDa. As with tetanospasmin, the chains remain connected by a disulfide bond. The A fragment of the nicked toxin, on a molecular weight basis, becomes the most potent toxin found in nature.

Structure of the botulinum toxin

Toxin Action

The botulinum toxin is specific for peripheral nerve endings at the point where a motor neuron stimulates a muscle. The toxin binds to the neuron and prevents the release of acetylcholine across the synaptic cleft.

The heavy chain of the toxin mediates binding to presynaptic receptors. The nature of these receptors is uncertain; different toxin types seem to utilize slightly different receptors. The binding region of the toxin molecule is located near the carboxy terminus of the heavy chain. The amino terminus of the heavy chain is thought to form a channel through the membrane of the neuron allowing the light chain to enter. The toxin (A fragment) enters the cell by receptor mediated endocytosis. Once inside a neuron, the toxin types probably differ in mechanisms by which they inhibit acetylcholine release, but a mechanism similar to or identical to tetanospasmin has been reported (i.e., proteolytic cleavage of synaptobrevin II). The affected cells fail to release a neurotransmitter, thus producing paralysis of the motor system. Once damaged, the synapse is rendered permanently useless. The recovery of function requires sprouting of a new presynaptic axon and the subsequent formation of a new synapse.

As stated above, the mechanism by which acetylcholine release is prevented is not known. However, recent evidence suggests that both botulinum toxin as well as tetanus toxin are zinc-dependent endopeptidases that cleave specific proteins that are involved in excretion of neurotransmitters. Both toxins cleave a set of proteins called synaptobrevins. Synaptobrevins are a set of proteins found in synaptic vesicle of neurons, the vesicles responsible for release of neurotransmitters. Presumably, proteolytic cleavage of synaptobrevin II would interfere with vesicle function and release of neurotransmitters.

Immunity

On the average there are about 25 cases of botulism annually in the U.S. Prior to the advent of critical care, the case fatality rate exceeded 60%, but currently it is about 20%. The first (or only) patient in an outbreak has a 25% chance of death, whereas subsequent cases which are diagnosed and treated more quickly, carry only a 4% risk.

The toxins that cause botulism are each specifically neutralized by its antitoxin. Botulinum toxins can be toxoided and make good antigens for inducing protective antibody. As with tetanus, immunity to botulism does not develop, even with severe disease, because the amount of toxin necessary to induce an immune response is toxic. Repeated occurrence of botulism has been reported.

Once the botulinum toxin has bound to nerve endings, its activity is unaffected by antitoxin. Any circulating ("unfixed") toxin can be neutralized by intravenous injection of antitoxin. Individuals known to have ingested food with botulism should be treated immediately with antiserum.

A multivalent toxoid evokes good protective antibody response but its use is unjustified due to the infrequency of the disease. An experimental vaccine exists for laboratory workers.

Prevention

The most important aspect of botulism prevention is proper food handling and preparation. The spores of C. botulinum can survive boiling (100 degrees at 1 atm) for more than one hour although they are killed by autoclaving. Because the toxin is heat-labile boiling or intense heating (cooking) of contaminated food will inactivate the toxin. Food containers that bulge may contain gas produced by C. botulinum and should not be opened or tasted. Other foods that appear to be spoiled should not be tasted.

Botulinum Toxin in Biowarfare

The use of botulinum toxin in biowarfare has been considered because botulinum toxin is the most potent poison known for humans; 10 grams is a lethal dose for the human population of Los Angeles. Below is an interesting anecdote that appeared in JAMA Vol. 285, No. 21, June 6, 2001

To the Editor:
A historical incident illustrates a number of features of botulinum toxin not discussed in the review of bioweaponry by Dr Arnon and colleagues.

During World War II, the US Office of Strategic Services (OSS) developed a plan for Chinese prostitutes to assassinate high-ranking Japanese officers with whom they sometimes consorted in occupied Chinese cities. Concealing traditional weapons on the women at the appropriate time would obviously be difficult. Therefore, under the direction of Stanley Lovell, the OSS prepared gelatin capsules "less than the size of the head of a common pin" containing a lethal dose of botulinum toxin. Wetted, a capsule could be stuck behind the ear or in scalp hair, later to be detached and slipped into the officer's food or drink. The OSS recognized that the normal background of botulism cases would deflect suspicion from the women.

The capsules were shipped to Chunking, China. The Navy detachment there, taking nothing for granted, tested the capsules on stray donkeys. The donkeys lived. Lovell was informed that the capsules were faulty, and the project was abandoned. Much later, Lovell learned of the donkey test with, one imagines, some consternation, since "donkeys are one of the few living creatures immune to botulism."

This incident has been retold in other publications. No source for the donkey-resistance information is ever given. More recent experience shows that botulism can occur in mules and donkeys (R. H. Whitlock, DVM, PhD, written communication, April 27, 2001).

Nevertheless, this incident raises 2 points: (1) botulism need not occur in epidemics when it is being used as a bioweapon, and (2) botulism in animals may be a sign of biowarfare.

Links

CDC - Botulism: Disease Information

CDC - Disease Listing, Botulism, General Information

CDC - Facts About Botulism

CDC - Technical Information About Botulism

CDC - Video: "The History of Bioterrorism: Botulism"

CDC - What treatment is available for botulism?

IL Public health - Botulism Fact Sheet

JAMA - Botulinum Toxin as a Biological Weapon: Medical and Public Health Management

Infectious Diseases Society of America: Botulism Resource List