|Year : 2001 | Volume
| Issue : 2 | Page : 35-43
Botulism: An update
S Mohanty , B Dhawan , R Chaudhry
Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 111 029,, India
Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 111 029,, India
Botulism is a paralytic illness caused by the action of a neurotoxin elaborated by Clostridium botulinum. Other clostridial bacteria, like C.butyricum and C.baratii can also produce the toxin leading to signs and symptoms of botulism. Though rare, the illness is potentially fatal and can masquerade as other illnesses making diagnosis difficult. Physicians need to familiarize themselves with the disease as prompt recognition and early treatment can considerably curtail the fatal outcome in the affected and prevent additional cases in the unaffected. New diagnostic, therapeutic and preventive modalities to tackle the disease have come into focus. Botulinum toxin, generally considered a potent poison, is successfully being used for treatment of various neuromuscular disorders representing one of the most dramatic role reversals of modern times.
|How to cite this article:|
Mohanty S, Dhawan B, Chaudhry R. Botulism: An update. Indian J Med Microbiol 2001;19:35-43
More than hundred and fifty years back, in 1817, the German physician and poet, Justinus Kerner (1786-1862) published the earliest systematic clinical descriptions of a paralytic illness affecting 230 patients. , The disorder was called botulism because sausages (botulus in latin) was found to be a source of infection. Kerner believed that contaminated sausages contained a toxic substance, “fatty acid” which was responsible for the illness. Being a district health officer, he made sausage poisoning or botulism, a notifiable disease. Later, in 1897, van Ermengen gave the first description of the anaerobic bacterium causing the outbreaks and the pathogenesis of the disease. Initially known as Bacillus botulinus, the nomenclature of the causative agent was changed to Clostridium botulinum >to acknowledge both its spindle shape (kloster is Greek for spindle) and its anaerobic metabolism (bacilli were by definition aerobes). 
Botulism is a potentially lethal paralytic illness resulting from the action of a potent neurotropic exotoxin elaborated by Clostridium botulinum, an anerobic, spore forming, gram positive rod. Outbreaks of botulism are a public health emergency that require prompt recognition and effective treatment to prevent additional cases in the community. The disease can masquerade as other illness, and seemingly unlikely food items can harbour the toxin. In the event of possible use of botulinum toxin as a biological warfare agent, early recognition becomes an even more critical component. Infant and wound botulism which were rare till the last quarter of 20th century have risen to usurp a position comparable to that of food borne botulism. New modes of transmission and newer aspects of pathogenesis have come to light. In addition, new diagnostic, therapeutic and preventive modalities are being developed to tackle this fatal disease of mankind.
| ~ The organism|| |
Clostridium botulinum is not a single species. Instead it a constellation of culturally distinct groups of organisms that among them, produce seven (A, B, C1, D, E, F, G) serologically distinct toxins, all with similar pharmacological action.  In addition, two more toxins, C2 and C3 of unknown significance are also produced by some species. 
Human botulism is primarily caused by toxin types A, B or E. Toxin types C and D mostly cause outbreaks of botulism in animals and birds. Strains of C.botulinum producing type G toxin though not definitely implicated in human or animal botulism, have, however been isolated from five autopsy samples.  Clostridium botulinum producing type G toxin was discovered by Gimenez and Ciccarelli from soil samples in western Argentina. Since it appeared to be different from other C.botulinum strains in that it did not produce lipase, this bacterium was later renamed from C.botulinum type G to Clostridium argentinense.  Recently, neurotoxigenic strains of C.butyricum , (which produce type E toxin) and C.baratii  (which produce type F toxin) have been shown to be capable of causing human botulism [Table:1]. Neurotoxigenic strains of C.butyricum have been shown to be capable of causing food-borne botulism. Both C.butyricum  and C.baratii  have been implicated in cases of infant botulism as well. In addition, C.baratii has also been implicated in cases of wound botulism. 
| ~ Epidemiology|| |
Clinical forms of botulism described in humans include : food-borne botulism, infant botulism, wound botulism, adult infectious botulism (adult intestinal colonization), inadvertent botulism and classification undetermined. In the United States, toxin type A is responsible for approximately half of the cases of food borne botulism, remaining cases almost equally divided between toxin types B and E.  Most of the type E outbreaks are associated with consumption of fish or fish products. In France, toxin type B has been identified in 97% of cases, rest being of type A or rarely of type E.  Among cases of infant botulism, approximately half are caused by toxin type A and half by toxin type B  with occasional cases caused by toxin types C, E, F and G. ,,, Among cases of wound botulism, approximately 80% are caused by toxin type A and 20% by toxin type B. 
Food borne botulism
Food borne botulism is the classic form of botulism in which preformed toxin is ingested. The ubiquitous presence of C.botulinum spores in soil and agricultural products makes contamination of human foods by C.botulinum spores and ingestion of spores not an unlikely occurrence . However, simple ingestion of spores is generally not sufficient to cause botulism due to the presence of the normal anaerobic flora of the GI tract that prevents C.botulinum colonization. Botulism results when the heat resistant spores survive food preservation methods that kill nonsporulating organisms, subsequently germinating and producing a potent neurotoxin under anaerobic, low acid (pH >4.6), and low solute conditions., Growth is most likely to occur in foods that have been heated and then cooled and stored for long periods at room temperature. Botulism, however, need not result in all such cases, because boiling the prepared food for 10 to 15 minutes before consumption inactivates the toxin.
Cases of botulism for the most part are associated with consumption of contaminated home-canned foods and foods fermented or preserved with traditional methods before eating. Recently identified vehicles for food-borne botulism include sevu, home cured ham,  commercial cheese sauce,  home canned bamboo shoots,  vacuum packaged hot smoked white fish,  locally made cheese  matambre in heat shrunk plastic wrap,  home preserved tomato sauce  and baked potatoes sealed in aluminium foil.  Canned food is not always the culprit. Insufficient cooking time and temperatures, storage in heat-shrinked plastic wrap; and inadequate refrigeration may also contribute to C.botulinum spore survival, germination and toxin production.
Infant botulism as a distinct clinical entity was first appreciated only in 1976, when two cases were reported by Pickett in California.  Statistics from the CDC show that this has been the predominant form of botulism recognised in the United States since then.  The yearly number of infant botulism cases now exceeds the number of foodborne and wound botulism combined. This special form of botulism occurs in children less than 1 year of age who have not yet developed a complete colonic microflora thereby promoting colonization with live C.botulinum bacteria and subsequent toxin production. This form of botulism is therefore not a true example of a preformed toxin. Infant botulism has been reported from countries on all the inhabited continents except Africa.  Approximately 250 cases occur in the United States each year, majority being reported from California, Utah and Pennsylvania,,,
Several risk factors have been associated with the development of infant botulism. , They include age (median age of onset 2 to 4 months), ingestion of honey and ingestion of corn syrup. Because bees moving from plant to plant accumulate spores along with pollen, honey frequently contains C.botulinum spores and has been implicated as the source in about 15% of cases of infant botulism. The source of C. botulinum spores in remainder of cases is unknown, but is presumed to be soil or house dust.
A link between infant botulism and sudden infant death syndrome (SIDS) came to light when a similarity was noted between the sudden respiratory arrest of an infant with botulism and SIDS. Two series have identified C.botulinum infection in 4.3% and 15% of SIDS cases.  A recent prospective study done on 248 victims over a 10-year period from 1981 to 1990 in Australia, however failed to implicate botulism as a significant risk factor in the causation of SIDS. 
Botulism in patients with wounds occurs when anaerobic conditions within an abscessed wound allow germination of C.botulinum spores, subsequent multiplication of the organisms and production and absorption of toxin in vivo. Cases of wound botulism developing as a complication of open fracture,  lacerating wounds of hands and wrist, head injury,  and tooth abscess  have been reported in literature. This form of botulism has increased dramatically in the last few years in California among injecting drug abusers acquiring an epidemic proportion during the period 1994-1996. Wound botulism attributable to drug injection was first reported in 1982 in new York City  and has been reported only sporadically since then. However, since 1990, the number of wound botulism cases reported annually in California has increased steadily. Many cases involved “skin popping” (injecting the drug subcutaneously or intramuscularly rather than intravenously) of a form of heroin called “black tar heroin”. Spores inoculated into subcutaneous tissue-either from the drug or from the skin after inadequate skin disinfection can germinate and produce toxin. 
Adult infectious botulism (Adult intestinal colonization)
In rare instances, a disease similar to that of infant botulism can occur in adults as a result of intestinal colonization with C. botulinum and in vivo toxin production. Such patients often have a history of abdominal surgery, gastrointestinal tract abnormalities, or recent antibiotic treatment that may disrupt the natural gartrointestinal flora. Cases have been caused by C. botulinum producing toxin types A and B, C. baratii producing toxin type F and C.butyricum producing toxin type E. ,, Voluminous Meckel's diverticula were resected from the patients infected with C. butyricum producing toxin type E, leading to ongoing speculation for causal relationship between Meckel's diverticula and C. butyricum producing type E toxin. ,
Recently, the CDC established another category- classification undetermined- which includes those cases of botulism in persons older than one year of age for which no vehicle is identified. 
This type of botulism occurs in patients who have been treated with injections of botulinum toxin for dystonic and other movement disorders.
| ~ Botulinal toxins and pathogenesis|| |
Though a given strain of C. botulinum produces only one serologically distinct toxin, some, such as types C and D strains  and types A and B strains  producing multiple toxins have been documented. The nucleotide sequences for all toxin types have been sequenced.  Comparison of nucleotide sequences between the botulinum toxin and tetanus toxin genes suggest that clostridial neurotoxins employ similar mechanism of action and the neurotoxin genes are probably derived from a common ancestor.  The fact that hitherto saprophytic clostridia such as C.butyricum and C.baratii can produce a botulinum neurotoxin suggest that the toxin genes can be transferred between different clostridium strains.  The genes for type A, B, E and F toxins, seem to be chromosomally located, genes for types C and D are carried by pseudolysogenic bacteriophages and that for type G is reported to be on an 81-Mda plasmid. , Recently, the transfer of toxin type E gene in C. butyricum has been demonstrated to be phage mediated as well. 
Botulinum toxins are synthesized as a single polypeptide chain with a molecular mass of 150 kDa. Proteolytic cleavage of this parent chain results in a heavy chain (approximately 100 kDA) linked by a disulfide bond to a light chain (approximately 50 kDa). The light chain is the catalytic subunit of the toxin, whereas the heavy chain contains an N-terminal translocation domain and a C-terminal binding domain. After binding, internalization takes place via a receptor mediated endocytic pathway; membrane acceptors probably consist of membrane gangliosides and proteins. Subsequently, the disulfide bond is cleaved by an unknown mechanism and the light chain is translocated across the endosomal membrane.
The light chain of botulinum toxins like tetanus toxin has been shown to be zinc endopeptidases.  But whereas the peripheral muscle spasms which characterize tetanus are due to a blockade of inhibitory (GABAergic and glycinergic) synapses in the central nervous system, botulism symptoms are only peripheral, consequent to a near irreversible and highly selective inhibition of acetylcholine release at the motor nerve endings innervating skeletal muscles. This occurs when the endopeptidases very specifically cleave one or more proteins of the neuroexocytosis apparatus. Tetanus toxin and botulinum toxins serotypes B,D,F, and G cleave at single sites, which differ for each neurotoxin, VAMP/synaptobrevin, a protein associated with the synaptic vesicle. Toxin types A and E, cleave SNAP-25, a protein associated with the presynaptic membrane at two different carboxyl terminal peptide bonds. Toxin type C seems to be able to cleave both SNAP-25 and a protein on the target membrane, syntaxin. ,
Toxins C2 and C3
Along with botulinal toxin, two additional toxins C2 and C3 of unknown significance are found in some strains of C.botulinum. C2 toxin ADP-ribosylates intracellular actin leading to disruption of normal microfilament formation, however, it does not cause paralysis and is not a neurotoxin. Probably, it aids the dissemination of botulinal toxin by increasing vascular permeability. C3 toxin ADP-ribosylates a 21-kDa GTP - binding protein of unknown function in the host cell; it possibly exerts a toxic effect by interfering with signal transduction. 
| ~ Clinical signs and symptoms|| |
Regardless of the manner in which the toxin enters into the body, it must reach the general circulation for its lethal effect to be manifest. The clinical picture is dominated by a constellation of neurologic signs and symptoms resulting from toxin-induced blockade of acetylcholine release at the voluntary motor and autonomic cholinergic junctions.
Although the syndrome is similar for each toxin type, that due to type A is reported to be more severe and fatal than type B or E.  Classically, food-borne botulism begins with a prodrome of nausea, vomiting, constipation, or cramps followed by gradual onset of neurologic symptoms 1 to 2 days after ingesting contaminated food. Neurologic symptoms at the onset include dryness of the mouth, dysphagia, dysphonia and inability to produce sweat or tears. ,, Later on, dysarthria, dyspnoea and peripheral muscle weakness supervenes., Ocular symptoms are observed in 90-92% cases and consist of diplopia, ptosis and loss of accommodation associated with mydriaris. , The illness is characterized by symmetric descending weakness or paralysis in the absence of sensory abnormalities. Fever is typically absent and consciousness remains completely preserved during the paralytic period. The cerebrospinal fluid protein level is normal and edrophonoum chloride (Tensilon) test is negative.
Infant botulism is characterized by constipation, poor feeding, lethargy, a weak cry, difficulty in sucking and swallowing and poor muscle tone. The baby often appears “floppy”. , Infant botulism may have a delayed onset because absorption of toxin from colon is slow as compared to that from stomach. The clinical manifestations of wound botulism are similar to food borne botulism, except that gastrointestinal symptoms are absent and the median incubation period is longer [7 days (range, 4 to 14 days)].  In severe cases of botulism, paralysis of respiratory muscles may lead to ventilatory failure and death. Before mechanical ventilation and intensive supportive care were available, up to 60% of patients died; since the 1950s, however, the mortality rate from botulism has steadily decreased.  Death now occurs in 7.5% cases of food borne botulism and 5% cases of infant botulism, Apart from respiratory compromise, other complications include syndrome of inappropriate antidiuretic hormone (SIADH) production, urinary tract infections, seizures and cardiac arrhythmias. , Following the illness, restoration of neuromuscular functions occurs gradually over days to weeks owing to axon terminal sprouting. Formation of new presynaptic end plates and neuromuscular junctions leads to certain amount of clinical improvement though complete recovery rarely occurs. 
| ~ Diseases and conditions confused with botulism|| |
Several disorders may mimic botulism and must be considered in the evaluation of afebrile, mentally intact patients with symmetric descending paralysis. These include Guillaine-Barre syndrome (especially the Miller Fisher Variant), myasthenia gravis, poliomyelitis, the Eaton_Lambert syndrome, the stroke syndrome, organophosphate or atropine intoxication, carbon monoxide poisoning, tick paralysis, acute intermittent porphyria (AIP) and paralytic shellfish poisoning.,,, 
| ~ Diagnosis of botulism|| |
Confirmatory tests of botulism involve tests for the detection of toxin by bioassay in mice, but the test may be negative in infant and wound botulism. Therefore, a search for clostridial bacteria, including C.butyricum and C.baratii, in addition to C. botulinum is also warranted. Before confirmatory tests, ancillary tests such as Ct or MRI of the head, Tensilon test and electromyogram may be performed. Recently, single fibre electromyography has been advocated to be of much more value in determining neuromuscular transmission abnormalities.  Nerve conduction studies are found to be normal. 
For toxin assay and anaerobic culture, suitable samples include serum, stool, the epidemiologically implicated food items and when possible, gastric aspirate, enema fluid and autopsy sections of the small and large intestine. The mouse bioassay can be performed at the CDC or some state public health laboratories. Botulinum toxin type is determined by neutralizing the biological activity of toxic samples injected into mice with type specific botulinum anti-toxin. ,  Although several alternative methods for toxin detection, such as enzyme-linked immunosorbent assay  or polymerase chain reaction  have been described, these methods remain largely experimental. Clostridial organisms can be isolated from clinical specimens by using anaerobic procedures and special enrichment techniques. -
| ~ Treatment|| |
Mechanical ventilation together with meticulous supportive care remain the cornerstone of management of patients with botulism till today. Patients should be hospitalized and monitored closely in an intensive care unit for incipient respiratory failure. Tube feeding may be necessary if swallowing mechanisms are impaired. Gastric lavage should be attempted if the potential food exposure was recent; catharctics and enemas may be given to eliminate toxin from the colon if there is no ileus. 
For wound botulism, thorough wound debridement should be done and an antibiotic such as penicillin should be given to eradicate C.botulinum from the wound site. In food borne and wound botulism, early administration of antitoxin is indicated which neutralizes toxin molecules not yet bound to nerve endings. The currently available licenced product is a trivalent equine antitoxin against toxin types A, B and E. Since 1996, the dose has been reduced from two to four 10 ml vials to one 10 ml vial (7500 IU of type A, 5500 IU of type B and 8500 IU of type E antitoxin) per patient administered intravenously.  The antitoxin was first made commercially available in 1964 by Connaught Laboratories, Toronto.  When foodborne, wound or adult infectious botulism is suspected, antitoxin is released from CDC quarantine stations.
In infant botulism, neither equine antitoxin nor antibiotics have been shown to be beneficial; thus supportive care remains the mainstay of treatment. The value of a human-derived product (human botulism immune globulin) is being tested under an Investigational New Drug protocol via the California Department of health. , 
| ~ Vaccines: A New Frontier|| |
Because botulism is a rare disease, development and universal administration of a vaccine is unwarranted. At times, however wide-scale immunization may be desirable, as for example, to protect armed forces and to counter bioterrorist attacks. Currently, a pentavalent (A-E) botulism toxoid is available for immunization of very high risk personnel, such as laboratory workers who handle botulinum toxin. Historically, the approach for vaccine production has been to grow toxigenic strains of C. botulinum, isolate the crude toxin, and then inactivate it by treating it with formalin or a similar agent.  The inactivated toxin would then be given in a sequence of three injections to induce active immunity.
Advances in molecular biology have given rise to more desirable approaches. One that is currently under investigation involves cloning and expression of a terminal highly immunogenic fragment, the 50 kDa carboxyl fragment (Hc) of the toxin.  Mice given three subcutaneous vaccinations were protected against an intraperitoneal administration of 10 50% lethal doses (LD50) of serotype A and 3LD50 of botulinum toxin serotype E but died when challenged with 10LD50 of serotype E or 3LD50 of serotype B.  This recombinant protein product has many properties that make it a good candidate for human use. The main drawback is that it would still have to be parenterally injected.
Another molecular biologic approach holds promise for development of an oral vaccine. The gene for botulinum toxin, and thus the gene product, might be modified to alter its toxicity, without changing its penetrability, specificity, or immunogenicity.  Such a gene product would be safe, effective, and easy to administer. The nontoxic preparation that was tested evoked a striking response - protection against subsequent challenge by more than 10,000 lethal doses of unmodified toxin. 
| ~ Clinical applications of botulinum toxin|| |
The clinical use of purified botulinum toxin represents one of the most dramatic role reversals in modern times : a potent poison transformed into a potent medicine. The toxin by blocking acetylcholine release provides symptomatic relief in various spastic and dystonic disorders. Broadly speaking, the toxin has been tested or adapted for therapeutic use in four clinical areas : ophthalmology, neurology, otolaryngology, and areas of medicine, such as gastroenterology, that focus on smooth muscle and sphincter control , , [Table:2].
Several factors explain why botulinum toxin has proved to be such a valuable drug. First, the toxin is highly selective in acting on cholinergic cells, a characteristic that diminishes the chances of adverse side effects. Second, the toxin has a long duration of action, lasting from several months to more than a year. Finally, the dose of toxin can be individually titrated for each patient to ensure maximal benefits. 
Botulinum toxin A is marked worldwide under the name BOTOX (Allergan) and in Europe as Dysport (Speywood). BOTOX is approved by the United States Food and Drug Administration (FDA) and is labeled for the treatment of various disorders.  Concerns regarding toxin therapy include lack of response in certain patients, limited duration of response in some and development of refractoriners after initial response to therapy in other. Several hypotheses, such as absence of toxin receptors in nerve endings, metabolism or elimination of toxin and formation of antibodies to the toxin have been forwarded to explain these occurrences,  but none has proved to be definitely correct.
| ~ Conclusions|| |
Botulism is a rare but potentially fatal disease. Clinically suspected cases of botulism should be reported immediately to local or state public health agencies to facilitate laboratory confirmation of the diagnosis, release of antitoxin, if clinically indicated; and containment of the infection. Future studies required to be done include development of new diagnostic modalities to replace the mouse bioassay for toxin detection, search for organisms other than C.botulinum that produce botulinum toxin and the availability of a human botulism immune globulin.
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