Botulism

Overview

Last updated Apr 9, 2012

Agent and Pathogenesis

Agent
Pathogenesis

Agent

Botulinum Toxin

Botulism is an intoxication caused by botulinum toxin, which is produced by Clostridium botulinum and, rarely, by other Clostridium species. Seven antigenically distinct toxin types (A, B, C, D, E, F, G) have been identified.

The following are key characteristics of botulinum toxins (CDC 1998, Hatheway 1998, Lacy 1998, Montal 2010, Schiavo 1994, Sneath 1986).

  • Botulinum toxins are the most lethal toxins known. For type A toxin, the toxic dose is estimated at 0.001 mcg/kg (Franz 1997); the lethal dose for a 70-kg person by the oral route is estimated at 70 mcg, by the inhalational route 0.80 to 0.90 mcg, and by the intravenous route 0.09 to 0.15 mcg (Sobel 2005). The toxins are identified by neutralization with type-specific antitoxin; minor cross-neutralization between types C and D and between types E and F has been observed (Smith 1988).
  • The toxins are produced by vegetative cells (ie, germination of spores) and released by cell lysis.
  • Some toxins are fully activated by the bacteria that produce them (proteolytic strains of type A, B, and F), and some require exogenous proteolytic activation (types E and non-proteolytic types B and F).
  • Types A, B, E, and F cause natural disease in humans. The vast majority of disease is caused by types A, B, and E; type F rarely occurs (ie, about 1% of US cases [Gupta 2005]).
  • In one study, a novel in vivo mouse assay was used to correlate toxin type and dosage with the duration of muscle paralysis for types A, B, and E (Keller 2006).
    • Botulinum toxin A produced longer paralysis than botulinum toxin B, consistent with human observations.
    • For type A, duration of paralysis was exponentially related to toxin dose; the paralysis time doubled with every 25% increase of the toxin concentration.
    • For type B, the duration of paralysis was linear relative to the toxin dose.
    • Type E toxin had the shortest duration of action, but unlike the other two toxins, the dose of toxin did not influence recovery time.
  • Types C and D cause natural disease in birds, horses, and cattle; strains that produce these types reside in the intestinal tract of certain animals. Contaminated silage has been reported to cause botulism outbreaks among cattle (Myllykoski 2008).
  • Toxin type G has never clearly been shown to cause human disease.
  • Toxin types C, D, and G cause botulism in primates when administered through aerosol challenge. As a result of these experiments, experts generally believe that humans also are susceptible to these types.
  • Botulinum toxins are colorless, odorless, and presumably tasteless.
  • Aerosolized particles of toxin are approximately 0.1 to 0.3 mcm in size (Shapiro 1997).
  • The toxins are inactivated by heating (>85°C for 5 minutes) (Siegel 1993).
  • In the event of an intentional release of botulinum toxin, the causative organisms may or may not be present.

Clostridium botulinum

The following are key microbiologic characteristics of C botulinum (CDC 1998, Hatheway 1998, Smith 1988, Sneath 1986).

  • Gram-positive spore-forming bacillus (may stain poorly)
  • Somewhat varying strain sizes but generally in the range of 0.5 to 2.0 mcm in width and 1.6 to 22.0 mcm in length (CDC 1998)
  • Straight to slightly curved, with a peritrichous flagellum
  • Spores are oval, eccentric to subterminal, and usually swell the bacterial cell
  • Strict anaerobe
  • "Sluggishly" motile
  • Produce lipase on egg-yolk agar
  • Ferment glucose and liquefy gelatin (all strains)
  • Commonly isolated from soil and marine and lake sediments

The classification of C botulinum strains is based on metabolic activity(groups I to IV) and on toxin types (types A to G) (Hatheway 1998, Smith 1988, Sneath 1986):

  • Group I includes type A strains and proteolytic strains of types B and F.
  • Group II includes type E strains and nonproteolytic strains of types B and F
  • Group III includes nonproteolytic strains of types C and D.
  • Group IV includes only strains that produce type G.
  • Strains that produce more than one toxin type or have genetic sequences encoding more than one toxin have been identified (Barash 2004, Fathalla 2008, Kirma 2004).
  • Each group has a different optimal growth temperature, but there are no colonial morphology features that allow distinction between groups or antigenic types.
  • Genetic homology has been demonstrated within antigenic groups of C botulinum, and there is minimal antigenic cross-reactivity between groups.
  • Antimicrobial susceptibilities of C botulinum strains vary somewhat by group, but most strains are susceptible to penicillin, metronidazole, rifampin, and erythromycin (Smith 1988).

C botulinum spores have the following features (Smith 1988):

  • Spores may survive boiling for up to 3 to 4 hours or temperatures of 105oC for 100 minutes.
  • Spores are readily killed by chlorine (either as chlorinated water or as diluted solutions of hypochlorite).
  • Spores undergo maximum germination when activated by heat. For example, type A strains undergo maximum germination by heat treatment (or "heat shocking") at 80°C for 10 to 20 minutes.
  • Spores are resistant to desiccation and can survive in the dry state for 30 years or more.
  • Spores are resistant to ultraviolet light, alcohols, and phenolic compounds. They are relatively resistant to irradiation.

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Pathogenesis

Exposure to botulinum toxin occurs through the following mechanisms (toxin is not absorbed through intact skin):

  • Ingestion of preformed toxin
  • Inhalation of preformed toxin
  • Local production of toxin by C botulinum organisms in the gastrointestinal tract
  • Local production of toxin by C botulinum organisms in devitalized tissue at the site of a wound
  • Iatrogenic exposure caused by injection of botulinum toxin for cosmetic purposes or to treat certain musculoskeletal disorders, such as spasticity or blepharospasm (Coban 2010)

Following exposure, pathogenesis includes the following steps (Arnon 2001, CDC 1998, Halpern 1995, Schiavo 1995, Simpson 2004):

  • Botulinum toxin is activated by proteolytic cleavage; the activated structure is a 150-kd polypeptide comprising two chains (a heavy chain [100 kd] and a light chain [50 kd]) that are connected by a single disulfide bond.
  • Botulinum toxin enters the circulation and is transported to the neuromuscular junction.
  • At the neuromuscular junction, the heavy chain of the toxin binds to the neuronal membrane on the presynaptic side of the peripheral synapse.
  • The toxin then enters the neuronal cell via receptor-mediated endocytosis.
  • The light chain of the toxin crosses the membrane of the endocytic vesicle and enters the cytoplasm.
  • Once inside the cytoplasm, the light chain of the toxin (which is a zinc-containing endopeptidase) cleaves some of the proteins that form the synaptic fusion complex. The synaptic proteins, referred to as SNARE proteins, include synaptobrevin (cleaved by toxin types B, D, F, and G), syntaxin (cleaved by toxin type C), and synaptosomal-associated protein (SNAP-25; cleaved by toxin types A, C, E) (Arnon 2001). The clostridial neurotoxin apparently first binds to the SNARE complex before cleavage occurs (Breidenbach 2004).
  • The synaptic fusion complex allows the synaptic vesicles (which contain acetylcholine) to fuse with the terminal membrane of the neuron. Disruption of the synaptic fusion complex prevents the vesicles from fusing with the membrane, which in turn prevents release of acetylcholine into the synaptic cleft.
  • Without neuronal acetylcholine release, the affiliated muscle is unable to contract and becomes paralyzed.
  • The blockade of acetylcholine release lasts up to several months; normal functioning slowly resumes either through turnover of SNARE proteins within the cytoplasm or through production of new synapses.
  • Death from botulism results acutely from airway obstruction or paralysis of respiratory muscles. Death also can result from complications related to prolonged ventilatory support and intensive care.
  • Botulinum toxin apparently does not cross the blood-brain barrier; therefore, central nervous system functions remain intact.

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Epidemiology

Foodborne Botulism
Wound Botulism
Infant Botulism
Adult Intestinal Toxemia Botulism
Inhalation Botulism
Iatrogenic Botulism
Botulism Caused by Other Clostridium Species

Foodborne Botulism

  • Foodborne botulism is caused by ingestion of food contaminated with preformed botulinum toxin and subsequent absorption of toxin through the gastrointestinal tract. The following steps are necessary for a food item to cause botulism (CDC 1998):
    • The food item must be contaminated with C botulinum spores, which are normally found in soil (and may be found in water).
    • The spores must survive food preservation methods.
    • Adequate conditions for spore germination and neurotoxin production must be present.
    • The food must not be reheated adequately (>85°C for 5 minutes) to inactivate the heat-labile toxin before the food is consumed (Siegel 1993).
  • Generally, adequate conditions for germination and neurotoxin production include the following, although various caveats exist (CDC 1998, Smith 1988,Solomon 2001):
    • An anaerobic environment
    • Nonacidic pH (generally 4.6 to 4.8; pockets of different pH may be present within a single food source and allow toxin to be produced in a food that overall has an acidic pH)
    • Minimum temperature of 10°C (the optimum temperature for growth of proteolytic strains is close to 35°C; some nonproteolytic strains of types B, E, and F can produce toxin at refrigeration temperatures [3°C to 4°C])
    • Availability of water with limited solute concentration
  • Toxin types A, B, and E account for most cases of foodborne botulism, and toxin types tend to be geographically distributed within the United States. The outbreaks reported to the Centers for Disease Control and Prevention (CDC) between 1950 and 1996 (CDC 1998) were distributed as follows:
    • 144 (86%) of 167 type A outbreaks occurred west of the Mississippi River
    • 37 (61%) of 61 type B outbreaks occurred east of the Mississippi River
    • 56 (84%) of 67 type E outbreaks occurred in Alaska
  • Type F foodborne botulism has rarely been reported in humans (CDC 1998, Midura 1972).
  • Botulism can be recurrent, although only a few such cases have been reported. One case arose from repeated ingestion of home-prepared hot chili pepper sauce (Bilusic 2008). Another report describes recurrent wound botulism among injecting drug users in California (Yuan 2011). 
  • The median number of cases of foodborne botulism reported to the CDC annually between 1973 and 1996 was 24 (range, 8 to 86 cases) (Shapiro 1998).
  • The mean number of foodborne botulism outbreaks per year between 1950 and 1996 was 9.4, with a mean number of 2.5 cases per outbreak (CDC 1998).
  • Between 1990 and 2000, the median number of botulism events per year was 14 (range, 9 to 24) and the median number of cases per event was 1 (range, 1-17) (Sobel 2004). During this time period, the highest incidence rates were in Alaska (19 per million population), Idaho (0.6 per million population), and Washington (0.3 per million population).
  • Improperly home-canned or home-prepared foods (particularly vegetables) continue to account for most of the food vehicles associated with foodborne botulism in the United States (Sobel 2004).
  • Over the past 20 years, a wide variety of commercially produced (preserved and nonpreserved) foods have caused botulism outbreaks. Examples include foil-wrapped baked potatoes, sauteed onions held under a layer of butter, garlic in oil, commercially produced cheese sauce, commercially prepared chili, hazelnut yogurt, jarred peanuts, matambre (Argentine meat roll) sealed in heat-shrinked plastic wrap, commercially prepared carrot juice, green-olive paste, and canned chili sauce (Angulo 1998, CDC 2007, Chou 1988, Kalluri 2003, MacDonald 1985: Type A botulism from sauteed onions, O'Mahony 1990, Pingeon 2011, St Louis 1988, Sheth 2008, Townes 1996, Villar 1999).
  • A variety of salted, fermented, smoked, and canned fish sources have been implicated in type E botulism outbreaks in the United States and elsewhere (King 2009, Lindstrom 2006, Sobel 2007, Telzak 1990).
  • Foodborne botulism is a significant public health problem among Alaskan natives and is usually associated with consumption of fermented meat from aquatic mammals (eg, whales, seals, walruses, and beavers) and fish (Fagan 2011, McLaughlin 2004, Shaffer 1990, Wainwright 1988). The incidence of disease among Alaskan natives appears to be decreasing but continues to be more than 800 times higher in this population compared with the general US population (Fagan 2011).
  • Occasionally, unusual food preparation methods (particularly for home-prepared products) can lead to botulism. For example, an outbreak in Turkey (eastern Anatolia) in 2005 was associated with eating suzme (yogurt buried under soil) (Akdeniz 2007). Outbreaks of botulism in prisons have been attributed to drinking pruno (an alcoholic beverage concocted by prisoners from food scraps such as potato peelings and apples that are allowed to ferment unrefrigerated) (Vugia 2009).
  • Sales of minimally heated, chilled foods have grown recently in Western countries, such as the United States and the United Kingdom, and have raised concerns about the potential for foodborne botulism (Peck 2006).
  • Waterborne botulism has not been reported, most likely because botulinum toxin is rapidly inactivated by standard treatment of potable water and a very large amount of toxin would be needed to contaminate a water supply because of the dilution factor (Arnon 2001, Siegel 1993). However, water may serve as a source of contamination for other food items. For example, an investigation by the US Food and Drug Administration (FDA) of a canning facility in Michigan found that some cans of green beans were contaminated with viable neurotoxin-producing C botulinum. Further investigation demonstrated that C botulinum spores were present in the cooling water system (Sachdeva 2010).

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Wound Botulism

  • Wound botulism is caused by infection of a contaminated wound with C botulinum and subsequent absorption into the circulation of locally produced toxin.
  • C botulinum is a natural contaminant of soil throughout the United States (Smith 1978).
  • Wound botulism has been recognized with increasing frequency among injecting drug users, particularly in California, where the disease has been associated with use of black tar heroin (Davis 2008, MacDonald 1985: Botulism and botulism-like illness in chronic drug users, Passaro 1998, Werner 2000, Yuan 2011). Similarly, in the United Kingdom, bacterial infections (particularly wound botulism) have increased markedly since 2000 among injecting heroin users (Brett 2005: Soft tissue infections caused by spore-forming bacteria in injecting drug users in the United Kingdom). The authors of this study observed that the major risk factor was skin- or muscle-popping. Cases also have been reported in Germany (Preuss 2006, Schroeter 2009) and in Sweden, where real-time polymerase chain reaction (PCR) was used to diagnose a case of type E wound botulism (Artin 2007).
  • Wound botulism in injecting drug users can be misdiagnosed as drug intoxication (Royl 2007); however, presenting features can alert physicians to the correct diagnosis (Sam 2010, Wenham 2008). Botulism should be considered in injecting drug users who present with dysarthria and dysphagia (Preuss 2006).
  • Wound botulism may occur following traumatic injury to an extremity, such as a compound fracture, laceration, puncture wound, gunshot wound, severe abrasion ("road rash"), or crush injury (Merson 1973, Werner 2000).
  • Sinusitis associated with intranasal cocaine use has been the source of wound botulism in a few cases (Kudrow 1988, MacDonald 1985: Botulism and botulism-like illness in chronic drug users, Roblot 2011, Werner 2000).
  • A few cases have occurred postoperatively (usually following intra-abdominal procedures) and an abscessed tooth was the source of C botulinum infection in one case (Nystrom 2011, Weber 1993).
  • Between 1943 (when the condition was first recognized) and 1985, 33 cases of wound botulism were reported to the CDC. Between 1986 and 1996, 78 cases were reported and most were associated with injecting drug use (CDC 1998).

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Infant Botulism

  • Most pediatric cases of botulism occur in infants (ie, infant botulism), although foodborne and wound botulism also can affect the pediatric population.
  • Infant botulism is caused by ingestion of C botulinum spores. The spores subsequently colonize the gastrointestinal tract, germinate, and produce toxin, which is absorbed into the circulation.
  • Most infants are well before illness onset (Wigginton 1993). The disease characteristically begins with lethargy and poor feeding (with or without constipation), followed by neuromuscular paralysis, hypotonia, or weakness (Clemmens 2007). Constipation may be subtle or overt.
  • The source of spores for most cases remains unknown, although the most common sources of infection for infants appear to be honey and environmental exposure (Arnon 1979, Brook 2007, Nevas 2005). Infant formula was postulated to be the source for one case (Brett 2005: A case of infant botulism with a possible link to infant formula milk powder). Other risk factors identified in one study for infants 2 months of age and older included breast-feeding, less than one bowel movement per day in the 2 months before illness onset, and ingestion of corn syrup (Spika 1989). In that study, the only identified risk factor among infants less than 2 months old was living in a rural area or on a farm.
  • Between 1976 (when infant botulism was first recognized) and 1996, 1,442 cases were reported to the CDC (CDC 1998).
    • Cases were reported from 46 states, with Delaware, Hawaii, Utah, and California having the highest incidence rates (9.0, 8.8, 6.3, and 5.7 per 100,000 live births, respectively).
    • Almost half of all cases were reported from California (680 cases; 47.2%).
    • The mean age at onset was 13 weeks (range, 1 to 63 weeks).
  • Analysis of infant botulism cases occurring globally from 1996 through 2008 revealed 524 cases in 26 countries representing five continents. The fact that most countries have not reported cases of infant botulism suggests that the disorder is underreported, under-recognized, or both, because the organism is present worldwide and cases of foodborne botulism have been reported in many of these countries (Koepke 2008).
  • Five cases of infant botulism caused by C baratii type F have been identified; the youngest patient was just 38 hours old at presentation (Barash 2005).
  • A review of charts of infant patients in California who were treated with the orphan drug Human Botulism Immune Globulin on the basis of clinical presentation but did not ultimately have laboratory-confirmed botulism (32 of the 681 who were treated) demonstrated that these patients fell into five categories: spinal muscular atrophy type I (five patients), metabolic disorders (eight patients), infectious diseases (three patients), miscellaneous (seven patients; includes Miller Fisher variant of Guillain-Barre syndrome, neuroblastoma stage III, and cerebral infarctions, among others), and probable infant botulism lacking laboratory confirmation (nine patients) (Francisco 2007).

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Adult Intestinal Toxemia Botulism

  • The pathogenesis of intestinal botulism in adults is similar to that of infant botulism. Disease is caused by ingestion of C botulinum spores, with subsequent colonization of the gastrointestinal tract. Spores germinate and produce toxin, which is then absorbed into the circulation.
  • Only a few cases have been recognized, and most have occurred postoperatively or in adults with underlying pathology of the gastrointestinal tract such as Crohn's disease (Bartlett 1986, Chia 1986, Griffin 1997, Shapiro 1998, Sheppard 2012). As of early 2012, cases had been reported from Canada, Iceland, Italy, Japan, and the United States (Sheppard 2012).
  • Several cases caused by type F toxin produced by C baratii have been reported to the CDC (McCroskey 1991), and cases caused by C butyricum producing type E toxin also have been recognized (Fenicia 1999).
    • A review of type F adult botulism in the United States between 1981 and 2002 demonstrated the following findings (Gupta 2005):
      • Thirteen cases of adult type F botulism were reported to the CDC during the study period, representing 1% of US cases.
      • A toxigenic C baratii organism producing type F toxin was isolated in 8 (80%) of 10 positive stool cultures. Type F toxin was identified in serum for nine of the cases.
      • In 5 (42%) of 12 cases, a history of gastrointestinal disease or an invasive gastrointestinal procedure was present before illness onset. Also in 5 (42%) of 12 cases, antimicrobials were reportedly taken before illness onset. A possible food source was only identified in one instance.

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Inhalational Botulism

  • Disease is caused by inhalation of aerosolized preformed botulinum toxin with subsequent absorption through the lungs into the circulation.
  • Three cases of inhalational botulism were reported in 1962 in veterinary technicians in Germany who were working with aerosolized botulinum toxin in animals (Arnon 2001). Symptoms occurred about 72 hours after exposure.
  • Inhalational disease also has been produced experimentally in animals. One study, involving primates, demonstrated that illness occurred 12 to 80 hours after exposure (Franz 1993). Another study, involving mice, demonstrated that following inhalational challenge, the maximum concentration of botulinum toxin in blood occurred at 2 hours postexposure (Park 2003).
  • A mouse study characterized the pathological consequences of inhalational botulinum toxin exposure in mice given prophylactic pentavalent (ABCDE) toxoid. The authors found that the mice sustained severe histopathological lung damage despite protection from the lethal neurotoxic effects. Signs included "thickening of the alveolar septa and perivascular areas with a generalized spreading interstitial edema and a moderate intra-alveola/intrabronchiola hemorrhage" (Taysse 2005). These findings suggest a direct toxic affect of botulinum toxin on lung tissues; however, more research is needed to better define this potential effect.

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Iatrogenic Botulism

  • Iatrogenic botulism is caused inadvertently following injection of botulinum toxin for therapeutic or cosmetic reasons (Sobel 2005). See the section: Therapeutic Botulinum Toxin for more information.
  • Four cases of iatrogenic botulism occurred in December 2004 in Florida following cosmetic injection with a botulinum toxin that was not approved for use in humans (see Dec 15, 2004, CIDRAP News story). The injections contained much higher concentrations of botulinum toxin than the FDA-approved product Botox. A research firm in Arizona sold the raw botulinum toxin to healthcare practitioners as a Botox substitute.
  • Another report identified four patients who developed iatrogenic botulism after receiving therapeutic doses of botulinum toxin for spasticity and blepharospasm; all recovered (Coban 2010).

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Botulism Caused by Other Clostridium Species

  • C butyricum–producing type E toxin has been reported to cause intestinal botulism in infants and young adults in Italy and foodborne botulism in Asia (Aureli 1986, Fenicia 1999, Schechter 1999).
  • C baratii–producing type F toxin has caused intestinal botulism in infants and adults; in the latter it is usually associated with gastrointestinal pathology, recent gastrointestinal surgery, or recent use of antimicrobial agents (Barash 2005, Gupta 2005, McCroskey 1991, Schechter 1999).

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Botulinum Toxin as a Biological Weapon

Historical Perspective
Mechanisms and Outbreak Features
Pediatric Considerations

Historical Perspective

Botulinum toxin poses a significant bioweapon threat "because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons" (Arnon 2001). However, some experts believe that the potential of botulinum toxin as a bioweapon is limited because of challenges with stabilizing the toxin for aerosol dissemination (Arnon 2001).

Past efforts to weaponize botulinum toxin include the following:

  • The United States produced botulinum toxin as a potential biological weapon beginning in World War II; however, the US offensive biological weapons program ended after the 1972 Biological and Toxin Weapons Convention (BTWC).
  • The former Soviet Union conducted research on use of botulinum toxin as a biological weapon as late as the early 1990s, despite having signed the BTWC.
  • At the time of the Gulf War, Iraq had produced 19,000 L of concentrated botulinum toxin, some of which was loaded into military weapons (Zilinskas 1997).
  • The Japanese cult Aum Shinrikyo attempted to use aerosolized botulinum toxin in Japanese cities on at least three occasions between 1990 and 1995. The C botulinum used in these attempts was collected from soil in northern Japan. These attacks failed because of faulty microbiological technique, deficient aerosol-generating equipment, or internal sabotage (Arnon 2001).

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Mechanisms and Outbreak Features

The two most likely mechanisms for use of botulinum toxin as a terrorist weapon include deliberate contamination of food or beverages or via an aerosol release (Villar 2006).

  • Because food products are often widely distributed, contamination of a commercially produced food or beverage product could result in a high number of casualties and fatalities across the country. In addition, such a bioterrorist act would produce severe civic disruption, economic loss, and social anxiety. According to the CDC, potentially contaminated food or beverage items need to be heated at 85°C (185°F) for 5 minutes prior to consumption to ensure that toxin is destroyed (CDC: Botulism: control measures overview for clinicians). Concern has been raised that typical temperatures employed for pasteurization of commercially available beverage products (such as milk) may not sufficiently denature all botulinum toxin in the product.
    • Mathematical modeling suggests that 1 g of botulinum toxin added to commercially distributed milk consumed by 568,000 people could result in 100,000 cases of botulism (Wein 2005). Ten grams of toxin added to the same quantity of milk could result in over 500,000 cases in the exposed population.
    • One study reported that conventional milk pasteurization (63°C, 30 min) inactivated botulinum toxin serotype A but did not inactivate botulinum toxin serotype B, indicating that serotype B toxin is potentially heat stable in milk (Rasooly 2010).
    • However, another study found that standard high-temperature short-time (HTST) pasteurization (heating milk to 72°C and holding it steady at this temperature for at least 15 seconds) inactivates at least 99.99% of botulinum toxin types A and B, suggesting that standard pasteurization conditions would reduce activity of these toxins much more dramatically than originally thought (Weingart 2010).
  • An aerosol release could also lead to high numbers of casualties, although the event would be more localized. Experts have estimated that 1 g of aerosolized botulinum toxin could kill up to 1.5 million people (Shapiro 1997). Aerosolized particles of botulinum toxin are approximately 0.1 to 0.3 mcm in size (Shapiro 1997). Despite these estimates, some experts discount the potential of botulinum toxin as a bioweapon because the toxin may not be very stable in an aerosolized form (Arnon 2001).

Although contamination of a water supply is feasible, this approach is unlikely since a large amount of toxin would be needed to initially contaminate water. In general, deliberated contamination of water with potential bioterrorism agents may not be very effective for the following reasons: dilution of the agent in a large body of water; direct inactivation from chlorine or other disinfectants; nonspecific inactivation by other mechanisms (such as hydrolysis, sunlight, or microbes); filtration; and the relatively small amount of water that is actually ingested from the source (Khan 2001).

  • Botulinum toxin is naturally inactivated in fresh water within 3 to 6 days, and toxin is rapidly (within 20 minutes) inactivated by standard potable water treatment (Siegel 1993).
  • A 2005 study found that two of seven small-scale water purification devices tested were able to effectively eliminate botulinum toxin from water. Those based on filtration (pore size 0.2 to 0.4 mcm) or irradiation from a UV-lamp (254 nm) failed to remove the toxin from inoculated water. Reverse osmosis and experimental sand filtration effectively eliminated the toxin (Horman 2005).

It is unlikely that therapeutic botulinum toxin could be used in a terrorist attack, because a vial of the currently licensed preparation contains only about 0.3% of the estimated human lethal inhalational dose and 0.005% of the estimated lethal oral dose (Arnon 2001).

The following features of a botulism outbreak would suggest deliberate toxin release (Arnon 2001).

  • An outbreak involving a larger number of cases than previous outbreaks
  • An outbreak caused by an unusual toxin type (ie, C, D, F, or G) or an outbreak involving type E toxin without an apparent aquatic source
  • Multiple simultaneous outbreaks with or without an apparent source.
  • For aerosol release, cases would not have a common food exposure but would have been in a common geographic location during the week before symptom onset

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Pediatric Considerations

  • In the event of an aerosol release of botulinum toxin, children may be at an even greater level of risk than adults, since children have a higher number of respirations per minute and consequently could have an increased level of exposure to toxin (AAP 2000).
  • Signs and symptoms of botulism in children following a bioterrorist attack (ie, aerosol or foodborne exposure) would be similar to those seen in adults.
  • Ensuring adequate intensive care resources for the pediatric population in the event of a bioterrorism attack involving an agent such as botulinum toxin should be an important priority in bioterrorism preparedness planning.

However, these analyses pertain to military uses of botulinum toxin to immobilize an opponent (William C. Patrick, unpublished data, 1998). In contrast, deliberate release of botulinum toxin in a civilian population would be able to cause substantial disruption and distress. For example, it is estimated that a point-source aerosol release of botulinum toxin could incapacitate or kill 10% of persons within 0.5 km downwind (William C. Patrick, unpublished data, 1998). In addition, terrorist use of botulinum toxin might be manifested as deliberate contamination of food. Misuse of toxin in this manner could produce either a large botulism outbreak from a single meal or episodic, widely separated outbreaks (Arnon 2001). In the United States, the CDC maintains a well-established surveillance system for human botulism based on clinician reporting that would promptly detect such events (Arnon 2001).

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Emergency Response

Botulism Surveillance
Botulism Outbreak or Intentional Dissemination
Emergency Response to a Mass Exposure
International Public Health Concerns

Botulism Surveillance

  • The CDC maintains an intensive surveillance system for botulism in the United States. Cases are identified through follow-up of requests for botulinum antitoxin.
  • Cases also may come to detection through requests for laboratory testing of food or clinical specimens. Arrangements for laboratory testing are made through state public health laboratories. These laboratories either have the capability to test specimens directly or they collect and submit specimens to another laboratory for testing (usually at the CDC). All positive specimens identified through state public health laboratories are reported to the CDC on at least an annual basis.
  • All state health departments have 24-hour emergency phone lines for reporting cases of botulism (CDC: Emergency response). Requests to the CDC for antitoxin are usually made through the state epidemiology offices, although some requests are made directly to the CDC by clinicians caring for suspect botulism patients.
  • The authors of a report published in 2012 observed: "The identification of epidemiologic linkages between foodborne botulism cases is a critical part of diagnostic evaluation and outbreak detection. Investigation of an intentionally contaminated food item with a long shelf life and widespread distribution may be delayed until an astute physician suspects foodborne botulism; suspicion of foodborne botulism occurs more frequently when more than one case is hospitalized concurrently. In an effort to augment national botulism surveillance and antitoxin release systems and to improve food defense and public health preparedness efforts, medical organizations and Homeland Security officials should emphasize the education and training of medical personnel to improve foodborne botulism diagnostic capabilities to recognize single foodborne botulism cases and to look for epidemiologic linkages between suspected cases" (Newkirk 2012).

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Botulism Outbreak or Intentional Dissemination

  • A single case of foodborne botulism (or botulism from an unknown source) is considered an outbreak (MacDonald 1986) and is a public health emergency. Suspected cases should be reported immediately to state or local public health officials.
  • Public health officials will: (1) assist with appropriate laboratory testing to confirm the diagnosis, (2) authorize use of antitoxin, (3) conduct aggressive surveillance for other cases, and (4) immediately begin an epidemiologic investigation to identify the source or vehicle (such as a contaminated commercial product) or to determine if there is evidence to suggest a bioterrorism-related event.
  • Original specimens should be preserved and their custody documented, pursuant to public health and regulatory investigation procedures as well as potential criminal investigation procedures (ASM 2013).
  • Public health officials will coordinate notification of local FBI agents as appropriate.
  • If available evidence suggests the potential for a continued increase in cases while the investigation proceeds, involved hospitals should establish communication networks between the emergency department, the intensive care unit, and those services likely to be involved in managing cases (eg, infectious disease, pulmonary, respiratory therapy, critical care, neurology). These networks should focus on establishing policies and procedures for handling large numbers of patients (see below).

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Emergency Response to a Mass Exposure

In the event of a mass exposure, such as a widespread aerosol release of botulinum toxin, the following steps would be necessary.

  • Rapid administration of antitoxin to ill persons: Although antitoxin does not reverse existing paralysis, once administered it binds to any toxin remaining in the circulation and, therefore, can mitigate progression of disease, increase the likelihood of survival, and decrease the duration of mechanical ventilatory support (if respiratory failure occurs). Release of antitoxin and coordination of administration would be performed by local/state public health officials in conjunction with the CDC.
  • Rapid mobilization of mechanical ventilators: Adequate supportive care resources, including those for infants and children, would be critical to successful management of any mass-exposure botulism outbreak.

Two articles published in 2009 provide tools for management of botulism mass casualty incidents. One involves an algorithm for the evaluation and management of botulism patients in a triage setting (Rega 2009), and the other offers a short questionnaire that can assist with screening of potential casualties (Burkholder-Allen 2009). 

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International Public Health Concerns

A large outbreak in Thailand (209 cases) in 2006 emphasized the need for addressing global policy issues concerning outbreaks in developing countries, including health infrastructure, communication and response systems, stockpiles of medication and supplies, decision algorithms for notification, and international response to public health emergencies (Ungchusak 2007). 

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Therapeutic Botulinum Toxin

Therapeutic Uses
Special Considerations

Therapeutic Uses

  • Patients with a range of spastic or autonomic neuromuscular disorders may benefit from small amounts of purified botulinum toxin injected into affected muscles (Schantz 1992). There are two types of therapeutic botulinum toxin:  purified botulinum toxin type A (Botox, produced by Allergan, Inc) (Allergan, Inc) and purified botulinum toxin type B (Myobloc, produced by Elan Pharmaceuticals, Inc) (FDA: Myobloc labeling information).

Examples of conditions that can be treated with botulinum toxin include:

  • Spasmodic torticollis
  • Strabismus
  • Blepharospasm
  • Laryngeal dystonia
  • Focal dystonias of the hand
  • Limb spasticity
  • Hemifacial spasm
  • Cerebral palsy
  • Migraine headache
  • Hyperhydrosis (severe underarm sweating)
  • Post-stroke spasticity
  • Urinary incontinence in adults with overactive bladder caused by neurologic disease
  • In April 2002, the FDA approved use of botulinum toxin type A for cosmetic purposes (Allergan, Inc, FDA: Botox Cosmetic labeling information). 

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Special Considerations 

Therapeutic botulinum toxin contains about 0.3% of the estimated lethal human inhalational dose and only 0.005% of the estimated lethal human oral dose; therefore, this form of toxin is not likely to be used as a bioterrorist weapon (Arnon 2001). However, iatrogenic cases of botulism have been reported.

  • A report published in 2010 identified four patients who developed iatrogenic botulism following treatment with botulinum toxin for musculoskeletal disorders (Coban 2010). One patient required intensive care, but all four survived.
  • An unlicensed, highly concentrated preparation of botulinum toxin caused botulism in four adult patients undergoing cosmetic procedures. Affected patients may have received doses 2,857 times the estimated human lethal dose by injection. Pretreatment serum levels in three of the four patients were from 21 to 43 times the estimated human lethal dose (Chertow 2006). Following protracted hospital courses, prolonged mechanical ventilation, and physical rehabilitation, all four of these patients survived.

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Clinical Features and Differential Diagnosis

Clinical Features
Differential Diagnosis

Clinical Features

  • Botulism is characterized by acute afebrile descending symmetric paralysis. Recovery occurs over weeks to months and often requires extensive supportive care.
  • Disease generally begins with evidence of cranial nerve dysfunction and then progresses to muscle weakness (proximal muscle groups are affected first and may be more severely involved).
  • Severity of disease ranges from mild cranial nerve dysfunction to complete flaccid paralysis. Paralysis of pharyngeal or respiratory muscles may result in the need for prolonged mechanical ventilation.
    • Severity of disease correlates with the amount of toxin absorbed into the circulation.
    • Several studies have shown that a shorter incubation period correlates with more severe disease (MacDonald 1985: Type A botulism from sauteed onions, Tacket 1984). Similarly, a study of botulism cases in Japan revealed that patients who had shorter incubation periods had a significantly higher risk of death (Nishiura 2007).
    • Disease caused by toxin type A tends to be more severe than disease caused by toxin type B or E (Shapiro 1998).
    • Among more than 200 patients in an outbreak in Thailand, respiratory failure was less likely to develop in those who did not manifest nausea or vomiting and did not have urinary retention requiring catheterization. Nausea or vomiting and any cranial neuropathy with urinary retention or difficulty swallowing were symptoms most predictive of respiratory failure (Wongtanate 2007).
    • One study of injecting drug users who had wound botulism found that longer time from presentation in the emergency department to administration of antitoxin and longer time from presentation to wound drainage were independently associated with increased length of stay in intensive care (Offerman 2009).
  • Death can result from airway obstruction or paralysis of respiratory muscles. Death also can result from complications related to prolonged ventilatory support and intensive care, such as aspiration pneumonia and other infectious conditions.
    • Before mechanical ventilation was widely available, the case-fatality rate was about 60% (Shapiro 1998).
    • The case-fatality rate currently is low owing to adequate supportive care; overall the rate is 5% to 10% for foodborne disease and somewhat higher for wound botulism (Shapiro 1998, Werner 2000).
    • In the event of a mass exposure (such as a bioterrorism attack), clinical resources could be overwhelmed rapidly and the case-fatality rate could be much higher.
    • A retrospective study of hospitalized foodborne botulism cases in the Republic of Georgia, 1980-2002, found that patients with shortness of breath and impaired gag reflex and without diarrhea were 23 times more likely to die than were patients without this syndrome (Varma 2004). In this case series, the incubation period was similar among those who died and those who survived, as was the likelihood of receiving antitoxin.
Clinical Features of Foodborne and Wound Botulism

Note: Information presented is for foodborne and wound botulism; infant botulism is not included, since that condition is distinct from what would be expected in a bioterrorism attack. The presenting features of inhalational botulism likely would be comparable to those of foodborne and wound botulism.

Characteristic
Features

Incubation perioda

—Dependent on level of toxin exposure
—For foodborne botulism, 2 hr–8 days
—For wound botulism, 4-14 days
—Unknown for inhalational botulism; estimated to be 24-36 hr; the only three reported cases in humans had an incubation period of 72 hr

Symptoms (compiled from reports of foodborne botulism outbreaks caused by toxin types A, B, and E)b

—Nausea (88%)c
—Dry mouth (82%)
—Blurred vision (78%)
—Dysphonia (76%)
—Dysphagia (75%)
—Weakness (72%)
—Fatigue (69%)
—Dyspnea (65%)
—Dysarthria (63%)
—Double vision (60%)
—Dizziness (56%)
—Vomiting (52%)c
—Constipation (related to autonomic dysfunction) (45%)
—Sore throat (40%)
—Abdominal cramps or abdominal pain (40%)d
—Diarrhea (35%)c
—Paresthesias (29%)

Signs (compiled from cases of types A and B botulism reported to CDC in 1973 and 1974)d

—Alert mental status (90%)
—Weakness of upper extremities (75%)
—Ptosis (73%)
—Weakness of lower extremities (69%)
—Extraocular muscle weakness (65%)
—Diminished gag reflex (65%)
—Facial nerve dysfunction (63%)
—Dilated or fixed pupils (44%)
—Diminished or absent deep tendon reflexes in affected groups (40%)
—Nystagmus (22%)
—Ataxia (17%)
—Other considerations:
  ~Patients generally afebrile
  ~Mental status generally intact, although patients may appear lethargic
   or have difficulty communicating because of bulbar dysfunction
  ~Sensory exam generally normal

Laboratory features

—Normal CSF glucose, protein, cell count
—Normal CBC
—Normal imaging of brain and spine (ie, CT scan or MRI)
—Characteristic EMG findingse:
  ~Incremental response (facilitation) to repetitive stimulation (not
   always present and often seen only at 50 Hz)
  ~Short duration of motor unit potentials (MUPs); polyphasic MUPs
  ~Decreased amplitude of compound muscle action potentials
   (CMAPs) after a single nerve stimulus (most prominent in proximal
   muscle groups)
  ~Normal sensory nerve function
  ~Normal nerve conduction velocity (motor and sensory)

Complications

—Respiratory failure (which may require prolonged ventilatory support); in some outbreak settings, up to 30%-40% of patients required mechanical ventilation
—Aspiration pneumonia (among patients with respiratory failure)f
—Residual fatigue, dry mouth or eyes, dyspnea on exertion up to several years after initial presentationg

Case-fatality rateh

—5%-10% for foodborne botulismi
—15%-44% for wound botulismj

Abbreviations: CSF, cerebrospinal fluid; CT, computed tomography; MRI, magnetic resonance imaging; CBC, complete blood count.

aArnon 2001, Franz 1997.
bThe percentages were derived from compiling information available from published reports of large foodborne outbreaks caused by toxin type A, B, or E. The number of cases in each denominator ranged from 30 to 180. Angulo 1998, Wainwright 1988, Weber 1993.
cGastrointestinal symptoms are uncommon in patients with wound botulism (Merson 1973) and likely would be uncommon in the setting of inhalational exposure.
dHughes 1981.
eCherington 1998, Maselli 2000.
fSchmidt-Nowara 1983.
gMann 1981, Mann 1983, Wilcox 1989.
hBefore mechanical ventilation was widely available, the case-fatality rate was much higher (about 60%). In the setting of a mass exposure, where intensive-care resources could rapidly be overwhelmed, the case-fatality rate may be higher than that currently observed.
iShapiro 1998.
 jMerson 1973, Shapiro 1998, Werner 2000.

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Differential Diagnosis

Differential Diagnosis of Botulism

Note: This differential diagnosis applies to botulism in adults and older children; infant botulism is not included, since that condition is distinct from what would be expected during a bioterrorism attack.

Condition
Features that distinguish each condition from botulisma

Guillain-Barre syndrome (GBS) (particularly Miller Fisher variant)

—Classic GBS results in ascending paralysis
—Miller Fisher variant may be descending and may have pronounced cranial nerve involvement; it usually includes a triad of ophthalmoplegia, ataxia, and areflexia (5% of GBS cases are of the Miller Fisher variant)b
—Abnormal CSF protein 1-6 wk after illness onset (although may be normal early in clinical course)
—Paresthesias commonly occur (often stocking/glove pattern)
—EMG shows abnormal nerve conduction velocity; facilitation with repetitive nerve stimulation does not occur (as with botulism)
—History of antecedent diarrheal illness (suggestive of Campylobacter infection, which accounts for about one third of GBS cases)
—Outbreaks of GBS do not occur (unlike botulism)

Myasthenia gravis

—Dramatic improvement with edrophonium chloride (ie, a positive Tensilon test), although some botulism patients may exhibit partial improvement following administration of edrophonium chloride (ie, a borderline Tensilon test)
—EMG shows decrease in muscle action potentials with repetitive nerve stimulation

Tick paralysisc

—Ascending paralysis
—Paresthesias are common
—Careful examination reveals presence of tick attached to skin
—Recovery occurs within 24 hr after tick removal
—EMG shows abnormal nerve conduction velocity and unresponsiveness to repetitive stimulation
—Usually does not involve cranial nerves

Lambert-Eaton syndrome

—Commonly associated with carcinoma (often oat cell carcinoma of lung)
—Although EMG findings are similar to those in botulism, repetitive nerve stimulation shows much greater augmentation of muscle action potentials, particularly at 20-50 Hz
—Increased strength with sustained contraction
—Deep tendon reflexes often absent; ataxia may be present
—Usually does not involve cranial nerves

Stroke or CNS mass lesion

—Paralysis usually asymmetric
—Brain imaging (CT or MRI) usually abnormal
—Sensory deficits common
—Altered mental status may be present

Poliomyelitis

—Febrile illness
—CSF shows pleocytosis and increased protein
—Altered mental status may be present
—Paralysis often asymmetric

Paralytic shellfish poisoning or ingestion of puffer fish

—History of shellfish (ie, clams, mussels) or puffer fish ingestion within several hours before symptom onset
—Paresthesias of mouth, face, lips, extremities commonly occur

Belladonna toxicity

—History of recent exposure to belladonna-like alkaloids
—Fever
—Tachycardia
—Altered mental status

Aminoglycoside toxicity

—History of recent exposure to aminoglycoside antibiotics
—More likely to occur in the setting of renal insufficiency
—Most commonly seen with neomycin
—Most commonly associated with other neuromuscular blocking agents such as succinylcholine and paralytics

Other toxicities (hypermagnesemia, organophosphates, nerve gas, carbon monoxide)

—History of exposure to toxic agents
—Carbon monoxide toxicity: altered mental status may occur, cherry-colored skin
—Hypermagnesemia: history of use of cathartics or antacids may be present, elevated serum magnesium level
—Organophosphate toxicity: fever, excessive salivation, altered mental status, paresthesias, miosis

Other conditions

—CNS infections (particularly brainstem infections)
—Inflammatory myopathy
—Hypothyroidism
—Diabetic neuropathy
—Viral infections
—Streptococcal pharyngitis (pharyngeal erythema and sore throat can occur in botulism owing to dryness caused by parasympathetic cholinergic blockade)

Abbreviations: CSF, cerebrospinal fluid; EMG, electromyogram; CT, computed tomography; MRI, magnetic resonance imaging.

aArnon 2001, Campbell 1981, Cherington 1998, Werner 2000.
bDorr 2006, Sobel 2005.
cFelz 2000.

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Laboratory Diagnosis

Specimen Collection and Transport
Laboratory Biosafety
Laboratory Response Network
Diagnostic Tests for Detection of Botulinum Toxin and C botulinum

Specimen Collection and Transport

Specimen collection and transport procedures for testing related to diagnosing botulism are outlined in the following table.

Collection and Transport of Laboratory Specimens for the Diagnosis of Botulism

Note: A list of patient medications should accompany specimens, since some medications may be toxic to mice and can be removed by dialysis before testing is performed.a

Specimen
Clinical Indication
Collection and Transport

Serum

Intentional release, foodborne botulism, autopsy specimens

—Collect >20 mL whole blood before administration of antitoxin using red-top or separator tube (no anticoagulant)
—Ship >10 mL serum at 4oC
—Do not ship whole blood, which tends to become hemolyzed during transit
—Notify testing lab if patient has received "stigmine drugs" or a Tensilon test
—Keep specimen refrigerated at all times

Wound botulism (critical specimen for confirmation)

—Collect 30 cc whole blood (before antitoxin administration)
—Ship at 4oC
—Sera submitted for toxin detection should not be hemolyzed
—Notify testing lab if patient has received "stigmine drugs" or a Tensilon test
—Keep specimen refrigerated at all times

Wound/tissue

Wound botulism

—Collect exudate, tissue, or swabs
—Ship at room temperature in anaerobic transport system

Stool, enema fluid, intestinal fluid

Intentional release, foodborne botulism, infant botulism, wound botulismb

—Obtain 10-50 g of stool (as little as "pea-size" for infant botulism); transport at 4oC
—Enema fluid (20 cc) can be collected as an alternative to stool, using minimal amount of sterile nonbacteriostatic water; ship at 4oC
—Intestinal fluid collected at autopsy (20 cc); ship at 4oC

Gastric fluid, vomitus

Foodborne botulism, intentional release

—Collect within 72 hr of symptom onset
—Obtain 20 cc of vomitus; ship at 4oC
—Obtain 20 cc of gastric fluid (living cases or at autopsy); ship at 4oC

Specimens to collect at autopsy

Intentional release, foodborne botulism, infant botulism

—Serum, according to methods outlined above
—Contents from different sections of small and large intestines (10 g per sample in separate containers)
—Gastric contents as indicated, according to methods outlined above
—Tissue samples as indicated, according to methods outlined above

Food samples (epidemiologically implicated)

Intentional release, foodborne botulism, infant botulism

—Obtain 10-50 g of implicated or suspect food; ship at 4oC in original container
—Place individually in leak-proof sealed transport devices

Nasal swab

Intentional releasec

—Obtain anaerobic swab; ship at room temperature

Environmental sample

Intentional release, infant botulism

—Collect as appropriate:
  ~Environmental swab; ship at room
   temperature
  ~Soil (50-100 g)
  ~Water (>100 mL)

aArnon

 2001
bA wound may not be the actual source of infection/intoxication.
cToxin may be present on nasal mucosa for up to 24 hr after inhalational exposure (Franz 1997).

Adapted from the following sources: ASM 2013: Sentinel laboratory guidelines for suspected agents of bioterrorism: botulinum toxin, CDC 1998, CDC: Specimen selection table.

Guidelines have been published for packing and shipping of infectious substances, diagnostic specimens, and biological agents from suspected bioterrorism (ASM 2012). C botulinum is classified under World Health Organization (WHO) risk group 4. Cultures that are reasonably suspected to contain C botulinum must be transported as "infectious substances." In addition, the US Department of Transportation (DOT) regulations and International Air Transport Association (IATA) rules require training of all individuals involved in the transport of dangerous goods, including infectious substances (DOT 2008, IATA 2012). Once botulinum toxin is identified, samples may be regulated as select agents and subject to additional transport requirements (see below). Chain of custody should be documented for material that may constitute evidence of criminal activity.

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Laboratory Biosafety

  • Botulinum toxin and Clostridium species that produce botulinum toxin are classified as select agents and therefore are regulated under 42 CFR part 73 (Possession, Use, and Transfer of Select Agents and Toxins), which was published in final form in the Federal Register in March 2005 (HHS 2005). As specified in the Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 42 CFR part 73 provides requirements for laboratories that handle select agents (including registration, security risk assessments, safety plans, security plans, emergency response plans, training, transfers, record keeping, inspections, and notifications).
  • C botulinum toxin detection should be performed only by trained individuals at laboratory response network (LRN) reference or higher laboratories.
  • Sodium hypochlorite (0.1%) or sodium hydroxide (0.1 N) inactivate the toxin and are recommended by the CDC for decontaminating work surfaces and spills of cultures or toxin (CDC 2009).
  • Biosafety recommendations from the FDA for laboratories that test for C botulinum include the following (Solomon 2001):
    • Place biohazard signs on doors to restrict entrance and keep the number of people in the laboratory to a minimum.
    • All workers should wear laboratory coats and safety glasses.
    • Never pipette anything by mouth; use mechanical pipettes.
    • 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.
    • Allow no eating or drinking in the laboratory.
    • In a very visible location, list phone numbers where therapeutic antitoxin can be obtained.
    • Reduce clutter in the laboratory to a minimum and keep all equipment and other materials in their proper place.

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Laboratory Response Network

The LRN is a network of more than 150 national and international laboratories. The network includes federal, state and local public health, military, food testing, environmental, veterinary, and international laboratories (CDC: Facts about the Laboratory Response Network, CDC: The Laboratory Response Network).

The LRN structure for bioterrorism designates laboratories as sentinel, reference, or national. Designation depends on the types of tests a laboratory can perform and how it handles infectious agents to protect workers and the public.

  • Sentinel laboratories, formally called level A laboratories represent an estimated 25,000 hospital-based laboratories that have direct contact with patients. In an unannounced or covert terrorist attack, sentinel laboratories could be the first facilities to encounter suspicious specimens. These laboratories generally have at least BSL-2 containment capabilities. A sentinel laboratory's responsibility is to rule out B anthracis or refer a suspicious sample to the nearest LRN reference laboratory. Sentinel laboratories use the ASM [American Society for Microbiology] Sentinel Level Clinical Microbiology Laboratory Guidelines to rule out microorganisms that might be suspected as agents of bioterrorism (ASM).
  • Reference laboratories, sometimes referred to as "confirmatory reference," can perform tests to detect and confirm the presence of a threat agent. These laboratories ensure a timely local response in the event of a terrorist incident. Rather than having to rely on confirmation from laboratories at the CDC, reference laboratories are capable of producing conclusive results; this allows local authorities to respond quickly to emergencies. These are mostly state or local public health laboratories but also include military, international, veterinary, agriculture, and food- and water-testing laboratories. Reference laboratories operate with BSL-3 containment facilities that have been given access to nonpublic testing protocols and reagents. One of the roles of the LRN reference laboratories is to provide guidance, training, outreach, and communications to the sentinel laboratories in their jurisdictions.
  • National laboratories have unique resources to handle highly infectious agents and the ability to identify specific agent strains through molecular characterization methods. These laboratories also are responsible for methods development, bioforensics, and select-agent activity.

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Diagnostic Tests for Detection of Botulinum Toxin and C botulinum

According to the ASM guidelines, LRN sentinel laboratories "should not attempt to culture, identify the organism, or attempt to perform toxin analysis." Furthermore, LRN sentinel laboratories should not accept environmental or animal specimens; such specimens should be forwarded directly to the state health department laboratory (ASM 2012). Only certain LRN reference laboratories have the capability to perform mouse bioassay testing. 

  • The mouse bioassay is currently the primary diagnostic method used for detection and identification of botulinum toxin. Other methods (see below) are still considered investigational.
    • Mice are injected intraperitoneally with the patient sample, stool or food extract, culture filtrate, or other sample and observed for up to 4 days.
    • Control mice are injected with a mixture of the sample combined with neutralizing antibody to different toxin types.
    • Signs of botulism intoxication usually are evident in 6 to 24 hours.
    • As little as 0.03 ng of toxin can be detected by this method (CDC 1998, Shantz 1992).
    • One report of a cohort of clinically defined wound botulism cases found that the serum mouse bioassay was only 68% sensitive in confirming infection (Wheeler 2009). The authors pointed out that physicians should be aware of the test's limitations and base their final diagnosis on clinical criteria when the mouse bioassay produces negative results.
  • Culture for C botulinum for stool or gastric specimens has been used for diagnosis, in addition to toxin testing (CDC 1998). Isolates are tested for neurotoxin by the mouse bioassay. An activation step with trypsin is required to detect toxin from some group II strains. Isolation of C botulinum from stool or a wound is considered diagnostic in patients with signs and symptoms of botulism.
  • Nasal swabs could potentially be collected in the event of an aerosol exposure (CDC: Specimen selection table, Franz 1997). As with other types of potential bioterrorism exposures, the sensitivity and diagnostic value of nasal culture is unknown. Nasal swabs should only be used as part of an epidemiologic investigation or on the basis of recommendations made by the CDC in the event of a bioterrorist attack.
  • Serological assays for botulinum toxin antibody are not useful as a measure of exposure, which does not typically induce an antibody response.
  • Detailed methods for testing food samples have been published by the FDA's Center for Food Safety and Applied Nutrition (CFSAN) (Solomon 2001). Detection of botulinum toxin in an epidemiologically implicated food item confirms the diagnosis of botulism. Since C botulinum is widely distributed in nature, the organism may be present in food without producing toxin or causing disease. Therefore, positive culture results from food, in the absence of detectable toxin, must be interpreted within the context of other epidemiological findings.
  • Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA analysis, and automated ribotyping methods have been compared for epidemiological typing of C botulinum type E using clinical and food isolates associated with four botulism outbreaks that occurred in the Canadian Artic. A modified PFGE protocol was judged to be the most useful method for typing epidemiologically related type E strains, based on its ability to type all strains reproducibly and with an adequate level of discrimination (Leclair 2006)
  • Investigators have identified high-affinity monoclonal antibodies (mAbs) that specifically bind botulism toxins type A and B. These have been used to develop highly sensitive sandwich immunoassays, which appear to be promising alternatives to the mouse bioassay (Scotcher 2010, Stanker 2008, USDA 2009).
  • A "ruggedized" real-time PCR assay called R.A.P.I.D. for use by first-responders and in military field hospitals and other rough environments is commercially available but not FDA approved (Idaho Technology).
  • Other tests for botulinum toxin (considered investigational):
    • Other enzyme-linked immunoassays (ELISA) (Dezfulian 1991, Ferreira 2001, Ferreira 2003, Wictome 1999)
    • An immuno-PCR assay that measures antigen-antibody reactions using a conjugated reporter DNA molecule followed by PCR amplification (Chao 2004)
    • Time-resolved fluorescence assays for C botulinum A/B neurotoxin (Peruski 2002)
    • Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) (Barr 2005, Cruzan 2006, Darby 2001, Wilkes 2006)
    • An optical immunoassay for rapid detection of neurotoxins A, B, E, and F (Ganapathy 2008)
    • A micromechanosensor for detection of botulinum toxin type B (Liu 2003)
    • Lateral flow devices for environmental testing (Alexeter Technologies,New Horizon Diagnostics, Osborn Scientific Group)
    • A botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate (Bagramyan 2008)
  • Other tests for C botulinum (organism)
    • PCR assays have been used for the detection of C botulinum toxin genes in animal, food, and fecal samples (Craven 2002, Dahlenborg 2001, Fenicia 2007, Franciosa 1994, Lindstrom 2001). One report published in 2009 described a set of real-time PCR tests for detecting botulinum neurotoxin genes for A, B, E, and F toxins produced by C botulinum, C baratii, and C butyricum (Fach 2009). PCR-based assays detect genetic sequences of the organism, not the toxin molecule itself. This is important to consider, since the organism may not be present in clinical specimens or may not be involved in an intentional release of botulinum toxin.
    • Subtyping methods for C botulinum, such as ribotyping, have been described (Skinner 2000).
    • An amplification method that analyzes variable number tandem repeat regions in C botulinum has been shown to be capable of discriminating among type A strains and may provide laboratories with a rapid, highly discriminatory diagnostic tool for use in botulism outbreaks (Macdonald 2008).

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Prevention and Treatment Issues

Therapy for Botulism
Botulinum Toxoid
Research on New Therapies and Vaccines

Therapy for Botulism

  • Supportive care is the mainstay for treatment of botulism; prolonged intensive care, mechanical ventilation, and parenteral nutrition may be required.
  • Botulinum antitoxin can be administered to treat forms of botulism other than infant botulism and is most effective if given early in the clinical course (Sobel 2009: Diagnosis and treatment of botulism). Although antitoxin will not reverse existing paralysis, it will prevent additional nerve damage if given before all circulating toxin is bound at the neuromuscular junction.
  • For botulism cases other than infant botulism, the CDC provides heptavalent botulinum antitoxin (HBAT, Cangene Corporation) through a CDC-sponsored FDA Investigational New Drug (IND) protocol. HBAT replaced bivalent botulinum antitoxin AB in March 2010 (CDC 2010).
    • The HBAT FDA IND treatment protocol includes specific, detailed instructions for intravenous administration of antitoxin and return of required paperwork to the CDC.
    • HBAT contains equine-derived antibody to the seven known botulinum toxin types (A through G) with the following nominal potency values: 7,500 U anti-A; 5,500 U anti-B; 5,000 U anti-C; 1,000 U anti-D; 8,500 U anti-E; 5,000 U anti-F; and 1,000 U anti-G.
    • In the setting of a bioterrorist attack, where cases may have been exposed to unusually large amounts of toxin, additional doses of antitoxin may be necessary. Alternatively, the patient's serum could be retested for the ongoing presence of circulating toxin (Arnon 2001); however, this process would take time. The scarcity of antitoxin would limit the capacity to provide additional doses.
  • In cases of wound botulism, the wound should be surgically debrided and antibiotics should be administered (usually penicillin).
  • Botulism immune globulin–intravenous (human) (BIG-IV) for treatment of infant botulism was licensed by the FDA in October 2003 as BabyBIG.
    • A 5-year randomized, double-blind, placebo-controlled trial of BIG-IV treatment for infant botulism in California demonstrated that it significantly: (1) shortened duration of hospitalization (from a mean of 5.7 weeks to 2.6 weeks), (2) shortened time spent in intensive care (from 5.0 weeks to 1.8 weeks), and (3) decreased mean hospital costs per patient by $88,000 (Arnon 2006).
    • BIG-IV is available as a public-service orphan drug and may be obtained by contacting the California Department of Human Services, Infant Botulism Treatment and Prevention Program (Arnon 2006, California Department of Health Services). The circumstances that enabled the creation of BIG-IV have been presented as a possible paradigm for development of other "orphan" drugs (drugs used to treat relatively few patients) (Arnon 2007).

 Availability of Botulinum Antitoxin

  • Antitoxin should be requested as soon as the diagnosis of botulism is suspected, since confirmation of botulism may take several days and antitoxin is most effective if given within 24 hours after symptom onset (Tacket 1984).
  • Antitoxin for use in the United States is of equine origin and only available through the CDC via state and local health departments (except in California and Alaska, where antitoxin release is controlled by the state health departments).
  • Requests for antitoxin usually are made through contact with state epidemiology offices. In addition to resources at the state level, epidemiologists at the CDC are available 24 hours a day to provide advice regarding use of antitoxin.
  • Antitoxin (supplied by the CDC) is maintained at quarantine stations located in airports in various metropolitan areas, Once antitoxin is requested for a patient with suspected botulism, it generally can be delivered within a few hours (Shapiro 1997, Sobel 2009: Diagnosis and treatment of botulism). 

Long-term Outcome Following Treatment

A case-control study of 217 botulism patients provided details about long-term outcome of treated patients (Gottlieb 2007).

  • Of the 211 patients who survived, 68% reported having worse health at the time of interview than 6 years earlier, compared with 17% of 656 controls (matched odds ratio, 17.6; 95% confidence interval, 10.9-28.4).
  • Nearly twice as many patients as controls (49% vs 25%) reported their current health as fair or poor. Significantly more botulism patients than controls reported fatigue, dizziness, dry mouth, and difficulty lifting objects.
  • Botulism patients were significantly more likely than controls to report difficulty breathing with moderate exertion and were also more likely to report being limited in vigorous activities, walking 3 blocks, and climbing 3 flights of stairs.

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Botulinum Toxoid

For years, the CDC recommended immunization with pentavalent (ABCDE) botulinum toxoid (PBT) for vaccination of workers at risk for occupational exposure to botulinum serotypes A, B, C, D, and E. PBT was available from the CDC under an investigational new drug protocol.

However, as of November 30, 2011, the CDC no longer offers PBT (CDC 2011). According to the CDC, "This decision was based on an assessment of the available data, which indicated a decline in immunogenicity of some of the toxin serotypes."

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Research on New Therapies and Vaccines

Antibody Therapeutics

Humanized monoclonal antibodies, small peptides, peptide mimetics, receptor mimics, and small molecules targeting active sites are candidates for inhibiting botulinum toxin and may eventually be used in treatment strategies (Adekar 2008, Cai 2007, Nowakowski 2002).

Protein and Peptide Vaccines

Various recombinant vaccines are currently under investigation in animal models (Baldwin 2008, Boles 2006, Lee 2007, Pier 2008, Webb 2007, Yu 2008. Vaccines based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin appear to be the most promising (Rusnak 2009, Smith 2009, Yu 2009, Zichel 2010).

A recombinant botulinum vaccine (rBV A/B) is being developed to protect adults 18 to 55 years of age against types A and B botulism. Toxicity has been evaluated in mice; the rBV A/B vaccine produced no apparent systemic or neurobehavioral toxicity and only transient mild inflammation at the injection site in the mice studies. These results indicate a favorable safety profile and support its use in a phase I clinical trial (Shearer 2012).

Viral Vector Vaccines

Vectored vaccines also have been studied; one report involved Venezuelan equine encephalitis virus as the vector (Lee 2006) and others have involved adenovirus (Xu 2009, Zeng 2007).

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Infection Control (Including Autopsies and Burial)

Isolation Precautions
Issues Related to Autopsies and Burial

Isolation Precautions

  • In the hospital setting, Standard Precautions are adequate for patients with botulism, since person-to-person transmission does not occur.
  • In the laboratory setting, sodium hypochlorite (0.1%) or sodium hydroxide (0.1 N) inactivate the toxin and are recommended by CDC for decontaminating work surfaces and spills of cultures or toxin (CDC 1999: Biosafety in microbiological and biomedical laboratories).

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Issues Related to Autopsies and Burial

  • Recent guidelines from CDC indicate that Standard Precautions should be used for postmortem care. These include using a surgical scrub suit, surgical cap, impervious gown or apron with full sleeve coverage, a form of eye protection (eg, goggles or face shied), shoe covers, and double surgical gloves with an interposed layer of cut-proof synthetic mesh (CDC 2004: Medical examiners, coroners, and biologic terrorism).
  • In addition, autopsy personnel should wear N-95 respirators during all autopsies, regardless of suspected or known pathogens. Powered air-purifying respirators (PAPRs) equipped with N-95 or high-efficiency particulate air (HEPA) filters should be considered.
  • Bodies infected with biological terrorism agents should not be embalmed (CDC 2004: Medical examiners, coroners, and biologic terrorism).

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Case Definitions and Public Health Reporting

Botulism Case Definitions
Public Health Reporting

Botulism Case Definitions

The following case definitions were published in the CDC's Morbidity and Mortality Weekly Report in 1997 (CDC 1997).

Foodborne Botulism

  • Clinical description: Ingestion of botulinum toxin results in an illness of variable severity. Common symptoms are diplopia, blurred vision, and bulbar weakness. Symmetric paralysis may progress rapidly.
  • Laboratory criteria for diagnosis:
    • Detection of botulinum toxin in serum, stool, or patient's food or
    • Isolation of C botulinum from stool
  • Probable case: A clinically compatible case with an epidemiologic link to a food source (eg, ingestion of a home-canned food within the previous 48 hours)
  • Confirmed case: A clinically compatible case that is laboratory-confirmed or that occurs among persons who ate the same food as persons who have laboratory-confirmed botulism

Infant Botulism

  • Clinical description: An illness of infants, characterized by constipation, poor feeding, and "failure to thrive" that may be followed by progressive weakness, impaired respiration, and death.
  • Laboratory criteria for diagnosis:
    • Detection of botulinum toxin in stool or serum or
    • Isolation of C botulinum from stool
  • Confirmed case: A clinically compatible case that is laboratory-confirmed, occurring in a child less than 1 year of age

Wound Botulism

  • Clinical description: An illness resulting from toxin produced by C botulinum that has infected a wound. Common symptoms are diplopia, blurred vision, and bulbar weakness. Symmetric paralysis may progress rapidly.
  • Laboratory criteria for diagnosis:
    • Detection of botulinum toxin in serum or
    • Isolation of C botulinum from a wound
  • Confirmed case: A clinically compatible case that is laboratory confirmed in a patient who has no suspected exposure to contaminated food and who has a history of a fresh, contaminated wound during the 2 weeks before onset of symptoms.

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Public Health Reporting

According to state disease-reporting requirements, all confirmed and suspected cases of botulism must be reported immediately to state or local public health officials, even after normal working hours. Public health officials will then contact the CDC through the CDC Emergency Operations Center (telephone number: 770-488-7100) to arrange for clinical consultation if necessary and for the release of botulinum antitoxin as appropriate (CDC 2003).

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Recent Literature

Barash JR, Arnon SS. A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins. J Infect Dis 2013 (published online Oct 7)

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MacDonald E, Arnesen TM, Brantsaeter AB, et al. Outbreak of wound botulism in people who inject drugs, Norway, October to November 2013. Euro Surveill 2013;18(45):pii=20630

Slotved H-C, Tanassi JT, Sparding N, et al. Botulinum toxin field assays evaluated using cosmetic botox preparations. Biosecur Bioterror 2013 (published online Nov 12)

WHO. Botulism fact sheet. Updated Aug 8, 2013 

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