BW-ch16.pdf

Botulinum Toxin

Chapter 16

BOTULINUM TOXIN

Zygmunt F. Dembek, P h D, mS, mPH * ; LeonarD a. SmitH, P h D † ; and Janice m. ruSnak, mD ‡

INTRODUCTION

HISTORY

DESCRIPTION OF THE AGENT

Botulinum Neurotoxin Production

PATHOGENESIS

CLINICAL DISEASE

DIAGNOSIS

Foodborne Botulism

Toxin Assays in Foodborne Botulism

Cultures in Foodborne Botulism

Inhalation-acquired Botulism

TREATMENT

Antitoxin

Clinically Relevant Signs of Bioterrorist Attack

Preexposure and Postexposure Prophylaxis

New Vaccine Research

SUMMARY

* Lieutenant Colonel, Medical Service Corps, US Army Reserve; Chief, Biodefense Epidemiology and Training and Education Programs, Operational

Medicine Department, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702

† Chief, Department of Molecular Biology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702

‡ Lieutenant Colonel, US Air Force (Ret); Research Physician, Special Immunizations Program, Division of Medicine, US Army Medical Research

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Deputy Director of Special Immunizations Program, US

Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

337

Medical Aspects of Biological Warfare

INTRODUCTION

the neurotoxins produced by Clostridia species are

among the most potent toxins known. because of their

extreme toxicity, botulinum ( C botulinum ) neurotoxins

were one of the first agents to be considered as a bio-

logical weapons agent. botulinum neurotoxin has been

developed as a biological weapon by many countries, in-

cluding Japan, germany, the united States, russia, and

iraq (Figure 16-1). botulism is a neuroparalytic disease,

most commonly caused by foodborne ingestion of neu-

rotoxin types a, b, and e, and is often fatal if untreated.

HISTORY

in the early 1930s, during its occupation of manchu-

ria, Japan formed a biological warfare command called

unit 731. general Shiro ishii, the military medical com-

mander of unit 731, admitted to feeding lethal cultures

of C botulinum to prisoners. 1 uS researchers began work-

ing on weaponization of botulinum toxin in the 1940s,

and allied intelligence indicated that germany was

attempting to develop botulinum toxin as a weapon

to be used against invasion forces. 2 at the time, neither

the composition of the toxic agent produced by C botu-

linum nor its mechanism of injury were fully known.

therefore, the earliest research goals were to isolate

and purify the toxin and to determine its pathogenesis.

the potential of botulinum neurotoxin as an offensive

biological weapon was also investigated 3–5 (the uS code

name for botulinum neurotoxin was “agent X”).

Following President richard m nixon’s executive

orders in 1969–1970, all biological agent stockpiles in

the uS offensive biological program, including botuli-

num neurotoxin, were destroyed. the 1975 convention

on the Prohibition of the Development, Production

and Stockpiling of bacteriological (biological) and

toxin Weapons and on their Destruction prohibited

the production of offensive toxins.

although the Soviet union signed and ratified the

convention, 6 its biowarfare program, including botuli-

num neurotoxin research, weapons development, and

production, continued and was even expanded in the

post-Soviet era. 7,8 the Soviet union reportedly tested

botulinum-filled weapons at the Soviet site aralsk-7

on Vozrozhdeniye (renaissance) island in the aral

Sea 8,9 and also attempted to use genetic engineering

technology to transfer complete toxin genes into other

bacteria. 10 in april 1992, President boris yeltsin pub-

licly declared that his country had covertly continued

a massive offensive biological warfare buildup, which

included developing botulinum toxin as a weapon.

that same year, colonel kanatjan alibekov (kenneth

alibek), the former deputy director of biopreparat (a

Soviet agency whose primary function was to develop

and produce biological weapons of mass casualties),

defected to the united States and described in detail

the Soviet biological weapons program. 10

iraq, which also signed the 1975 convention, ex-

panded its biowarfare program in 1985. ten years later,

it admitted to the united nations Special commission

inspection team to having produced 4,900 gallons of

concentrated botulinum neurotoxin for use in specially

designed missiles, bombs, and tank sprayers in 1989

and 1990. 7,11 of this preparation, 2,600 gallons were

used to fill 13 ScuD missiles with a 600-km range

and 100 400-lb (r-400) bombs (each bomb could hold

22 gallons of toxin solution). However, iraq did not

use biological agents during the Persian gulf War or

operation iraqi Freedom, and it has maintained that

its biological weapon stockpiles were destroyed. 12

Fig. 16-1. botulinum neurotoxin a is composed of an ~50

kDa light chain (Lc-red) and an ~100 kDa heavy chain

linked by a single disulfide bond. the Lc functions as a zinc-

dependent endopeptidase, whereas the heavy chain contains

two functional ~50 kDa domains: a c-terminal ganglioside

binding domain (Hc-purple), and an n-terminal transloca-

tion domain (Hn-blue). a belt portion of Hn (green) wraps

around Lc. the active site zinc is shown as a purple sphere.

this figure is based on the structure determined by Lacy

and colleagues. Data source: Lacy Db, tepp W, cohen ac,

Dasgupta br, Stevens rc. crystal structure of botulinum

neurotoxin type a and implications for toxicity. Nat Struct

Biol . 1998;5:898–902.

courtesy of S ashraf ahmed, mD, integrated toxicology

Division, uS army medical research institute of infectious

Diseases, Fort Detrick, maryland.

338

Botulinum Toxin

the aum Shinrikyo, a Japanese cult formed in 1987

by Shoko asahara, attempted to develop biological

weapons after its political party was defeated in the

1990 election campaign. known for its deadly 1995

sarin attack in the tokyo subway, aum Shinrikyo also

attempted to produce botulinum neurotoxin. before

the sarin attack, three briefcases containing portable

disseminating devices generating water vapor were

found in the subway station. at his 1996 trial, asa-

hara said he believed the cases contained botulinum

neurotoxin, although the toxin was not detected in

the devices. With 50,000 followers worldwide and

an estimated $1 billion in financial resources, the cult

had the capability to develop biological toxins for use

as weapons, and the intent to do so. 13 although no

cult members were specialists in biological weapons

development, microbiologists, medical doctors, and

other scientists were among the followers. it is not fully

understood why the biological assaults failed, but in-

formation from asahara’s trial indicated that the cult’s

scientists had difficulty overcoming technical barriers

in isolating and cultivating C botulinum . 13

a successful bioterrorist attack on large numbers

of people with botulinum neurotoxin would likely

overwhelm the public health system. the medical

intervention required to assist patients with botulism

includes mechanical ventilation and urgent attendant

healthcare. if the rajneeshee cult had used a colorless,

odorless, and tasteless solution of botulinum toxin

instead of Salmonella typhimurium on salad bars in its

1984 attack in the Dalles, oregon, 14–16 many of the

751 persons who contracted Salmonella gastroenteritis

would likely have died; the neurological sequelae of

hundreds of patients with botulinum toxin poisoning

would have quickly overwhelmed community medi-

cal resources. 17

in 2005 Wein and Liu 18 described in detail how

a bioterrorism attack using botulinum neurotoxin

could be perpetrated upon the nations’ milk supply.

they describe a mathematical model representative of

california’s dairy industry with milk traveling from

cows to consumer in a supply chain: milk is processed

from cows; picked up by tanker truck; piped through

milk silos; processed via separation, pasteurization,

homogenization, and vitamin fortification; and even-

tually distributed to the public. 18 naturally occurring

salmonellosis outbreaks from milk and milk products

affecting over 200,000 persons have already occurred,

leading to a realistic assessment of such vulnerabil-

ity in the national milk distribution system. 19,20 the

ability to spread botulinum neurotoxin via a liquid

media, if present in sufficient concentration, makes

this agent a logical choice for such a scenario. model-

ing of botulinum in a liquid dispersal medium is not

new, and has been posited for terrorist use in a water

fountain, 21 based upon microbiological contamination

at a recreational facility 22 ; however, Wein and Liu’s

modeling goes much further than tocsin generation,

pinpointing critical entry points of neurotoxin into

the milk supply, estimating the amount of toxin

required, and pointing out weaknesses in current

detection technology. 18 the paper has generated de-

bate. 23 Stewart Simonson, former assistant secretary

for public health emergency preparedness at the uS

Department of Health and Human Services, has re-

gretted the publication decision. 24

DESCRIPTION OF THE AGENT

Clostridium species bacteria are sporulating, obli-

gate anaerobic, gram-positive bacilli. the spores of C

botulinum are ubiquitous, distributed widely in soil

and marine sediments worldwide, and often found

in the intestinal tract of domestic grazing animals. 25–29

under appropriate environmental or laboratory condi-

tions, spores can germinate into vegetative cells that

will produce toxin. C botulinum grows and produces

neurotoxin in the anaerobic conditions frequently en-

countered in the canning or preservation of foods. the

spores are hardy, and special efforts in sterilization are

required to ensure that the spores are inactivated. mod-

ern commercial procedures have virtually eliminated

food poisoning by botulinum toxin; most cases today

are associated with home-canned foods (particularly

vegetables such as beans, peppers, carrots, and corn

that are associated with a higher pH) or food items

prepared by restaurants. 30,31

C botulinum produces seven antigenic types of neu-

rotoxins, denoted by the letters a through g. all seven

neurotoxins are structurally similar (approximately 150

kd in mass) but immunologically distinct. 32 However,

there is some serum cross-reactivity among the sero-

types because they share some sequence homology

with one another as well as with tetanus toxin. 33 the

unique strain C baratii produces only serotype F, 34 and

the C butyricum strain, serotype e. 35

botulism is a neuroparalytic disease. Human botu-

lism cases are caused primarily by neurotoxin types

a, b, and e, 30 and occasionally by type F. 36 C argen-

tinense produces type g, which has been associated

with sudden death, but not neuroparalytic illness, in

a few patients in Switzerland. 37 types c and D cause

disease in animals. all seven toxins are known to cause

inhalational botulism in primates, 38 and therefore could

potentially cause disease in humans. clostridial c2

cytotoxin is an enterotoxin, but not a neurotoxin. it

affects multiorgan vascular permeability via cellular

339

Medical Aspects of Biological Warfare

damage from its action on actin polymerization in the

cellular cytoskeleton, and has been implicated in a fatal

enteric disease in waterfowl. 39,40

rorist with the proper expertise and resources could

obtain a toxin-producing strain of C botulinum . Vari-

ous scientific journals, textbooks, and internet sites

provide information on how to isolate and culture

anaerobic bacteria and, specifically, how to produce

botulinum toxin. the major cause of botulism is the

ingestion of foods contaminated with C botulinum

and preformed toxin. the food supply remains vul-

nerable to a botulinum toxin attack (discussed later

in this chapter).

Botulinum Neurotoxin Production

Spore germination and subsequent growth of tox-

in-producing bacteria occur in improperly preserved

foods, 41–48 decaying animal carcasses and vegetable

matter, 49–53 and microbiology laboratories. 54–58 a ter-

PATHOGENESIS

the seven neurotoxins have different specific toxici-

ties 59–61 and durations of persistence in nerve cells. 62,63

all botulinum toxin serotypes inhibit acetylcholine

release, but they act through different intracellular

protein targets, exhibit different durations of effect, and

have different potencies. 64 all seven toxins may poten-

tially cause botulism in humans given a large enough

exposure. botulinum neurotoxin can enter the body

via the pulmonary tract (inhalational botulism), the

gastrointestinal tract (foodborne and infant botulism),

and from infected wounds (wound botulism). upon

absorption, the circulatory system transports the toxin

to peripheral cholinergic synapses, primarily targeting

neuromuscular junctions. 65 the toxin binds to high-af-

finity presynaptic receptors and is transported into the

nerve cell through receptor-mediated endocytosis. in

the nerve cell, it functionally blocks neurotransmitter

(acetylcholine) release, thereby causing neuromuscular

paralysis. other neurotransmitters co-located with ace-

tylcholine may also be inhibited, 66,67 and noncholinergic

cells may also be affected. 68 the estimated human dose

(assuming a weight of 70 kg) of type a toxin lethal to

50% of an exposed population (the LD 50 ) is estimated,

based on animal studies, to be approximately 0.09 to

0.15 µg by intravenous administration, 0.7 to 0.9 µg

by inhalation, and 70 µg by oral administration. 69–72

CLINICAL DISEASE

untreated botulism is frequently fatal. the rapidity

of the onset of symptoms, as well as the severity and

duration of the illness, is dependent on the amount and

serotype of toxin. 30,73 in foodborne botulism, symptoms

appear several hours to within a few days (range 2

hours to 8 days) after contaminated food is consumed. 30

in most cases the onset of symptoms occurs within 12

to 72 hours postexposure. in one study, the median

incubation period for the onset of symptoms from all

toxin serotypes was 1 day. 73 However, the median time

to onset of symptoms for serotype e was much shorter

(range 0–2 days) compared to toxin serotypes a (range

0–7 days) and b (range 0–5 days); most individuals

with toxin serotype e had symptoms within 24 hours

of ingestion. Symptoms from foodborne botulism from

toxin serotype a generally are more severe than from

toxin serotypes b and e. 73

as a neuroparalytic illness, botulism presents as

an acute, symmetrical, descending, flaccid paralysis.

However, early symptoms may be nonspecific and

difficult to associate with botulinum intoxication.

individuals with foodborne botulism often pres-

ent initially with gastrointestinal symptoms such as

nausea, vomiting, abdominal cramps, and diarrhea.

initial neurologic symptoms usually involve the cranial

nerves, with symptoms of blurred vision, diplopia,

ptosis, and photophobia, followed by signs of bulbar

nerve dysfunction such as dysarthria, dysphonia,

and dysphagia. onset of muscle weakness ensues in

the following order: muscles involving head control,

muscles of the upper extremities, respiratory muscles,

and lastly muscles of the lower extremities. Weakness

of the extremities generally occurs in a proximal-to-

distal pattern, and is generally symmetric. 31 However,

asymmetric extremity weakness may occasionally be

observed, occurring in 9 of 55 botulism cases in one

review. 74 respiratory muscle weakness can result in

respiratory failure, which may be abrupt in onset.

in one study, the median time between the onset of

intoxication symptoms and intubation was 1 day. 73

other commonly reported symptoms include fatigue,

sore throat, dry mouth, constipation, and dizziness. 74

botulism is not associated with sensory nerve deficits.

However, one review of botulism from toxin serotype

a or b showed that 8 of 55 cases reported symptoms

of paresthesias. 74 Death is usually the result of respi-

ratory failure or secondary infection associated with

prolonged mechanical ventilation. in general, intoxi-

cation with toxin serotype a results in a more severe

disease, often with bulbar and skeletal muscle impair-

ment, and thus the need for mechanical ventilation. 73–75

intoxication with toxin serotype b or e is more often

340

Botulinum Toxin

associated with symptoms of autonomic dysfunction,

such as internal ophthalmoplegia, nonreactive dilated

pupils, and dry mouth.

Paralysis from botulism can be long lasting. me-

chanical ventilation may be required for 2 to 8 weeks

with foodborne botulism, with paralysis lasting as long

as 7 months. 74 Symptoms of cranial nerve dysfunction

and mild autonomic dysfunction may persist for more

than a year. 76–78

the following symptom triad should suggest the

diagnosis of botulism: (1) an acute, symmetric, de-

scending, flaccid paralysis with prominent bulbar

palsies in (2) an afebrile patient with (3) a normal

sensorium. the bulbar palsies of botulism consist of

the “four Ds”: diplopia, dysarthria, dysphonia, and

dysphagia. Five classic symptoms have also been used

to diagnose botulism: (1) nausea and vomiting, (2)

dysphagia, (3) diplopia, (4) dry mouth, and (5) fixed

dilated pupils. 74 However, individuals may not exhibit

all five symptoms; a recent review from the republic

of georgia reported that only 2% of patients (13/481)

presented with all five criteria. 48

although foodborne botulism is the most likely

route of exposure for botulism from natural causes or a

bioterrorist event, botulism acquired on the battlefield

is most likely to occur from inhalation of botulinum

toxin, a route of exposure that does not naturally occur.

the duration from exposure to the onset of symptoms

for inhalational botulism is similar to that observed

with ingestion of botulinum toxin, generally ranging

from 24 to 36 hours to several days postexposure. 73,79

clinical symptoms resulting from inhalational intoxi-

cation are similar to botulism acquired from ingestion

of the toxin.

the only reported inhalation-acquired botulism

in humans occurred in 1962 in a german research

laboratory. 80 three laboratory workers experienced

symptoms of botulinum intoxication after conducting

a postmortem examination of laboratory animals that

had been exposed to botulinum toxin type a. Hospi-

talized 3 days after their exposure, the workers were

described as having ( a ) a “mucous plug in the throat,”

( b ) difficulty in swallowing solid food, and ( c ) “the

beginning of a cold without fever.” the symptoms

had progressed on the 4th day, and the patients com-

plained of “mental numbness,” extreme weakness, and

retarded ocular motions. their pupils were moderately

dilated with slight rotary nystagmus, and their speech

became indistinct and their gait uncertain. the patients

were given antibotulinum serum on the 4th and 5th

days. between the 6th and 10th days after exposure,

the patients experienced steady reductions in their

visual disturbances, numbness, and difficulties in swal-

lowing. they were discharged from the hospital less

than 2 weeks after the exposure, with a mild general

weakness as their only remaining symptom. 80

DIAGNOSIS

the differential diagnosis of botulism includes other

diseases with symptoms of paralysis:

does not involve cranial nerves; electromyo-

gram findings similar to botulism).

• Stroke or central nervous system mass lesion

(paralysis usually asymmetric, brain imaging

abnormal).

• Paralytic shellfish poisoning (history of shell-

fish ingestion; paresthesias of mouth, face,

lips, and extremities common).

• belladonna toxicity, such as atropine (history

of exposure, tachycardia, and fever).

• aminoglycoside toxicity (drug history of

aminoglycoside therapy).

• other neurotoxins, such as snake toxin (history

of snake bite, presence of fang punctures).

• chemical nerve agent poisoning (often asso-

ciated with ataxia, slurred speech, areflexia,

cheyne-Stokes respiration, and convulsions).

• guillain-barré syndrome (usually ascending

paralysis, paresthesias common, elevated

cerebrospinal fluid (cSF) protein [may be

normal early in illness], electromyogram

findings). note: the cSF findings are usually

normal in botulism, but mild elevation of cSF

protein between 50 and 60 mg/dL has been

noted in a minority of botulism patients. 74

• myasthenia gravis (dramatic improvement

with edrophonium chloride, autoantibodies

present, electromyogram findings). note:

botulism cases may have a positive response

to edrophonium chloride (26%), but the re-

sponse is generally not dramatic. 74

• tick paralysis (ascending paralysis, paresthe-

sias common, usually does not involve cranial

nerves; detailed exam often shows presence

of tick).

• Lambert-eaton syndrome (commonly associ-

ated with carcinoma, particularly lung carci-

nomas; deep tendon reflexes absent; usually

the clinical presentation of an afebrile patient with an

acute, symmetric, descending, flaccid paralysis (without

sensory deficits) with a normal sensorium suggests

the diagnosis of botulism. any occurrence of botulism

requires notification of public health officials and an

epidemiological evaluation. electrophysiological studies

341

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