DTIC ADA501673: Mechanism of Action of Botulinum Neurotoxin and Overview of Medical Countermeasures for Intoxication

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Mechanism of action of botulinum neurotoxin and overview of medical countermeasures for
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Adler, Oyler, G, Apland, JP, Deshpande, SS, Nicholson, JD,
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Published in Chemical Warfare Agents: Chemistry, Pharmacology, Toxicology, and Therapeutics (eds. J.A. Romano, Jr., B.J. Lukey, H.
Salem), 2nd ed. CRC Press: Boca Raton, FL, pp. 389-422, 2008.
14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Botulinum intoxication, medical countermeasures, treatment, prophylaxis, pharmacology, toxicology
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16 Mechanism of Action
of Botulinum Neurotoxin
and Overview of Medical
Countermeasures
for Intoxication
Michael Adler, George Oyler, james P. Apland,
Sharad S. Deshpande, james D. Nicholson,
jaime Anderson, Charles B. Millard,
and Frank j. Lebeda
CONTENTS
I. Introduction 390
A. Characteristics ofBoNTIntoxication 391
B. Symptomology 392
C. Functional Domains ofBoNT 392
n. Manifestations ofBotulism '" 393
A. FoodbomeBotulism 394
B. Wound Botulism 395
C. Intestinal Botulism 395
1. InfantBotulism 395
2. AdultIntestinalBotulism 396
D. Inhalation Botulism 396
m. Prophylaxis andTreatmentOptions 397
A. Antitoxin 398
1. Equine Botulinum Antitoxin 398
2. RecombinantMonoclonal Antibodies 398
B. Prophylaxis 399
C. Pharmacological Intervention 399
1. PotassiumChannel Blockers 400
2. Inhibitors for SpecificStages ofIntoxication 401
IV. Conclusions and FutureResearch 411
Acknowledgments 413
References 413
Theopinionsorassertionscontainedhereinaretheprivateviewsoftheauthors.andarenottobeconstruedasreflectingthe
viewoftheDepartmentoftheAnnyortheDepartmentofDefense.
389
390 ChemicalWarfareAgents: Chemistry, Pharmacology,Toxicology, andTherapeutics
I. INTRODUCTION
Thebotulinumneurotoxins(BoNTs)* compriseafamilyofsevendistinctneurotoxicproteins(A-G)
producedbyimmunologicallydiscretestrainsoftheanaerobicbacteriumClostridiumbotulinumand
in rare cases by Clostridium baratii and Clostridium butyricum (Habermann and Dreyer, 1986;
HarveyetaI., 2002; Simpson,2004).Thesetoxinsactonperipheralcholinergicsynapsesto inhibit
spontaneousandimpulse-dependentreleaseofacetylcholine(ACh)(Brooks, 1956;Kaoetal., 1976).
Intoxication by BoNT results in muscle weakness, which can be fatal when the diaphragm and
intercostal muscles become sufficiently compromised to impair ventilation (Dickson and Shevky,
1923).TheBoNTsarethemostpotentsubstancesinnature,andexposuretoaslittleas1-3ng/kgmay
besufficienttocausehumanlethality(Gill, 1982;MiddlebrookandFranz, 1997;ArnonetaI.,200I).
Thepurposeofthischapteristousetheinsightsgainedinourunderstandingofthemechanism
ofBoNT action to establish a conceptual framework within which to develop effective treatment
strategies for intoxication. The chapter is organized into three major topics: (l) an overview of
BoNT action, (2) a description of the manifestations of botulism, and (3) an evaluation of
conventional and emergent treatmentoptions. From the first description ofbotulism in 1793 until
the mid-1950s, BoNT was primarily viewed as apublic health problem because ofits association
with food poisoning (Gill, 1982; Hatheway, 1988; Shapiro et aI., 1998). Although implicated in
onlyasmaIlfractionoffoodbomeillnesses(<0.1%),theseverityoftheclinicalsyndromeproduced
byBoNTandthepotentialfornumerouscasualtiesledeachoutbreaktobeconsideredasapotential
health crisis (Hatheway, 1988; Smith and Sugiyama, 1988; Snydman, 1989; Shapiroetal., 1998).
Duetoitsselectivetargetingofperipheralcholinergicsynapses,BoNThasalsobeenusedasatool
to study cholinergic pathways, especiaIly to explore the influence ofsynaptic inactivity and ACh
deprivation on muscle function (Drachman and Johnston, 1975; Kao et al., 1976;Thesleff, 1989;
ThesleffetaI., 1990).
DuringWorldWarn,BoNTwasdevelopedasabiological weapon,becauseofitspotentialto
createmasscasualtieson thebattlefield(Franz, 1997;Grace, 2003). ThebattlefielduseofBoNTis
now viewed as less likelyfoIlowing adoptionofthe 1972BiologicalandToxinWeaponsConven
tion and the dissolution ofthe Soviet Union. However, the Iraqi stockpiling ofBoNT before the
PersianGulfWarof1991 revealstheabilityofadeterminednationtoacquirebiologicalweaponsin
relative secrecy(Shoham, 2000; Arnon etaI., 2001).
With the rise of global terrorism, exemplified by organizations such as the Japanese Aum
ShinrikyocultandalQaeda,thepotentialuseofBoNTasabioterroristweaponhasbecomeamore
immediate threat (Franz et al., 1997; Middlebrook and Franz, 1997; Arnon et al., 2001; Grace,
2003).BoNTiswellsuitedforthisrolebecauseofitsextraordinarylethality,capacitytoelicitpanic,
and potential to disrupt the public health system (Atlas, 1998). Additional attributes of BoNT
include widespread availability, low cost, and ease of production, transport, and concealment
(ArnonetaI.,2001).TheseconsiderationshaveledBoNTtobeclassifiedasaCategoryAbiothreat
agent by the Centers for Disease Control and Prevention (CDC) (Lohenry and Foulke, 2(06).
Bioterroristattacks aregenerally thoughtto involvedispersal ofBoNTas an aerosol, butthe toxin
can also be used to contaminatethefood supply (Wein andLiu, 2005).
Systematic research on the mechanism of action of BoNT began with Emile Pierre van
Ermengem's historic isolation and characterization ofC. botulinum folIowing a large outbreak in
EllezeIIes, Belgium, in 1895 and has continued with increasing interest and enthusiasm to the
presenttime(Simpson, 2004;GrumellietaI.,2005;Rossettoetal.,2006;Singh,2006). Early work
on BoNT intoxication revealed the existence ofmultiple serotypes, localized the site ofaction to
peripheral cholinergic synapses, proposed the mechanism ofimpaired ACh release, and ruled out
noncholinergic, sensory, and central nervous system (CNS) involvement (Dickson and Shevky,
* In this chapter, BoNTis used todesignateboth purebotulinumneurotoxinaswell as theneurotoxincomplex.Some
authorspreferthedesignationofBoTxforthelatter.
Botulinum Neurotoxin: Mechanismand Countermeasures 391
1923;GuytonandMacDonald, 1947;Burgenetai., 1949;Ambache, 1951;Brooks, 1956).Mostof
these findings wereestablishedbythe mid-1950sand refinedduring thenext threedecades (Lundh
etai., 1977; Simpson, 1981;Thesleff, 1989).
Theremarkablespecificityforperipheralcholinergicsynapsesandlongdurationofactionledto
I
theuseofBoNT Aforagrowing numberoffocal dystonias and movementdisorders following its
approval in 1989 as an "orphan drug" by the U.S. Food and Drug Administration (FDA) for the
treatment ofstrabismus, blepharospasm, and hemifacial spasm (Jankovic and Brin, 1997; Schantz
and Johnson, 1997).Theattributes thatrenderBoNTadeadly poison also makethe neurotoxin an
ideal therapeutic agent to treat diseases ofmuscle hyperactivity. In addition to its original indica
tions, BoNTs are also used for treatment of spasticity following brain and spinal cord injuries,
stroke, multiplesclerosis, cerebral palsy, and numerous otherdisorders. Expansion and refinement
in its clinical use constitute the most active focus of current BoNT research, and a number of
excellent reviews have been published (Jankovic and Brin, 1997; Schantz and Johnson, 1997;
Tugnoli et al., 1997; Johnson, 1999; Aoki, 2002; Charles, 2004; Chaddock and Marks, 2006;
Eleopraetal., 2006; Dutton and Fowler, 2(07).
In addition to discovering additional indications for the native neurotoxin, a promising new
approach has been to alter the BoNT-binding domain to retarget the modified toxins to noncholi
I
nergic sites. Notable examples include anovel conjugateofBoNT A, whose binding domain was
replaced by Erythrina cristagalli lectin for targeting to pain fibers (Chaddock et al., 2004), and a
modified BoNT/CI in which the binding domain was replaced by epidermal growth factor for
targeting to epithelial cells to inhibitexcess mucus secretion (Fosteret ai., 2006). The former has
potential for relief ofchronic pain, whereas the latter may be useful for treatment ofasthma and
chronicobstructivepulmonary disease.
During the last two decades, enormous progress has been madein understanding theaction of
BoNTatthemolecularlevel.Thiswasspurredbyanumberofcrucialdevelopments:(1)elucidation
oftheaminoacidsequenceleadingtorecognitionofthezinc-bindingmotif(Jongeneeletal., 1989),
(2)demonstrationofzincmetalloproteaseactivitywithidentificationofsubstratesandcleavagesites
(Schiavo et ai., 1992a, 1992b, 1993, 1994; Blasi et al., 1993a, 1993b; Montecucco et al., 1994;
Yamasaki et al., 1994), (3) solution of the crystal structure for BoNT beginning with serotypes
A and B (Lacy et al., 1998; Swaminathan and Eswaramoorthy, 20(0), and (4) elucidation ofthe
proteinreceptorforBoNTjB,BoNT/G,andBoNTIA(Nishikietal., 1994;Dongetal.,2003,2006;
Rummel et ai., 2004, 2007; Chai et ai., 2006; Jin et al., 2006). These developments provided a
detailedunderstandingofthemechanismsofactionsofBoNTandopenedthepossibilityforrational
studies ofpharmacological antagonists forBoNTtoxicity.
A. CHARACTERISTICS OF BoNT INTOXICATION
The typical manifestation of botulism is a flaccid paralysis that is bilateral and descending,
involvingskeletal muscleand structuresinnervatedby autonomicganglia(Habermann andDreyer,
1986;SmithandSugiyama, 1988;Merzetai.,2003). HumanintoxicationiscausedbyserotypesA,
B, E, and, to a much lesser extent, F, and is generally manifested as foodborne, wound, and
intestinal (infant) botulism (Simpson, 1981). Wound and infant botulism are usually mediated by
serotypesAandB(Pickettetal., 1976;Arnon, 1995).Twoadditional forms ofbotulismhavebeen
observedthatdonotoccurin nature: inhalation botulismand iatrogenicbotulism. Theformer is so
rare in humans that only one occurrence, a laboratory accident, has ever been reported (Holzer,
1962). An outbreak ofinhalation botulism would be suspected as a terrorist incident unless other
causeswerefound (Arnonetal., 2001;ParkandSimpson, 2003;Adler, 2(06). Iatrogenic botulism
stemsfrom overdoseofclinicallyorcosmeticallyusedBoNT(Klein,2004).Arecentcasein which
fourindividualswereinjectedwith multiplelethaldosesofanonapprovedpreparationofBoNTIA
during a cosmetic procedure illustrates the potential hazard of this otherwise safe use ofBoNT
(Chertow etal., 2006; Souayah et al., 2006).
392 ChemicalWarfareAgents: Chemistry, Pharmacology,Toxicology, andTherapeutics
C. botulinumsporesarewidely distributedinsoils, seasediments,decaying vegetation,animal
carcasses, and sewage (Smith, 1978). The intestinal tracts ofbirds, mammals, and fish may also
acquire C. botulinum as a transient member of their intestinal flora. The hosts do not exhibit
botulism since growth of these anaerobic bacteria is suppressed when there is competition from
other organisms and a functional immune system (Smith, 1978; Smith and Sugiyama, 1988;
Snydman, 1989). The resistance ofclostridial spores to harsh environmental conditions enables
theirdissemination by aircurrentsand dustparticles, leading to surfacecontamination ofexposed
food products (Stingeretal., 2(05). Botulismis notcontagious, however, andcontactwith spores
does not usually lead to disease except in young infants under 1 year in age (infant botulism),
in adults with altered gastrointestinal (Gn anatomy and microflora (adult intestinal botulism), or
followinggerminationinwounds(woundbotulism)(MershonandDowell, 1973;MacKenzieetaI.,
1982; Arnon, 1995;Shapiroetal., 1998).
B.
SVMPTOMOLOGV
Theclinicalsyndromeofbotulismreflects toxin-inducedblockadeofAChreleasefrom neuromus
cular and neuroeffectorjunctions (Burgen et aI., 1949; Ambache, 1951). The basic syndrome of
BoNT intoxication is similar for foodbome, intestinal, and wound botulism and does not vary
appreciably among toxin serotypes (Sobel, 2005). The earliest symptoms generally include visual
disturbances (diplopia, blurred vision) and xerostomia(Hughes andTacket, 1983). With low-level
exposure, these symptoms may gradually resolve, even in the absence ofmedical intervention. In
more severe cases, the initial symptoms are followed by dysphasia, dysphonia, and dysarthria,
reflecting an especially high susceptibility ofcranial efferent terminals to BoNT action (Shapiro
etal., 1998).Adescendinggeneralizedskeletalmuscleweaknessmaythendevelop,progressingfrom
theuppertothelowerextremities.Involvementofthediaphragmandintercostalmusclescanleadto
ventilatory failure and death, unless appropriate supportive care is provided (Cherington, 1998;
Robinson andNahata, 2003; Sobel, 2005). Although motorfunction is severely impaired, there is
littleornosensoryalterationorCNSinvolvementinbotulinumintoxication(Simpson, 1981).
Symptomsareusuallyobserved 12-36hafterexposure,although onsettimesasshortas4hor
aslongas8dayshavebeenreported(RobinsonandNahata,2003;Sobel,2005).Thepreponderance
ofsymptoms, including thepotentially lethal respiratory collapse, stems from inhibition ofneuro
muscular transmission (Burgen et aI., 1949; Brooks, 1956; Kao et al., 1976; Simpson, 1981).
Parasympatheticdysfunctionisresponsibleforblurredvision,xerostomia,constipation,andurinary
retention (Ambache, 1951; MacKenzieetaI., 1982; Menetal., 2003).
C. FUNCTIONAL DOMAINSOF BoNI
The BoNTs are synthesized as -150 kDa single-chain protoxins (range, 140-167 kDa). They are
proteolytically activated (nicked) to form dichain molecules consisting ofa-50 kDa light chain
(LC) and a -100 kDa heavy chain (HC) (DasGupta and Sugiyama, 1972; Bandyopadhyay et aI.,
1987). The two chains arecoupled by a singledisulfidebond and by noncovalent forces. In their
natural state, BoNTs exist as complexes consisting of -150 kDa neurotoxin associated with a
group of nontoxic proteins. The latter are designated as neurotoxin-associated proteins (NAPs),
some of which possess hemagglutinin activity (Sakaguchi, 1982; Shanna et aI., 2(06). NAPs
associate with BoNT in the bacterial culture medium by noncovalent interactions and protect the
neurotoxin from proteolytic and low pH-mediated inactivation. They have also been suggested to
facilitateabsorptionofBoNTfromtheGItractintothebloodstream(ShannaandSingh, 1998).The
ability of BoNT to manifest oral toxicity has generally been attributed to the presence of these
proteins; conversely, the inability ofthe related tetanus neurotoxin (TeNT) to produce foodbome
intoxication has been ascribed to the absence ofsuch NAPs (Singh et al., 1995). Maksymowych
etal. (1999) have raised somequestions on the importanceofNAPs in BoNTtoxicity, especially
Botulinum Neurotoxin: Mechanismand Countermeasures 393
withregardtotheirroleintranscytosisoftheneurotoxin.Theseinvestigatorsdemonstratedthatpure
BoNT/A lacking NAPs was still toxic to mice following intragastric administration, although to a
lesserextentthan the toxin complex. Whenexaminedatelevatedconcentrations, thedifferences in
efficacy between pure and NAP-containing neurotoxin were progressively reduced. These results
indicate that pure neurotoxin does not require accessory proteins for absorption from the GI tract.
Moreover, even though the NAPs areclearly protective, sufficientpureneurotoxin can survivethe
inhospitableenvironmentofthe GI tract to produce lethality.
In conformity with the sequential processing of bacterial protein toxins such as diphtheria or
cholera toxin, the action of BoNT involves multiple discrete steps: binding to surface receptors,
internalization via receptor-mediated endocytosis, and translocation from endosome to cytosol. For
BONT, the final step is cleavage of soluble N-ethylmaIeimide-sensitive fusion protein attachment
protein receptors(SNAREs)in thecytosol (Simpson, 1981,2004;Montecuccoetal., 1994).Binding
and translocation are mediated by the C- and N-terminal domains of the BoNT HC, respectively
(Daniels-HolgateandDolly, 1996;KoriazovaandMontal,2003; Simpson, 2004;FisherandMontal,
2(06).TheLChaszincmetaIloproteaseactivity,targetedtooneofthethreeSNAREproteins(SNAP
25, synaptobrevin,orsyntaxin)thatarerequiredforthedocking and fusion ofsynapticvesicles with
activezonesatthecytoplasmicsurfaceofthenerveterminal(SchiavoetaI., I992a;Montecuccoand
Schiavo, 1993;MontecuccoetaI., 1994;Schiavoetal.,2(00).
Serotypes B, D, F, and G cleave different sites on the synaptic vesicle protein, synaptobrevin
(VAMP), whereasserotypesAandEcleavethepresynapticmembrane-associatedproteinSNAP-25
(Schiavo et aI., 2000; Simpson, 2(04). Serotype Cl is unique in that it cleaves two cytoplasmic
proteins, syntaxin and SNAP-25 (Williamson et aI., 1996). Interaction ofthese SNAREs on the
surfaceofsynapticvesiclesandactivezonemembranesisrequiredforvoltage-andCa2+-dependent
releaseofneurotransmitter;cleavagebyBoNTinhibitsthisprocess,leadingtomuscleweaknessand
paralysis (Sutton et aI., 1998; Schiavo et aI., 20(0). Cleavage ofSNARE proteins appears to be
sufficient to account for all actions ofthe BoNTs, and the SNARE hypothesis has received near
universal acceptance since its introduction in the early 1990s.
ForeachBoNTserotype,thedichainformconstitutestheactiveconfigurationoftheneurotoxin;
the isolated LC and HC are devoid ofsystemic toxicity. Theabsenceoftoxicity isconsistent with
findings thattheLCcannotgainaccess tothecytosolunlessitiscoupledtotheHCandthattheHC
lacks the ability to inhibitneurotransmitter release (Stecheret al., 1989; Goodnough et al., 2002).
The isolated LC does, however, remain enzymatically active as evidenced by its ability to inhibit
exocytosis from permeabilized chromaffin cells (Stecher et aI., 1989), by its ability to cleave
SNAREproteins in cell-free assays (Adleretal., 1998),and by its capacity to inhibitACh release
inskeletal musclewhendeliveredby liposomes (dePaivaandDolly, 1990).Itisnotclearwhether
any portion of the HC is translocated along with the LC, and if so, whether it exerts a role in
enhancing the catalytic activity orstability ofthe LC.
All BoNTserotypes suppressACh release, show high specificity forcholinergic synapses, and
share the same overall mode ofaction; they differ, however, in potency and in duration ofaction.
Type A neurotoxin exhibits the highest potency (Gill, 1982), and types A and C1 produce the
longest intoxication times (Eleopraet aI., 1998; Keller et al., 1999; Adleret al., 2001; Keller and
Neale, 2001; Foran et al., 2003; Keller, 2(06). Other differences include targeting of different
functional surface receptors on the motor nerve terminal (Black and Dolly, 1986; Montecucco,
1986;Daniels-HolgateandDolly, 1996;Rummeletal.,2007),andcleavinguniquepeptidebondsin
the appropriateSNAREproteins (Schiavoetal., 2000; Simpson, 2004; Rossetto etaI., 2006).
II. MANIFESTAliONS OF BOTULISM
Botulinum intoxication generally results from ingestion ofpreformed toxin elaborated in contam
inated foods (foodborne) or from colonization by C. botulinum ofdeep wounds with subsequent
productionoftoxin (woundbotulism)(MershonandDowell, 1973;Snydman, 1989).A third form,
394 ChemicalWarfareAgents: Chemistry, Pharmacology,Toxicology, andTherapeutics
tenned intestinal botulism, is observed in young infants (infant botulism), or less commonly
in adults with altered GI anatomy or microflora (adult intestinal botulism), and originates from
colonization ofthe large intestine by C. botulinum with subsequent production and absorption of
toxin(picketteta!., 1976;Amon, 1995).Twoadditionalfonnsofbotulismareiatrogenicbotulism,
from accidental overdoses following clinical orcosmetic procedures (Klein, 2004), and inhalation
botulism(Franz etal., 1993).
A.
FOODBORNE BOTULISM
ElaborationofBoNTinfoodsrequirescontactwith C. botulinumsporesunderconditionsthatallow
bacterial cell proliferation and toxin production. These consist of an anaerobic environment,
temperatures between 4°C and 40°C, pH above 4.6, water activity greater than 0.94 « 10%
NaCI), and lack ofadequate preservatives (Baird-Parker and Freame, 1967; Stingeret aI., 2005).
The requirements for growth of C. botulinum are stringent, especially anaerobiosis, making out
breaks relatively rare; nevertheless, episodes offoodbome botulism constitute a persistent public
health threat (Sobel et aI., 2004). In fact, food-related botulism outbreaks in the United States
have shown no significant reduction during the past century, with an average ofapproximately
24cases/year(ShapiroetaI., 1998).
Theprimary vehicleforfoodbomebotulismpresentlyandduringmostofthetwentiethcentury
hasbeenimproperlypreparedhome-preservedfoodproducts,ofteninvolvingvegetableswithalow
acid content (Smith and Sugiyama, 1988; Snydman, 1989). Other sources are food consumed in
restaurants that use unsafe procedures and contaminated commercially canned food products; the
latter has become rare since the introduction ofmodem methods (O'Mahoney et al., 1990). Data
compiled for foodbome botulism during the decade 1990-2000in the United States indicate that
serotypeAwasresponsiblefor50%ofallcases,whereasserotypesBandEaccountedfor10%and
37%, respectively, of intoxications in which serotype involvement was established (Sobel et aI.,
2004). Human foodbome intoxication by BoNT/F is exceedingly rare; between 1981 and 2002,
only asinglecase was reported to theCDC (Guptaeta!., 2005).
Although the number ofoutbreaks has been relatively constant, the case to fatality ratio has
improved markedly. From 1899 to 1950, foodbome botulism was associated with 60% mortality;
from 1950to 1996,theaverageannualmortalityfellto 15.5%(Shapiroeta!., 1998),anddecreased
to4%during the lastdecade (Sobel eta!., 2(04). Theseadvances in survival havecomeprimarily
from improvements in critical care (TacketetaI., 1984; Sobel etaI., 2004). Further reductions in
morbidity and mortality from botulinumintoxication will require bettermethods for detection and
diagnosis ofBoNToutbreaks and availabilityofspecific pharmacologicaltreatments (FranzetaI.,
1997; Dickerson andJanda, 2006).
PerhapsthelargestoutbreakoffoodbomebotulismrecordedtodateoccurredinNanProvince,
Thailand during 14-18 March 2006 (Ungchusak et a!., 2007). The outbreak was traced to con
sumptionofcontaminatedhome-cannedbambooshootsservedatareligiousfestival.Thesuccessful
handlingofthisoutbreakbytheThaiMinistryofPublicHealthhasimplicationsfortheappropriate
management of a small-scale bioterrorism attack involving deliberate contamination of the food
supply or an aerosol attack. A total of 209 people exhibited signs and symptoms of botulism,
with abdominal pain, dry mouth, and nausea being the most frequently reported; 134 villagers
requiredhospitalizationand42requiredmechanicalventilation.Botulismwas suspectedassoonas
more specific signs such as bulbar muscle paralysis were observed, especially when coupled
with respiratory depression. Due to familiarity with botulism in Thailand, the correct diagnosis
wasreached, andemergencyprocedureswereimplementedpromptly, whichallowedallpatients to
survive the outbreak. After initial triage, patients were flown to hospitals that had adequate
emergency care facilities, including ventilators. In countries where botulism outbreaks are less
frequent, health care personnel are often unfamiliar with its clinical presentation (Ruthman et aI.,
1985). As a result, botulism may not be diagnosed in a timely fashion, leading to delays in
Botulinum Neurotoxin: Mechanismand Countermeasures 395
treatment,andinamuchpoorerprognosis.Thelessonslearnedfromthislargeoutbreak.suggestthat
successful management ofa bioterrorist attack involving botulism would requireearly recognition
andaccuratediagnosisofsignsandsymptoms,arealisticplanforallocatingresources, adequacy of
thoseresources, andcoordinationamongthehealthcarefacilities,responsiblegovernmentagencies,
and international partners (Ungchusak et al., 2007). For this outbreak, antitoxin was obtained
through informal government channels, and although responses were expeditious, antitoxin from
theUnitedStatesandtheUnitedKingdomdidnotreachThailanduntil5-9daysaftertheoutbreak.
Thisdelay wouldhaveresultedinpotentialdeaths inthemoreseveregroupwereitnotforaccessto
ventilators and emergency care. Formal international arrangement for antitoxin delivery is prefer
able to informal mechanisms, but local strategic stockpiles ofcritical medicines and supplies are
essential to avoid delays in treatment.
B. WOUND BOTULISM
Woundbotulismisrelativelyrare,accountingforonly5%ofalloutbreaks.Themajorityoftheseare
caused by serotype A, and the remainder by serotype B (Shapiro et al., 1998). The neurological
symptoms ofwound botulism differ little from those offoodborne botulism except for the general
absence of GI symptoms. Historically, this form of botulism was so uncommon that it was not
even recognized until the last halfofthe twentiethcentury. From its discovery in 1943 until 1990,
only 47 incidences ofwound botulism were documented (WeberetaI., 1993). An examination of
these cases indicated that wounds susceptible to C. botulinum are generally deep with avascular
areas butneed notappearobviouslyinfectedornecrotic. Additional risk factors includecompound
fractures andextensivecrush injuries(MershonandDowell, 1973).Contaminationofwoundswith
C. botulinum spores leads to germination and colonization at the site of infection. Localized
weakness results from production of toxin at the wound, and systemic botulism can occur from
toxin transmitted via the bloodstream to distant targets (WeberetaI., 1993).
From 1980 to the present time, wound botulism has been observed predominantly in illicit
drug users following repeated subcutaneous administration of black tar heroin, or in individuals
with nasal or sinus lesions from chronic cocaine abuse (Anderson et al., 1997). During the
last decade alone, wound botulism from black tar heroin has exceeded the total reported wound
botulism cases during the preceding 40 years by a factor of almost three (Sandrock and
Murin, 2001).
Forreasons that are not completely understood, wounds are much more likely to be contami
nated by Clostridium tetani than with C. botulinum. Although an aggressive vaccination program
has nearlyeliminated tetanus in developed nations, the absence ofuniversal tetanus vaccination in
manydevelopingcountriesresults insubstantialmortality (VandelaeretaI., 2003). Alargenumber
ofTeNT intoxications occurin neonates, often by infection ofthe umbilical stump. The mortality
ratesindevelopingcountrieswerereportedtobe85%forneonataltetanusand50% fornonneonatal
tetanus during the mid-1980s (Schofield, 1986). For the year 2002, deaths from TeNT were
estimated by the World Heath Organization (WHO) at 213,000 worldwide, of which 198,000
occurred inchildren under5 yearsofage (Vandelaeret al., 2003).
C. INTESTINAL BOTULISM
1. Infant Botulism
Infant botulism is a consequence of intoxication by BoNT following ingestion or inhalation of
clostridial spores that colonizethe large intestine; young infants, especially those between 2 and 4
months of age, are susceptible to this form of botulism (Pickett et aI., 1976; Amon, 1995).
Germination ofspores and growth ofvegetative cells lead to production ofBoNT; the neurotoxin
thus elaborated crosses the intestinal wall and reaches susceptible targets such as skeletal muscle