Botulinum Toxin

1. Clostridium botulinum: Botulinum ToxinClostridium botulinum: Botulinum Toxin
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. • The genus consists of G+ve, anaerobic, Spore
forming bacilli.
• Highly pleomorphic, straight or slightly curved
rods with slightly curved ends.
• 3-8 x 0.4-1.2 µm in size
• Spores are wider than bacillary body, giving
bacillus a swollen appearance resembling
spindle; hence named so (Kolster meaning
spindle ).
• Motile (except Cl. tetani Type VI and Cl.
perfringens).
• Cl. perfringens and Cl. butyricum are capsulated;
others are non-capsulated.
2

3. SPORESSPORES
 The shape and position of spores varies in different spp. & thus useful in
their identification.
 Spores may be;
Central or equatorial in Cl. bifermentans (Spindle shaped)
Sub terminal in Cl. perfringens (club shaped)
Oval or terminal in Cl. tertium (resembling tennis racket)
Spherical and terminal in C. tetani (drum sticks )
3

4. Clostridium spp. Anaerobic Gram-Positive Spore-Forming Bacilli
Four broad types of pathogenesis:
1. Histotoxic group — tissue infections
(C. perfringens type A, exogenously acquired more commonly than endogenously)
(C. septicum; endogenously-acquired)
a. cellulilitis
b. myonecrosis
c. gas gangrene
d. fasciitis
2. Enterotoxigenic group — gastrointestinal disease
a. clostridial foodbome disease (8-24h after ingestion of large numbers of organisms on con-taminated
meat products, spores germinate, enterotoxin produced (C. perfringens type A)
b. necrotizing enteritis (beta toxin-producing C.perfringens type C)
(C. difficile endogenously-acquired or exogenously-acquired person-to-person in hospital)
c. antibiotic-associated diarrhea
d. antibiotic-associated pseudomembrane colitis
3. Tetanus (exogenously acquired) — C. tetani neurotoxin
a. generalized (most common)
b. cephalic(primary infection in head, comnnonly ear)
c. localized
e. neonatal (contaminated umbilical stump)
4. Botulism (exogenously acquired) — C. botulinum neurotoxin
a. foodborne (intoxication,1-2days incubation period)
b. infant (ingestion of spores in honey)
c. wound (symptoms similar to foodborne, but 4 or more days incubation)

5. Clostridia of medical importanceClostridia of medical importance
5

6. Clostridium Associated Human Disease

7. Clsotridium botulinum
Important note:
• The toxin is NOT secreted by C. Botulinum,
instead, it is produced during the bacteria’s
autolysis.

8. Introduction to botulism
 Neuroparalytic disease caused by neurotoxins (BTX or BoNTs)
produced by bacteria Clostridium botulinum.
 BoNT is broken into 7 neurotoxins (labeled as types A, B, C [C1,
C2], D, E, F, and G), which are antigenically and serologically distinct
but structurally similar.
 7 different BoNTs (A-G) produced by different strains of C.
botulinum.
 BoNTs A, B, E, and F outbreaks in humans
 BoNT C in birds
 BoNT D in cattle
 BoNT G isolated from soils
 BoNT A most common and most potent toxin, followed type B.
 Human botulism is caused mainly by types A, B, E, and (rarely) F.
 Types C and D cause toxicity only in animals.
 BoNTs also produced by other members of Clostridia family.

9. The BONT
Structure:
 The BoNT molecule is
synthesized as a single chain
precursor (pretoxin) of 150 kDa
and which is cleaved to form fully
active neurotoxin; the di-chain
molecule composed of a light
chain (LC) and heavy chain (H)
linked by single disulfide bridge.
 Light chain acts as an Zn-
endopeptidase.
 When bridge is intact, BoNT
has no catalytic activity.

10. The BoNT…..
The heavy chain (~100 kD)
provides cholinergic
specificity and is
responsible for binding the
toxin to presynaptic
receptors, it also promotes
light-chain translocation
across the endosomal
membrane.
The light chain (~50 kD)
acts as a zinc (Zn2+
)
endopeptidase similar to
tetanus toxin with
proteolytic activity located
at the N-terminal end.

11. Structure
Turton, K., J.A. Chaddock, and K.R. Acharya (2002). “Botulinum and tetanus neurotoxins: structure, function
and therapeutic utility.” TRENDS in Biochemical Sciences 27(11): 552-558.

12. 3D Structure
Turton, K., J.A. Chaddock, and K.R. Acharya (2002). “Botulinum and tetanus neurotoxins: structure, function
and therapeutic utility.” TRENDS in Biochemical Sciences 27(11): 552-558.

13.  Release of acetylcholine
at the neuromuscular
junction is mediated by the
assembly of a SYNAPTIC
FUSION COMPLEX.
 The synaptic fusion
complex is a set of SNARE
proteins, which include 3
different types of SNARE
proteins
Synaptobrevins/VAMPs
SNAP-25
Syntaxin
 SNARE proteins allows
the membrane of the
synaptic vesicle containing
acetylcholine to fuse with
the neuronal cell membrane
 neurotransmitter is
released.
 After membrane fusion,
acetylcholine is released
into the synaptic cleft and
then bound by receptors on
Release of acetylcholine at the neuromuscular junction

14. MECHANISM OF ACTION
Botulinum toxins act at four different sites in the body:
 The neuromuscular junction
 Autonomic ganglia
 Postganglionic parasympathetic nerve endings
 Postganglionic sympathetic nerve endings that release
acetylcholine.

15. BoNTs block Acetylcholine release in
three steps
• Binding
– BoNT binds by Hc to receptor
on cell
• Internalization
– Toxins internalized via
receptor-mediated endocytosis
• THE POINT OF NO RETURN:
Once endocytosed, the
toxins can no longer be
neutralized by antisera
• Translocation
– Heavy and light chains
separate; light chain enters the
cytosol and cleaves SNAREs
 SNARE complex is non-
functional and Ach is not
released
SNARE: Soluble NSF-Attachment protein REceptor; NSF: N-ethylmaleimide-Sensitive
Fusion protein; SNAP-25: Synaptosomal-associated Protein of 25 kd.
BoNTs prevent neurotransmitter release

16. BoNTs cleave SNAREs
Humeau, Y., F. Doussau, et al. (2000). “How botulinum and tetanus neurotoxins block
neurotransmitter release.” Biochimie 82: 427-446.
 The light chain of
botulinum toxin cleaves
specific sites on the SNARE
proteins, preventing
complete assembly of the
synaptic fusion complex
thereby blocking
acetylcholine release.
 Botulinum toxins
 types B, D, F, and G cleave
VAMP/synaptobrevin 2
 types A, C, and E cleave
SNAP-25
 type C cleaves syntaxin.
 Without acetylcholine
release, the muscle is unable
to contract.

17. BoNTs prevent neurotransmitter release

18. BoNTs cleave SNARE proteins and prevent Ach release

22. Effects of BoNT
Induces weakness of striated muscles by inhibiting transmission
of alpha motor neurones at the neuromuscular junction. This
has led to its use in conditions with muscular over-activity, such
as dystonia.
Transmission is also inhibited at gamma neurones in muscle
spindles, which may alter reflex over-activity.
Also inhibits release of acetylcholine in all parasympathetic and
cholinergic postganglionic sympathetic neurons. This has
generated interest in its use as a treatment for overactive
smooth muscles (for eg., achalasia) or abnormal activity of
glands (for eg., hyperhidrosis).
The toxin requires 24-72 hours to take effect, reflecting the time
necessary to disrupt the synaptosomal process.
In very rare circumstances, some individuals may require as
many as five days for the full effect to be observed.
Peaking at about 10 days, the effect of botulinum toxin lasts
nearly 8-12 weeks.

23. Clinical Perspective of Clostridium botulinum
 The toxin cannot pass through the skin, thus, transmission
requires a break in the skin or direct absorption through mucus
membranes in the lungs or GI tract.
 There are four types of botulism, characterized by the method
of delivery of the toxin.
1. Foodborne botulism is the result of the ingestion of food
contaminated with Clostridium botulinum containing the pre-
formed toxin.
Note: Ingestion of the toxin makes a person ill, not Clostridium
botulinum itself.
2. Wound botulism: Occurs when a break in the skin becomes
infected with Clostridium botulinum which then multiply and
release botulism toxin into the blood.
4. Infant botulism: is the result of the infestation of the digestive
tract with Clostridium botulinum.
5. Inhalation botulism: Occurs when aerosolized botulism toxin
enters the lungs.

25. • In infant botulism, illness results from
infestation of the GI tract with Clostridium
botulinum.
• Such infestation is generally not an issue in
individuals older than one year due largely
to the large number of competing
microorganisms found in the mature GI
tract.
• Roughly 100 cases of botulism are reported
in the U.S. each year.
• Approximately 25% are foodborne, 72% are
infant botulism, and the remaining 3% are
wound botulism.
• Inhalation cases do not occur naturally.
• Wound botulism is on the rise due to an
increase in the use of black tar heroin.
• The source of the botulism could be the drug
itself, a cut in the drug, dirty injection
equipment, or contamination during the
preparation process.

26. • The incubation period varies according to the mode of
transmission, rate of absorption of the toxin, total amount and
type of toxin.
• Foodborne botulism usually takes 24-36 hours to manifest itself.
• Wound botulism often takes 3 or more days to appear.
• Inhalation botulism has occurred very rarely, but incubation
times may range from several hours to perhaps days, again
depending upon the type and amount of toxin inhaled.
Incubation period

27.  All four types of botulism result in SYMMETRIC DESCENDING FLACCID
PARALYSIS of motor and autonomic nerves always beginning with the
cranial nerves.
 These symptoms are preceded by constipation in cases of infant
botulism.
 Symptoms include:
Double or blurred vision
Drooping eyelids
Dry mouth
Difficulty in Swallowing
Muscle weakness
 If left untreated symptoms may expand to include paralysis of
respiratory muscles as well as the arms and legs.
 Asphyxiation due to respiratory paralysis is the most common cause of
death in botulism cases.
 Botulism results in death in approximately 8% of documented cases.
 The key to survival is early diagnosis.
 For the period 1899-1949 the case fatality ratio was approximately 60%.
For the Period 1950-1996 the case-fatality ratio was 15.5%.
 This improvement is largely attributable to improvements in respiratory
intensive care and availability and prompt administration of the
antitoxin.
Symptoms

28. Treatment
 Antitoxin can halt the progress of symptoms if administered
early to victims of food and wound botulism.
 Antitoxin is not given to victims of infant botulism because
when this is diagnosed, it is generally too late for the antitoxin
to do any good.
 Wound botulism is treated surgically to remove the
Clostridium colony.
 Artificial respiration is required if paralysis reaches the lungs.
Such respiratory assistance may be required for weeks to
months.
 The paralysis induced by the toxin slowly improves over the
course of many weeks.
 Many patients make close to a full recovery following weeks
to months of intensive care, however, lingering effects such as
fatigue and shortness of breath may linger for years.

29. Immuno-prophylaxis
• Attempts to develop an effective botulism vaccine
date back to the 1940’s.
• One current effort (now moving into clinical trials)
uses injection of a non-toxic carboxy-terminus
segment of the botulism toxin to confer immunity to
the toxin.

30. Center for Food Security and Public Health
Iowa State University 2004
Animals
 Botulism affects animal species such as cattle, sheep, horses,
wild birds and poultry, and mink and ferrets.
 Dogs and pigs can also be affected, however they seem to be
more resistant to the disease - Fairly resistant; therefore, cases
are much more uncommon compared to the previously
mentioned species.
 To date no natural cases have been documented in cats.

31. Cattle and Sheep
 In cattle and sheep, disease is usually caused by ingestion of neurotoxin in
contaminated feed stuffs.
 Most cattle cases involve type B, C, and D toxin while most sheep cases involve
type C toxin.
 The incubation time is 24 hours to 7 days.
 Common sources of the toxin include improperly stored silage or spoiled
brewer’s grains.
 Silage incorporating poultry litter or poultry products can also be a source of
botulism toxin.
 Cattle with phosphorus deficiency can obtain the toxin via ingestion of soil
while enacting pica.
 Finally, carcasses unintentionally baled into hay or chopped into hay cubes or
pellets may potentially contribute to botulism in ruminants. This later source
was responsible for the deaths of 400 dairy cattle in a California herd in 1998.
Following the outbreak, it was discovered that the unintentional contamination
occurred from the carcass of a dead cat in the feed.

32. Ruminants: Clinical Signs
 Clinical signs in ruminants include progressive ascending
ataxia from the hindlimbs to the forelimbs.
 Animals are usually recumbent and cattle will turn their heads
into their flanks.
 Signs of cranial nerve dysfunction are present, such as
dysphagia, drooling, tongue paresis, and facial muscle paresis.
 Eye effects include decreased pupillary light reflex, ptosis and
mydriasis.
 Additionally, rumen stasis and bloat can occur, as well as an
atonic bladder with loss of urination.

33. Cattle and Sheep: Diagnosis
 Diagnosis of botulism in ruminants can be determined by
obtaining a good history.
 Bloodwork and CSF taps are usually normal.
 An ELISA test is available for types C and D toxin.
 The definitive diagnosis comes from demonstration of the
toxin in serum, gut contents or organs.
 Additionally, electromyography (EMG) results may be
diagnostic.

34. Center for Food Security and Public Health
Iowa State University 2004
Cattle and Sheep: Treatment
• Treatment for ruminants includes symptomatic and supportive
treatment- includes general nursing care, fluids and nutrition.
• Ventilator support may be needed.
• Metronidazole may be useful, however avoid aminoglycosides,
tetracyclines and procaine penicillin as they have been
associated with neuromuscular weakness.
• Antitoxin may be given when diagnosed in an early stage.
• It is usually ineffective by the time clinical signs are present, but
it can block further uptake of the toxin.

35. Horses
 Horses, especially foals, are highly sensitive to botulism toxin.
 The most common toxin in equines are types B and C.
 The incubation period in equines is 24 hours to 7 days.
 In horses, the most common sources of botulism occur from
contaminated feed or infection by the organism in open
wounds.

36. Center for Food Security and Public Health
Iowa State University 2004
Adult Horses
 In adult horses, botulism is also called “forage poisoning”.
 Horses ingest the preformed toxin from contaminated feed.
 Clinical signs include dyspnea, a flaccid tail and muscle tremors.
 They will have severe paresis which progresses to rapid
recumbency.
 Adult horses will be unable to retract their tongue and will
therefore, drool.

37. Center for Food Security and Public Health
Iowa State University 2004
Foals
 In foals, botulism causes “Shaker Foal Syndrome” and typically
affects them from 2 weeks to 8 months old.
 Foals that are on a high plane of nutrition are more susceptible.
 Foals typically consume spores in contaminated feed which
germinate and release the neurotoxin within the
gastrointestinal tract.
 Type B toxin is the most common form found in foals.
 Shaker foal syndrome is most common in Kentucky and the
eastern seaboard.
 Clinical signs in foals include paresis, recumbency, and muscle
tremors.
 Cranial nerve signs also exist, such as dysphagia, ptosis,
mydriasis, and decreased pupillary light reflex.
 Additional signs include ileus, constipation and urine retention.
 Death is usually due to respiratory paralysis.
 Mortality can be greater than 90%.

38. Birds and Poultry
• Botulism infection in avian species is commonly
referred to as “limber neck” disease.
• The most common neurotoxin type found in
birds is type C for waterfowl and shorebirds
(especially ducks in the western U.S.) and
poultry. Type E neurotoxins affect gulls and
loons.
• Wild birds can be a good sentinel species.
• The most common source for wild birds is
decomposing (anaerobic conditions)
vegetation and invertebrates.
• The birds ingest the toxin or invertebrates with
the toxin.
• Outbreaks occur from coast to coast in the
United States and Canada, generally from July
through September.
• Thousands of birds may die during a single
outbreak.
• Botulism toxin can be common in the gut of
poultry and wild birds as well as the litter, feed
and water of chickens.

39. Center for Food Security and Public Health
Iowa State University 2004
Birds and Poultry: Clinical Signs
• Avian species typically show clinical signs of botulism 12-48
hours after ingestion of the toxin.
• They will have a ‘limber neck’, with a droopy head and appear
drowsy.
• Infection makes these birds unable to use their wings or legs or
to hold their heads up, so they drown.
• Death can also result from water deprivation, electrolyte
imbalance, respiratory failure and predation.
• Eyelid paralysis

40. Mink and Ferrets
• Mink and ferrets are extremely susceptible to botulism.
• They are usually affected by type C toxin, occasionally types A
and E can be isolated.
• The most common sources are from chopped raw meat or fish
or can come from improper storage of meat by-products.
• A vaccine is available for these animals.
• Annual vaccination of kits and breeding animals with botulism
(type C) toxoid is recommended to prevent outbreaks.
• Kits should be vaccinated after 6 weeks of age.

41. Dogs
• Botulism in dogs is RARE however, the majority of cases of
canine botulism are caused by neurotoxin type C; a few are
caused by type D.
• Cases are typically caused by ingestion of the toxin.
• This may come from the ingestion of contaminated carrion, or
in hunting breeds exposed to wetland areas with avian
botulism epizootics.
• The incubation period in dogs ranges from a few hours to 6
days.
• Duration of illness is from 14-24 days.
• Clinical signs involve progressive symmetric ascending
weakness from rear to forelimbs that can result in quadriplegia.
• Cranial nerves are also affected causing decreased pupillary
light reflexes, jaw tone, and gag reflexes.
• Pain perception is still maintained and the dog is alert.
• Death from respiratory paralysis can occur.
• Dogs can also lose their ability to urinate and defecate.

42. Dogs
 The diagnosis of botulism in dogs can be difficult.
 History of carrion ingestion and physical exam can be helpful.
 Bloodwork and CSF taps are usually within normal limits.
 A electromyography (EMG) can be diagnostic.
 Additionally, demonstration of the toxin in serum, vomitus, feces or
the suspected food/carrion can also be diagnostic.
 The preferred method is the mouse neutralization test.
 Treatment involves supportive and nursing care because dogs will
not be able to swallow, eat or drink well.
 Additionally, loss of urination and defecation ability may require
assistive measures.
 Antibiotics are usually not indicated, since a toxin is the cause of the
clinical signs.
 Antitoxin can be administered, but it is usually not effective once the
toxin has bound to neuromuscular junctions. It can however prevent
further binding of any toxin remaining in the system.
 There is a potential risk of anaphylactic shock with the antitoxin.

43. Prevention and control: Human
 Prevention of botulism in humans includes educating yourself
and clients about this disease.
 Because HONEY can contain botulinum spores and is not
chemically treated/boiled/pasteurized before consuming, it is
recommended that children under one year of age should not
eat honey.
 Other recommendations include proper home canning and
food preservation methods which will destroy the spores.
 Prompt refrigeration of foods will also help.
 Boiling foods, especially those that are home canned, for over
10 minutes to destroy the toxin.
 Before discarding any suspected food be sure to boil it for the
appropriate time to detoxify it.
 Boil or chlorine disinfect any utensils that were in contact with
the suspected food.
 Finally, report any suspect cases to the state or local health
authorities.

44. Center for Food Security and Public Health
Iowa State University 2004
Ruminants: Prevention
• The best prevention against botulism in ruminants includes good
husbandry practices.
• Rodent and vermin control will minimize potential carcass
sources.
• Prompt disposal of carcasses will be helpful.
• Avoid spoiled feedstuffs or poor quality silage.
• Vaccines can be used in cattle, sheep and goats in endemic areas.

45. Equine: Prevention
• Prevention of botulism in horses includes good husbandry
practices.
• Rodent and vermin control will help to reduce carcass
contamination of feed.
• Avoid feeding spoiled feed stuffs.
• A prophylactic vaccine is available for pregnant mares.
However, only type B botulinum toxoid is available for horses.
• Initially, mares should be vaccinated during gestation with a
series of three doses administered 1 month apart, with the last
dose 2-4 weeks before foaling to ensure optimal protection of
the foal via colostrum.
• Vaccination of horses with type B toxoid will not induce
protection against other neurotoxin types, since there is no
cross-protection between them.
• Currently there is no approved equine vaccine for protection
against type C botulism.

46. Diagnosis
 The symptoms of botulism are similar to those of Guillain-Barré
syndrome, stroke, and myasthenia gravis.
 As a result, botulism is probably substantially under-diagnosed.
 Serum electrolytes, renal and liver function tests, complete blood
tests, urinalysis, and electrocardiograms will all be normal unless
secondary complications occur.
 A brain scan, spinal fluid examination, electromyograph, or
tensilon test (edrophonium) may be required to positively identify
botulism.
 The most effective test comes from the identification of botulism
toxin in serum or stool. The test is most often carried out by
injecting samples into a mouse and observing whether symptoms of
botulism develop.
 However, the false negative rate for this test can be as high is 60%
for serum samples and near 80% for stool samples in individuals
clinically diagnosed with botulism.
 Collection of samples early in the progression of the illness may be
helpful, however, large outbreaks have occurred in which none or a
very low percentage of victims produced a positive test result.

47. Diagnosis
 In vitro methods utilizing ELISA are under development but are
not yet validated.
 Isolation of Clostridium botulinum from the patient’s feces or
gastric sample is a good confirmation of botulism as the
organism is rarely found in humans in the absence of the
botulism poisoning, however, poisoning can occur without
ingestion of the microorganism at all.
 If botulism poisoning is suspected clinicians are advised to
contact local and state health authorities who should then
contact the CDC.

48. CULTURAL CHARACTERISTICS
 Clostridia are anaerobic.
 Optimum temp. for growth is 37°C; pH 7-7.4.
 Robertson’s cooked meat broth is useful medium.
 Most species produce gas in this medium
 Saccharolytic species turn meat pink.
 Proteolytic species turn meat black with foul smell
48

49. A long history
 Botulism originally known as “sausage poisoning” in late 18th
century and throughout 19th
century.
 From Latin botulus = sausage
Bacterial etiology recognized at end of 19th
century
 Outbreak of botulism in Belgium 1895 revealed cause as
neuroparalytic toxin produced by anaerobic bacterium.
 Probably Type B
 Outbreak in Germany several years later
 Bacterium isolated; different from that in Belgium
 Probably Type A
The 20th
Century and Beyond
 1949 Burgen et al. determines botulinum toxin blocks
neurotransmitter release.
 1979 Simpson proposes cellular mechanism in 3 steps: binding,
internalization, and intraneuronal action.
 1990 a.a. sequence of one BoNT determined in Niemann’s laboratory
 21st
century – 3D structure of BoNT A resolved.

50. Historical Aspect
A German poet, doctor and scientist , Dr. Justinus Kerner of
Wurttemberg, first explained the disease called botulism (1817 to
1822) caused by ‘sausage poison’.
Already imagined that the toxin that caused such a serious disease,
could be used to treat diseases like muscular spasms.
Dr Emile Pierre van Ermengem (belgium) in 1895 successfully isolated
this bacterium, named it Bacillus botulinus.
Botulinum toxin was first used to treat human disease (1980) by Drs
Alan Scott (opthalmologist) and Edward Schantz, in treating
strabismus.
In 1987, ophthalmologist Jean Carruthers observed that frown lines
disappeared after the use of botulinum toxin A for blepharospasm.
In 1996, they published the first paper on the use of Botox for
cosmetic purposes.
In 2002, the FDA announced the approval of BOTOX® Cosmetic to
temporarily improve the appearance of moderate-to-severe frown
lines between the eyebrows (glabellar lines).
In July 2004, the FDA approved BOTOX® to treat severe underarm
sweating, known as primary axillary hyperhidrosis, that cannot be
managed by topical agents.

51. The History Continues…
• First biological toxin used for treatment of human disease
• Manufactured for medical use in 1989 under name “Oculinum”
– Licensed for treatment of strabismus and blepharospasm
(eye conditions characterized by excessive muscle
contraction)
Vangelova, Luba. “Botulinum Toxin: A Poison that Can Heal.” Available at:
http://www.fda.gov/fdac/features/095_bot.html.
Blepharospasm treated with Oculinum

52. Medical uses of BoNT
• Now manufactured under the name “Botox”.
• Experimentally used for treating migraine headaches, chronic
low back pain, stroke, cerebral palsy, and dystonias (neurologic
diseases involving abnormal muscle posture and tension).
• Frequent injections allows an individual to develop antibodies
– Studies carried out to determine feasibility of other strains of BoNT
• BoNT B manufactured for treatment of cervical dystonia in
2000 as “Myobloc”.

53. Cosmetic Use of BoNT
• 1994 FDA denounced the promotion of BoNT use for
wrinkles as "an egregious example of promoting a
potentially toxic biologic for cosmetic purposes."
• Botox approved for Cosmetic use in April, 2002
• Myobloc not approved for cosmetic use, but is used
experimentally in many cosmetic procedures anyway.

54. BoNT A (Botox)
Botox injection patient 13 weeks after injection
Sadick, N. and A.R. Herman (2003). “Comparison of Botulinum Toxins A and B in the
Aesthetic Treatment of Facial Rhytides.” Dermatologic Surgery 29:340-347.

55. BoNT B (Myobloc)
Myobloc injection patient 11 weeks after
procedure
Sadick, N. and A.R. Herman (2003). “Comparison of Botulinum Toxins A and B in the
Aesthetic Treatment of Facial Rhytides.” Dermatologic Surgery 29:340-347.

56. Preparations of BoNTs
Serotype A is the only commercially available form of
botulinum toxin for clinical use, although experience is
emerging with development of other serotypes B, C, and F .
The products and their approved indications include the
following:
BotulinumtoxinA (Botox, Botox Cosmetic)
Botox - Cervical dystonia, severe primary axillary hyperhidrosis,
strabismus, blepharospasm, neurogenic detrusor overactivity,
chronic migraine, upper limb spasticity
Botox Cosmetic - Moderate-to-severe glabellar lines
BotulinumtoxinA (Dysport) - Cervical dystonia, moderate-to-
severe glabellar lines
BotulinumtoxinA (Xeomin) - Cervical dystonia,
blepharospasm
Botulinumtoxin B (Myobloc) - Cervical dystonia

59. Potency of BoNT
• The potency of BoNT-A is measured in mouse units (MU).
• One MU of BoNT-A is equivalent to the amount of toxin that
kills 50% of a group of 20 g Swiss-Webster mice within 3 days of
intraperitoneal injection (LD50).
• According to one report, 1 nanogram of toxin contains
approximately 20 U of BOTOX® (ie, 1 U of BOTOX® is equal to
approximately 0.05 nanogram of the toxin).
• One unit of BOTOX®
has a potency that is approximately equal
to 4 unit of Dysport®
.
• One unit of Xeomin®
is equal to 1 unit of Botox®

60. Weaponization of Botulinum Toxin
 1 mg of Botulinum Toxin can kill
around one million people if evenly
distributed.

61. What does it mean to “weaponize” a biological agent?
The "weaponization" of a microbial pathogen or toxin involves:
• Rendering the agent resistant to standard antibiotic drugs
• Freeze-drying and milling the agent into an extremely fine
powder, consisting of particles tiny enough to become readily
airborne and inhaled into the victims' lungs to cause infection
• Stabilizing the agent so that it will remain infectious for a
longer period after release
• Treating the powder with chemical additives that absorb
moisture and reduce clumping, so as to facilitate
aerosolization.
Answer provided by Jonathan B. Tucker, Ph.D. an expert on chemical and biological weapons in
the Washington, D.C and a biological weapons inspector in Iraq under the auspices of the United
Nations Special Commission in 1995.

62. History of Botulinum Toxin as bio-weapon
Early primitive example:
1910s Mexico1
• Supporters of Pancho Villa used Botulinum toxin against
Mexican federal troops.
• Buried pork and green beans for several days.
• Then used the mixture to contaminate food or smeared on
knife-like weapons.
• Result in food and wound botulism
Easy to make!

63. History of Botulinum Toxin as bio-weapon
Organized weapon programs:
World War II
• Japanese invaders (Biological Warfare group Unit 731) fed cultures of
Clostridium Botulinum to prisoners and caused lethal effect
• Later, Germany, US and USSR were all producing weaponized Botulinum Toxin
1970s
• Biological and Toxin Weapons Convention banned bio-weapon
research and production
• Iraq and USSR continued to produce Botulinum Toxin as potential weapon
• Soviets  splice the Botulinum toxin gene into other bacteria
• Iran, North Korea and Syria are also believed to go on with developing
Botulinum toxin in weapons

64. History of Botulinum Toxin as bio-weapon
1990s Persian Gulf War
• Iraq produced 19,000 L of concentrated Botulinum Toxin
• ~10,000 L were loaded into military weapons
• The 19,000 L of Botulinum Toxin is around 3 times the amount needed to
kill the entire human population by inhalation.
Note: Iraq chose to weaponize more Botulinum Toxin than any other biological
agents

65. Contemporary attempt of the toxin in attack (However, unsuccessful)
1990-95 Japan
• By Japanese Cult Aum Shinrikyo
• Obtained C. Botulinum from soil collected in
Northern Japan
• Dispersed Botulinum Toxin as aerosols (spray)
- In downtown Tokyo
- US military bases in Japan
 Why failed? Unknown!
But suspect:
• Particles were not refined enough
• Strain of bacteria used were not virulent
 However their release of SARIN gas succeeded
• Killed and injured many in a subway attack
• Sarin is an extremely water soluble chemical
agent (can diffuse thru eyes, skin etc)
So after all there were no actual record of a successive attack or an
intentional outbreak by terrorist or armed forces using botulinum toxin.
However, it still has great potential being a biological weapon

66. Bioweapon Potential
Botulinum Toxin is a major threat because
 Extreme potency and lethality
 Ease of production
 Ease of transport
 Need for prolonged intensive care

67. Bioweapon Potential
Extreme potency and lethality
One of top 6 potential biological warfare agents
Listed as Category A agent by CDC i.e. Highest priority
The most toxic substance known
→ toxic dose ~0.001µg/Kg body weight
→ 15,000 times more toxic than nerve agent VX
→ 100,000 times more toxic than sarin
→ 1 gram of crystalline toxin can kill >1 million people if dispersed
and inhaled evenly

68. Ease of production
• Recipes for home brews of Botulinum toxin can be easily found
on internet
• C. Botulinum can be grown on fairly basic media and lab
protocols can be accessed in many books.
• Toxin is easily purified using general biochemical purification
techniques.
– salting out, acid precipitation, gel filtration chromatography etc..
• Industrial-scale fermentation can produce large quantities of
the toxin for use as a biological agent.

69. Ease of transport
•It can be easily transported if not made into aerosolized
form.
•C. Botulinum grows naturally in soil and BoNT can stay
stable in still water for weeks
•Not contagious
•Does not spread by inhalation naturally
•Does not enter through skin

70. Need for Prolonged intensive care
After intoxication and treating w/ antitoxin:
• Ventilatory support and 24-hr nursing care is commonly needed
for 2-8 weeks.
• Some might require up to 7 months before the return of
muscular function.
• However, no municipality has sufficient ventilators to provide
intensive care for mass victims of a terrorist attack by aerosolized
BoNT.

71. Bioterrorism: routes of intoxication
Types of Botulism:
• Food/waterborne
• Inhalational
• Wound (least likely)
Potential Victims:
Persons of all age and sex.
Lethal Dosage:
For a 70 Kg human
- 0.09-0.15 µg intravenously or intramuscularly
- 0.70-0.90 µg inhalationaly
- 70 µg orally

72. Wound Botulism
• Least effective thus not practical to use in bioterrorism
• Extremely small scale
• Requires direct contact between wound and spores of C.
Botulinum
• Very primitive (may involve using knives)
Jermann M, Hiersemenzel LP, Waespe W. Drug-dependent patient with multiple cutaneous
abscesses and wound botulism. Schweiz Med Wochenschr 1999, 129:1467

73. Food Botulism
Carried out by deliberate contamination of food or water supply:
•In commercial production facilities or restaurants or reservoirs.
•Produces either large epidemic (area) outbreak or separated episodic (time)
outbreaks.
Recognition of intentional outbreaks:
•Victims all share common dietary exposure
Average Incubation Period:
•Neurological symptoms occurs ~12-36 hrs after ingestion
•BoNT can stay in untreated water or uncooked food for up to days
•It is colorless, odorless and tasteless
However, BoNT:
•Can be inactivated by heat (>85°c for 5 min)
•Can be rapidly inactivated by standard potable water treatments (e.g.
Chlorination, aeration)
•Large capacity reservoirs requires large* quantities of BoNT in order to cause
severe contamination
*difficult for terrorists to make and carry around
There are obstacles in order to produce an effective large scale food botulism

74. Inhalational Botulism
• Man-made (does not occur naturally)
• Utilizes aerosolized Botulinum toxin
– May involve freeze-drying and milling the toxin into an extremely fine
powder
• Absorption of toxin through mucosal surface in the lung
Incubation Period:
• Neurological symptoms usually occurs 24-72 hrs after aerosol
exposure

75. Inhalational Botulism: Aerosols, Missiles and Bombs
 Large scale
 More efficient way of bio-terrorism
 Can equip war-heads of missiles or bombs and grenades - As white powder or
liquid slurry
 Can be sprayed as aerosols
 Point-source aerosol release can incapacitate or kill 10% of persons within 500
meters downwind.
1 gram of crystalline toxin can kill >1 million people if dispersed and inhaled
evenly
More deadly than food botulism (because smaller lethal dose) but technically
and financially difficult to carry out:
Instability
Inactivated by sunlight within 1-3 hours
Detoxified in air within 12 hours
Technically difficult and complicated for the insufficiently funded terrorist to:
Make the powder form for efficient dispersal
obtain the accurate dispersal equipment
This is illustrated by the lack of actual cases
(However we can never underestimate the ability
of terrorists).
Even though studies are continually performed on animals, there were no
actual historical records of successful attacks using airborne botulinum
toxin.
Only record being an accidental human inhalation of aerosolized BoNT in a
veterinary facility in 1962 Germany
3 ppl were affected - and prolly didn’t die

76. Potential danger: Botulinum Superbug?!
WHY?
• BoNT is non-contagious (just a toxin)
• If it can be made contagious, it will be even more deadly
One known study:
Gene splicing experiments in Soviet Union during 1970s
May have involved:
• Splicing the BoNT gene into other contagious bacteria (e.g. Ebola) to
increase the transmission rate.
• Genetic modifications that removes the effectiveness of possible vaccines or
immune responses
• Genetic engineering to produce new virulent strains or new toxic genes
This is actually a secret research performed by USSR….and revealed by
former bioweapon program scientists..therefore no hard evidence can be
found..

77. Good bioweapon?
• Most poisonous poison
• Easy to make
• Can effectively immobilize military opponent
• Takes long time to recover and cure
• Not easy to diagnose
• Confirmation tests are unreliable
• Insufficient emergency care facilities available if there is a
massive attack

78. Poor bioweapon?
• Non contagious
• Does not pass through skin
• Very unstable
• Food/waterborne botulism can be greatly prevented by cooking
and water treatment
• Airborne botulism is technically difficult to achieve
• Clinical treatment can greatly reduce mortality rate

79. Thanks
Acknowledgement: All the presentations available online on the
subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the
source and authenticity of the content.