Important note:

The toxin is NOT secreted by C.

Botulinum, instead, it is produced during
the bacterias autolysis.

Clostridium Botulinum
Catherine Chung
Colin Brinkman
Pam Chao

Introduction to botulism
Neuroparalytic disease caused by neurotoxins
(BoNTs) produced by bacteria Clostridium
botulinum.
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

BoNTs also produced by other members of Clostridia
family.

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
Niemanns laboratory
21st century 3D structure of BoNT A resolved.

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)

Blepharospasm treated with Oculinum

Vangelova, Luba. Botulinum Toxin: A Poison that Can Heal. Available at:
http://www.fda.gov/fdac/features/095_bot.html .

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

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

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.

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.

The Smart Stuff

Structure:
Translated as a single chain precursor
(pretoxin)
Cleaved to generate fully active neurotoxin
composed of a light chain (LC) and heavy chain
(H)
Light and heavy chains linked by single
disulfide bridge
Light chain acts as an endopeptidase
When bridge is intact, BoNT has no catalytic
activity

More on 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.

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.

BoNTs prevent neurotransmitter

release
SNARE proteins form complex to allow
synaptic vesicles to fuse with plasma
membrane neurotransmitter is released
3 different types of SNARE proteins
VAMPs
SNAP-25 (cleaved by BoNT A)
Syntaxin

BoNTs prevent neurotransmitter

release

BoNTs cleave SNAREs

Humeau, Y., F. Doussau, et al. (2000). How botulinum and tetanus neurotoxins block
neurotransmitter release. Biochimie 82: 427-446.

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

BoNTs block Acetylcholine release

in 3 steps
Translocation
Heavy and light chains separate; light chain
enters the cytosol and cleaves SNAREs
SNARE complex is non-functional and Ach is
not released

BoNTs cleave SNARE proteins and

prevent Ach release

Clinical Perspective of Clostridium

botulinum
There are four types of botulism, characterized
by the method of delivery of the toxin.
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.
Foodborne botulism is the result of the
ingestion of food contaminated with Clostridium
botulinum containing the preformed toxin.
Note: Ingestion of the toxin makes a person ill,
not Clostridium botulinum itself.

 Wound botulism occurs when a break in

the skin becomes infected with
Clostridium botulinum which then
multiply and release botulism toxin into
the blood.
Inhalation botulism occurs when
aerosolized botulism toxin enters the
lungs.
Infant botulism is the result of the
infestation of the digestive tract with
Clostridium botulinum.

 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.

 The incubation period varies according to the

mode of transmission, rate of absorption of the
toxin, and the 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.

 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 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.

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.

Treatment
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.

Treatment
Attempts to develop an effective botulism
vaccine date back to the 1940s. 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.

Diagnosis
The symptoms of botulism are similar to
those of Guillain-Barr 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.

Diagnosis
A brain scan, spinal fluid examination,
electromyograph, or tensilon test 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.

Diagnosis
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.

Diagnosis
In vitro methods utilizing ELISA are under
development but are not yet validated.
Isolation of Clostridium botulinum from the
patients 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

Prevention
Proper food preparation is one of the most
effective ways to limit the risk of exposure to
botulism toxin.
Boiling food or water for ten minutes can
eliminate some strains of Clostridium botulinum
as well as neutralize the toxin as well. However,
this will not assure 100% elimination.
Limiting growth of Clostridium botulinum and the
production of botulism toxin is an alternative to
their outright destruction.

 Temperature, pH, food preservatives,

and competing microorganisms are
among the factors that influence the
rate and degree of Clostridium
botulinum growth.
Growth of most strains of Clostridium
botulinum will not occur below 10 or
above 50 degrees Celsius.

 Clostridium botulinum will not grow in

media with pH values lower than about
5.
Food preservatives such as nitrite,
sorbic acid, parabens, phenolic
antioxidants, polyphosphates, and
ascorbates inhibit the growth of the
microorganism.

 Lactic acid bacteria including

Lactobacillus, Pediococcus, and
Pactococcus can inhibit the growth of
Clostridium botulinum by increasing the
acidity of the medium.
While the cause of roughly 85% of infant
botulism cases is unknown, in up to 15%
of infant botulism cases the causes was
ingestion of honey. Infants younger than
one year old should not be fed honey.

Avoiding Exposure
Avoid home-processed foods if at all possible,
especially those with a low salt and acid content.
Botulism toxin is destroyed at a temperature of
176 F, thus if you must eat home-processed
foods, boil them for 10 minutes before eating if
at all possible.
If canning vegetables, use a pressure cooker, as
it will kill any spores because it can reach
temperatures above boiling.

Weaponization of Botulinum Toxin

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

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!

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

History of Botulinum Toxin as bio-weapon

Organized weapon programs:
1970s
Biological and Toxin Weapons Convention banned bioweapon 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

History of Botulinum Toxin as bio-weapon

Organized weapon programs:
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

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)

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

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

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 ~0.001g/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

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

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.

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

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

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

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

Bioterrorism: routes of intoxication

Types of Botulism:
Wound (least likely)
Food/waterborne
Inhalational

Potential Victims:
Persons of all age and sex.

Lethal Dosage:
For a 70 Kg human
- 0.09-0.15 g intravenously or intramuscularly
- 0.70-0.90 g inhalationaly
- 70 g orally

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

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

Food Botulism
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 (>85c 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 poisoning

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

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

Inhalation Botulism
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..)

Potential danger: Botulinum Superbug?!

WHY?
BoNT is non-contagious (just a toxin)
If it can be mae 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
Heres an Interview of a former Soviet scientist who was in the
bioweapon program:
http://video.pbs.org:8080/ramgen/wgbh/mediastorage/nova/bioterror/popo
v_220.rm

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

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

A video clip about Botulism

Video: "The History of Bioterrorism" from CDC

http://video.cdc.gov/ramgen/phepr/historyofbt/realonecc/05_botulism_cc.rm