Botulism, pathogenesis, diagnosis, clinical

manifestation, treatment
 Introduction to botulism

Neuroparalytic disease caused by neurotoxins BoNT produced by

bacteria Clostridium botulinum.
 7 different neurotoxins (A-G) produced by different strains of C. botulinum.
 A, B, E, and F outbreaks in humans
 C in birds
 D in cattle
 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 Niemann’s 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”
 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.
 Organism

Clostridium botulinum
 Gram positive
 Obligate anaerobic bacillus
 Spores
 Ubiquitous
 Resistant to heat, light, drying and radiation
 Specific conditions for germination
 Anaerobic conditions

 Warmth (10-50oC)

 Mild alkalinity

Center for Food Security and Public Health Iowa State University
2004
 The various species of toxigenic clostridia

 C. botulinum groups I, II, and III;

C. argentinense (toxin type G);
C. baratii (toxin type F);
C. butyricum (toxin type E)
Can be differentiated on the basis of phenotypic characteristics,
including specific biochemical properties and morphologic appearance
on egg yolk agar.
Certain strains may produce more than one toxin serotype.
 Neurotoxins

Seven different types: A through G

 Different types affect different species
 All cause flaccid paralysis
 Only a few nanograms can cause illness
 Binds neuromuscular junctions
Toxin: production requires spore germination, which occurs only with a
rare confluence of circumstances: an anaerobic atmosphere, a pH of
>4.5, low salt and sugar concentrations, and temperatures of 4–120°C .
Destroyed by boiling
Spores: Higher temperatures to be inactivated

Center for Food Security and Public Health Iowa State University
2004
 Neurotoxins
Neurotoxin A B C D E F G
Human X X X X
Horses X X
Cattle X X X
Sheep X
Dogs X X
Avian X X
Mink & Ferret X X X

Center for Food Security and Public Health Iowa State University
2004
• Toxin type A produces the most severe syndrome, with the greatest
• proportion of patients requiring mechanical ventilation.
• Toxin type B appears to cause milder disease than type A.
• Two cases of human illness due to toxin type C and one outbreak caused by toxin
type D were reported more than 50 years ago.
• The reasons for the rarity of cases due to types C and D are not known; all four
serotypes that affect humans produce botulism in experimental models.
• Toxin type E, most often associated with foods of aquatic origin, produces a
syndrome of variable severity.
• The rare cases of illness caused by toxin type F are characterized by rapid
progression to quadriplegia and respiratory failure but also by relatively rapid
recovery.
 Transmission

Ingestion
 Organism
 Spores

 Neurotoxin

Wound contamination
Inhalation
Person-to-person not documented

Center for Food Security and Public Health Iowa State University
2004
 Epidemiology

In U.S., average 110 cases each year

 Approximately 25% food-borne
 Approximately 72% infant form
 Remainder wound form
Case-fatality rate
 5-10%
Infective dose- few nanograms

Center for Food Security and Public Health Iowa State University
2004
 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
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
 Toxin binding is irreversible, but nerve terminals do regenerate.
In the United States, 95% of patients recover fully, but this process
may take many months and often requires extended outpatient
rehabilitation therapy.
 Syndromes of botulism

(1) food-borne illness due to ingestion of toxin in contaminated food; (2)

wound infection due to wound colonization by toxigenic clostridia with in
situ toxin production;
(3) infant botulism due to colonization of the infant intestine by toxigenic
clostridia with in situ toxin production;
and
(4) adult intestinal toxemia, a rare form of colonization with
similarities to infant botulism.
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.
 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.
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.
 Foodborne Botulism

Preformed toxin ingested from contaminated food (3- to 5-day food

history)
Most common from home-canned foods
 Asparagus, green beans, beets, corn, baked potatoes, garlic, chile peppers, tomatoes;
type A
 Improperly fermented fish (Alaska); type E

Center for Food Security and Public Health Iowa State University
2004
 Infant Botulism

Most common form in U.S.

Spore ingestion
 Germinate then toxin released and colonize
large intestine
Infants < 1 year old
 94% < 6 months old
Spores from varied sources
 Honey, food, dust, corn syrup

Center for Food Security and Public Health Iowa State University
2004
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.
 Wound Botulism

Organism enters wound

 Develops under anaerobic conditions
 It does not penetrate intact skin
 Associated with addicts of black-tar heroin

Center for Food Security and Public Health Iowa State University
2004
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.
Wound Botulism
 Adult intestinal toxemia botulism

results from absorption of toxin produced in situ after rarely occurring

intestinal colonization with toxigenic clostridia.
Typically, patients have some anatomic or functional bowel
abnormality or have recently used antibiotics that may protect normally
fastidious Clostridium species from competing components of the
bowel flora.
Despite antitoxin treatment, protracted symptoms or relapse due to
ongoing intraluminal production of toxin may be observed.
 Clinical course

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.
 Adult Clinical Signs

Nausea, vomiting, diarrhea

Double vision
Difficulty speaking or swallowing
Descending weakness or paralysis
 Shoulders to arms to thighs to calves
Symmetrical flaccid paralysis
Respiratory muscle paralysis

Center for Food Security and Public Health Iowa State University
2004
 Symptoms
Cranial nerve involvement, which almost always marks the onset of symptoms of botulism,
usually produces diplopia, dysarthria, dysphonia, and/or dysphagia. Ptosis and facial
paralysis; fixed or dilated pupils.
Weakness progresses, often rapidly, from the head to involve the neck, arms, thorax, and
legs; occasionally, weakness is asymmetric;
Followed by flaccid, descending, completely symmetric paralysis of voluntary muscles;
Autonomic symptoms may include anhidrosis, dry mouth, and very dry, occasionally sore
throat with pronounced mucosal erythema and pain mimicking pharyngitis, and postural
hypotension.
Patients are usually afebrile.
Deep tendon reflexes may be normal or may progressively disappear.
Patients usually exhibit no sensory or cognitive deficits.
They are generally alert and oriented
 Infant Clinical Signs

Constipation
Lethargy
Poor feeding
Weak cry
Bulbar palsies
Failure to thrive

Center for Food Security and Public Health Iowa State University
2004
 Diagnosis

Clinical signs
Toxin in serum, stool, gastric aspirate, suspected food
Culture of stool or gastric aspirate
 Takes 5-7 days
Electromyography also diagnostic
Mouse neutralization test
 Results in 48 hours

Center for Food Security and Public Health Iowa State University
2004
 Treatment

Intensive care immediately

 Ventilator for respiratory failure
Botulinum antitoxin
 Derived from equine source
 CDC distributes
 Used on a case-by-case basis
Botulism immune globulin
 Infant cases of types A and G

Center for Food Security and Public Health Iowa State University
2004
 Treatment

If diagnosed early, foodborne and wound botulism can be treated with
an antitoxin which blocks the actions of toxin circulating in the blood.
This prevents patients from worsening in condition.
If respiratory failure and paralysis occur, the patient may be put on a
breathing machine for weeks plus intensive medical and nursing care.
Paralysis will improve after several weeks.
Antitoxin is currently not routinely given for treatment of infant
botulism.

48
 Human: Prevention

Do not feed honey to children <1 yr of age

Proper food preservation methods
 Proper time, temperature and pressure
 80oC for 30 min or 100oC for 10 min

Prompt refrigeration of foods

Boil foods for > 10 minutes
Decontamination
 Boil suspected food before discarding
 Boil or chlorine disinfect utensils used

Center for Food Security and Public Health Iowa State University
2004
 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

Here’s an Interview of a former Soviet scientist who was in the

bioweapon program:
http://video.pbs.org:8080/ramgen/wgbh/mediastorage/nova/bioterror/p
opov_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