1. As a Bioterrorism Agent
Botulism
As a Bioterrorism Agent

2. Botulism History Germany (1793) earliest recorded human outbreak
Organism isolated in 1895
Mortality rate of 5-50%; long recovery period
Weaponized by several nations including the U.S., Japan, and Soviet Union, beginning in the 1930’s
Iraq (1980’s) produced 19,000 L of concentrated botulism toxin
Japan (1990’s) Aum Shinrikyo cult
The first known outbreak of botulism occurred in Germany, 1793 caused by spoiled sausage, and consequently, the name botulism comes from the Latin word “botulus” meaning sausage. The organism which causes botulism, Clostridium botulinum, was first isolated in the late 19th century.
Botulism is a serious condition that can cause paralysis and death. Although the mortality rates for botulism have declined significantly in the 20th century (~50% to ~5%) it is still a life-threatening disease that requires intensive care and months of rehabilitation. The potential mortality rates of a bioterrorist attack of aerosolized botulism toxin are unknown but are estimated to be great.
The concept of using botulism toxin as a weapon is not new. Botulism toxin has been part of several nations’ weapons programs since the 1930’s, including the U.S., Japan, and the former Soviet Union. Iraq admitted to the UN in 1991 that it had produced 19,000 L of concentrated botulism toxin. If aerosolized, this amount would be sufficient to kill the entire human population 3x over. To date, a significant amount of this botulism toxin is not accounted for. The Japanese cult Aum Shinrikyō made at least three attempts to release aerosolized botulism toxin into the community.
References:
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):
Botulism in the United States Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases   Park JB, Simpson LL. Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component. Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment

3. What Makes Botulism Toxin a Good Weapon?
Botulism toxin is the most poisonous substance known
High lethality: 1 aerosolized gram could potentially kill 1 million people
Isolated fairly easily from soil
Could be released as an aerosol or as a contaminant in the food supply
Expensive, long-term care needed for recovery
Botulism toxin is an agent of concern for bioterrorism because it has the potential to cause high morbidity and mortality. It has been estimated that one aerosolized gram could kill 1 million people. Spores can be isolated from the soil, making the agent easily attainable. Botulism could be released into the food supply, where it would initially be difficult to differentiate a bioterrorism outbreak from a naturally occurring foodborne outbreak. Finally, the time and resources needed to care for large numbers of botulism patients as a result of a bioterrorism event would be staggering.
References:
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment
Renee FR, and Milap CN. Management of Botulism. The Annals of Pharmacotherapy: Vol. 37, No. 1, pp. 127–131.
Botulinum Toxin: Overview
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):

4. Botulism Microbiology
Toxin produced by the bacterium Clostridium botulinum
Anaerobic, gram positive, rod-shaped bacteria
Bacteria are 0.5 to 2.0 mcm in width and 1.6 to 22.0 mcm in length
Create spores that can remain dormant for 30 years or more
Spores extremely resistant to environmental stressors, such as heat and UV light
Botulism toxin is produced by Clostridium botulinum, an anaerobic, gram positive, rod-shaped bacterium. The size of bacteria range from 0.5 to 2.0 mcm in width and 1.6 to 22.0 mcm in length. C. botulinum are spore forming bacteria. The spores are the dormant form of the organism and can last many years, waiting for environmental conditions to become favorable for germination. The spores are extremely hearty and require temperatures greater than boiling to destroy.
References:
Botulism in the United States Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.
C. botulinum

5. Left to right: vegetative cells, lipase on egg-yolk agar, vegetative cells and spores

6. Clostridium botulinum
7 types of botulism A through G, based on the antigenic properties of the toxin produced
toxins A, B, E and F cause illness in humans
toxins C and D cause illness in birds and mammals
toxin G
There are 7 types of botulism, distinguishable by the antigenic properties of the neurotoxin produced. Toxins A, B, E, and F are pathogenic to humans, although nearly all of human illness is caused by toxins A, B, and E. C and D cause illness in birds and other mammals, and toxin G is still under investigation for its pathogenesis.
References:
Botulism in the United States Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment

7. Categories of Botulism
Foodborne botulism
caused by eating foods that contain botulism toxin
Intestinal botulism (infant and child/adult)
caused by ingesting spores of the bacteria which germinate and produce toxin in the intestines
Wound botulism
C. botulinum spores germinate in the wound
Inhalation botulism
Aerosolized toxin is inhaled
does not occur naturally and may be indicative of bioterrorism
There are three naturally occurring routes of botulism transmission. Foodborne, intestinal, and wound botulism. The fourth route of transmission, inhalation, does not occur naturally and could be indicative of a bioterrorist event.
The most commonly reported form of botulism is infant (intestinal) botulism. An average of 79 cases are reported in the U.S. annually. Infants <12 months of age are particularly susceptible to C. botulinum spores because their digestive tracts are not fully developed and therefore not able to prevent the germination and subsequent toxin production in the intestines. Adults or children >12 months rarely develop intestinal botulism, but may be more susceptible if they have pre-existing intestinal conditions.
Foodborne botulism occurs when food is contaminated with the botulism toxin and absorbed through the gastrointestinal tract. An average of 28 cases of foodborne botulism are reported in the U.S. annually. Outbreaks have been associated with a variety of foods such as garlic packed in oil, baked potatoes wrapped in aluminum foil, home-canned vegetables, jerky, and fermented fish.
Wound botulism occurs when C. botulinum spores infect and germinate in the wound, producing toxin which is absorbed into the bloodstream. Typically, there are very few cases of wound botulism reported each year in the U.S. However, the increased use of black tar heroin (which can contain C. botulinum spores) in California and other western states has resulted in increased cases of wound botulism.
There have only been 3 documented cases of human inhalation botulism, all of which occurred in West Germany in 1962 as the result of working with C. botulinum toxin in a laboratory.
References:
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment

8. Botulism Pathogenesis
Incubation period
ingestion: unknown
foodborne: 6 hours-8 days
wound: 4-14 days
inhalation: (estimated) hours
Toxin enters bloodstream from mucosal surface or wound
Binds to peripheral cholinergic nerve endings
Inhibits release of acetylcholine, preventing muscles from contracting
Symmetrical, descending paralysis occurs beginning with cranial nerves and progressing downward
The incubation period for botulism varies depending on amount of exposure and route of transmission. Incubation for ingestion botulism, for either infant or child/adult, is undeterminable because the date of spore ingestion is usually unknown. The incubation period for foodborne botulism can range from 6 hours to 8 days and 4-14 days for wound botulism. There have only been three documented cases of human inhalation botulism, all resulting from a laboratory accident. Incubation for inhalation botulism has been estimated from these cases and primate studies.
Botulism toxin enters the bloodstream via mucosal surface, such as the lungs or intestine, or wound. The toxin causes paralysis by binding to peripheral cholinergic nerve endings and inhibiting the release of acetylcholine, which prevents muscles from contracting. Botulism-related paralysis presents as symmetrical, descending progression from the cranial nerves downward.
References:
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment
Botulism in the United States, Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.

9. Botulism Pathogenesis (cont.)
Can result from airway obstruction or paralysis of respiratory muscles
Secondary complications related to prolonged ventilatory support and intensive care
Death from botulism usually occurs as a result of respiratory distress. Secondary infections from prolonged mechanical ventilation and intensive care may also be fatal.
References:
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment

10. Botulism Toxin Mechanism
“Botulinum toxin is activated by proteolytic cleavage; the activated structure is a 150-kd polypeptide comprising two chains (a heavy chain [100 kd] and a light chain [50 kd]) that are connected by a single disulfide bond.
Botulinum toxin enters the circulation and is transported to the neuromuscular junction.
At the neuromuscular junction, the heavy chain of the toxin binds to the neuronal membrane on the presynaptic side of the peripheral synapse.
The toxin then enters the neuronal cell via receptor-mediated endocytosis.
The light chain of the toxin crosses the membrane of the endocytic vesicle and enters the cytoplasm.
Once inside the cytoplasm, the light chain of the toxin (which is a zinc-containing endopeptidase) cleaves some of the proteins that form the synaptic fusion complex. These proteins, referred to as SNARE proteins, include synaptobrevin (cleaved by toxin types B, D, F, and G), syntaxin (cleaved by toxin type C), and synaptosomal-associated protein (SNAP-25; cleaved by toxin types A, C, E).
The synaptic fusion complex allows the synaptic vesicles (which contain acetylcholine) to fuse with the terminal membrane of the neuron. Disruption of the synaptic fusion complex prevents the vesicles from fusing with the membrane, which in turn prevents release of acetylcholine into the synaptic cleft.
Without neuronal acetylcholine release, the affiliated muscle is unable to contract and becomes paralyzed.
The blockade of acetylcholine release lasts up to several months; normal functioning slowly resumes either through turnover of SNARE proteins within the cytoplasm or through production of new synapses. “
References:
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment

11. Botulism Clinical Presentation
Classic symptoms of botulism poisoning include:
blurred/double vision
muscle weakness
drooping eyelids
slurred speech
difficulty swallowing
patient is afebrile and alert
Infants with botulism will present with:
weak cry
poor feeding
constipation
poor muscle tone, “floppy” baby syndrome
Regardless of the route of transmission (foodborne, inhalation, wound, ingestion), the clinical presentation of botulism is similar. Classic symptoms of botulism poisoning include blurred or double vision, muscle weakness, drooping eyelids, slurred speech, and difficulty swallowing. Clinical signs of an infant with botulism include a weak cry, poor feeding, constipation, and “floppiness” due to poor muscle tone.
References:
Botulism in the United States, Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):

12. These photographs were taken of a 17-yr old male with mild botulism poisoning. Figure A shows the patient at rest, while Figure B shows the patient giving his “maximum smile”. The dilated pupils, drooping eyelids, and lack of smile creases around the eyes are consistent with symptoms of botulism.
References:
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):

13. Possible Case of Botulism
Call MDH immediately (24/7) at or if a case of botulism is suspected.
Botulism is considered a medical emergency. All confirmed and suspected cases of botulism should be reported immediately to the Minnesota Department of Health. Minnesota public health officials will then contact CDC through the CDC Emergency Operations Center.

14. Botulism Clinical Treatment
Antitoxin administration
Supportive Care
mechanical ventilation
body positioning
parenteral nutrition
Elimination
Induced vomiting
High enemas
Once botulism is suspected, the antitoxin should be administered as quickly as possible. Almost all cases of confirmed botulism require hospitalization and supportive care. Because of the paralysis of respiratory muscles, botulism patients may require mechanical ventilation for weeks to months. Supportive body positioning may aid respiratory mechanism if patient is nonventilated. Nutrients administered intravenously (parenteral nutrition) may be necessary for severe cases of botulism.
For foodborne or ingestion botulism, elimination by induced vomiting or enemas may reduce the amount of toxin the patient is exposed to. This is not recommended for infants.
References:
Renee FR, and Milap CN. Management of Botulism. The Annals of Pharmacotherapy: Vol. 37, No. 1, pp. 127–131.
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):
Red Book, 26th Edition Report of the Committee on Infectious Disease. American Acacdemy of Pediatrics.

15. Supportive body positioning for botulism patients.
References:
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):

16. Botulism Transmission
Home-canned goods (foodborne)
particularly low-acid foods such as asparagus, beets, and corn
Honey (ingestion)
can contain C. botulinum spores
not recommended for infants <12 months old
Crush injuries, injection drug use (wound)
Canned food goods are a potential source of foodborne botulism if proper canning procedures are not adhered to. Particular care when canning alkaline (low-acid) foods needs to be taken due to the affinity of C. botulinum for such environments. Pressure cookers are recommended for most home-canning because they can reach higher temperatures than boiling, sufficiently killing any spores contaminating the food.
Although many cases of ingestion botulism are not associated with honey consumption, it is the only food identified as high risk for containing C. botulinum spores. Therefore, honey is not recommended for infants <12 months old.
Wound botulism has been associated with injection drug use and traumatic injuries to the extremities, such as crush wounds.
References:
Botulism: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment
Botulism in the United States, Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.

17. Examples of sources of botulism
Examples of sources of botulism. Honey (intestinal), canned goods (foodborne), black tar heroin (wound), and symbol representing potential bioterrorist attack (inhalation).

18. Botulism Infection Control
Botulism cannot be transmitted person-to-person
Standard precautions should be taken when caring for botulism patients
There is no secondary transmission of botulism, therefore standard precautions should be sufficient when caring for patients with botulism.
References:
Botulism in the United States, Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.

19. Botulism Laboratory Procedures
Toxin neutralization mouse bioassay
serum, stool, gastric aspirate, suspect foods
Isolation of C. botulinum or toxin
feces, wound, tissue
The most sensitive laboratory procedure for the diagnosis of botulism is a toxin neutralization bioassay in mice. Botulism toxin can be extracted from serum, stool, gastric aspirate, or suspect food samples. The neutralized toxin is then injected into mice, which are then observed for signs of botulism. The bioassay procedure can take up to 4 days, so if clinical presentation indicates botulism, it is recommended to administer the antitoxin without waiting for results. It is also possible to diagnose botulism by demonstrating C. botulinum or its toxin in stool, wounds or tissue.
References:
Botulism in the United States, Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.
Red Book, 26th Edition Report of the Committee on Infectious Disease. American Acacdemy of Pediatrics.

20. Botulism Antitoxin Equine antitoxin
Trivalent and bivalent antitoxins available through the CDC
Licensed trivalent antitoxin neutralizes type A, B, and E botulism toxins
Effective in the treatment of foodborne, intestinal, and wound botulism
Effectiveness for inhalation botulism has not been proven
Does not reverse current paralysis, but may limit progression and prevent nerve damage if administered early
A trivalent and bivalent equine antitoxins are available from the CDC via state or local health departments for treatment of botulism. The trivalent antitoxin neutralizes botulism toxin types A, B, and E which are the antigenic types most likely to cause illness in humans. The bivalent antitoxin neutralized botulism toxin types A and B. The effectiveness of the equine antitoxins to treat foodborne, intestinal, and wound botulism has been established. Their effectiveness to treat inhalation botulism is not known.
The antitoxin should be administered as early as possible once symptoms of botulism present. Current paralysis will not be reversed, but the progression of paralysis and nerve damage may be prevented with early administration.
References:
Renee FR, and Milap CN. Management of Botulism. The Annals of Pharmacotherapy: Vol. 37, No. 1, pp. 127–131.
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):
Botulism in the United States. Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.

21. Botulism Antitoxin (cont.)
Hypersensitivity to equine antitoxin
9% of people experience some hypersensitivity
Approximately 9% of persons treated experience hypersensitivity to the equine antitoxin. Few of these cases have severe hypersensitivity reactions.

22. Botulism Differential Diagnoses
Guillain-Barré syndrome
Myasthenia gravis
Stroke
Tick paralysis
Lambert-Eaton syndrome
Psychiatric illness
Poliomyelitis
Diabetic Complications
Drug intoxication
CNS infection
Overexertion
Several conditions that affect the central nervous system are differential diagnoses for botulism. Some distinguishing features of these conditions can help to prevent misdiagnosis. Patients with Guillain-Barré syndrome, for example, tend to have ascending paralysis and/or a recent history of infection. Myasthenia gravis is associated with recurrent paralysis. Stroke victims often have asymmetrical paralysis. Tick paralysis is also ascending, and the tick may be found on the patient’s body. Lambert-Eaton syndrome is associated with lung carcinoma and increased strength with sustained contraction.
References:
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):
Botulism in the United States. Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.

23. Botulism Vaccine
A toxoid vaccine (antigen types A, B, C, D, and E) is available for laboratory workers at high risk of exposure
Limited supplies of this vaccine available
There is a vaccine for botulism that protects against type A, B, C, D, and E available for those at high risk of exposure. In the event of a bioterrorist attack of botulism toxin, however, reserves would quickly be depleted.
References:
Arnon SS, Schechter R, Inglesby TV et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: medical and public health management. JAMA Feb 28;285(8):
Botulism in the United States, Handbook for Epidemiologists, Clinicians, and Laboratory Workers. CDC National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases 1998.

24. Therapeutic Uses of Botulism Toxin
Focal dystonias - involuntary, sustained, or spasmodic patterned muscle activity
Spasticity - velocity-dependent increase in muscle tone
Nondystonic disorders of involuntary muscle activity
Strabismus (disorder of conjugate eye movement) and nystagmus
Disorders of localized muscle spasms and pain
Smooth muscle hyperactive disorders
Cosmetic use
Sweating disorders
Botulism toxin has numerous therapeutic uses including treatment for involuntary muscle disorders, migraine headaches, sweating disorders, and cosmetic uses. The dose needed for these treatments is a very small fraction of the human lethal dose, and there are minimal to no side effects.
References:
Botulinum Toxin: Overview