Death

Death results from loss of diaphragmatic and accessory respiratory muscle function insufficient tidal volume, dyspnea or ventilary failure

Death may also result from loss of pharyngeal muscle function airway muscle paralysis, airway obstruction

Lethal Factor

Foodborne 6% mortality rate in treated patients Early deaths – failure to diagnose/recognize severity of disease Death after 2 weeks from complications of long-term mechanical

ventilatory management Wound

15% case fatality rate in treated patients Lethal dose on the order of ng per kg body weight .7-.9 μg = lethal dose for 70 kg individual if inhaled; ricin: 3-5 μg/kg Cited as 100,000 to 3,000,000 times as potent as nerve gas sarin

(WWII) Serotypes A, B, E most associated with botulism outbreaks in humans,

but also F

Differential Diagnosis:Botulism is commonly

misdiagnosed as

Guillan-Barre syndrome Myasthenia gravis Eaton-Lambert syndrome Stroke syndrome Carbon monoxide intoxication Tick paralysis Food poisoning

Differential Diagnosis: Ancillary Testing

Procedure: Result Consistent

with Botulism

Eliminates possibility of:

MRI/CT scan (brain imaging)

Normal Stroke syndrome

Lumbar puncture Normal Guillain-Barre syndrome

Edrophonium chloride test

Negative Myasthenia gravis

Electromyography Decreased amplitude of action potentials in involved muscle groups

N/A

Rapid repetitive electromyography (20-50 Hz)

Facilitation (increasing pattern of action potential amplitude)

N/A

Confirmatory Tests for C. Botulinum / BoNT

• Mouse Bioassay for toxin - Sample from serum, feces, or food in question- Administer sample to mouse with and without type-specific anti-toxin- Determine response level- Results in 1-2 days, can determine presence/absence and type of BoNT by seeing which anti-toxin type neutralizes biological activity of toxic sample- Cumbersome, and with animal subjects

Confirmatory Tests for BoNT

Culturing of stool specimens for C. botulinum Other prospects include PCR, ELISA, time-

resolved fluorescence

PCR of various strains of C. botulinum

MW A B E F A B A -

B E B

F F E,F

Botulism as a Biological Weapon

Starting in the 1930’s, the Japanese during their occupation of Manchuria admitted to feeding cultures of C. botulinum to prisoners.

The US had produced botulism toxin for use in warfare during WWII and were equipped with the vaccine during the D-Day invasion.

Despite the 1972 Biological and Toxin Weapons Convention prohibition of offensive research and production of biological weapons, the Soviet Union and Iraq have successfully created these types of weapons.

Botulism as a Biological Weapon

The Soviet Union had attempted to splice out the botulinum toxin gene into other bacteria.

Iran, Iraq, North Korea, and Syria are all believed by the US to be researching or have developed botulinum toxin as a weapon.

Botulism as a Biological Weapon

After the Persian Gulf War, Iraq admitted to having produced 19,000L of the concentrated toxin, 10,000L of which were fitted to military weapons.

Iraq had also outfitted several missiles with botulinum toxin, anthrax, and aflatoxin; (likely to be aerosolized and enter the bloodstream through the lungs)

Botulinum: The Deadly Assassin

Botulinum toxin can be implicated with the assassination of Reinhard Heydrich, head of the Gestapo and the SS during WWII

Shrapnel injuries from a bomb delivered by Czech patriots

Post-operation, his condition was satisfactory; until after the seventh day where his condition suddenly became worse and he died the next morning.

Botulinum: The Deadly Assassin

Czech patriots were trained and equipped by the British government, who were developing botulinum toxin as a weapon.

Heydrich’s chart suggest massive pulmonary embolism, however his heart and lungs were normal.

The head of the British biological warfare program has made remarks that imply that he had a hand in the assassination.

The bomb was specially modified, possibly to include the botulinum toxin.

Botulinum: The Deadly Assassin

During WWII, the US Office of Strategic Services has developed a plan for Chinese prostitutes to assassinate high-ranking Japanese officers in occupied Chinese cities.

Gelatin capsules with a lethal dose of botulinum toxin were to be slipped into food or a drink.

Purification of Botulinum Toxin

Toxin is easily purified using general biochemical purification techniques. Toxin separated from extract by salting out, acid precipitation, gel

filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second purification step of gel filtration.

10 L of culture produced 22.9 mg of toxin with1.1x108 LD50.

Botulinum Toxin

LD50 is approximately 1ng/kg via intravenously, subcutaneously,or intraperitonealy and about 3ng/kg by inhalation.

The average person is 70-100kg, so a lethal dose would require only 70-300ng per person.

Botulinum toxin as a bioterrorist weapon.

Splicing the toxin gene into other bacteria to allow for growth in more permissive conditions or to increase the transmission rate.

Genetic modifications that removes the effectiveness of the vaccine (altering the antigens by mutagenic PCR assay)

Botulinum toxin as a bioterrorist weapon.

The goal of bioterrorism is to create terror, not necessarily with casualties.

A bioterrorist group could easily purify botulinum toxin and contaminate food in order to affect thousands of people.

Hospital care is required for weeks to months which would quickly exceed the capacity for proper care.

Medical Uses for Botox

Temporary removal of wrinkles Treatments for muscular spasms Therapy for cerebral palsy, parkinsons,

migraines, and overactive bladder.

Other Interesting Botox Facts

Botox from drugstore.com One vial $442.68 Three vials $1,327.03

Recently, “Botox Parties” have become popular means for doctors to increase their clients and “it’s a great way for people to meet each other and it’s a comfortable way for patients to get Botox.”

Prophylaxis Passive immunity – pentavalent (ABCDE)

botulinum toxoid Distributed by CDC for laboratory workers at

high risk of exposure to botulinum toxin Distributed by military for protection of troops

against attack Used for more than 30 years to immunize more

than 3000 laboratory workers in many countries Monovalent vaccine that protects against BoNT

serotype F also available as Investigational New Drug

Therapy: Step 1 – Antitoxin

Passive immunization with equine trivalent antitoxin Antibodies for BoNTs/A, B, E Neutralizes toxin molecules that are not yet bound to

nerve endings Therefore should be administered early (within 24 hours

of toxin exposure) and Will minimize subsequent nerve damage and severity of

disease but will not reverse existent paralysis Available from CDC via state and local health

departments

Therapy: Step 1 – Antitoxin (cntd.)

Investigational heptavalent (ABCDEFG) antitoxin held by US Army for dissemination to patients affected by botulinum toxin types other than A, B, E

Amount of neutralizing AB in both antitoxins far exceeds (over 100-fold greater than) highest amount of circulating antitoxin ever measured at CDC

Single, 10mL vial; no additional doses necessary

As of June 1998, human botulism immune globin (a human-derived antitoxin product) is available only for infant botulism patients, under a Treatment Investigational New Drug protocol

Therapy: Step 2 – Supportive Care

Mechanical ventilation

Treatment of secondary infections

Therapy: Step 2 – Supportive Care (cntd.)

Reverse Trendelenburg positioning of nonventilated patients for reduced oral secretions in airway and improved respiratory mechanics

Recovery

Time can range from several days to several weeks or longer, depending on extent of toxin activity

Involves regeneration of motor nerve endings/ axons of affected neurons

Recovery: Regeneration of Axon

Terminals

Decontamination

Foodborne: heating to internal temperature of 85˚C for 5 minutes will detoxify food or drink Aerosolized:

Persistence at site of intentional release determined by atmospheric conditions and particle size of aerosol

Toxin degraded by extremes of temperature and humidity

Decay rate weather dependent, but estimated at less than 1%-4% per minute

At 1% decay rate, toxin significantly inactivated 2 days after aerosolization

Mass Immunization

Pro argument: could theoretically eliminate threat posed by botulinum toxins A through E

Con argument: Scarcity of toxoid Rarity of natural disease Elimination of potential therapeutic benefits of

medicinal botulinum toxin 3 injections + annual booster delivers immunity

Current Vaccine Production: Shortcomings

C. botulinum is a spore-former; need to renovate or

build facility for manufacture toxin-based product – many resources needed

Yields of toxin production from C. botulinum relatively low Safety precautions to handle large quantities of toxin increase cost

of manufacturing Pentavalent toxin consists of relatively crude extract of clostridial

proteins that may influence immunogenicity/reactivity of vaccine Formaldehyde used to inactivate toxin; residual amounts used to

prevent reactivation of toxin, but formaldehyde is reactogenic

Current Research/Future Strategies in Vaccine

Development Recombinant vaccine

No new research facility needed, no culturing of large quantities of hazardous toxin-producing bacteria fewer manufacturing costs less expensive vaccine

3 functional domains: binding, internalization, catalytic; fragment would not possess all three no need for formalin to maintain deactivation

Synthetic vaccine – Atassi and coworkers Investigators synthesized peptides overlapping Hc

region of BoNT serotype A BoNT/A antibodies and T-lymphocytes used to map

these epitopes (of Hc region) Synthesized BoNT epitopes could potentially be

delivered as vaccine, to induce endogenous immunity

Mike Meagher, contracted by US Army to develop BoNT vaccine

Current Research/Future Strategies in Vaccine Development (cntd.)

Use of Venezuelan equine encephalitis virus replicon vector (Lee and coworkers) Introduction of nontoxic Hc region of BoNT/A into vector yields high

levels of Hc (demonstrated in mice) Protective antigens against BoNT produced in vivo

Design of synthetic genes encoding non-toxic, carboxy-terminal fragments of C. botulinum neurotoxins (Byrne and coworkers) gene products used to illicit immunity demonstrated protective immunity in mice and non-human primates

against high levels of toxin Oral vaccine – gene/product modified to alter toxicity without

changing penetrability, specificity, immunogenicity; fraction of oral dose would be degraded, but enough absorbed

Research Need: Alternate Therapy via Enteral

detoxification Distribution of antitoxin to local hospitals takes

several hours Standard detoxification can be administered

immediately Reduce absorption through GI tract or reduce

circulating levels of botulinum toxin through osmotic catharsis

Perhaps more useful in foodborne than in aerosolized botulism limited value

Structure-based drug design

Figure from Arnon, et al, 2001

Targets: Binding Site of BoNT or Gangliosides

Themselves

Binding

Targets: cleavage sites on SNARE proteins or BoNT

catalytic site

Cleavage of SNARE proteins by zinc-dependent catalytic site

Research Need: Immunity via

Recombinant oligoclonal antibodies

Half life of 1 month, as opposed to 5-8 days (for equine)

Oligoclonal human antibodies = theoretic protection against toxins ABCDEFG for months

Not entirely foreign source perhaps less reactogenicity than equine products

Already shown effective against HIV and Anthrax Stockpiles could deter terrorist attacks Prophylaxis or treatment

This Just In: Botulism Beaten!

USCF Research Team led by James Mark, August, 2002, published in Nature Reviews Immunology

3 recombinant, oligoclonal antibodies that neutralize toxin

Will protect against toxin within 1-2 hours after delivered Effective up to 2 days after exposure to toxin Protection can last 3-6 months “The drug neutralizes the toxin better than the most

potent natural immune response.” – Marks, BBC News Online