Important note:The toxin is NOT secreted by C. Botulinum, instead, it is produced during the bacterias autolysis.

Clostridium Botulinum

Catherine ChungColin BrinkmanPam Chao

Introduction to botulismNeuroparalytic 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 humansBoNT C in birdsBoNT D in cattleBoNT G isolated from soilsBoNT A most common and most potentBoNTs also produced by other members of Clostridia family.

A long historyBotulism originally known as sausage poisoning in late 18th century and throughout 19th century.From Latin botulus = sausageBacterial etiology recognized at end of 19th centuryOutbreak of botulism in Belgium 1895 revealed cause as neuroparalytic toxin produced by anaerobic bacteriumProbably Type B Outbreak in Germany several years laterBacterium isolated; different from that in BelgiumProbably Type A

The 20th Century and Beyond1949 Burgen et al. determines botulinum toxin blocks neurotransmitter release1979 Simpson proposes cellular mechanism in 3 steps: binding, internalization, and intraneuronal action.1990 a.a. sequence of one BoNT determined in Niemanns laboratory21st century 3D structure of BoNT A resolved.

The History ContinuesFirst biological toxin used for treatment of human diseaseManufactured for medical use in 1989 under name OculinumLicensed 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 BoNTNow manufactured under the name BotoxExperimentally 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 antibodiesStudies carried out to determine feasibility of other strains of BoNTBoNT B manufactured for treatment of cervical dystonia in 2000 as Myobloc

Cosmetic Use of BoNT1994 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, 2002Myobloc not approved for cosmetic use, but is used experimentally in many cosmetic procedures anyway

BoNT A (Botox) Botox injection patient 13 weeks after injectionSadick, 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 procedureSadick, 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 bridgeLight chain acts as an endopeptidaseWhen bridge is intact, BoNT has no catalytic activity

More on StructureTurton, 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 StructureTurton, 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 releaseSNARE proteins form complex to allow synaptic vesicles to fuse with plasma membrane neurotransmitter is released3 different types of SNARE proteinsVAMPsSNAP-25 (cleaved by BoNT A)Syntaxin

BoNTs prevent neurotransmitter release

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

BoNTs block Acetylcholine release in three stepsBindingBoNT binds by Hc to receptor on cellInternalizationToxins internalized via receptor-mediated endocytosisTHE POINT OF NO RETURN: once endocytosed, the toxins can no longer be neutralized by antisera

BoNTs block Acetylcholine release in 3 steps TranslocationHeavy and light chains separate; light chain enters the cytosol and cleaves SNAREsSNARE complex is non-functional and Ach is not released

BoNTs cleave SNARE proteins and prevent Ach release

Clinical Perspective of Clostridium botulinumThere 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 visionDrooping eyelidsDry mouthDifficulty SwallowingMuscle 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.

TreatmentAntitoxin 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.

TreatmentWound 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.

TreatmentAttempts 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.

DiagnosisThe 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.

DiagnosisA 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.

DiagnosisHowever, 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.

DiagnosisIn 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

PreventionProper 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 ExposureAvoid 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 drugsfreeze-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 infectionstabilizing the agent so that it will remain infectious for a longer period after releasetreating 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-weaponEarly primitive example:

1910s Mexico1supporters of Pancho Villa used Botulinum toxin against Mexican federal troops Buried pork and green beans for several daysThen used the mixture to contaminate food or smeared on knife-like weaponsResult in food and wound botulism

Easy to make!

History of Botulinum Toxin as bio-weaponOrganized weapon programs:

World War II Japanese invaders (Biological Warfare group Unit 731) fed cultures of Clostridium Botulinum to prisoners and caused lethal effectLater, Germany, US and USSR were all producing weaponized Botulinum Toxin

History of Botulinum Toxin as bio-weaponOrganized weapon programs:

1970sBiological and Toxin Weapons Convention banned bio-weapon research and productionIraq and USSR continued to produce Botulinum Toxin as potential weaponSoviets splice the Botulinum toxin gene into other bacteriaIran, North Korea and Syria are also believed to go on with developing Botulinum toxin in weapons

History of Botulinum Toxin as bio-weaponOrganized weapon programs:

1990s Persian Gulf WarIraq produced 19,000 L of concentrated Botulinum Toxin~10,000 L were loaded into military weaponsThe 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 JapanBy Japanese Cult Aum ShinrikyoObtained C. Botulinum from soil collected in Northern JapanDispersed Botulinum Toxin as aerosols (spray)- In downtown Tokyo- US military bases in Japan

Why failed? Unknown! But suspect:Particles were not refined enoughStrain of bacteria used were not virulent

However their release of sarin gas succeededKilled and injured many in a subway attackSarin is an extremely water soluble chemical agent (can diffuse thru eyes, skin etc)

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

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

Extreme potency and lethalityOne of top 6 potential biological warfare agentsListed as Category A agent by CDC i.e. Highest priorityThe 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 sarin1 gram of crystalline toxin can kill >1 million people if dispersed and inhaled evenly

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

Ease of productionRecipes for home brews of Botulinum toxin can be easily found on internetC. 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 PotentialBotulinum Toxin is a major threat because-Extreme potency and lethality-Ease of production -Ease of transport -Need for prolonged intensive care

Ease of transportIt 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 weeksNot contagiousDoes not spread by inhalation naturallyDoes not enter through skin

Bioweapon PotentialBotulinum 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 careAfter intoxication and treating w/ antitoxin:Ventilatory support and 24-hr nursing care is commonly needed for 2-8 weeksSome might require up to 7 months before the return of muscular functionHowever, no municipality has sufficient ventilators to provide intensive care for mass victims of a terrorist attack by aerosolized BoNT

Bioterrorism: routes of intoxicationTypes of Botulism:Wound (least likely)Food/waterborneInhalational

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 BotulismLeast effective thus not practical to use in bioterrorismExtremely small scaleRequires direct contact between wound and spores of C. BotulinumVery 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 BotulismCarried 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 BotulismBoNT can stay in untreated water or uncooked food for up to daysIt 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 BotulismMan-made (does not occur naturally)Utilizes aerosolized Botulinum toxinMay involve freeze-drying and milling the toxin into an extremely fine powderAbsorption 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 BombsLarge scaleMore efficient way of bio-terrorismCan equip war-heads of missiles or bombs and grenades- As white powder or liquid slurryCan be sprayed as aerosolsPoint-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 BotulismMore deadly than food botulism (because smaller lethal dose) but technically and financially difficult to carry out:InstabilityInactivated by sunlight within 1-3 hoursDetoxified in air within 12 hoursTechnically difficult and complicated for the insufficiently funded terrorist to:Make the powder form for efficient dispersalobtain 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 1970sMay 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 responsesGenetic 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/popov_220.rm

Good bioweapon?Most poisonous poisonEasy to makeCan effectively immobilize military opponent Takes long time to recover and cureNot easy to diagnoseConfirmation tests are unreliableInsufficient emergency care facilities available if there is a massive attack

Poor bioweapon?Non contagiousDoes not pass through skinVery unstableFood/waterborne botulism can be greatly prevented by cooking and water treatmentAirborne botulism is technically difficult to achieveClinical treatment can greatly reduce mortality rate

A video clip about BotulismVideo: "The History of Bioterrorism" from CDChttp://video.cdc.gov/ramgen/phepr/historyofbt/realonecc/05_botulism_cc.rm

*ADD CITATION

*Add citationAdd labels of Heavy and Light chainsBoNT A

*As mentioned earlier, 1 mg of Botulinum Toxin can kill around one million people if evenly distributed.In the following slides I will talk about how it is used in history and *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 *I will not go into detail since that can prolly take another hrImportant point is the knowledge of production is readily available in the internet.

**As mentioned by Colin, the recovery takes up to months*Cannot find sources as for exactly how to equip the agents onto a bomb or missileNext slide will explain*Side story: An article from Nature MagazineUS army filed and published patent for a rifle-launched non-lethal cargo dispenser that can be filled with different agents.Basically the patent outlines the steps to make a potential bio-agent projectileThe patent is no longer accessibleProlly the reason why we cant find information regarding military equipment of these biological agents

*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 didnt die *This is actually a secret research performed by USSR.and revealed by former program scientists..therefore no hard evidence can be found..*So heres a little movie about the Toxin as a Biological Weapon, including a brief history