Botulinum Toxin: A Bioterrorism Weapon

Botulinum toxin, one of the most toxic poisons known, is derived from the spore-forming bacteria Clostridium botulinum. This bacteria occurs naturally in forest soils, lake and stream sediments and the intestinal tracts of some fish and animals. Botulinum toxin gained public attention after a vial of the bacteria was found by investigators in Iraq after the ouster of Saddam Hussein. At the end of the 1991 Gulf War, Iraq claimed it had produced 19,000 liters of the toxin; if true, it was an amount capable of killing the entire population of the earth.1 Botulinum toxin is also in the news because it is the ingredient in Botox, the much-publicized injection used to remove wrinkles and reduce aging effects.

What Is Botulinum Toxin?

Clostridium botulinum belongs to a family of Gram-positive bacteria that are capable of existing both as rod-shaped bacterial cells and as spores. Spores are heat-resistant and can survive in food that is improperly or minimally processed. The toxin produced by the bacteria is an enzymatic zinc-containing protein that breaks up one of the proteins involved in the release of acetylcholine into the neuromuscular junction. It is thus a potent and fast-acting neurotoxin. It is listed as "the most toxic substance known."2 It is estimated that a single gram of pure crystalline toxin, evenly dispersed and inhaled, could kill as many as one million people.3 However, such a dissemination would be technically unfeasible, since an effective mechanism for producing a large aerosol of botulinum has not been successfully developed.3

Botulinum toxin can be absorbed through the gastrointestinal or respiratory tracts; however, it is not absorbed by intact skin. The estimated lethal dose for a 91-kg (200-pound) person is 0.9–1.2 micrograms per kilogram by inhalation, and approximately 90 micrograms per kilogram by oral ingestion.4 Symptoms of botulinum toxin poisoning can occur within as little as two hours of exposure, or as late as eight days after.4

The toxin is a white crystalline solid; in liquid form, it is odorless, colorless and (as far as known) tasteless.3 The toxin is a protein and is sensitive to heat; it is destroyed when exposed to temperatures above 176ºF (80ºC) for a

10-minute period.5 Thus the toxin does not survive in foods that are properly cooked during preparation. The spores are more heat-resistant and can survive higher temperatures and longer periods of heating. Most naturally occurring outbreaks have resulted from eating improperly cooked foods like home-preserved foods, canned meats and cream cheese, or raw vegetables and fruits. Natural outbreaks are rare, but occur in the U.S. an average of 20 times per year and are distributed throughout all 50 states.6

Based on antigenic specificity, seven subtypes of botulinum toxin are recognized. Types A, B, E and F cause disease in humans. Types C and D cause cases mostly in animals and poultry. Type G, isolated from soil samples in Argentina, is not known to have caused any outbreaks to date.5

Botulinum Toxin as a Weapon

Since the 9/11 and anthrax letter events in 2001, the possibility of a biological attack on this country by terrorists has become an unfortunate reality. Botulinum toxin has always been on the list of top bioweapon candidate agents, because of its extremely high toxicity: 50–100 times more toxic orally than sodium cyanide. The bacteria are easy to grow, and the toxin is relatively easy to produce in large quantities. This makes it a prime candidate for the bioterrorist's arsenal. The U.S. and Russia gave up trying to utilize botulinum because it could not be effectively "weaponized" like anthrax and smallpox. Weaponizing a bio-agent is a complex, multi-stage process. The final stage of weaponizing entails binding the bacteria to ultra-finely powdered materials like bentonite or silica gel. This allows the material to stay suspended, forming a stable aerosol vehicle for dissemination.7 However, studies done by the U.S. military indicate that a point-source aerosol release of weaponized botulinum toxin could incapacitate or even kill 10% of the people downwind for a distance of one-third of a mile (1.760 feet).7 Such a release in a subway, shopping mall or a large enclosed event like a basketball game would have a major impact on a city, especially if multiple releases occurred at different locations simultaneously.

However, we should not necessarily expect that terrorist groups will have the wherewithal or even bother to develop refined preparations of botulinum or other agents. Relatively "crude" preparations of botulinum toxin delivered by homemade systems can cause significant exposure of the public. In March 1995, a radical Japanese cult, Aum Shinrikyo, placed packages containing plastic bags full of liquid sarin on five subway trains in different locations around Tokyo. The bags were broken by piercing them with umbrellas. This released the liquid, which quickly evaporated, filling the train cars with sarin gas, a deadly nerve agent. Close to 4,000 people were injured in the attack, and 12 died.3 The anthrax episode in 2001 utilized a weaponized form of the bacteria, and the method of dissemination-letters in the U.S. mail-represented a crude but effective delivery system which was able to disseminate an agent to a variety of locations in a short time period. The release of a crude preparation of toxin at multiple locations would effectively injure or kill some people while causing mass panic in the population. This type of attack can throw a city, or the whole country, into panic mode and would certainly tax local EMS and emergency department personnel and resources.

Terrorists have used botulinum toxin in the past. Aerosols of it were dispersed by Aum Shinrikyo at multiple sites in downtown Tokyo and U.S. military installations in Japan on three occasions between 1990–95.3 No one was injured or killed, and the reason these attacks failed is uncertain. It may have been due to use of a botulinum strain that produced an ineffective toxin subtype, faulty aerosol-generating equipment, or possibly internal sabotage by a cult member. The German Red Army was found to have manufactured the toxin in a safe house in Paris in the 1980s; however, it was never used in an attack.

In the 1930s, Japan developed an extensive bioweapons research complex at the village of Pingfan in Manchuria. Called Water Purification Supply Unit 731, or simply Unit 731, experiments on animal and human studies were performed using a wide variety of agents. Dr. Shiro Ishii, the director of the facility, admitted exposing Chinese, Korean and American prisoners to botulinum toxin.8 There are suggestions it was also used by the Japanese to poison streams used by the Soviet Union for water sources. At the end of World War II, the U.S. and England took all of the data developed at Japan's Unit 731 for use in their own bio-agent development programs.8

The United States produced botulinum toxin during WWII, and designated it "Agent X." More than one million doses of antitoxin were available for allied troops preparing to invade Normandy on D-Day. Although the public reason given was a concern that Germany had weaponized botulinum toxin, private speculation was that the United States was prepared to use botulinum toxin if chemical agents were used against allied forces in the invasion.

Another potential target for the toxin is our food supply. Many parts of the U.S. food production and distribution system are potential focal points for sabotage. Terrorists generally consider these "soft" targets, since they are almost impossible to guard at all times. In October 2003, the FDA posted a warning about the potential for bioterrorist attacks on the nation's food and water supplies.9 Contamination of the food supply could come through spraying the agent on fruits and vegetables, which come to the marketplace unregulated, with a number of middlemen in the shipping process. Contamination might also occur through processed foods such as dairy products, like ice cream and cheese, or through hamburger, canned meats, etc. In January 2003, hamburger in a Michigan supermarket was found to be laced with a nicotine pesticide, causing severe illness in more than 100 people. A disgruntled employee in a single store caused this incident; however, it demonstrated the relative ease with which foods can be contaminated. This is a clear example of how a relatively crude preparation of a bio-agent can be delivered by low-tech means.

Recognizing a Terrorist Attack

An outbreak with a large number of individuals presenting acute flaccid paralysis and significant bulbar palsies, all with histories of recent consumption of a similar food or food from a single location (a party, sporting event, supermarket, etc.), could be the result of a terrorist attack. Consumption of the same food from a variety of supermarkets might also indicate that a foodborne attack has occurred. An outbreak of symptoms similar to those above occurring with a common geographic location (subway, airport, office building or sporting event), but no common food exposure, would suggest an aerosol attack. In each case, an attempt to find out from victims if other individuals were present and may have left the scene after being exposed would be essential to identifying all exposed individuals.

Symptoms and Treatment Steps

The signs of botulinum poisoning are essentially the same regardless of the route of exposure. Since the toxin preferentially affects the nervous system, paralysis generally starts at the head and moves down the body, affecting the limbs and eventually the lungs. Early signs of exposure include weakness, lassitude and vertigo; these are followed by facial diplegia, ptosis, disconjugate gaze and dysarthria. As the lungs are affected, there is difficulty breathing, and the victim eventually requires mechanical ventilation (see Table I).4

The differential diagnosis of botulinum poisoning includes tick paralysis, myasthenia gravis, diphtheria, pontine infarction and Guillain-Barré Syndrome (Miller variant).4 Diagnosis on symptoms alone would make it difficult to differentiate from these other illnesses if seen in a single patient. However, encountering a large number of patients presenting the same symptoms would indicate exposure to some toxic agent. Confirmation generally requires laboratory analysis of serum or feces to demonstrate presence of the toxin. Laboratory analysis of the food eaten in a foodborne episode may also reveal its presence.9 A rapid, qualitative handheld field test for the toxin is available from several companies, including Alexeter Technologies10 and Osborn Scientific Group.11 In the event of a terrorist attack where large numbers of individuals are affected, a positive result with this type of kit can provide an immediate indication of the nature of the biological agent involved.

Treatment of toxin victims would include standard procedures:?Start an IV line, administer oxygen, take an EKG, obtain vital signs and check oxygen saturation levels with a fingertip monitor. If the EKG indicates irregular results, start a code of drugs as appropriate. If exposure occurred at a sufficiently high dose, swallowing difficulty or respiratory failure may already be present and necessitate breathing assistance, and possibly intubation, before the ED is reached. An antitoxin that provides passive immunization to botulinum toxin is available, but it must be administered relatively close to the time of exposure to be effective.12 However, most hospitals do not stock this item. In any event, while this may improve the overall recovery of the victim, paralysis usually persists for several weeks after treatment.

Botulinum toxoid vaccine is available.12,13 A pentavalent toxoid of type A, B, C, D and E is available for pre-exposure prophylaxis. This toxoid is distributed to laboratory workers at high risk of exposure and by the military for the protection of troops.

It is unlikely that the F and G type toxins would be used in warfare because the strains of C. botulinum that produce these toxins are difficult to grow in large quantities. If new techniques allow production of toxins F and G in large quantities, the pentavalent toxoid will be useless.

The pentavalent toxoid is available as an IND only. This product has been given to several thousand volunteers and workers at risk from their occupations. It induces sufficient serum antitoxin levels; it requires three injections, followed by a yearly booster, for complete protection. The quantity of vaccine is quite limited, and it would most likely be reserved for groups judged at high risk for exposure, such as investigative agents.

Protection for the EMS Responder

In a foodborne attack, victims and foodstuffs should be handled with latex gloves. It is unlikely that significant toxin would be released into the air from the contaminated food materials. In a suspected aerosol attack, a full-face respirator should be worn to protect from exposure to residual aerosol. In an attack, it is difficult to determine the persistence of the aerosol after the initial release(s). Temperature, humidity and the size of the aerosol particles all determine the rate of dissipation into the atmosphere. An enclosed site should be considered contaminated for at least 48 hours after exposure. The toxin does not penetrate intact skin, so protective suits would not normally be required, but this is at the discretion of individual EMS teams. Any clothing, skin or hair exposed to toxin should be washed thoroughly with soap and water. The toxin is heat-sensitive, so decontamination of equipment can be accomplished by heating to 80ºC for 10 minutes or by washing in 0.1% hypochlorite bleach solution.3

References

1. Statement by David Kay on the Interim Progress Report of the Activities of the Iraq Survey Group (ISG), before the House Permanent Select Committee on Intelligence and Senate Select Committee on Intelligence, October 2, 2003.
2. National Institute of Occupational Safety and Health Registry of the Toxic Effects of Chemical Substances (R-TECS), keyword "Botulinum Toxin."
3. Arnon SS, Schechter R, Inglesby TV, et al. Botulinum toxin as a biological weapon: Medical and public health management. JAMA 285:1,059, 2001.
4. Martin CO, Adams HP. Neurological aspects of biological & chemical terrorism. Archives of Neurology 60:21–25, 2003.
5. U.S. FDA. Bad Bug Book, Chapter 2, vm.cfsan.fda.gov/~mow/chap2.html.
6. Botulism in the United States 1899–1996: Handbook for Epidemiologists, Clinicians and Laboratory Workers. Centers for Disease Control and Prevention (CDC), Atlanta, GA, 1998. Available at www.cdc.gov/nci dod/dbmd/diseaseinfo/botulism.pdf.
7. William C. Patrick III, former Chief of Product Development, U.S. Army Biological Warfare Laboratories, Ft. Detrick, MD (private communication with Richard Preston, quoted in The Demon in the Freezer, Random House, 2002).
8. Goebel G. History of Biological Weapons, www.vectorsite.net/twgas3.html.
9. FDA Center for Food Safety & Applied Nutrition, vm.cfsan.fda.gov/~dms/rabtact.html.
10. Alexeter Technologies, Inc. Chicago, IL. www.alexeter.com.
11. Osborn Scientific Group, Lakeside, AZ. www.osborn-scientific.com.
12. Biological Defense Vaccine Information Summaries. Publication of USAMRIID, Fort Detrick, Maryland, 1994.
13. Weiner SL. Strategies for prevention of a successful biological warfare aerosol attack. Mil Med 161:251–256, 1996.