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Reasonable Rascal
12-03-01, 23:44
MEDICAL MANAGEMENT OF BIOLOGICAL CASUALTIES HANDBOOK

Third Edition

U.S. ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES

FORT DETRICK FREDERICK, MARYLAND

July 1998

Editors:

COL Edward Eitzen
MAJ Julie Pavlin
LTC Ted Cieslak
LTC George Christopher
CDR Randall Culpepper

Comments and suggestions are solicited and should be addressed to:

Operational Medicine Division

Attn: Mr. Paul Porreca

U.S. Army Medical Research
Institute of Infectious Diseases
Fort Detrick, Maryland 21702-5011


DISCLAIMER

The purpose of this Handbook is to provide concise supplemental reading material to assist in education of biological casualty management. Every effort has been made to make the information in this handbook consistent with official policy and doctrine. The information contained in this handbook is not official Department of the Army policy or doctrine, and it should not be construed as such.

ACKNOWLEDGMENTS

This handbook would not be possible without the generous assistance and support of COL David Franz, COL Gerald Parker, LTC Gerald Jennings, SGM Raymond Alston, COL James Arthur, COL W. Russell Byrne, LTC Les Caudle, Dr. John Ezzell, COL Arthur Friedlander, Mr. Darren Gerlach, SGT KevinGianunzio, Dr. Robert Hawley, LTC Erik Henchal, COL(ret) Ted Hussey, Dr. Peter Jahrling, LTC Ross LeClaire, Dr. George Ludwig, Mr. William Patrick, Dr. Mark Poli, Mr. Paul Porreca, Dr. Fred Sidell, Dr. Jonathon Smith, Mr. Richard Stevens, COL Stanley Wiener, Mr. Benjamin Wilson and others too numerous to mention. The exclusion of anyone on this page is purely accidental and in no way lessens
the gratitude we feel for contributions received.

INTRODUCTION

Medical defense against biological warfare is an area of study for military health care providers which does not apply readily to the day to day mission of caring for patients in peacetime. However, during Operations Desert Shield/Desert Storm, it became obvious that the threat of biological attacks against our soldiers was real, and that we could do more to educate our medical professionals about how to prevent and treat biological warfare casualties. Many of our medical personnel who deployed for the Gulf War had less than an optimal understanding of the biological threat and of the medical means available to counter it. Since Desert Storm, there has been a renewed emphasis placed on making sure that our health care professionals gain the necessary background in this important area of military medicine.

In fact, our training efforts have significantly intensified over the past eighteen months following increased incidents and threats of domestic terrorism (e.g., New York City World Trade Center bombing, Tokyo subway sarin release, Oklahoma City federal building bombing, Atlanta Centennial Park bombing). Additionally, the recent escalation of tensions in Iraq and subsequent deployment of military troops to the Persian Gulf region underscored the importance of force protection from biological threats. The Secretary of Defense announced in November 1997 that all U.S. military troops will be immunized against anthrax. Finally, the disclosure of a sophisticated offensive biological warfare program in the Former Soviet Union (FSU) and subsequent media attention has reinforced the need for increased training and education.

The Medical Management of Chemical and Biological Casualties Course taught at both USAMRIID and USAMRICD was revised in March 1998 by doubling its class capacity providing education in both biological and chemical medical defense to over 560 military medical professionals per calendar year. Also, the highly successful 3-day USAMRIID satellite course on the Medical Management of Biological Casualties presented in September 1997 reached over 5600 military and other government health care professionals throughout the United States.

Through this handbook and the training courses noted above, military medical professionals will learn that effective medical countermeasures are available against many of the bacteria, viruses, and toxins which might be used as biological weapons against our military forces. The importance of this education cannot be overemphasized and it is hoped that our physicians, nurses, and allied medical professionals will develop a solid understanding of the biological threats we face and the medical armamentarium for defending against these threats.

The global biological warfare threat is taken seriously by our leaders. The United States was willing to return to war against Iraq in February 1998 to preserve the integrity and the independence of the UNSCOM inspectors such that they would have unconditional, unfettered and unrestricted access to all suspected sites in Iraq in their search for weapons of mass destruction. The threat is indeed serious, and the potential for devastating casualties is high for certain biological agents. However, with appropriate use of medical countermeasures either already developed or under development, many casualties can be prevented or minimized, and the fighting strength of our forces can be maintained.


The purpose for this handbook is to serve as a small and concise manual for medical personnel to carry in their BDU pocket as a guide to medical prophylaxis and management of biological casualties. It is designed as a quick reference and overview, and is not intended as a definitive text on the medical management of biological casualties.

HISTORY OF CHEMICAL WARFARE AND CURRENT THREAT

The use of biological weapons and efforts to make them more useful as a means of waging war have been recorded numerous times in history. Two of the earliest reported uses occurred in the 6th century BC, with the Assyrians poisoning enemy wells with rye ergot, and Solon’s use of the purgative herb hellebore during the siege of Krissa. In 1346, plague broke out in the Tartar army during its siege of Kaffa (at present day Feodosia in Crimea). The attackers hurled the corpses of those who died over the city walls; the plague epidemic that followed forced the defenders to surrender, and some infected people who left Kaffa may have started the Black Death pandemic which spread throughout Europe. Russian troops may have used the same plague-infected corpse tactic against Sweden in 1710.

On several occasions, smallpox was used as a biological weapon. Pizarro is said to have presented South American natives with variola-contaminated clothing in the 15th century, and the English did the same
when Sir Jeffery Amherst provided Indians loyal to the French with smallpox-laden blankets during the French and Indian War of 1754 to 1767. Native Americans defending Fort Carillon sustained epidemic casualties which directly contributed to the loss of the fort to the English.

In this century, there is evidence that during World War I, German agents inoculated horses and cattle with glanders in the U.S. before the animals were shipped to France. In 1937, Japan started an ambitious biological warfare program, located 40 miles south of Harbin, Manchuria, in a laboratory complex code named "Unit 731". Studies directed by Japanese General Ishii continued there until 1945, when the complex was leveled by burning it. A post World War II investigation revealed that numerous organisms had received Japanese research attention, and that experiments had been conducted on prisoners of war. Slightly less than 1,000 human autopsies apparently were carried out at Unit 731, most on victims exposed to aerosolized anthrax. Many more prisoners and Chinese nationals may have died in this facility - some have estimated up to 3,000 human deaths. In 1940, a plague epidemic in China and Manchuria followed reported overflights by Japanese planes dropping plague-infected fleas. By 1945, the Japanese program had stockpiled 400 kilograms of anthrax to be used in a specially designed fragmentation bomb.

In 1943, the United States began research into the offensive use of biological agents. This work was started, interestingly enough, in response to a perceived German biological warfare (BW) threat as opposed to a Japanese one. The United States conducted this research at Camp Detrick (now Fort Detrick), which was a small National Guard airfield prior to that time, and produced agents at other sites until 1969, when President Nixon stopped all offensive biological and toxin weapon research and production by executive
order. Between May 1971 and May 1972, all stockpiles of biological agents and munitions from the now defunct U.S. program were destroyed in the presence of monitors representing the United States Department of Agriculture, the Department of Health, Education, and Welfare, and the states of Arkansas, Colorado, and Maryland. Included among the destroyed agents were Bacillus anthracis, botulinum toxin, Francisella tularensis, Coxiella burnetii, Venezuelan equine encephalitis virus, Brucella suis, and
Staphylococcal enterotoxin B. The United States also had a medical defensive program, begun in 1953, that continues today at USAMRIID.

In 1972, the United States and many other countries signed the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction, commonly called the Biological Weapons Convention. This treaty prohibits the stockpiling of biological agents for offensive military purposes, and also forbids research into such offensive employment of biological agents. The former Soviet Union and the government of Iraq were both signatories to this accord. However, despite this historic agreement among nations, biological warfare research continued to
flourish in many countries hostile to the United States. There were also several cases of suspected or actual use of biological weapons. Among the most notorious of these were the "yellow rain" incidents in Southeast Asia, the accidental release of anthrax at Sverdlovsk, and the use of ricin as an assassination weapon in London in 1978.

Testimony from the late 1970's indicated that the countries of Laos and Kampuchea were attacked by planes and helicopters delivering aerosols of several colors. After being exposed, people and animals became disoriented and ill, and a small percentage of those stricken died. Some of these clouds were thought to be comprised of trichothecene toxins (in particular, T2 mycotoxin). These attacks are lumped under the label "Yellow Rain". There has been a great deal of controversy about whether these clouds were truly biological warfare agents: some have argued that the clouds were nothing more than bee feces produced by swarms of bees.

In late April of 1979, an incident occurred in Sverdlovsk (now Yekaterinburg) in the former Soviet Union which appeared to be an accidental release of anthrax in aerosol form from the Soviet Military Compound 19, a microbiology facility. Residents living downwind from this compound developed high fever and difficulty breathing, and a large number died. The final death toll was estimated at the time to be between 200 and 1,000. The Soviet Ministry of Health blamed the deaths on the consumption of contaminated meat, and for years controversy raged in the press over the actual cause of the outbreak. All evidence available to the United States government indicated a massive release of aerosolized anthrax. In the summer of 1992, U.S. intelligence officials were proven correct when new Russian President Boris Yeltsin acknowledged that the Sverdlovsk incident was in fact a large scale accident involving the escape of an aerosol of anthrax spores from the military research facility. In 1994, Meselson and colleagues published an in-depth analysis of the Sverdlovsk incident (Science 266:1202-1208). They documented that all of the 1979 cases occurred within a narrow zone extending downwind in a southerly direction from Compound 19. A total of 77 patients were identified by Meselson's team, including 66 fatalities and 11 survivors.

Before the Sverdlovsk incident, in 1978, a Bulgarian exile named Georgi Markov as attacked in London with a device disguised as an umbrella which injected a tiny pellet filled with ricin toxin into the subcutaneous tissue of his leg while he was waiting for a bus. He died several days later. On autopsy, the tiny pellet was found and determined to contain the toxin. This assassination, it was later revealed, was carried out by the communist Bulgarian government, and the technology to commit the crime was supplied to the Bulgarians by the former Soviet Union.

In August of 1991, the first United Nations inspection of Iraq's biological warfare capabilities was carried out in the aftermath of the Gulf War. On August 2, 1991, representatives of the Iraqi government
announced to leaders of United Nations Special Commission Team 7 that they had conducted research into the offensive use of Bacillus anthracis, botulinum toxins, and Clostridium perfringens (presumably one of its toxins). This was the first open admission of biological weapons research by any country in recent memory, and it verified many of the concerns of the U.S. intelligence community publicly. Iraq had extensive and redundant research facilities at Salman Pak and other sites, many of which were destroyed during the war.

In 1995, further information on Iraq's offensive program was made available to United Nations inspectors. Iraq conducted research and development work on anthrax, botulinum toxins, Clostridium perfringens, aflatoxins, wheat cover smut, and ricin. Field trials were conducted with Bacillus subtilis (a simulant for anthrax), botulinum toxin, and aflatoxin. Biological agents were tested in various delivery systems, including rockets, aerial bombs, and spray tanks. In December 1990, the Iraqis filled 100 R400 bombs with botulinum toxin, 50 with anthrax, and 16 with aflatoxin. In addition, 13 Al Hussein (SCUD) warheads were filled with botulinum toxin, 10 with anthrax, and 2 with aflatoxin. These weapons were deployed in January 1991 to four locations. All in all, Iraq produced 19,000 liters of concentrated botulinum toxin (nearly 10,000 liters filled into munitions), 8,500 liters of concentrated anthrax (6,500 liters filled into munitions) and 2,200 liters of aflatoxin (1,580 liters filled into munitions).

The threat of biological warfare has increased in the last two decades, with a number of countries working on offensive use of these agents. The extensive program of the former Soviet Union is now controlled largely by Russia. Russian president Boris Yeltsin has stated that he will put an end to further offensive biological research; however, the degree to which the program has been scaled back, if any, is not known. Recent revelations from a senior BW program manager who defected from the FSU in 1992 outlined a
remarkably robust biological warfare program including active research into genetic engineering, binary biologicals and chimeras. There is also growing concern that the smallpox virus, eliminated from the face of the earth in the late 1970's and now stored in only two laboratories at the CDC in Atlanta and the Institute for Viral Precautions in Moscow, Russia, may have been "bargained" away by desperate Russian scientists seeking money.

There is intense concern in the West about the possibility of proliferation or enhancement of offensive programs in countries hostile to the western democracies, due to the potential hiring of expatriate Russian scientists. It was reported in January 1998 that Iraq had sent about a dozen scientists involved in BW research to Libya to help that country develop a biological warfare complex disguised as a medical facility in the Tripoli area. In a report issued in November 1997, Secretary of Defense William Cohen singled out Libya, Iraq, Iran, and Syria as countries "aggressively seeking" nuclear, biological, and chemical weapons.

There is also an increasing amount of concern over the possibility of terrorist use of biological agents to threaten either military or civilian populations. There have been cases of persons loyal to extremist groups trying to obtain microorganisms which could be used as biological weapons. The Department of Defense is leading a federal effort to train the first responders in 120 American cities to be prepared to act in case of a domestic terrorist incident involving WMD.

Certainly the threat of biological weapons being used against U.S. military forces is broader and more likely in various geographic scenarios than at any point in our history. Therefore, awareness of this potential threat and education of our leaders and medical care providers on how to combat it are crucial.

ANTHRAX

SUMMARY

Signs and Symptoms: Incubation period is 1-6 days. Fever, malaise, fatigue, cough and mild chest discomfort is followed by severe respiratory distress with dyspnea, diaphoresis, stridor, and cyanosis. Shock and death occurs within 24-36 hours after onset of severe symptoms.

Diagnosis: Physical findings are non-specific. A widened mediastinum may be seen on CXR. Detectable by Gram stain of the blood and by blood culture late in the course of illness.

Treatment: Although effectiveness may be limited after symptoms are present, high dose antibiotic treatment with penicillin, ciprofloxacin, or doxycycline should be undertaken. Supportive therapy may be
necessary.

Prophylaxis: An FDA licensed vaccine is available. Vaccine schedule is 0.5 ml SC at 0, 2, 4 weeks, then 6, 12, and 18 months for the primary series, followed by annual boosters. Oral ciprofloxacin or doxycycline
for known or imminent exposure.

Isolation and Decontamination: Standard precautions for healthcare workers. After an invasive procedure or autopsy is performed, the instruments and area used should be thoroughly disinfected with a sporicidal agent (chlorine).

OVERVIEW

Bacillus anthracis, the causative agent of Anthrax, is a rod-shaped, gram-positive, sporulating organism with the spores constituting the usual infective form. Anthrax is primarily a zoonotic disease of herbivores, with cattle, sheep and horses being the usual domesticated animal hosts, but other animals may be infected. Human disease may be contracted by handling contaminated hair, wool, hides, flesh, blood and excreta of infected animals and from manufactured products such as bone meal, as well as by purposeful dissemination of spores. Infection is introduced through scratches or abrasions of the skin, wounds, inhalation of spores, eating insufficiently cooked infected meat, or by flies. All human populations are susceptible. Recovery from an attack of the disease may be followed by immunity. The spores are very stable and may remain viable for many years in soil and water. They will resist sunlight for varying periods.

HISTORY AND SIGNIFICANCE

Anthrax spores were weaponized by the United States in the 1950's and 1960's before the old U.S. offensive program was terminated. Other countries have weaponized this agent or are suspected of doing so. The anthrax bacterium is easy to cultivate and spore production is readily induced. Spores are highly resistant to sunlight, heat and disinfectants - properties which could be advantageous when choosing a bacterial weapon. Iraq admitted to a United Nations inspection team in August of 1991 that it had
performed research on the offensive use of B. anthracis prior to the Persian Gulf War of 1991, and in 1995 Iraq admitted to weaponizing anthrax. This agent could be produced in either a wet or dried form, stabilized for weaponization by an adversary and delivered as an aerosol cloud either from a line source such as an aircraft flying upwind of friendly positions, or as a point source from a spray device. Coverage of a large ground area could also be theoretically facilitated by multiple spray bomblets disseminated from a missile warhead at a predetermined height above the ground.

CLINICAL FEATURES

Anthrax presents as three distinct clinical syndromes in man: cutaneous, inhalational, and gastrointestinal disease. The cutaneous form (also referred to as malignant pustule) occurs most frequently on the hands and forearms of persons working with infected livestock. It begins with a papule followed by formation of a blister-like fluid-filled vesicle. The vesicle typically dries and forms a coal-black scab, hence the term
anthrax (Greek for coal). Sometimes this local infection will develop into a systemic infection which is often fatal. Endemic inhalational anthrax, known as woolsorters disease, is a rare infection contracted by inhalation of the spores. It occurs mainly among workers handling infected hides, wool, and furs. The intestinal form, which is also very rare in man, is contracted by the ingestion of insufficiently cooked meat from infected animals. In man, the mortality rate of cutaneous anthrax ranges up to 25 per cent; in inhalational and intestinal cases, the case fatality rate is almost 100 percent.

DIAGNOSIS

After an incubation period of 1-6 days, presumably dependent upon the dose and strain of inhaled organisms, the onset of inhalation anthrax is gradual and nonspecific. Fever, malaise, and fatigue may be present, sometimes in association with a nonproductive cough and mild chest discomfort. These initial symptoms are often followed by a short period of improvement (hours to 2-3 days), followed by the abrupt development of severe respiratory distress with dyspnea, diaphoresis, stridor, and cyanosis. Shock and death usually follow within 24-36 hours after the onset of respiratory distress. Physical findings are typically non-specific. The chest X-ray may reveal a widened mediastinum ± pleural effusions late in the disease in about 55% of the cases, but typically is without infiltrates. Bacillus anthracis will be detectable by Gram stain of the blood and by blood culture with routine media, but often not until late in the course of the illness. Only vegetative encapsulated bacilli are present during infection. Spores are not found within the body unless it is open to ambient air. Studies of inhalation anthrax in non-human primates (rhesus monkey) showed that bacilli and toxin appear in the blood late on day 2 or early on day 3 post-exposure. Toxin production parallels the appearance of bacilli in the blood and tests are available to rapidly detect the toxin. Concurrently with the appearance of anthrax, the WBC count becomes elevated and remains so until death.

MEDICAL MANAGEMENT

Almost all inhalational anthrax cases in which treatment was begun after patients were significantly symptomatic have been fatal, regardless of treatment. Penicillin has been regarded as the treatment of choice, with 2 million units given intravenously every 2 hours. Tetracyclines and erythromycin have been recommended in penicillin allergic patients. The vast majority of naturally-occurring anthrax strains are
sensitive in vitro to penicillin. However, penicillin-resistant strains exist naturally, and one has been recovered from a fatal human case. Moreover, it might not be difficult for an adversary to induce resistance to penicillin, tetracyclines, erythromycin, and many other antibiotics through laboratory manipulation of organisms. All naturally occurring strains tested to date have been sensitive to erythromycin, chloramphenicol, gentamicin, and ciprofloxacin. In the absence of information concerning antibiotic sensitivity, treatment should be instituted at the earliest signs of disease with intravenous ciprofloxacin (400 mg q 8-12 hrs) or intravenous doxycycline (200 mg initially, followed by 100 mg q 12 hrs). Supportive therapy for shock, fluid volume deficit, and adequacy of airway may all be needed.

Standard Precautions should be practiced. After an invasive procedure or autopsy, the instruments and area used should be thoroughly disinfected with a sporicidal agent. Iodine can be used, but must be used at disinfectant strengths, as antiseptic-strength iodophors are not usually sporicidal. Chlorine, in the form of sodium or calcium hypochlorite, can also be used, but with the caution that the activity of hypochlorites is greatly reduced in the presence of organic material.

PROPHYLAXIS

Vaccine: A licensed vaccine is derived from sterile culture fluid supernatant taken from an attenuated strain. The vaccination series consists of six 0.5 ml doses SC at 0, 2, and 4 weeks, then 6, 12 and 18 months, followed by yearly boosters. Limited human data suggest that the vaccine protects against cutaneous anthrax. There is insufficient data to know its efficacy against inhalational anthrax in humans, although studies in rhesus monkeys indicate that good protection can be afforded after only two doses (15 days apart) for up to 2 years. However, it should be emphasized that the vaccine series should be completed according to the routine 6 dose primary series. As with all vaccines, the degree of protection depends upon the magnitude of the challenge dose; vaccine-induced protection could presumably be overwhelmed by extremely high spore challenge.

Contraindications for use of this vaccine include hypersensitivity reaction to a previous dose of vaccine and age < 18 or > 65. Reasons for temporary deferment of the vaccine include pregnancy; active infection with fever; or a course of immune suppressing drugs such as steroids. Reactogenicity is mild to moderate. Up to 6 percent of recipients will experience mild discomfort at the inoculation site for up to 72 hours (e.g., tenderness, erythema, edema, pruritus), while less than 1 percent will experience more severe local reactions, potentially limiting use of the arm for 1-2 days. Modest systemic reactions (e.g., myalgia, malaise, low-grade fever) are uncommon, and severe systemic reactions such as anaphylaxis, which precludes additional vaccination, are rare. The vaccine should be stored between 2-6 oC (refrigerator temperature, not frozen).

Antibiotics: The choice of antibiotics for prophylaxis is difficult to make; for example, it seems relatively easy to induce penicillin and tetracycline resistance in the laboratory. Therefore, prophylaxis with ciprofloxacin (500 mg po bid) or doxycycline (100 mg po bid) is recommended. If personnel are unvaccinated, a single 0.5 ml dose of vaccine should also be given subcutaneously. Should the attack be confirmed as anthrax, antibiotics should be continued for at least 4 weeks in all those exposed, and until all those exposed have received three doses of the vaccine. Two additional 0.5 ml doses of vaccine should be given 2 weeks apart in the unvaccinated; those previously vaccinated with fewer than three doses should
receive a single 0.5 ml booster, while vaccination probably is not necessary for those who have received the initial three-doses of the primary series, within the previous six months. Upon discontinuation of antibiotics, patients should be closely observed; if clinical signs of anthrax occur, patients should be treated as indicated above. If vaccine is not available, antibiotics should be continued beyond 4 weeks and withdrawn under medical observation. Optimally, patients should have medical care available upon discontinuation of antibiotics, from a fixed medical care facility with intensive care capabilities and infectious disease consultants.

BACTERIAL AGENTS

Bacteria are unicellular organisms. They vary in shape and size from spherical cells - cocci - with a diameter of 0.5-1.0 m m (micrometer), to long rod-shaped organisms - bacilli - which may be from 1-5 m m in size. Chains of bacilli may exceed 50 m m. The shape of the bacterial cell is determined by the rigid cell wall. The interior of the cell contains the nuclear material (DNA), cytoplasm, and cell membrane, that are necessary for the life of the bacterium. Many bacteria also have glycoproteins on their outer surfaces which aid in bacterial attachment to surface receptors on cells and are of special importance in their ability to cause disease. Under special circumstances some types of bacteria can transform into spores. The spore of the bacterial cell is more resistant to cold, heat, drying, chemicals and radiation than the bacterium itself. Spores are a dormant form of the bacterium and, like the seeds of plants, they can germinate when conditions are favorable.

Bacteria can cause diseases in human beings and animals by means of two mechanisms which differ in principle: in one case by invading the tissues, in the other by producing poisons (toxins). In many cases pathogenic bacteria possess both properties. The diseases they produce often respond to specific therapy with antibiotics. This manual will cover several of the bacteria or rickettsia considered to be potential BW threat agents: Bacillus anthracis (Anthrax), Brucella spp. (Brucellosis), Vibrio cholerae (Cholera), Burkholderia mallei (Glanders), Yersinia pestis (Plague), Francisella tularensis (Tularemia), and Coxiella burnetii (Q Fever).

Reasonable Rascal
12-03-01, 23:50
BIOLOGICAL TOXINS

Toxins are defined as any toxic substance of natural origin produced by an animal, plant, or microbe. They are different from chemical agents such as VX, cyanide, or mustard in that they are not man-made. They are non-volatile, are usually not dermally active (mycotoxins are an exception), and tend to be more toxic per weight than many chemical agents. Their lack of volatility also distinguishes them from many of the chemical threat agents, and is very important in that they would not be either a persistent battlefield threat or be likely to produce secondary or person to person exposures. Many of the toxins, such as low molecular weight toxins and some peptides, are quite stable, as where the stability of the larger protein bacterial toxins is more variable. The bacterial toxins, such as botulinum toxins or shiga toxin, tend to be the most toxic in terms of dose required for lethality (Appendix C), whereas the mycotoxins tend to be among the least toxic compounds, thousands of times less toxic than the botulinum toxins. Some toxins are more toxic by the aerosol route than when delivered orally or parenterally (ricin, saxitoxin, and T2 mycotoxins are examples), whereas botulinum toxins have lower toxicity when delivered by the aerosol route than when ingested. However, botulinum is so toxic inherently that this characteristic does not limit its potential as a biological warfare agent. The utility of many toxins as military weapons is potentially limited by their inherent low toxicity (too much toxin would be required), or by the fact that some, such as saxitoxin, can only feasibly be produced in minute quantities. The relationship between aerosol toxicity and the quantity of toxin required to provide an effective open-air exposure is shown in Appendix D. The lower the lethal dose for fifty percent of those exposed (LD50), in micrograms per kilogram, the less agent would be required to cover a large battlefield sized area. The converse is also true, and means that for some agents such as ricin, very large quantities (tons) would be needed for an effective open-air attack.

Where toxins are concerned, incapacitation as well as lethality must be considered. Several toxins cause significant illness at levels much lower than the level required for lethality, and are thus militarily significant in their ability to incapacitate soldiers.

This manual will cover four toxins considered to be among the most likely toxins which could be used against U.S. forces: botulinum toxins, staphylococcal enterotoxin B (SEB), ricin, and T-2 mycotoxins.

BOTULINUM TOXINS

SUMMARY

Signs and Symptoms: Ptosis, generalized weakness, dizziness, dry mouth and throat, blurred vision and diplopia, dysarthria, dysphonia, and dysphagia followed by symmetrical descending flaccid paralysis
and development of respiratory failure. Symptoms begin as early as 24-36 hours but may take several days after inhalation of toxin.

Diagnosis: Clinical diagnosis. No routine laboratory findings. Biowarfare attack should be suspected if multiple casualties simultaneously present with progressive descending bulbar, muscular, and respiratory weakness.

Treatment: Intubation and ventilatory assistance for respiratory failure. Tracheostomy may be required. Administration of heptavalent botulinum antitoxin (IND product) may prevent or decrease progression to respiratory failure and hasten recovery.

Prophylaxis: Pentavalent toxoid vaccine (types A, B, C, D, and E) is available as an IND product for those at high risk of exposure.

Isolation and Decontamination: Standard Precautions for healthcare workers. Toxin is not dermally active and secondary aerosols are not a hazard from patients. Hypochlorite (0.5% for 10-15 minutes) and/or soap and water.

OVERVIEW

The botulinum toxins are a group of seven related neurotoxins produced by the bacillus Clostridium botulinum. These toxins, types A through G, could be delivered by aerosol over concentrations of troops. When inhaled, these toxins produce a clinical picture very similar to foodborne intoxication, although the time to onset of paralytic symptoms
may actually be longer than for foodborne cases, and may vary by type and dose of toxin. The clinical syndrome produced by one or more of these toxins is known as "botulism".

HISTORY AND SIGNIFICANCE

Botulinum toxins have caused numerous cases of botulism when ingested in improperly prepared or canned foods. Many deaths have occurred secondary to such incidents. It is feasible to deliver botulinum toxins as a biological weapon, and other countries have weaponized or are suspected to have weaponized one or more of this group of toxins. Iraq admitted to a United Nations inspection team in August of 1991 that it
had done research on the offensive use of botulinum toxins prior to the Persian Gulf War, which occurred in January and February of that year. Further information given in 1995 revealed that Iraq had not only researched the use of this toxin as a weapon, but had filled and deployed over 100 munitions with botulinum toxin.

TOXIN CHARACTERISTICS

Botulinum toxins are proteins of approximately 150,000 kD molecular weight which can be produced from the anaerobic bacterium Clostridium botulinum. As noted above, there are seven distinct but related neurotoxins, A through G, produced by different strains of the clostridial bacillus. All seven types act by similar mechanisms. The toxins produce similar effects when inhaled or ingested, although the time course may vary depending on the route of exposure and the dose received. Although an aerosol attack is by far the most likely scenario for the use of botulinum toxins, theoretically the agent could be used to sabotage food supplies; enemy special forces or terrorists might use this method in certain scenarios to produce foodborne botulism in those so targeted.

MECHANISM OF TOXICITY

The botulinum toxins as a group are among the most toxic compounds known to man. Appendix C shows the comparative lethality of selected toxins and chemical agents in laboratory mice. Botulinum toxin is the most toxic compound per weight of agent, requiring only 0.001 microgram per kilogram of body weight to kill 50 percent of the animals studied. As a group, bacterial toxins such as botulinum tend to be the most lethal of all toxins. Note that botulinum toxin type A is 15,000 times more toxic than VX and 100,000 times more toxic than Sarin, two of the well known organophosphate nerve agents.

Botulinum toxins act by binding to the presynaptic nerve terminal at the neuromuscular junction and at cholinergic autonomic sites. These toxins then act to prevent the release of acetylcholine presynaptically, and thus block neurotransmission. This interruption of neurotransmission causes both bulbar palsies and the skeletal muscle weakness seen in clinical botulism.

Unlike the situation with nerve agent intoxication, where there is too much acetylcholine due to inhibition of acetylcholinesterase, the problem in botulism is lack of the neurotransmitter in the synapse. Thus, pharmacologic measures such as atropine are not indicated in botulism and would likely exacerbate symptoms.

CLINICAL FEATURES

The onset of symptoms of inhalation botulism may vary from 24 to 36 hours, to several days following exposure. Recent primate studies indicate that the signs and symptoms may in fact not appear for several days when a low dose of the toxin is inhaled versus a shorter time period following ingestion of toxin or
inhalation of higher doses. Bulbar palsies are prominent early, with eye symptoms such as blurred vision due to mydriasis, diplopia, ptosis, and photophobia, in addition to other bulbar signs such as dysarthria, dysphonia, and dysphagia. Skeletal muscle paralysis follows, with a symmetrical, descending, and
progressive weakness which may culminate abruptly in respiratory failure. Progression from onset of symptoms to respiratory failure has occurred in as little as 24 hours in cases of foodborne botulism.

Physical examination usually reveals an alert and oriented patient without fever. Postural hypotension may be present. Mucous membranes may be dry and crusted and the patient may complain of dry mouth or even sore throat. There may be difficulty with speaking and with swallowing. Gag reflex may be absent. Pupils may be dilated and even fixed. Ptosis and extraocular muscle palsies may also be observed. Variable degrees of skeletal muscle weakness may be observed depending on the degree of progression in an individual patient. Deep tendon reflexes may be present or absent. With severe respiratory muscle paralysis, the patient may become cyanotic or exhibit narcosis from CO2 retention.

DIAGNOSIS

The occurrence of an epidemic of cases of a descending and progressive bulbar and skeletal paralysis in afebrile patients points to the diagnosis of botulinum intoxication. Foodborne outbreaks tend to occur in small clusters and have never occurred in soldiers on military rations such as MRE's (Meals, Ready to Eat). Higher numbers of cases in a theater of operations should raise at least the consideration of a biological warfare attack with aerosolized botulinum toxin. Foodborne outbreaks are theoretically possible in troops on normal "A" rations.

Individual cases might be confused clinically with other neuromuscular disorders such as Guillain-Barre syndrome, myasthenia gravis, or tick paralysis. The edrophonium or Tensilon® test may be transiently positive in botulism, so it may not distinguish botulinum intoxication from myasthenia. The cerebrospinal fluid in botulism is normal and the paralysis is generally symmetrical, which distinguishes it from enteroviral myelitis. Mental status changes generally seen in viral encephalitis should not occur with botulinum intoxication.

It may become necessary to distinguish nerve agent and/or atropine poisoning from botulinum intoxication. Nerve agent poisoning produces copious respiratory secretions and miotic pupils, whereas there is if anything a decrease in secretions in botulinum intoxication. Atropine overdose is distinguished from botulism by its central nervous system excitation (hallucinations and delirium) even though the mucous membranes are dry and mydriasis is present. The clinical differences between botulinum intoxication and nerve agent poisoning are depicted in Appendix E.

Laboratory testing is generally not helpful in the diagnosis of botulism. Survivors do not usually develop an antibody response due to the very small amount of toxin necessary to produce clinical symptoms. Detection of toxin in serum or gastric contents is possible, and mouse neutralization (bioassay) remains the most sensitive test. Other assays include gel hydralization or ELISA. Serum specimens should be drawn from suspected cases and held for testing at such a facility.

MEDICAL MANAGEMENT

Respiratory failure secondary to paralysis of respiratory muscles is the most serious complication and, generally, the cause of death. Reported cases of botulism prior to 1950 had a mortality of 60%. With
tracheostomy or endotracheal intubation and ventilatory assistance, fatalities should be less than five percent. Intensive and prolonged nursing care may be required for recovery which may take several weeks or even months.

Antitoxin: In isolated cases of food-borne botulism, circulating toxin is present, perhaps due to continued absorption through the gut wall. Botulinum antitoxin (equine origin) has been used in those circumstances, and is thought to be helpful. Animal experiments show that after aerosol exposure, botulinum antitoxin can be very effective if given before the onset of clinical signs. Administration of antitoxin is reasonable if disease has not progressed to a stable state.

A trivalent equine antitoxin has been available from the Centers for Disease Control and Prevention for cases of foodborne botulism. This product has all the disadvantages of a horse serum product, including the risks of anaphylaxis and serum sickness. A "despeciated" equine heptavalent antitoxin against types A, B, C, D, E, F, and G has been prepared by cleaving the Fc fragments from horse IgG molecules, leaving F(ab) 2 fragments. This product is under advanced development, and is currently available under IND status. Its efficacy is inferred from its performance in animal studies. Disadvantages include a reduced, but
theoretical risk of serum sickness.

Use of the antitoxin requires skin testing for horse serum sensitivity prior to administration. Skin testing is performed by injecting 0.1 ml of a 1:10 dilution (in sterile physiological saline) of antitoxin intradermally in the patient's forearm with a 26 or 27 gauge needle. Monitor the injection site and observe the patient for allergic reaction for 20 minutes. The skin test is positive if any of these allergic reactions occur: hyperemic areola at the site of the injection > 0.5 cm; fever or chills; hypotension with decrease of blood pressure > 20 mm Hg for systolic and diastolic pressures; skin rash; respiratory difficulty; nausea or vomiting; generalized itching. Do NOT administer Botulinum F(ab&rsquo;)2 Antitoxin, Heptavalent (equine derived) if the skin test is positive. If no allergic symptoms are observed, the antitoxin is administered intravenously in a normal saline solution, 10 mls over 20 minutes.

With a positive skin test, desensitization is carried out by administering 0.01 - 0.1 ml of antitoxin subcutaneously, doubling the previous dose every 20 minutes until 1.0 - 2.0 ml can be sustained without any marked reaction.

PROPHYLAXIS

Vaccine: A pentavalent toxoid of Clostridium botulinum toxin types A, B, C, D, and E is available under an IND status. This product has been administered to several thousand volunteers and occupationally at-risk
workers, and induces serum antitoxin levels that correspond to protective levels in experimental animal systems. The currently recommended primary series of 0, 2, and 12 weeks, then a 1 year booster induces protective antibody levels in greater than 90 percent of vaccinees after one year. Adequate antibody levels are transiently induced after three injections, but decline prior to the one year booster.

Contraindications to the vaccine include sensitivities to alum, formaldehyde, and thimerosal, or hypersensitivity to a previous dose. Reactogenicity is mild, with two to four percent of vaccinees reporting erythema, edema, or induration at the local site of injection which peaks at 24 to 48 hours, then dissipates. The frequency of such local reactions increases with each subsequent inoculation; after the second and third doses, seven to ten percent will have local reactions, with higher incidence (up to twenty percent or so) after boosters. Severe local reactions are rare, consisting of more extensive edema or induration. Systemic reactions are reported in up to three percent, consisting of fever, malaise, headache, and myalgia. Incapacitating reactions (local or systemic) are uncommon. The vaccine should be stored at refrigerator temperatures (not frozen).

Three or more vaccine doses at 0, 2, and 12 weeks, then at 1 year if possible, all by deep subcutaneous injection are recommended for selected individuals or groups judged at high risk for exposure to botulinum toxin aerosols. There is no indication at present for use of botulinum antitoxin as a prophylactic modality except under extremely specialized circumstances.

BRUCELLOSIS

Summary

Signs and Symptoms: Incubation period from 5-60 days; average of 1-2 months. Highly variable. Acute and subacute brucellosis are non-specific. Irregular fever, headache, profound weakness and fatigue, chills,
sweating, arthralgias, mylagias. Depression and mental status changes. Osteoarticular findings (i.e., sacroiliitis, vertebral osteomyleitis). Fatalities are uncommon.

Diagnosis: Blood cultures require a prolonged period of incubation in the acute phase. Bone marrow cultures produce a higher yield. Confirmation requires phage-typing, oxidative metabolism, or genotyping procedures. ELISA's followed by Western blotting are used.

Treatment: Doxycycline and rifampin for a minimum of six weeks. Ofloxacin + rifampin is also effective. Therapy with rifampin, a tetracycline, and an aminoglycoside is indicated for infections with complications
such as endocarditis or meningoencephalitis.

Prophylaxis: No approved human vaccine is available. Avoid consumption of unpasteurized milk and cheese.

Isolation and Decontamination: Standard precautions for healthcare workers. Person-to-person transmission via tissue transplantation and sexual contact have been reported but are insignificant. Environmental decontamination can be accomplished with a 0.5% hypochlorite solution.

Overview

The Brucellae are a group of gram-negative cocco-baccillary organisms, of which four species are pathogenic in humans. Abattoir and laboratory worker infections suggest that Brucella spp. are highly infectious via the aerosol route. It is estimated that inhalation of only 10 to 100 bacteria is sufficient to cause disease in man. The relatively long and variable incubation period (5-60 days) and the fact that many infections are asymptomatic under natural conditions has made it a less desirable agent for weaponization, although large aerosol doses may shorten the incubation period and increase the clinical attack rate. Brucellosis infection has a low mortality rate (5% of untreated cases) with most deaths caused by endocarditis or meningitis. It is an incapacitating and disabling disease in its natural form.

History and Significance

Marston described disease caused by B. melitensis among British soldiers on Malta during the Crimean War as "Mediterranean gastric remittent fever". Work by the Mediterranean Fever Commission identified
goats as the source of human brucella infection on Malta, and restriction of the ingestion of unpasteurized goats milk and cheese soon decreased the number of cases of brucellosis among military personnel.

In 1997, most cases were associated with ingestion of unpasteurized dairy products and abattoir and veterinary work. In the United States most cases are reported from Florida, California, Virginia, and Texas. It is a rare disease in the United States with an incidence of 0.5 per 100,000 population.

In 1954, Brucella suis became the first agent weaponized by the U.S. in the days of its offensive BW program at the newly constructed Pine Bluff Arsenal. Despite this, B. melitensis actually produces more severe human disease.

Clinical Features

Brucellosis may present as a nonspecific febrile illness which resembles influenza. Fever, headache, myalgia, arthralgia, back pain, sweats, chills, and generalized weakness and malaise are common complaints. Cough and pleuritic chest pain may occur in up to twenty percent of cases, but these are usually not associated with acute pneumonitis. Pulmonary symptoms may not correlate with radiographic findings. The chest x-ray may be normal, or show lung abscesses, single or miliary nodules, bronchopneumonia, enlarged hilar lymph nodes, and pleural effusions. Gastrointestinal symptoms occur in up to 70 percent of adult cases, and less frequently in children. These include anorexia, nausea, vomiting, diarrhea and constipation. Ileitis, colitis and granulomatous or a mononuclear infiltrative hepatitis may occur. Lumbar pain and tenderness can occur in up to 60% of cases and is due to various osteoarticular infections of the axial skeletal system. Paravertebral abscesses may occur and can be imaged by CT scan or MRI. CT scans often show vertebral sclerosis. Vertebral and disc space destruction may occur in chronic cases. One or, less frequently, both sacroiliac joints may be infected causing low back and buttock pain that is intensified by stressing the sacroiliac joints on physical exam. Hepatomegaly and splenomegaly can occur in up to 45-63 percent of cases. Peripheral joint involvement may vary from pain on range of motion testing to joint immobility and effusion. Peripheral joint effusions usually show a mononuclear cell predominance and organisms can be isolated in up to 50% of cases. The hip joints are the most commonly involved peripheral joints, but ankle, knee, and sternoclavicular joint infection may occur. Plain radiographs of involved sacroiliac joints usually show blurring of articular margins and widening of the joint space. Technetium or Gallium-67 bone scans are 90% sensitive for detecting sacroileitis and will also detect other sites of bone and joint involvement; they are also useful for differentiating sacroiliac from hip joint involvement.

Meningitis occurs in less than 5% of cases and may be an acute presenting illness of a chronic syndrome occurring late in the course of a persistent infection. The cerebrospinal fluid contains an increased number of lymphocytes and a low to normal glucose. Culture of the CSF has sensitivity of 50%, and specific brucella antibodies can be detected in the fluid in a higher percentage of cases. Encephalitis, peripheral neuropathy, radiculoneuropathy and meningovascular syndromes have also been observed in rare cases. Behavioral disturbances in children and psychoses may occur in the meningoencephalitic form of the disease. Epididymo-orchitis may occur in men as the most frequent genitourinary form of brucellosis. Rashes occur in less than 5% if cases and include macules, papules, ulcers, purpura, petechiae, and erythema nodosum.

Diagnosis

The leukocyte count is usually normal but may be low. Anemia and thrombocytopenia may occur. Blood and bone marrow culture during the acute febrile phase of the illness will yield a positivity rate of 15-70% and 92% respectively. A biphasic culture method for blood (Castaneda bottle) may increase the number of isolates. The serum agglutination test (SAT) will detect both IgM and IgG antibodies. A titer of 1:160 or greater is indicative of active disease. The IgM titer can be measured by adding a reduced agent such as 2-mercaptoethanol to the serum. This will destroy the agglutinability of IgM allowing the IgM titer to be measured by subtracting the now lower titer from the total serum agglutinin titer. A dot-ELISA using an autoclaved extract of B. abortus has been found to be a sensitive and specific screening test for detection of Brucella antibodies under field conditions. ELISA tests for antibody detection require standardization using a specific antigen before they will be widely available. Antigen detection on DNA extracted from blood
mononuclear cells has been accomplished using PCR analysis of a target sequence on the 31-kilodalton B. abortus protein BCSP 31. This test has been proven to be rapid and specific and may replace blood culture in the future, since the latter may require incubation for up to 6 weeks. PCR for Brucella species is not available at this time except in research laboratories, but shows promise for future use.

Medical Management

Isolation is not required other than contact isolation for draining lesions. Person to person transmission is possible via contact with such lesions. Biosafety level 3 practices should be used for suspected brucella cultures in the laboratory because of the danger of inhalation infection. Antibiotic therapy is recommended as the sole therapy unless there are surgical indications for the treatment of localized diseases (e.g., valve replacement for endocarditis).

The treatment recommended by the World Health Organization for acute brucellosis in adults is doxycycline 200 mg/day p.o. plus rifampin 600-900 mg/day for a minimum of six weeks. The previously established regimen of intramuscular streptomycin along with an oral tetracycline may give fewer relapses but is no longer the primary recommendation. Ofloxacin 400 mg/day and rifampin 600 mg/day p.o. is also an effective combination. Combination therapy with rifampin, a tetracycline, and an aminoglycoside is indicated for infections with complications such as meningoencephalitis or endocarditis. Doxycycline clearance is increased in the presence of rifampin and plasma levels are lower than when streptomycin is used instead of rifampin.

Prophylaxis

Live animal vaccines are used widely. Consumption of unpasteurized milk and cheese should be avoided. No approved human brucella vaccine is available. An experimental human brucellosis vaccine has been tested on 271 subjects with a 25% rate of unpleasant acute side effects, but no long term adverse side effects.