Clinical manifestations

Presentation and course

Involvement of the nervous system is uncommon with most microorganisms that might reasonably be employed in biological warfare and bioterrorism.

Bacillus anthracis. Severe hemorrhagic meningitis regularly occurs with anthrax infection, often accompanied by severe systemic disease, most likely transmitted by spore inhalation.

All forms of anthrax infection (ie, cutaneous, gastrointestinal, inhalational, and injection) can be complicated by meningitis. With natural anthrax, anthrax bacilli most commonly enter the body via the skin and disseminate to the CNS via the hematogenous or lymphatic routes, leading to fatal bacterial meningitis. The interval from exposure to presentation of cardinal signs (ie, fever, dyspnea, and headache) varies from 2 to 60 days.

Botulinum toxin. At the end of the 18th century, outbreaks of "sausage poisoning" in southern Germany, especially in Württemberg, prompted early research on botulism. The German poet and district medical officer Justinus Kerner (1786-1862) published the first accurate descriptions of the symptoms of food-borne botulism between 1817 and 1822 (63). Kerner labeled the suspected poison "sausage poison" or "fatty poison."

Clostridium botulinum was first recognized and isolated in 1895 by Belgian bacteriologist Émile Pierre-Marie van Ermengem (1851-1932), a professor of bacteriology at the University of Ghent. van Ermengem investigated a botulism outbreak in the small Belgian village of Ellezelles that occurred after a funeral dinner with home-cured ham in which 34 people were poisoned (156). The isolate was originally named Bacillus botulinus, after the Latin word for sausage, botulus. However, isolates from subsequent outbreaks were always found to be anaerobic spore formers, so American bacteriologist Ida Albertina Bengtson (1881-1952) proposed that the organism be placed in the genus Clostridium as the genus Bacillus was restricted to aerobic spore-forming rods (11). Bengtson had earned her master's degree in 1913 and her PhD in 1919, both from the University of Chicago. In 1916, she became the first woman hired to work in the Hygienic Laboratory of the U.S. Public Health Service (later part of the National Institutes of Health).

Natural outbreaks of foodborne botulism are typically from ingestion of improperly prepared home-canned foods (41; 43; 140; 157; 93; 70), although botulism from drinking illicit alcohol has been reported (102). Botulism may also occur with the injection of illicit drugs, such as black tar heroin or methamphetamine (116; 159), and contamination of wounds. In foodborne outbreaks in North America in which the toxin type was determined, most were caused by type A, occasionally by type B or type E, and rarely by type F (140); however, in outbreaks among Alaska Native communities, particularly the Inuit of Nunavik in northern Quebec and the First Nations population of the Pacific coast of British Columbia, those affected are predominantly exposed to type E botulinum toxin through the consumption of traditionally prepared marine mammal and fish products (43; 93; 70). Other countries have somewhat different distributions of toxin types; in France, for example, type B has been the predominant toxin type, although a wider diversity of toxin types has been noted since 2000 (126).

Bioterrorist attacks with botulinum toxin could be either through oral ingestion or inhalation (59). Previous natural cases of inhalation botulism are rare (74; 130; 126; 131). The characteristic descending paralysis in humans typically starts in the extraocular and bulbar muscles, with associated autonomic features. Bilateral but asymmetric clinical signs, although rare in botulism, do not rule out that diagnosis; in particular, asymmetry may occur with demyelination of cranial nerves (64). Repetitive nerve stimulation usually shows an incremental muscle response. The differential diagnosis is from naturally occurring paralyzing illnesses (eg, Guillain-Barré syndrome, myasthenic crisis, diphtheria, tetrodotoxin, saxitoxin, snake envenomation) and from chemical warfare poisoning by organophosphates. Treatment is supportive.

Bioterrorists are more likely to contaminate victims’ inhaled air with botulinum toxin rather than the food supply. No immediate effects are noted after inhalation of botulinum toxin as the latent period varies from 1 to 5 days. Depending on the dissemination method, such attacks would likely affect both animals and humans.

The clinical features in humans are predominantly a combination of multiple cranial nerve palsies and skeletal muscular paralysis. The early manifestations of botulin intoxication are usually ophthalmological, with symptoms such as blurred vision, diplopia, and ptosis. Signs include nystagmus, mydriasis, and extraocular muscle dysfunction. Other symptoms, such as dysphagia and dysarthria, are associated with descending flaccid paralysis that typically develops 12 to 72 hours after exposure. The autonomic effects of botulism are manifested by anticholinergic signs and symptoms (eg, dry mouth, ileus, constipation, and urinary retention). Sensory symptoms are absent. Because botulinum toxins do not cross the blood-brain barrier and do not affect the brain, affected individuals are usually alert and oriented. With severe respiratory muscle paralysis, the patient may become cyanotic or exhibit narcosis from CO2 retention. Respiratory paralysis may lead to death.

Prognosis and complications

Anthrax. Untreated, the case fatality from cutaneous anthrax is about 20% to 30%, compared to 25% to 60% with gastrointestinal anthrax, and nearly 100% with inhalational anthrax. With treatment, mortality is less than 1% with cutaneous anthrax (47; 57; 166; 71), whereas mortality remains high, even with aggressive treatment, with gastrointestinal or inhalational anthrax (73; 71). Inhalational anthrax was generally thought to be fatal in 80% to over 90% of cases (119; 18; 01; 166), but the mortality in the 2001 U.S. outbreak has been much better than anticipated (5 of 11 cases or 45%) with the use of aggressive treatment and intensive care unit support, including mechanical ventilation and dialysis as necessary (27; 166). From 2001 to 2014, 8 of 15 (53%) patients with inhalation anthrax survived with early diagnosis, combination antimicrobial drug treatment to eradicate the bacteria and inhibit toxin production, and aggressive pleural effusion management (71). Initiation of antibiotic or anthrax antiserum therapy during the prodromal phase of inhalational anthrax is associated with markedly improved survival compared with initiation of treatment of fulminant cases (73). Nevertheless, anthrax survivors report significant health problems and poor life adjustment 1 year after the onset of bioterrorism-related anthrax infection (129).

Anthrax meningoencephalitis is usually rapidly fatal, with roughly two-thirds of affected patients dying within 24 hours of presentation (92; 112), although there are a small number of reported survivals following anthrax meningoencephalitis (51; 135; 160; 149; 151; 85; 147; 92; 13). In a systematic review, survival of anthrax meningitis was predicted by treatment with a bactericidal agent and the use of multiple antimicrobials (82). There is limited information on long-term outcomes among the few survivors, but several cases were reported to have fully recovered (149; 147; 92; 13).

Botulism. The advent of modern supportive care has improved botulism prognosis so that the case fatality rate for food-borne botulism has decreased from the 50% to 70% range to the 5% to 20% range. Although botulinum neurotoxins bind irreversibly to presynaptic endplates and impair them irreparably, axons can regenerate new endplates, and full recovery can occur, although it may take several months. The case fatality and outcomes from biological warfare or terrorism could easily be much worse than the results obtained with individual sporadic cases or cases from small natural outbreaks because the medical capacity could be overwhelmed and treatment supplies (eg, Botulism Antitoxin Heptavalent [A, B, C, D, E, F, G]) could be exhausted by sudden widescale demand.

Biological basis

Etiology and pathogenesis

Various agents used for biological and chemical warfare are listed in Table 1. Many of them can cause neurologic manifestations. Encephalomyelitic viruses, toxins, and chemicals are more likely to be associated with neurotoxicity. Some of the bacterial infections may be associated with neurologic complications.

Characteristics that make a pathogen a high risk for bioterrorism include a low infective dose, ability to be aerosolized, high contagiousness, and hardiness (ie, survival in various environmental conditions) (04).

Table 1. Potential Biological Warfare or Bioterrorism Agents with Frequent Involvement of the Nervous System

CDC categorization of bioterrorism agents and diseases. The Centers for Disease Control and Prevention (CDC) has a 3-tier categorization system for bioterrorism agents and diseases (CDC: Bioterrorism Agents and Diseases):

Category A. Category A is comprised of high-priority agents that pose a risk to national security because (1) they can be easily disseminated or transmitted from person to person, (2) they have the potential for major public health impact (eg, high case-fatality rates), (3) they will likely cause public panic and social disruption, and (4) they require special measures for public health preparedness. The two Category A diseases with the greatest potential to affect the nervous system as a major feature of the illness are anthrax (Bacillus anthracis) and botulism (Clostridium botulinum toxin). Other Category A diseases or agents include plague (Yersinia pestis), smallpox (variola major), tularemia (Francisella tularensis), and viral hemorrhagic fevers (eg, Ebola, Marburg) (08; 09; 136).

Category B. Category B is comprised of moderate-priority agents that (1) are moderately easy to disseminate, (2) result in moderate morbidity rates and low mortality rates, and (3) require specific enhancements for diagnostic capacity and warrant enhanced disease surveillance. Of these, only viral encephalitis viruses (alphaviruses, such as eastern equine encephalitis, Venezuelan equine encephalitis, and western equine encephalitis) are likely to primarily affect the nervous system (137).

Category C. Category C is comprised of lower-priority agents, including emerging pathogens that could potentially be engineered for mass dissemination because of availability, ease of production and dissemination, or potential for major health impact (eg, potential for high morbidity or mortality).

Bacillus anthracis (Category A). Insight regarding the mechanism for the spread of B anthracis by inhalation is provided by pathologic studies of victims from the Sverdlovsk epidemic of 1979. The contagion mechanism was traced to the release of aerosols containing this organism at a secret biological-agent production facility in Russia (68).

A hematogenous spread involved the central nervous system, resulting in vasculitis, hemorrhages, and edema due to the toxins. The risk of hemorrhagic meningitis after exposure to anthrax inhalation is estimated to be as high as 50% (76; 77).

Equine encephalitis (Category B). Venezuelan equine encephalitis virus could be developed for transmission by inhalation with secondary zoonotic transmission cycles sustained by horses and mosquitoes (59), as could eastern and western equine encephalitis viruses.

The Eastern equine encephalitis virus is spread to people by the bite of an infected mosquito (19).

In the United States, most cases occur in eastern or Gulf Coast states.

Although rare, approximately 30% of people with Eastern equine encephalitis die, and many survivors have residual neurologic problems. Eastern equine encephalitis, commonly called Triple E or sleeping sickness (not to be confused with African trypanosomiasis), is caused by a zoonotic mosquito-vectored Togavirus that is present in North, Central, and South America and the Caribbean.

In the United States, Eastern equine encephalitis is often associated with coastal plains, most commonly in East Coast and Gulf Coast states. No human vaccines or licensed therapeutic drugs are available for Eastern equine encephalitis in the U.S. Eastern equine encephalitis virus is a potential bioweapon, particularly because it is transmissible by aerosol.

The Western equine encephalomyelitis virus is an alphavirus of the family Togaviridae.

The Western equine encephalomyelitis virus is an arbovirus (arthropod-borne virus) transmitted by mosquitoes of the genera Culex and Culiseta.

The virus is transmitted to people and horses by bites from infected mosquitoes (Culex tarsalis and Aedes taeniorhynchus), particularly during wet summer months; birds are the natural reservoir of the virus.

In North America, Western equine encephalomyelitis occurs primarily in U.S. states and Canadian provinces west of the Mississippi River. The disease also occurs in South America. Western equine encephalomyelitis is commonly a subclinical infection in adult humans, but the disease can cause serious sequelae in infants, children, and the elderly. The overall mortality of Western equine encephalomyelitis is low (approximately 4%) and is associated mostly with infection in the elderly. Approximately 15% to 20% of horses that acquire the virus die or need to be euthanized.

No human vaccines or licensed therapeutic drugs are available in the U.S. for Western equine encephalomyelitis. Western equine encephalitis virus was one of more than a dozen agents the United States researched as potential biological weapons before the nation suspended its biological weapons program.

Venezuelan equine encephalitis is a mosquito-borne disease endemic in regions of Central and South America that causes sporadic outbreaks of equine and human encephalitis. The virus induces neural necrosis and edema in affected mammals.

Toxins. The term “toxin” refers to a toxic substance of biological origin, although simple toxins can be synthesized in the laboratory or produced by genetic modification in other species.

Bacterial toxins are of two general types: proteinaceous exotoxins (eg, tetanus, diphtheria, or botulinum toxins) are part of a defensive system of bacteria (to avoid capture, ingestion, etc.) and so are typically secreted into the surrounding medium, in contrast to endotoxins (eg, lipopolysaccharides in the outer membrane of gram-negative bacteria) that are only released when the bacterium disintegrates.

Mycotoxins are diverse and could be used by terrorist organizations to poison food and water sources, although dispersal in indoor air appears to be the most effective method for a bioterrorist attack (99). Crude concentrated or dried extracts of readily grown fungal cultures can simultaneously produce several toxins with synergistic actions, but mycotoxin bioterror weapons are more likely to involve the liver (eg, aflatoxins, fumonisin B1), kidneys (ochratoxin A, fumonisin B1), lungs (fumonisin B1), gastrointestinal tract (trichothecenes type A, eg, T-2 toxin; type B, eg, deoxynivalenol [DON]), and bone marrow (trichothecenes type A, eg, T-2 toxin) than the nervous system (98; 72; 142; 114; 99; 80).

As chemical products of living organisms, toxins, when employed by people for a nefarious purpose, occupy an ill-defined “no man’s land” between chemical warfare and biological warfare agents. From a pharmacological and toxicological point of view, toxins could be considered chemical weapons, but most experts and the U.S. Army classify toxins as biological weapons.

Toxins most relevant to bioterrorism include ricin, botulinum, Clostridium perfringens epsilon toxin, conotoxins, Shiga toxins, saxitoxins, tetrodotoxins, mycotoxins, nicotine (04), and brevetoxin. Of these, botulinum, brevetoxin, conotoxins, nicotine, saxitoxins, and tetrodotoxin are most likely to involve the nervous system.

(1) Botulinum toxin (Category A). Botulinum toxin is derived from anaerobic Clostridium botulinum bacteria. Clostridium botulinum bacteria are Gram-positive. On microscopic examination after appropriate histologic staining, Clostridium botulinum endospores may have the morphology of “safety pins,” ”bobby pins,” or “tennis rackets.”

It is feasible to deliver botulinum toxin as an aerosolized biological weapon. Botulinum toxins are a series of seven related protein neurotoxins, serotypes A through G.

Each serotype is a protein of approximately 150 kD produced by a separate strain of the gram-positive anaerobic bacterium Clostridium botulinum. Botulinum toxins block acetylcholine release from the presynaptic nerve terminal in the peripheral nervous system, leading to muscle paralysis. Botulinum toxin A is the most toxic serotype for humans. It can be readily aerosolized and persists in this state for weeks in still water and food but degrades on exposure to heat, acidity, or air for more than 12 hours.

Botulinum toxin binds to high-affinity recognition sites on the cholinergic nerve terminals (there may be a separate one for each serotype). The toxin enters the nerve terminal by receptor-mediated endocytosis but then is extruded into the cytoplasm. In the cytoplasm, the toxin cleaves SNARE proteins (ie, proteins that mediate vesicle fusion with the presynaptic membrane), preventing the cell from releasing vesicles of the neurotransmitter, interrupting neurotransmission, and causing paralysis (61; 44).

Botulinum toxin consists of two polypeptide subunits: A and B chains. The B subunit binds to receptors on the axons of motor neurons. The toxin is taken into the axon by receptor-mediated endocytosis, and then the A chain acts to prevent fusion of the synaptic vesicle with the cell membrane, thus preventing the release of acetylcholine. This mechanism of blocking neuromuscular transmission is called presynaptic inhibition. The presynaptic inhibition interrupts both cholinergic autonomic (muscarinic) and motor (nicotinic) neurotransmission, producing the cranial neuropathies and skeletal muscle paralysis seen in clinical botulism. Recovery only occurs after the neuron generates a new axon terminal, which can take months.

The estimated lethal dose of type A toxin is 1.3 to 2.1 ng/kg intravenously or intramuscularly, 10 to 13 ng/kg when inhaled, or 1000 ng/kg orally.

(2) Brevetoxins (Category C). Brevetoxins are a group of tasteless and odorless neurotoxic cyclic polyether compounds produced naturally by species of dinoflagellates (Karenia sp.). Brevetoxins bind to voltage-gated sodium channels in nerve cells, leading to disruption of normal neurologic processes and causing neurotoxic shellfish poisoning. The typical natural route of human exposure is by ingestion of contaminated shellfish. Ingestion of these toxins produces a combination of gastrointestinal and neurologic signs and symptoms; gastrointestinal symptoms include abdominal pain, vomiting, and diarrhea, and the most disabling neurologic signs and symptoms include vertigo and ataxia. Inhalational exposure may cause respiratory symptoms, such as cough, dyspnea, and bronchospasm. Toxicity can also result from dermal exposure.

(3) Conotoxins (Category C). Conotoxins, the venom of various species of cone snails, are pharmacologically and chemically diverse.

Conotoxins affect neurotransmitter receptors (eg, nicotinic, adrenergic, NMDA, and serotonergic) and ion channels (eg, sodium, potassium, and calcium).

The most lethal effect of conotoxins on humans is diaphragmatic paralysis and respiratory arrest. Most conotoxins are not a bioterrorism threat, but some could be weaponized and used as an aerosol (eg, a-conotoxins, k-conotoxins, and o-conotoxins).

(4) Saxitoxins (Category C). More than 50 structurally related neurotoxins (known collectively as "saxitoxins") are produced by protists, algae, and cyanobacteria. Saxitoxin, the best-known paralytic shellfish toxin, is one of the most potent known natural toxins. Saxitoxin acts as a selective, reversible, voltage-gated sodium channel blocker. Saxitoxin binds directly in the pore of the channel protein, occluding the opening, and preventing the flow of sodium ions through the membrane.

(5) Tetrodotoxin (Category C). Tetrodotoxin is an extremely potent toxin naturally found mainly in the liver and sex organs (gonads) of some fish (eg, puffer fish, globefish, and toadfish) and in some amphibian, octopus, and shellfish species.

Tetrodotoxin binds to the voltage-gated sodium channels in nerve cell membranes and blocks the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron (94). This mechanism of action was established in 1964 by Toshio Narahashi (1927-2013), John W Moore (1920-2019), and William R Scott at Duke University (108; 107).

Brevetoxins, saxitoxins, tetrodotoxin, and some conotoxins all act to block voltage-gated sodium channels.

Epidemiology

The most likely first indicator of a biological warfare event would be a marked increase in the number of patients presenting with clinical features caused by the disease agent, beyond what would be expected for natural outbreaks. Patients presenting en masse with a similar pulmonary syndrome should alert the clinician to the possibility of aerosolized toxin exposure (167).

If an attack with a toxin were to occur, epidemiological clues would include the clustering of cases (ie, more than expected with the natural occurrence of the disease), more severe disease than typical, and unusual routes of exposure (eg, inhalation rather than ingestion for some toxins).

Anthrax bioweapon or bioterrorism. Anthrax is rarely found in most developed countries because of vaccines for animals and at-risk people. There has been a dramatic reduction in livestock and human anthrax cases in the United States since 1900 because of livestock and at-risk human immunization, proper disposal of infected animals, and restriction of imported wool, hides, and other products (17; 18).

Because of the complexities of the development and dispersal of an anthrax bioweapon, it had been anticipated that small-scale sabotage or attempts at personal murder were more likely than large-scale attempts at inflicting mass casualties with anthrax and that crude dispersal in a close area was the most likely mode of attack. In the 2001 United States outbreak of anthrax acquired through intentional contamination of mail, most cases involved media, government, and postal service employees; several civilian cases remain unexplained (28).

Epidemiologic clues to bioweapon use include (1) an outbreak or multiple, simultaneous outbreaks with large numbers of patients complaining of respiratory symptoms; (2) high case fatality numbers; (3) sick or dead animals of different species; and (4) multidrug-resistant pathogens (47). A World Health Organization report estimated that the release of 50 kg of anthrax spores upwind of a city of 500,000 population would result in 125,000 infections and nearly 95,000 deaths (165). A report by the United States Congressional Office of Technology Assessment estimated between 130,000 and 3 million deaths following the aerosolized release of 100 kg of anthrax spores upwind of Washington, D.C. (110).

Botulism. An average of 110 cases of botulism are reported annually in the United States, about 25% of which are foodborne botulism, whereas 75% are infant botulism (25). Five western states account for more than half of reported foodborne outbreaks since 1950: California, Washington, Colorado, Oregon, and Alaska.

Foodborne botulism is a significant public health problem among Alaska Natives and is associated with improper preparation and storage of traditional foods. At least two thirds of foodborne outbreaks are due to home-processed foods, most of the remainder are from unknown sources, and only 7% are due to commercially processed foods (25).

Although infant botulism has been the most common form of botulism in the U.S. since 1980, infant botulism is a rare sporadic disease that occurs in isolated cases, has no epidemic potential, and generally does not pose a major public health threat.

Infant botulism is epidemiologically distinct from foodborne botulism because it results from colonization (infection) of the intestine by spores of C. botulinum with subsequent in vivo toxin production rather than from ingestion of toxin preformed in contaminated foods. Although infant botulism was first described in 1976 (105; 117), earlier cases have been identified retrospectively (25).

Prevention

Much work needs to be done internationally to develop capabilities to manage victims of biological or chemical terrorism (84). Most physicians and hospital emergency departments in the United States are ill-prepared to treat victims of such terrorism (83; 162; 143).

The United States military and the Department of Homeland Security regularly conduct bioterrorism response training, sometimes in conjunction with civilian HAZMAT firefighters and U.S. National Guard Weapons of Mass Destruction-Civil Support Teams (WMD-CST).

Some of the soldiers are Chemical, Biological, Radiological and Nuclear (CBRN) Marines. Scenarios included an aircraft leaking chemical agents, locating a chemical agent spill, and rescuing multiple casualties in the field or in a metro line (no doubt motivated by the deadly Tokyo subway sarin attack in 1995 conducted by the Aum Shinrikyo cult). Some of the training exercises are conducted in Northville, a fictional town at the Chemical, Ordnance, Biological, and Radiological (COBRA) Facility in Anniston, Alabama.