Biological Warfare: Infectious Disease and Bioterrorism

Biotechnology. 2016 : 687719.

Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA

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The term biological warfare typically conjures images of medieval warriors tossing dead cattle over city walls or clandestine government agents secretly releasing mysterious microbes into enemy territory. Of course, biological warfare does encompass such activity, but the vast majority of what constitutes biological warfare is far more mundane. Ever since life evolved on earth about 3.8 billion years ago, organisms have constantly devised new ways to kill each other. Any organism that makes use of toxinsfrom bacteria to snakesis engaging in a form of biological warfare. Humans who engage in biological warfare do so by taking advantage of these toxin-producing organisms.

Keywords: bacteriocins, biosensors, Black Death, botulinum toxin, bubonic plague, checkerboard hybridization, dominant-negative mutations, ergot, high-containment laboratories, incubation time, kappa particles, lysins, phage therapy, poison sequence, quorum sensing, ricin, siderophores, smallpox, toxins, weaponization

The term biological warfare typically conjures images of medieval warriors tossing dead cattle over city walls or clandestine government agents secretly releasing mysterious microbes into enemy territory. Of course, biological warfare does encompass such activity, but the vast majority of what constitutes biological warfare is far more mundane. Ever since life evolved on Earth about 3.8 billion years ago, organisms have constantly devised new ways to kill each other. Any organism that makes use of toxinsfrom bacteria to snakesis engaging in a form of biological warfare. Humans who engage in biological warfare do so by taking advantage of these toxin-producing organisms.

An entire textbook could be filled with examples of organisms that employ toxins to kill other organisms. We therefore touch only briefly on the natural history of biological warfare.

Bacteria are particularly adept at biological warfare. While humanity finds antibiotics incredibly useful in our battle against infectious disease, bacteria did not create them for our benefit. Instead, they make antibiotics to kill off other bacteria that are competing for the same habitat or resources. Similarly, bacteria synthesize toxic proteins known as bacteriocins to kill their relatives because closely related strains of bacteria are likelier to compete with each other. For example, many strains of Escherichia coli deploy a wide variety of bacteriocins (referred to as colicins) intended to kill other strains of E. coli. The genes for colicins are normally carried on plasmids, and many of these plasmids are commonly used in molecular biology and genetic engineering (see Chapter 3). Yersinia pestis, the plague bacterium, also makes bacteriocins (called pesticins in this case) designed to kill competing strains of its own species ().

Bacteriocins Inhibit Other Bacteria

A bacteriocin-producing strain of Lactococcus in a piece of cheese can inhibit the growth of a related microorganism.

From Garde S, etal. (2011). Outgrowth inhibition of Clostridium beijerinkii spores by a bacteriocin-producing lactic culture in ovine milk cheese. Int. J Food Microbiol150, 5965.

A point of clarification: The distinction between bacteriocin and toxin has to do with the target. Bacteria deploy bacteriocins against their fellowoften closely relatedbacteria with the deliberate intention of killing them. In contrast, proteins produced by bacteria that act against higher organisms are referred to as toxins. Perhaps counterintuitively, pathogenic bacteria do not usually intend to kill the organisms they infect. Rather, they want to manipulate them long enough to survive and reproduce. The longer the host stays alive, the longer it provides a home for the infecting bacteria. Just like antibiotics, some bacterial toxins are useful to humans. The bacterium Bacillus thuringiensis produces an insect-killing toxin that is harmless to vertebrates, and this Bt toxin has been used extensively in genetically modified crops. (See Chapter 15.)

Lower eukaryotes also regularly engage in biological warfare. Paramecium, a ciliated protozoan, carries symbiotic bacteria (Caedibacter) known as kappa particles that grow and divide inside the larger eukaryotic cell ().

Killer Paramecium Uses a Bacterial Toxin

(A) The kappa particles are found in the cytoplasm of the Paramecium. (B) Kappa particles are symbiotic Caedibacter that are found in many strains of Paramecium, yet they have their own DNA and divide like typical bacteria.

Strains of Paramecium with kappa particles are known as killers and, due to unknown genetic factors and resistance mechanisms, are naturally tolerant of them. Killer strains release kappa particles into the environment, and if a sensitive Paramecium (i.e., one lacking the ability to harbor kappa particles) eats and digests just a single kappa particle, a protein toxin is released and kills the Paramecium. Interestingly, the toxin is not encoded by a gene on the bacterial chromosome, but on a plasmid derived from a defective bacteriophage. So a toxin encoded by a virus infecting the kappa particle bacterium has been commandeered for the purpose of killing other strains of Paramecium.

This phenomenon is not at all unusual. Many toxins used by pathogenic bacteria that infect humans are actually encoded by foreign DNA of nonchromosomal origin, such as viruses, plasmids, or transposons. These elements are often integrated into the chromosome of pathogenic strains of bacteria. For example, the only strains of Corynebacterium diphtheriaethe causative agent of diphtheriathat are dangerous to humans are the ones that carry a toxin-encoding virus.

Higher eukaryotes can either create their own toxinssuch as the venom produced by snakes and scorpionsor expropriate toxins produced by other species. One species of caterpillar that feeds on tobacco plants can exhale noxious nicotine at spiders, chasing them away. Other insects rely on microbes to wage biological warfare. Certain parasitic wasps inject their eggs into the maggots (i.e., larvae) of plant-eating insects. After the eggs hatch, the newborn wasps eat the living maggots from the inside ().

Wasps Use Viruses against Maggots

Certain types of wasps lay their eggs inside tobacco hornworm larvae. The wasp lands on the back of the larva and injects the eggs plus adenovirus into the maggot through the ovipositor. The adenovirus prevents the larva from eating and therefore developing into a pupa. When the eggs hatch, the young use the insides of the larva as a food source, to grow and develop into adult wasps.

The maggots are eventually killed, and a new generation of wasps is released. The secret to the wasps success is the injection of an adenovirus along with the eggs. The virus targets the maggots fat body (vaguely equivalent to the liver of higher animals) and cripples the maggots developmental control system and immune system. The maggot loses its appetite for plants and is prevented from molting and turning into a pupa, the next stage in its life cycle.

Many different kinds of organisms engage in biological warfare. Bacteria kill other bacteria with antibiotics or bacteriocins. They also make toxins that are targeted at higher organisms. Eukaryotes can either make their own toxins or commandeer those produced by lower organisms.

Although we rarely perceive it this way, infectious disease is just another manifestation of biological warfare that is ubiquitous throughout life. The evolutionary relationship between hosts and pathogens is essentially a never-ending arms race. When a pathogen evolves a new toxin, the host evolves a response to it. Humanity has taken this arms race one step further by utilizing technology such as vaccines and industrial-scale manufacturing of antibiotics. However, the microbes are fighting back.

Perhaps the biggest problem plaguing medical microbiology today is the rise of antibiotic resistance. There are many reasons why bacteria have developed this resistance, but all of the explanations have one thing in common: the proliferation and misuse of antibiotics. For instance, medical doctors often prescribe antibiotics to patients who have an infection, even if it is unknown whether the disease is bacterial. Other times, the wrong antibiotic is prescribed. In many developing countries, antibiotics can be bought over the counter without a prescription. Compounding the dilemma, patients who receive antibiotics often do not comply with the recommended dose, ending treatment as soon as they feel better. This has the effect of selecting for the survival of the bacteria that have already developed a slight resistance to the drug. When the patient propagates the infection, he unintentionally passes on these toughened survivors. The widespread use of antibiotics in animal feedwhich farmers use to fatten up livestockis also a major contributor to the problem.

Today, many experts worry about incurable infections. Methicillin-resistant Staphylococcus aureus (MRSA) gets a lot of media attention, but it is not the only worrisome microbe. There have been reports from around the world of totally drug-resistant tuberculosis, which as the name implies, appears to be resistant to all treatment. In a 2013 report, the Centers for Disease Control and Prevention (CDC) issued an urgent warning about infections from (1) Clostridium difficile, which causes diarrhea and is often acquired by patients in health-care settings who were treated with antibiotics for other infections; (2) Carbapenem-resistant Enterobacteriaceae (CRE), such as Klebsiella and E. coli, which also cause health-care-associated infections and may be resistant to all known antibiotics; and (3) Neisseria gonorrhoeae, the etiologic agent of gonorrhea, which is growing in resistance to several antibiotics.

While these developments are alarming, much research is being done to combat the rise of antibiotic resistance. Although microbes have responded to our antibiotic assault, we are developing some new weapons to regain the upper hand.

Although there has been speculation of an inevitable post-antibiotic era, there are still plenty of opportunities for the development of novel antibiotics.

One strategy is to attack previously unexploited vulnerable spots in a bacteriums metabolism or life cycle, preferably those that bacteria cannot easily defend by acquiring resistance. For instance, bacteria use iron chelators, known as siderophores, to bind iron and extract it from host proteins. Siderophores are excreted, bind iron, and are then taken back into bacteria by specialized transport systems. Absence of high-potency siderophores largely abolishes virulence in both plague and tuberculosis. Because mammals do not make siderophores, their unique biosynthetic pathways provide an attractive target for development of novel antibiotics. Yersiniabactin, the siderophore of several pathogenic Yersinia species, is capped by a salicyl group ().

Salicyl-AMS Inhibits the Production of Yersiniabactin

The structure of yersiniabactin shows the salicyl group in red. The precursor, salicyl-AMP, is made by activating salicylate with ATP. The sulfamoyl analog, salicyl-AMS, inhibits the incorporation of the salicyl group into yersiniabactin.

The intermediate in the pathway, produced when ATP activates salicylate, is salicyl-AMP. A chemically synthesized analog of salicyl-AMP, called salicyl-AMS, replaces the phosphate with a sulfamoyl group. The compound is highly active and specifically inhibits siderophore synthesis. This prevents the growth of Yersinia under iron-limiting conditions, such as encountered in the human body.

Another strategy is to screen novel microbes for antibiotics. As discussed earlier, bacteria produce antibiotics for the explicit purpose of killing other bacteria. Since most microbes that exist in nature have neither been cultured nor identified, it is likely that many natural antibiotics have yet to be discovered. In 2013, a new antibiotic, called anthracimycin, was isolated from an Actinomycete that lives in the ocean. The new antibiotic is active against Bacillus anthracis and MRSA, and modifying it with chlorine groups expanded its spectrum of activity.

Yet another strategy is to identify and clone potential antimicrobial biosynthetic pathways. For example, based on its DNA sequence, one research group cloned a biosynthetic gene cluster from an Actinomycete called Saccharomonospora that was predicted to produce an antimicrobial lipopeptide. Expressing the gene cluster resulted in the discovery of a new antibiotic, taromycin A. The major advantage of this technique is that it can be applied to microbes that are difficult to culture in the laboratory.

A different approach is to disrupt existing antibiotic resistance, rather than developing new antibiotics. For example bacteriophage, such as those that live in the human gut, can shuttle antibiotic resistance genes between bacteria. Consequently, developing drugs that kill or disable bacteriophage is an innovative way to combat the spread of antibiotic resistance. Additionally, disrupting bacterial quorum sensing has been suggested. Bacteria use quorum sensing as a communication system in order to coordinate behavior ().

Quorum Sensing

Bacteria can coordinate behavior by detecting the presence of a signal molecule that indicates the density of the population.

From Boyen F, etal. (2009). Quorum sensing in veterinary pathogens: mechanisms, clinical importance and future perspectives. Vet. Microbiol135, 187195.

By releasing particular chemical compounds into the environment, bacteria can detect when a threshold population density, or quorum, has been reached. Many pathogens construct antibiotic-resistant biofilms after the population has reached a particular density. Disrupting their communication system would cripple their ability to coordinate behavior and keep the bacteria more vulnerable to antibiotics.

The history of phage therapythat is, using bacteriophage (also called phage) to treat bacterial infectionsbegins in France in 1921. That year, microbiologist Felix dHrelle used phage to treat patients suffering from dysentery ().

Felix dHrelle

Microbiologist Felix dHrelle helped pioneer phage therapy.

In 1927, he also used phage therapy to treat cholera victims in south Asia. Unfortunately, many other scientists in the United States and elsewhere were unable to replicate his work, and when the widespread production of antibiotics started in 1945, the scientific community mostly lost interest in phage therapy. The French, however, enthusiastically practiced phage therapy into the 1990s and, during those seven decades, there were reports of successful treatment of typhoid fever, colitis, septicemia, skin infections, and various other bacterial diseases. Other countries that embraced phage therapy include Poland, Russia, and Georgia. Today, patients there can receive phage therapy for chronic and antibiotic-resistant bacterial infections.

Since the 1990s, the Western scientific community has renewed its interest in phage therapy. One benefit of using phage, as opposed to antibiotics, is their specificity. Antibiotics kill many different types of bacteriawhich is harmful if they destroy helpful gut bacteriabut individual phage species infect only a group of very closely related bacteria. Every bacterial infection could, in theory, be targeted by a highly specific phage.

As predicted, however, bacteria also can develop resistance to phage, mainly through thwarting viral attachment. Now, researchers are investigating the use of lysins, a class of toxins that phage use to dismantle bacterial cell walls as part of their lytic cycle (). Because lysins target conserved regions within peptidoglycan, it is believed that bacteria will be less able to develop resistance. Lysins work best against Gram-positive bacteria, but genetic engineering can expand the spectrum of activity to include Gram-negative bacteria also.

Bacteriophage Tsamsa Kills Bacillus anthracis

The lysin isolated from the bacteriophage Tsamsa kills Bacillus anthracis and other closely related species.

From Ganz HH, etal. (2014). Novel giant Siphovirus from Bacillus anthracis features unusual genome characteristics. PLoS One9(1), e85972.

As an alternative to phage, it may be possible to deploy predatory bacteria against human pathogens. Bdellovibrio, which invades other bacteria rather like a virus, and Micavibrio, which attaches to bacterial cell surfaces, have been shown to kill antibiotic-resistant pathogenic bacteria invitro.

Because of a persistent fear that we will run out of novel antibiotics, many clever new technologies have been suggested to fight bacterial infections. Some of the most promising of these antibiotics utilize genetic engineering.

For example, many pathogenic Escherichia coli use the FimH adhesin to bind to mammalian cells via mannose residues on surface glycoproteins. Several alkyl- and aryl-mannose derivatives bind with extremely high affinity to the adhesin and block its attachment to the natural receptor. Such mannose derivatives, therefore, could serve as anti-adhesin drugs. However, manufacturing pharmaceuticals is quite expensive. It would be far cheaper to genetically engineer nonpathogenic strains of E. coli to express the mannose derivatives on their cell surfaces. Pathogenic bacteria would then bind to these decoys instead of to mammalian cells. This would also avoid the need for continuous administration of sugar derivatives because the decoy strains of E. coli would multiply naturally in the intestine. Alternatively, nonpathogenic strains of E. coli could be engineered with genes for adhesins that would allow them to compete with pathogens for mammalian cell receptors. (Such engineered strains would also have the advantage of being able to deliver protein pharmaceuticals or large segments of DNA for gene therapy into mammalian cells.)

A different approach is to generate altered toxins that interfere with their natural analogs. Typical A-B bacterial toxins are made from a single active A subunit, which carries out a toxic enzymatic reaction inside a target cell, and often several binding B subunits, which serve as a delivery system by attaching to the cell surface. Because several properly functioning binding subunits are required to deliver the active subunit, one approach to antitoxin therapy relies on utilizing dominant-negative mutations in the binding subunit of the toxin. The mechanism involves the binding of a defective protein subunit to functional subunits resulting in a complex that is inactive overall. (The term dominant-negative refers to mutations in which an abnormal gene product sabotages the activity of the wild-type gene product. Consequently, most dominant-negative mutations affect proteins with multiple subunits.) Dominant-negative mutations have been deliberately isolated in the B protein (called the protective antigen) of anthrax toxin. Mixing mutant subunits with wild-type ones resulted in the assembly of inactive heptamers that bind the A subunits (called lethal factor and edema factor) of anthrax toxin. As a result, the toxic A subunits cannot be transported into target cells (). This technique has been shown to protect both cultured human cells and whole mice or rats from death by lethal levels of anthrax toxin.

Dominant-Negative Mutations

For anthrax, the B subunit (called PA63 protein or protective antigen) binds the A subunits (called lethal factor, LF, and edema factor, EF) and transports them into the target cell cytoplasm via an endocytic vesicle. The dominant-negative inhibitory (DNI) mutant of the PA63 protein (purple) assembles together with normal PA63 monomers (pink) to give an inactive complex that cannot release the LF and EF toxins from the vesicle into the cytoplasm.

Many of the advances in nanotechnology aimed at fighting pathogens involve the creation of bactericidal surfaces (see Chapter 7 for more on nanotechnology). Several metals are inherently antibacterial. For instance, silver ions kill bacteria through several mechanisms, such as generating reactive oxygen species and disrupting protein disulfide bonds. Surfaces coated with silver, selenium, and copper nanoparticles all show antimicrobial activity.

Metals are not the only option. A substance known as black silicon is made of tiny nanopillars that are able to physically destroy bacteria, including endospores, through mechanical stress (). Antimicrobial activity has also been demonstrated with stacked carbon nanotubes called nanocarpets (see Chapter 7). Additionally, polymers of esters and cyclic hydrocarbons reduce attachment of bacteria. Such discoveries could allow for improved sanitation in health-care settings and the manufacture of antimicrobial medical devices.

Antibiotic resistance is a growing concern, but contrary to popular reports, it is not necessarily an intractable problem. Novel targets for antibiotics, phage therapy, genetic engineering, and nanotechnology provide multiple possibilities for fighting antibiotic-resistant pathogens.

Nanostructures Can Kill Bacteria

Scanning electron micrograph of black silicon surface showing its hierarchical structures. (A) Periodically arranged micropillar arrays; (B) a micropillar with nanostructures; (C) nanostructures formed on the top of the micropillar.

From He Y, etal. (2011). Superhydrophobic silicon surfaces with micro-nano hierarchical structures via deep reactive ion etching and galvanic etching. J Colloid Interface Sci364, 219229.

Throughout history, humans have devised new and innovative ways to kill other humans. When technology was primitive, warriors used whatever nature provided. Burning crops was probably the easiest and earliest form of warfare aimed at undermining an enemy, as was poisoning a communitys drinking water with dead or rotting animals.

Slightly more advanced forms of biological warfare emerged when soldiers began dipping spears in feces and throwing poisonous snakes. During the Black Death epidemic of the mid-1300s, the Tartars catapulted plague-ridden corpses over the walls into cities held by their European enemies. Although this is sometimes credited with spreading the plague, rats and their fleas were far more effective at spreading bubonic plague than contact with corpses ().

Bubonic Plague

This painting by Arnold Bcklin, simply titled Plague, depicts the fear that bubonic plague provoked in antiquity.

From ET Rietschal, etal. (2004). How the mighty have fallen: fatal infectious diseases of divine composers. Infect Dis Clin North Am18, 311339.

Given the state of hygiene in most medieval towns or castles, there was little need to provide an outside source of infection. With plague, typhoid, smallpox, dysentery, and diphtheria already around, all that was usually necessary was to let nature take its course. Similarly, a widespread myth exists that European settlers purposefully infected Native Americans with smallpox. While it is true that the British military attempted this strategy during the French and Indian War in the mid-1700s, the vast majority of Native American deathsperhaps as much as 95% of the populationwere due to inadvertent infection with smallpox and other diseases.

The truth is, until very recently, humans were not particularly hygienic. Consider, for instance, that antiseptic surgeryinvented by Joseph Lister and now considered a mainstay of modern medicinewasnt widely adopted until the late 1870s. Before then, armies and civilian populations were so dirty and disease-ridden that practicing germ warfare was like throwing mud on a pig. It is only in our modern hygienic age that biological warfare has become a more meaningful threat.

Modern biological warfare began during World War I. Although the Germans refused to use biological agents against people, they did use them against animals, infecting Allied horses with glanders (Burkholderia mallei) and anthrax. The French also employed glanders against German horses. During World War II, the infamous Japanese Unit 731 experimentally infected Chinese prisoners of war with horrifying diseases, such as cholera, epidemic hemorrhagic fever, and venereal disease. It was also responsible for dropping plague-infected flea bombs on cities in China, although this likely had little effect partly because plague was already endemic to the region ().

Unit 731

Japanese military Unit 731 killed thousands of Chinese people with experimental infections and biological warfare.

Source: Figure 6 from: Lpez-Muoz F, etal. (2007). Psychiatry and political-institutional abuse from the historical perspective: the ethical lessons of the Nuremberg Trial on their 60th anniversary. Prog Neuropsychopharmacol Biol Psychiatry31, 791780.

After World War II, particularly during the Korean War, the United States ratcheted up its biological weapons program. Perhaps the most controversial aspect of the program was the purposeful release of biological agents, such as the relatively harmless Serratia marcescens, over American cities to study weapons dispersal. The military unintentionally infected 11 civilians, one of whom died. By 1969, the U.S. had weaponized anthrax and tularemia. However, in 1975, the U.S. renounced all biological weapons by signing the Biological Weapons Convention (BWC).

The Soviet Union also signed the BWC but then deceitfully enlarged its efforts. The scope of the Soviet program was astonishing. The Soviets manufactured several hundred tons of anthrax, and an accidental release in 1979 killed 66 people. The former USSR also made thousands of pounds of smallpox and plague, and in 1989, they supposedly managed to weaponize Marburg virus, which causes a deadly hemorrhagic fever similar to Ebola. These allegations remain unconfirmed. Finally, under President Boris Yeltsin in 1992, Russia ended its biological weapons program, but the fate of the weapons stockpiles remains unclear.

Today, biological warfare is feared less from nations and more from terrorist groups or lone wolves. But there is disagreement over just how much of a threat this poses. Many believe that terrorists would be incapable of carrying out an effective, large-scale biological attack. For instance, in 1984, the Rajneesh cult gave food poisoning to about 750 citizens of a small Oregon town for political purposes by adding Salmonella to salad bars. Aum Shinrikyo, a Japanese cult that perpetrated a sarin gas attack in the Tokyo subway in 1995, experimented with biological weapons, but to no avail. The 2001 U.S. anthrax attack (discussed in more detail in the following section) killed only 5 people. Skeptics point to incidents like these as evidence that bioterrorists are incapable of inflicting widespread damage. Other analysts disagree ().

Bioterrorism

Some experts believe that a large-scale bioterrorist attack will occur in the not-too-distant future, but others say bioterrorism is an ineffective tactic. Attack methods include contamination of food and water supplies (A), bombs (B), using the mail (C), contamination of water (F), spraying aerosolized agents (E, G), direct injection (D), or the infiltration of suicide infectees (H).

From Osterbauer PJ, Dobbs MR (2005). Neurobiological weapons. Neurol Clin 23, 599621.

Some biological agents, such as anthrax, require little expertise to grow or weaponize. With microbiological information universally available on the Internet, some experts believe that it is just a matter of time before a large bioterrorist attack occurs. A small crop duster airplane loaded with anthrax and flown over a major city could potentially kill hundreds of thousands if not millions of people. Exacerbating the problem is the fact that a 2010 federal commission found the United States to be completely unprepared in the event of a bioterrorist attack.

During the Vietnam War, the Viet-Cong guerillas dug camouflaged pits as booby traps. Inside, they often positioned sharpened bamboo stakes or splinters smeared with human waste. Although it was possible to contract a nasty infection from these, the main purpose was psychological. The tactic worked. The response of American troops was to alter their movements in a way that was disproportionate to the actual threat. An analogous scenario played out following the 2001 anthrax attack in the United States in which there was a colossal disruption of postal services and massive new expenses. Yet, only 5 people died in the attack. (Compare that to the roughly 62,000 Americans who died from influenza and pneumonia that same year.)

Both of these examples serve to underscore two important points: First, biological warfare will almost certainly have a far greater psychological impact than direct impact; and second, protective measures against biological attacks are costly and inconvenient. For instance, giving soldiers vaccines against all possible biological agents would be impractical and possibly dangerous if they have been developed under emergency conditions without thorough testing. Also, vaccines have side effects. Consider the anthrax vaccine used by the U.S. army that was approved in 1971. Vaccination requires six inoculations plus annual boosters. It produces swelling and irritation at the site of injection in 5% to 8% and severe local reactions in about 1% of those inoculated, although major systemic reactions are rare. Although it works against natural exposure, it is uncertain whether it would protect against a concentrated aerosol of anthrax spores.

Or consider the smallpox vaccine (). For every 1 million people vaccinated, the CDC estimates that 1,000 people will have serious side effects, 14 to 52 people will have life-threatening side effects, and 1 or 2 people will die. Is it worth vaccinating an entire army or nationknowing ahead of time that many will die or become sickto protect them against an unlikely threat? From an epidemiological standpoint, the answer is clearly no, which explains why citizens do not receive smallpox vaccinations. The general rule in public health is to vaccinate only if the risk of the disease is greater than the risk of vaccination.

Smallpox Vaccine

How the normal skin reaction to smallpox vaccination progresses in two patients.

Source: Centers for Disease Control and Prevention.

Even if widespread vaccination is forgone in favor of other measures, such as protective clothing or respirators, there is still the financial cost. A nation that invests heavily in bioterrorism preparedness could have spent that money in more productive ways. Dressing troops in special clothing and equipment could promote heat stress or make them easier targets for conventional weaponry. Additionally, medications taken prophylactically to prevent infectious diseases are expensive, rarely 100% effective, and may have long-term negative health consequences.

Biological warfare has been practised since ancient times. However, it has only rarely been effective. Naturally occurring infectious diseases have killed far more people. Still, bioterrorism may pose a serious threat today. Even if an attack kills relatively few people, the psychological impact could be enormous.

Biological warfare is used to kill, injure, and psychologically intimidate enemies. Many naturally occurring diseases are effective agents, although it might be possible to improve them with genetic engineering, as discussed later.

What makes for an effective biological agent? Five major factors need to be considered.

Preparation. Some pathogenic microorganisms are relatively easy to grow in culture, whereas others are extremely difficult or expensive to manufacture in sizeable quantities. Viruses, for instance, can grow only inside host cells, and culturing animal cells is more complex than growing bacteria. Similarly, pathogenic eukaryotes such as Plasmodium (malaria) or Entamoeba (amoebic dysentery) are difficult to culture on a large scale, although some pathogenic fungi can be grown relatively easily. Bacteria are generally the easiest to manufacture on a large scale, but most bacterial infections can be cured with antibiotics. Viruses, though more difficult to grow, have the advantage of being largely incurable despite a small and growing range of specific antiviral agents.

Another factor is weaponization. The disease agent must be prepared in a manner that facilitates storage and dispersal. Because bacterial cells and spores tend to clump together spontaneously, they must be weaponized to allow effective delivery.

Dispersal. Dispersal is a particular challenge for biological weapons. The most likely option would be some form of airborne delivery. However, if applied outdoors, this tactic would be vulnerable to the whims of the weather. Not only is a pleasant breeze required, but also the wind needs to blow in the right direction! During the 1950s, the British government conducted field tests with harmless bacteria. When the wind blew them over farmland, many of the airborne bacteria survived the trip and reached the ground alive. In contrast, when the wind blew the bacteria over industrial areas, especially oil refineries or similar installations, the airborne bacteria were almost all killed. Ironically, air pollution may help protect an urban population from a bioterrorist attack. To aerosolize a biological agent for an indoor attack, a buildings ventilation system or a medical nebulizer could be used ().

Nebulizer

A medical nebulizer could be used to aerosolize a biological agent for an indoor attack. Two general types of nebulizer are in use: the jet nebulizer that uses pressurized gas and the ultrasonic nebulizer that relies on ultrasonic vibrations.

Persistence. Persistence may be the most difficult factor to consider. On the one hand, the biological agent should be able to persist in storage until it is ready to be deployed, and it must survive long enough in the environment to infect the enemy. On the other hand, it should not persist so long that the victor is unable to invade and conquer enemy territory.

Many infectious agents are sensitive to desiccation and become inactive if exposed to air for significant periods of time. Moreover, natural UV radiation from the sun also inactivates many bacteria and viruses. Thus, most biological warfare agents must be protected from this open air factor before use and then dispersed as rapidly as possible. For instance, many viruses last only a few days, if even that, outside their animal or human hosts. (However, infections due to these agents may persist among the local population.)

Anthrax is often chosen as a biological weapon because of its ability to persist for long periods of time. The bacterium Bacillus anthracis, which causes the disease, spreads by forming spores that are tough and difficult to destroy (). When suitable conditions return, for example, inside the lungs of a human, the spores germinate and resume growth as normal bacterial cells, releasing life-threatening toxins.

Spores of Bacillus anthracis

Anthrax spores, which are seen here forming inside bacterial cells, are difficult to destroy and last a very long time.

From Ringertz SH, etal. (2000). Injectional anthrax in a heroin skin-popper. Lancet356, 15741575.

Incubation time. A problem unique to biological warfare, compared to conventional weapons, is that death or incapacitation from infectious disease is a relatively slow process. Even the most virulent pathogens, such as Ebola virus or pneumonic plague, can take a few days to kill. An infected enemy would therefore still be capable of fighting for a significant period. Yet, a biological agent that kills too quickly may not have time to spread among the enemy population.

High-containment laboratories. High-containment laboratories are needed for research and development of infectious biological agents. Biological containment is rated on a scale with four levels. Biosafety level 1 (BSL-1) microbes are mostly harmless, such as nonpathogenic E. coli. BSL-2 organisms are human pathogens, but not easily transmitted in the laboratory, such as Salmonella. BSL-3 organisms are dangerous and often can be transmitted via aerosol, such as tuberculosis and SARS. BSL-4 laboratories are for extremely dangerous and easily transmissible microbes, such as Ebola.

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Biological Warfare: Infectious Disease and Bioterrorism