Alaine McGarry

Toxicology Term Paper

Bacillus anthracis: A Possible Biological Warfare Threat

Bacillus anthracis is a rod-shaped, nonmotile, aerobic, gram-positive bacterium that can be transmitted from herbivoric animals to humans (Figure 1). Before an animal’s death, its orifices exude blood containing huge quantities of the bacterium. Anthrax forms extremely resilient spores upon exposure to atmospheric conditions. Because of this spore-producing ability, even after the animal host dies the germs can sporulate again, persisting in the soil for up to several decades (1). No cases of human-to-human transmission have been reported for this zoonosis. Historically, human cases have been either industrial, as with woolsorter’s, or agricultural (2).

Anthrax can be transmitted via 3 routes: cutaneous anthrax, gastric anthrax, and inhalational anthrax. Of the three, cutaneous anthrax is clearly the most prevalent, accounting for about 95% of all cases (3). In cutaneous anthrax, the bacterium invades when broken skin contacts contaminated animal products (1). Within about 2 weeks, at the point of entry a localized sore develops which turns the skin black, becoming a large welt. This coal black lesion "gave rise to the term anthrax that is derived from the Greek anthrakos" which means ‘coal’ (3). While generally self-limited, large doses of oral antibiotics such as penicillin, ciprofloxacin, or doxycycline cure cutaneous anthrax rapidly and effectively. Antibiotic treatment speeds healing, and decreases the possibility of systemic diffusion and lethal toxemia (4). With both gastric and inhalational anthrax, on the other hand, large doses of intravenous antibiotics are necessary (2). Gastric anthrax is exceedingly rare, and results from consumption of contaminated, poorly cooked meat (1). The initial site of infection is probably the cecum or terminal ileum (2). Interestingly, no cases have ever been reported in the United States and gastric anthrax is more common in the developing world. Indeed, most recent outbreaks have occurred in southeastern Asia and Africa where anthrax is endemic, often in isolated villages, and therefore have been difficult to investigate.

Pulmonary anthrax (woolsorter’s disease) results from spore inhalation and like gastric anthrax, is very rare, but inhalation remains the primary infection route for weaponized anthrax, due to its nearly uniform fatality (Table 1). Pulmonary anthrax is approximately 90% fatal, and the US Department of Defense estimates that the human LD50 is between 8,000 and 10,000 spores (5). Some estimates, however, project that the minimum infectious dose could be as high as 80,000 spores (2). As shown in Table 2, early symptoms include malaise, myalgias, fatigue, occasional retronasal pressure, nonproductive cough, and a low-grade fever, and are followed by an acute phase; the acute phase involves respiratory distress, shock, and death (Table 3; 3). Because the early symptoms so closely resemble viral infections or the common cold, anthrax diagnosis proves difficult. While acute phase symptoms are more readily diagnosable, the disease’s advanced progression often makes treatments ineffective at this point (5). Moreover, in the US, there were only 11 reported cases of inhalational anthrax between 1945 and 1994, and so few health physicians have clinical experience with the disease (6). In inhalational anthrax, the pathological lesions "are the local effects of the massive bacterial growth and toxin production, edema, hemorrhage, and necrosis of mediastinal lymph nodes and soft tissues, and pleural effusions; and the systemic effects of toxemia…hematogenous spread results in hemorrhagic meningitis and multiple hemorrhagic foci in the submucosa of the gastrointestinal tract" (4). Studies in non-human primates indicate that alveolar macrophages engulf inhaled spores, transporting them through lymphatic vessels (4).

Historical descriptions of anthrax date back 3,500 years, and anthrax may have even caused two of the ancient Egyptian plaques (5). During the Middle Ages, anthrax was known as the ‘Black Bane’ (2). Its use as a tool of mass destruction is a much more recent phenomenon. Although there exist a diverse array of potential biological weapons candidates, experts generally agree that anthrax is "the microbe most likely to be selected in its highly resistant spore form for air-borne dissemination"(7). There have been several reported instances of the use of anthrax in war. During World War I, Germany began a secret biological warfare program employing anthrax. While humans were not the intended targets, reported cases of human anthrax increased during this period, prompting government investigations (8). In the 1940’s, the Japanese used anthrax in China, killing more than 1,000 people (9). During the Korean War, allegations surfaced that UN forces, under United States direction, used biological warfare against China and North Korea. Such use would clearly violate the 1925 Geneva Protocol, which prohibited chemical and bacteriological weapons usage (7). While the US dismissed these claims as Communist propaganda, contending that existing endemic agents were to blame for the disease outbreaks, Chinese officials (and even some scholars) still believe that the allegations are valid (8).

In 1969-1970, President Richard Nixon renounced offensive biological warfare in favor of defensive measures. He worked to hasten negotiations in the UN Conference on Disarmament, whose goal was to create an international treaty banning the possession of chemical or biological weapons, and calling for weapon stockpile destruction (7). The UN report, in conjunction with a subsequent World Health Organization (WHO) report, was important in the Biological Weapons Convention (BWC) of 1972 (7). The BWC banned both the use of biological weapons or ‘germ warfare’ and the development of such agents for hostile purposes; the treaty was ratified in 1975. Unfortunately, the BWC lacks the "structured organization, authority, and financial resources to enforce its provisions," which include routine inspections based on well-founded suspicions or allegations (7). While the treaty did ban biological weapons, it did not entirely ban associated research and thus may not have prevented anything other than large-scale weapon production.

Unexpectedly, in 1979, a human anthrax outbreak was reported in Sverdlovsk (Ekaterinburg), a city located east of Moscow. Because of this incident, questions arose regarding the Soviet Union’s compliance terms outlined during the Biological Weapons Convention of 1972. However, the Soviet Union maintained that the anthrax outbreak was an endemic one. The United States doubted this explanation, believing instead that the Soviets were secretly replenishing their biological weapons arsenal, as the presence of refrigerated storage bunkers and very high smokestacks would indicate (1). In spite of its suspicions, the US government delayed taking action until March 1980, probably in hopes that SALT ratification could still occur.

Obliged to respond to US accusations, Soviet officials condemned the allegations as US propaganda and asserted that local people developed gastrointestinal anthrax after unwittingly consuming meat from diseased animals. Indeed, at the time of the human anthrax outbreak, there was a reported outbreak of anthrax in local livestock. Such outbreaks occurred frequently, the Soviets claimed. Was this an outbreak of gastric anthrax or pulmonary anthrax? This question fueled much of the debate over the Sverdlovsk incident. New anthrax cases emerged over the course of a month (Figure 2), and experts initially believed that the meat explanation better fit this data. Figure 3 illustrates the "death-spread" argument, which contended that only gastric anthrax could have such prolonged incubation times. In 1992, years later, slides of lung tissue from infected individuals were uncovered. Since these slides showed advanced anthrax lesions in the lungs, primary infection had actually occurred via inhalation (1). Thus the gastric lesions were secondary to the pulmonary anthrax. Moreover, new research indicated that the anthrax incubation period could last much longer than initially believed, up to 30 days following exposure, further decreasing support for the gastric anthrax theory and the death-spread argument (1).

Although Soviet officials denied knowledge of any anthrax pathogen escape, further investigation revealed that windborne anthrax pathogen aerosol had indeed escaped and spread from the city’s military microbiology facility, Military Compound 19 (10). Figure 4 illustrates the aerosol’s deadly path. In fact, most if not all infections coincided with the pathogen’s release on one particular day. Secondary aerosols are unlikely to cause any inhalation infections, but nonhuman primate experiments illustrate the prolonged viability of inhaled anthrax spores (10). From this same accidental anthrax release, livestock had also been infected, up to 50 km away (4). Thus the livestock were victims, rather than the endemic source, of the anthrax pathogen. According to Meselson et al., the Sverdlovsk "epidemic is the largest documented outbreak of inhalation anthrax" (10). Hemorrhagic meningoencephalitis was the most common final cause of death (11). If not for the large-scale administration of vaccine and prophylactic antibiotics, as well as expert medical treatment, the number of fatalities would easily have exceeded sixty-six persons (4).

Russia continued the offensive biological weapons program begun by the Soviet Union. When the US and the United Kingdom challenged the Russians regarding this program, the Russians started to civilianize their former weapons establishments (12). In all likelihood though, the Russian program continues to this day. Unfortunately, many other countries are also suspected of developing biological weapons. In 1995, 17 countries were biological weapons suspects, including Israel, Iran, Libya, Syria, Iraq, North Korea, Taiwan, Egypt, Vietnam, Laos, Cuba, Bulgaria, India, South Korea, South Africa and China (13). Because of their past history of militant behavior, the first 5 countries are particularly troublesome.

During the 1991 Persian Gulf War, Iraq produced anthrax in large amounts for use as a biological weapon, and even admitted so to a UN inspection team. At one site, and probably more, the Iraqis had anthrax research facilities and production capacity (14). While UN weapons inspectors found no weapons per se, they did find that anthrax was being researched for purposes of warfare. Iraq claimed that it had manufactured 8,300 liters of the bacterium, and acknowledged that it had constructed around 200 warheads and aerial bombs containing biological weapons (9). This secret weapons program, Iraq stated, dated back to at least 1974 (12). By immunizing their troops against anthrax, the US government hoped to deter Iraq from employing the bacterium (15). In contrast, Iraqi soldiers received no such immunizations, which makes one questions whether the Iraqi government would have engaged in germ warfare (14). Nonetheless, as Picture 1 illustrates, the Israelis people took the Iraqi threat very seriously. While Iraq claimed that it terminated its weapons program in 1991, the United Nations Special Commission suspects that active preservation of weapons capability continued until at least 1995 (12).

Concerns continue to grow regarding the threat of a bioterrorist attack. In 1998, Larry Wayne Harris, a microbiologist with connections to white-supremacist groups, was arrested after threatening to release ‘military grade anthrax’ in Las Vegas. Even though Harris’ anthrax strain was a harmless veterinary vaccine one, the incident seems to have popularized anthrax as a potential agent in biological warfare (16). Anthrax is inexpensive and easy to acquire, produce and disperse, further adding to its popularity (18). There have been an increasing number of incidents involving apocalyptic religious cults, single-issue groups, and nationalist and separatist groups, although fortunately most have been hoaxes. In 1999, two anthrax hoaxes occurred in Salt Lake City only 8 days apart (17). Some incidents, unfortunately, have not been hoaxes. Aum Shinrikyo, an apocalyptic cult, carried out a 1995 sarin gas attack on the Tokyo subway system. More than 5,000 were injured, and 12 people were killed (9). Surprisingly, the cult had an extensive biological weapons program, which included anthrax; such large, well-funded organizations are the most worrisome (6). According to the World Health Organization, if 50 kg of anthrax were released upwind of a city of 500,000 people, there would be 95,000 resulting fatalities and an additional 125,000 incapacitations (Table 3). Until large numbers of people became seriously ill and presented with acute inhalational anthrax symptoms, an anthrax attack could go virtually undetected.

If a bioterrorist attack were to occur, the economic costs would be staggering. According to the CDC (Centers for Disease Control and Prevention), for every 100,000 persons exposed to anthrax, the economic loss would be around $26 billion. Kaufmann et al. agree with this assessment, adding that quick post-attack prophylaxis program implementation is the best way to reduce these economic losses (19). If multiple incidents were to occur in a major city, the pharmaceutical stockpile needed would cost approximately $270 million (20). While penicillin is a common and somewhat effective anthrax treatment, penicillin-resistant strains can be manufactured and would prove even more devastating in a biological attack (5). In poor countries, since animals are typically not vaccinated against biowarfare disease agents, they are even more vulnerable than humans, further compounding the economic devastation (Franz).

In anthrax infection, toxemia is critical. From Bacillus anthracis toxins, three proteins have been identified: protective antigen (PA), edema factor (EF), and lethal factor (LF). Individually, these proteins are nontoxic. However, protective antigen binds to target cell receptors and is proteolytically cleaved. This cleavage results in exposure of another binding domain, which can combine with either edema factor or lethal factor (2). Protective antigen has channel-forming activity, allowing edema factor and lethal factor to translocate into cells (4). Edema factor is a calmodulin-dependent adenylate cyclase and increases intracellular cAMP, thereby causing interstitial edema (4). In fact, edema factor and protective antigen compose edema toxin, which causes cutaneous edema and inhibits polymorphonuclear lymphocyte phagocytic and oxidative burst functions (5). Likewise, protective antigen and lethal factor comprise lethal toxin, which causes death in monkeys and other laboratory animals via selective macrophage toxicity. Lethal toxin’s toxic effects require calcium and migration through an acidic endocytic vesicle (4). In high doses lethal toxin kills macrophages, but in lower doses it causes macrophage IL-1 and TNF-a secretion (4). IL-1 has an important role in anthrax toxemia, since IL-1 antibodies protect mice against a normally lethal anthrax challenge (2).

These toxins and a poly-D-glutamic acid capsule, which might confer phagocyte resistance, are both B. anthracis virulence factors, and are encoded on plasmids pX01 and pXO2 (4). Both plasmids are necessary for virulence, and their large size suggests that other pathogenicity genes may also exist (5). Immunization with protective antigen prevents fatality, as does antitoxin if given early enough. Unlike antibodies to lethal toxin and edema toxin, antibodies to protective antigen are necessary, although not sufficient, for immunity (4). While antitoxin has fallen out of favor, it was reportedly used during the Sverdlovsk epidemic (2).

While military-grade masks and proper shelter can be effective in preventing disease, vaccine development is crucial in anthrax prevention and treatment. Vaccines can actually reduce the require length of antibiotic treatment (21). Without vaccination, patients exposed to inhalational anthrax undergo 8 weeks of antibiotic treatment, whereas with 3 doses of vaccine, only 4 weeks of such treatment are necessary (6).

In 1880, Pasteur developed an anthrax vaccine using a heat-attenuated, non toxin-producing strain; his sheep vaccination trial was quite successful. Today, anthrax vaccine research continues, although current vaccines are based upon Sterne’s attenuated live, toxin-producing vaccine. Because Sterne’s vaccine retained a degree of virulence however, it was used almost exclusively in animals (5). In 1943, the Soviets developed the first human vaccine, a live spore vaccine that has uncertain efficacy since few studies assess comparative vaccine efficacy. Cell-free bacilli filtrates with high protective antigen content are employed in US vaccines developed in the 1950s and 1960s (5). Filtrates are made from anthrax strains that lack the capsule plasmid pXO2. This vaccine, termed MDPH-PA or MDPH-AVA, has been licensed since 1970 by the FDA and will not cause disease in vaccinated individuals (22). Beginning in March 1998, the US government required that all active and reserve members of its armed forces receive this vaccine.

Although the MDPH US vaccine may prove lifesaving, it only exists in limited supply due to low production capacity, and would be totally depleted by the armed forces (21). In addition, stored vaccine may not retain its viability (6). Bioport is the sole supplier of the US human anthrax vaccine, and like its predecessors, the company has been guilty of quality control problems. In addition, several problems with the vaccine have stimulated continued research. For instance, the immunization schedule involves multiple doses and annual boosters; mild local reactions occur at a high frequency, in about 30% of recipients (22). Moderate local inflammatory reactions also occur in 4 % of recipients, but systemic reactions occur in less than 0.2% of recipients (22). As Table 5 shows, research in guinea pigs also indicates that the vaccine fails to confer protection against all natural anthrax strains (23). Indeed, "vaccine development has been hampered by limited understanding of anthrax pathogenicity and lack of knowledge of epitopes that contribute to the improved immunity conferred by live vaccines" (23).

In light of the afore-mentioned incidents, funding for the US Army Biological Research Program, which develops vaccines and treatments against potential biowarfare agents, increased dramatically (24l). The FDA also prepared to adjust its policies to meet the bioterrorist threat. For instance, the ‘animal efficacy rule’ allows the FDA to consider animal efficacy data when approving new vaccines, if human clinical trials are impossible (25). Of course, data would have to be reproducible across species, and the FDA still requires conventional data based upon testing of human volunteers (25).

The FDA and the CDC jointly manage the Vaccine Adverse Event Reporting System (VAERS), which tracks adverse vaccine-associated events. For the current US anthrax vaccine, 215 adverse events have been reported to be possibly associated with vaccination (22). Of the 215 adverse events, 22 were serious, but no definite cause and effect relationship can be determined (22). At-risk mill workers in the northeastern US received a similar vaccine, which had a 92.5% efficacy rate against cutaneous anthrax (2). Unfortunately, the efficacy against inhalational anthrax could not be determined. Any study of human vaccine potency and efficacy requires anthrax challenge studies, impossible due to the high mortality rate associated with inhalational anthrax. Because of the impracticality of human models, experimental animal models are the best alternative in vaccine efficacy evaluation. In guinea pigs, the AVA vaccine failed to confer adequate protection against aerosol challenge, but gave variable protection against an intramuscular challenge (22). Conversely, in nonhuman primates and rabbits, the AVA gave excellent protection against aerosol challenge (22). Since nonhuman primates are much more similar to humans than are guinea pigs, one could argue that the AVA vaccine would also provide humans adequate protection against inhalational anthrax.

In animal studies, addition of certain adjuvants greatly improves vaccine efficacy and leads to more rapid immunity (Tables 6, 7). Before deployment to the Persian Gulf, British troops were immunized with anthrax vaccine containing killed Bordetella pertussis adjuvant. Following the Gulf War, questions arose concerning the possible adverse effects of this adjuvant, as it causes aging in guinea pigs. According to Nass, B. pertussis might induce Gulf War illness by shifting the cytokine balance from T_H1 to T_H2. To date, the only adjuvants licensed for human use in the US are aluminum-based (23).

With recent advances in molecular biology, more effective vaccines will probably be developed. In the United States, the search for an improved vaccine is following several different paths. A chemically pure PA vaccine is currently in development, but it is unknown whether it will stimulate sufficient immunity (23). Researchers are also attempting to create a safe, live vaccine that will present antigens better than a chemical vaccine. By employing Lactobacillus casei as an expression vector for B. anthracis proteins, Zegers et al. are currently developing a safe oral vaccine against anthrax. Using this vector, they have achieved high protective antigen expression (26). In spite of these attempts, eight years after the Gulf War’s end, scientists have yet to develop a better vaccine, and so inhalational anthrax remains a major threat to public health.

Because bacillus anthracis is simple to acquire and disperse, it has become a popular potential agent of germ warfare. Of the three forms of anthrax, inhalational anthrax is the most deadly and thus the favored choice for biological weapons production. If a covert anthrax attack were to occur, there would be considerable devastation, especially since inhalational anthrax is difficult to diagnose prior to the acute stages. Although the Biological Weapons Treaty of 1975 banned the development of such agents for offensive purposes, many nations continue their weapons programs today, albeit in secret. Recognizing this noncompliance, the United States government mandated the vaccination of all members of its armed forces in 1998. In addition, scientists are continuing the search for new and better anthrax vaccines. Although their search has met with only marginal success, with improving technology, a safer, more efficacious vaccine may eventually become a reality.

Works Cited

1. Gordin, M. "The anthrax solution: The Sverdlovsk incident and the resolution of a biological weapons controversy." Journal of the History of Biology. 30 (1997): 441-80.

2. Pile, JC, Malone, JD, Eitzen, EM, and AM Friedlander. "Anthrax as a potential biological warfare agent." Archives of Internal Medicine. 158 (1998): 429-33.

3. McGovern, TW, Christopher, GW, and EM Eitzen . "Cutaneous manifestations of biological warfare and related threat agents." Archives of Dermatology. 135 (1999): 311-22

4. Walker, DH, Yampolska, O, and LM Grinberg. "Death at Sverdlovsk: What have we learned?" American Journal of Pathology. 144.6 (1994): 1135-41.

5. Shafazand, S, Doyle, R, Ruoss, S, et al. "Inhalational anthrax: Epidemiology, diagnosis, management." Chest. 116.5 (1999): 1369-76.

6. Kortepeter, MG, and GW Parker. "Potential biological weapons threats." Emerging Infectious Diseases. 5.4 (1999): 523-27.

7. Kaplan, M. "The efforts of WHO and Pugwash to eliminate chemical and biological weapons-a memoir." Bulletin of the World Health Organization. 77.2 (1999): 149-55.

8. Furmanski, M. "Anthrax." The New England Journal of Medicine. 342.1 (2000): 61-2.

9. Stephenson, J. "Confronting a biological Armageddon: Experts tackle prospect of bioterrorism." Journal of the American Medical Association. 276.5 (1996): 349-51.

10. Meselson, M, Guillemin, J, Hugh-Jones, M, et al. "The Sverdlovsk anthrax outbreak of 1979." Science. 266 (1994): 1202-08.

11. Grinberg, LM, Abramova, AA, Yampolskaya, O, et al. "Pathology of human inhalation anthrax." Laboratory Investigation. 78.1 (1998): 144A.

12. Davis, CJ. "Nuclear blindness: An overview of the biological weapons programs of the former Soviet Union and Iraq." Emerging Infectious Diseases. 5.4 (1999): 509-12.

13. Cole, LA. "The specter of biological weapons." Scientific American. 275.6 (1996): 60-65.

14. Goldsmith, MF. "Defensive biological warfare researchers prepare to counteract ‘natural’ enemies in battle, at home." JAMA. 266.18 (1991): 2522-23.

15. Marwick, C. "Defense appears to have advantage over offense presently in biological warfare." JAMA. 265.5 (1991): 700.

16. Tucker, JB. "Historical trends related to bioterrorism: An empirical analysis." Emerging Infectious Diseases. 5.4 (1999): 498-504.

17. Swanson, ER, and DE Fosnocht. "Anthrax threats: A report of two incidents from Salt Lake City." The Journal of Emergency Medicine. 18.2 (2000): 229-32.

18. Guidotti, TL. "Bioterrorism and the public health response." American Journal of Preventive Medicine. 18.2 (2000): 178-80.

19. Kaufmann, AF, Meltzer, MI, and GP Schmid. "The economic impact of a bioterrorist attack: Are prevention and postattack intervention programs justifiable?" Emerging Infectious Diseases. 3.2 (1997): 83-94.

20. Tarlach, GM. "The threat of biochemical attacks offer roles for R.Ph.s." Drug Topics. 142.6 (1998): 46.

21. Russell, PK. "Vaccines in civilian defense against bioterrorism." Emerging Infectious Diseases. 5.4 (1999): 531-33.

22. Friedlander, AM, Pittman, PR, and GW Parker. "Anthrax vaccine: Evidence for safety and efficacy against inhalational anthrax." JAMA. 282.22 (1999): 2104-06.

23. Nass, M. "Anthrax vaccine: Model of a response to the biologic warfare threat." Infectious Disease Clinics of North America. 13.1 (1999): 187-205.

24. Sidel, VW. "Weapons of mass destruction: The greatest threat to public health." JAMA. 262.5 (1989): 680-82.

25. Fox, JL. "Adjusting FDA policies to address bioterrorist threat." Nature Biotechnology. 17 (1999): 323-24.

26. Zegers, ND, Kluter, E, van der Stap, H, et al. "Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: Towards the development of an oral vaccine against anthrax." Journal of Applied Microbiology. 87 (1999): 309-14.

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