Understanding the pathogenesis, signs, and symptoms of the different forms of clinical anthrax may aid in providing appropriate therapy in the event of a large-scale exposure to pathogenic endospores
Figure 1. Gram stain of Bacillus anthracis.
Anthrax is a zoonotic disease (a disease of humans acquired from an animal source). Grazing herbivores such as cattle, sheep, horses, and goats are the animals most commonly infected with Bacillus anthracis, the causative microorganism. Human infection results, under natural circumstances, from contact with contaminated animals or animal products, most often by the cutaneous route and only rarely by the respiratory or gastrointestinal routes. There are no known cases of human-to-human transmission. The current interest in the disease is due to the recent use of B. anthracis as a biological weapon by terrorists and the potential for its use in warfare.
Anthrax may persist in the soil for many years after contamination of a pasture. Environmental persistence appears to be related to a number of factors, including high levels of soil nitrogen and organic content and ambient temperature greater than 15°C.1 Heavy rains after a drought trigger spore germination and bacterial multiplication, which appear to be important in maintaining the organism in potentially infectious quantities.2
Naturally occurring human cases of anthrax have been divided into two groups: agricultural and industrial. Agricultural cases of human anthrax result from direct contact with an animal dying from anthrax. Industrial cases result from contact with anthrax spores that contaminate raw materials such as hides, goat hair, wool, and bones that are used as part of a manufacturing process. Because B. anthracis spores can survive for long periods of time, the variety of products that have been linked to human infection is wide and varied and includes such items as shaving brushes, bongo drums, and saddle blankets.
Historical Perspective
B. anthracis is a soil bacterium that has probably been on earth longer than man, and the disease anthrax is one of the great infectious diseases of ancient times. In addition, the black bane, a disease that swept through Europe in the 1600s, causing large numbers of human and animal deaths, was probably anthrax. B. anthracis is unusually large and was the first bacterium shown to cause a disease. As early as 1850, it was seen in the blood of sheep dying of anthrax, and in 1877, Robert Koch grew it in pure culture, demonstrated its ability to form spores, and produced experimental anthrax by injecting it into animals.3 Around the same time, John Bell recognized woolsorter’s disease, or inhalational anthrax, and by setting standards for wool disinfection, he was able to reduce the incidence of the disease in England.4
In 1881, at the famous field trial at Pouilly-le-Fort, Pasteur vaccinated 24 sheep, one goat, and six cows with a culture of the bacillus attenuated by growth at 42°C. After 2 weeks, the vaccinated animals and the unprotected controls were injected with a virulent culture. Two days later, the unvaccinated animals either had died or were very ill, while all of the vaccinated animals remained unharmed. This dramatic demonstration provided a potent stimulus for the development of immunology.5
Large accidental anthrax outbreaks in humans have occurred throughout modern times. More than 6,000 cases of mostly cutaneous anthrax occurred in Zimbabwe between October 1979 and March 1980,6 and an unintentional release of anthrax spores in 1979 from a Soviet military research center in Yekaterinburg (Sverdlovsk), Russia, resulted in 41 documented cases of inhalational anthrax.7 Twenty-five cutaneous cases occurred in Paraguay in 1987 after the slaughter of a single infected cow.8
Morphology
B. anthracis is a large, gram-positive, nonmotile, spore-forming rod 1 to 1.5 mm in width and 4 to 10 mm in length (Figure 1). In smears from infected tissues, it appears singly or in short chains, and its capsule is readily demonstrable using stain or fluorescein-labeled anticapsular antibodies.
The bacterium does not form spores in the living animal. Spores are formed under conditions unfavorable for continued multiplication of the vegetative form. In the presence of high carbon dioxide concentrations, the organism forms capsules, and the colonies are smooth and mucoid in culture. Spores begin to appear at the end of the logarithmic phase of growth and are numerous after 48 hours. The oval spores (2 to 6 mm in diameter) are clearly visible in the centers of the bacilli. Anthrax spores are relatively resistant to heat and chemical disinfectants. They are usually destroyed by boiling for 10 minutes and by dry heat at 140°C for 3 hours. They may remain viable for months in animal hides and for years in dry earth.
Determinants of Virulence
B. anthracis possesses a unique cell-wall polysaccharide antigen, and it forms a single antigenic type of capsule consisting of a protein made up of poly-d-glutamic acid. All virulent strains of the bacillus form this capsule. The major virulence factors of B. anthracis are encoded on two virulence plasmids, pXO1 and pXO2.
Capsule production depends on the presence of pXO2. The capsule is nontoxic and protects the organism from destructive antibodies and bactericidal components of serum.9 It plays an important role in the establishment of infection and a less significant role in the terminal phases of the disease, which are mediated by the anthrax toxin.
In addition to the capsule, virulent strains of B. anthracis produce three proteins, all of which are components of a complex exotoxin (anthrax toxin) that plays a major role in virulence. Fatal bacterial infection may result from the synthesis of this complex exotoxin. Production of anthrax toxin is mediated by the plasmid pXO1. The anthrax toxin consists of a mixture of three temperature-sensitive, immunogenic proteins: edema factor (EF); protective antigen (PA), which is capable of inducing protective antibodies; and lethal factor (LF). The three exotoxin components combine to form two binary toxins, edema toxin and lethal toxin.
Edema toxin consists of EF, which is a calmodulin-dependent adenylate cyclase, and PA, the binding moiety that permits entry of the toxin into the host cell. EF increases cellular levels of cyclic adenosine monophosphate, which upsets water homeostasis and is believed to be responsible for the massive edema seen in cutaneous anthrax.10
Lethal toxin consists of LF, which is a zinc metalloproteinase, and PA, which acts as the binding domain.11 Lethal toxin stimulates macrophages to release proinflammatory cytokines such as tumor necrosis factor and interleukin-1b, which may be responsible for sudden death in patients with systemic anthrax.12
A secondary effect of the EF and LF is the depletion of adenosine triphosphate reserves in macrophages and neutrophils that are needed for the engulfment process. These two toxins, in the presence of PA, increase host susceptibility to infection by impairing the phagocytic activity of regional white blood cells during the infectious process. Both the capsule and the anthrax toxin are involved in the early stages of infection, through direct effects on phagocytic cells.13
B. anthracis coordinates the expression of its virulence factors in response to a specific environmental signal. Anthrax toxin proteins and the antiphagocytic capsule are produced in response to growth in the presence of increased atmospheric carbon dioxide. This carbon dioxide signal is thought to be of physiological significance for pathogens that invade mammalian host tissue.13
Figure 2. Transmission of anthrax.
Anthrax Pathogenesis
Anthrax infection is initiated by the endospores of B. anthracis. Endospores are introduced into the body through a break in the skin, inhalation, or ingestion (Figure 2). These endospores are then phagocytized by macrophages and carried to regional lymph nodes. The spores germinate inside the macrophages and become vegetative bacteria,14 which are, in turn, released from the macrophages. They then multiply in the lymphatic system and eventually enter the bloodstream.
Endospores that germinate into virulent anthrax bacilli multiply at the site of entry. Phagocytic cells migrate to the area, but the encapsulated organisms can resist engulfment, or, if engulfed, can resist killing and digestion. The production of the anthrax toxin further impairs phagocytic activity by disrupting cytokine production and producing lethal effects on white blood cells, including phagocytes.
Anthrax toxin also results in necrosis of lymphatic tissue, thereby allowing large numbers of organisms into the body. After the organisms and their toxins enter the circulation, a clinical syndrome reminiscent of septic shock ensues. Autopsy examination of patients who have died from anthrax shows widespread hemorrhage and necrosis involving multiple organs. Death is apparently due to oxygen depletion, secondary shock, increased vascular permeability, and, ultimately, respiratory and cardiac failure. Death from anthrax in humans or animals usually occurs suddenly and unexpectedly. The level of lethal anthrax toxin in the circulation increases rapidly quite late in the course of the disease, and it closely parallels the concentration of organisms in the blood.
The clinical manifestations of anthrax were described in the late 19th and early 20th centuries when the disease was more prevalent in its natural form. Three primary forms of the disease exist: cutaneous, inhalational, and gastrointestinal (including oropharyngeal). A fourth form, anthrax meningitis, has rarely been reported.
Cutaneous Anthrax
The cutaneous form of anthrax accounts for more than 95% of naturally occurring cases. It begins after introduction of spores through a break in the skin, such as a cut or abrasion (anthrax spores cannot penetrate intact skin). The skin lesions of cutaneous anthrax take on a predictable appearance and are easy to recognize.
The primary lesion of cutaneous anthrax usually develops at the site of a minor scratch or abrasion on an exposed area of the face, neck, or upper extremities into which anthrax spores have been accidentally inoculated. The spores germinate, and after an incubation period of 1 to 7 days, a small pruritic papule appears. Within a day or two, vesicles surround the papule or a single vesicle appears. The vesicular fluid is clear or bloody and contains numerous bacilli, but few leukocytes. The vesicle may become enlarged and may eventually rupture, leaving an ulcer covered by a characteristic dark-brown eschar. A gelatinous, nonpitting edema may surround the ulcer for a considerable distance. Eventually, the eschar becomes black (anthrax is derived from the Greek word for coal), dries, and falls off during the next 1 to 3 weeks, usually leaving no scar. At no stage is the lesion particularly painful.
Regional lymphadenopathy is often present during the first few days. In severe cases, the bloodstream may be invaded. As with all forms of anthrax during the initial stages of clinical infection, many patients experience fever, headache, and malaise. Lesions about the neck or face may produce mediastinal edema and respiratory distress. About 20% to 30% of untreated cases of cutaneous anthrax will result in death.15 Mortality in treated patients is less than 1%.16
Inhalational Anthrax
Inhalational anthrax is the most dreaded form of the disease. Until very recently, it was a disease of mainly historical interest. Now, with the first new cases seen in the United States since the 1970s, inhalational anthrax is receiving widespread attention.
Inhalational anthrax results from exposure to spore-bearing dust or aerosol. Aerosolized anthrax spores >5 mm in size are deposited in the upper airways and are effectively trapped or cleared by the mucociliary system. Spores 2 to 5 mm in size are able to reach the alveolar ducts and alveoli. These spores are engulfed by pulmonary macrophages and transported to mediastinal and hilar lymph nodes. Weapons-grade anthrax is a term referring to the presence of smaller spores, which can penetrate deep into the lungs and cause serious disease.
In order to produce fatal pulmonary infection, it is necessary to aerosolize a relatively large number of spores. The estimated infectious dose for 50% of individuals is 8,000 to 10,000 spores.17 Moreover, because only particles less than 6 mm in diameter are likely to penetrate to the alveoli, the average particle size of the aerosol is critical. In fact, the lethal dose varies directly with the median size of the spores.17 These limitations may help explain the rarity of naturally occurring human anthrax in most of the Western world, even in areas of high soil contamination.
The clinical syndrome resulting from inhalational anthrax usually follows a biphasic pattern. After an incubation period of 1 to 6 days, a nonspecific influenza-like illness characterized by fever, myalgia, headache, nonproductive cough, and mild chest discomfort ensues. A brief intervening period of improvement sometimes follows 1 to 3 days of these prodromal symptoms, but the regional lymph nodes are quickly overwhelmed, the anthrax toxin finds its way into the systemic circulation, and rapid deterioration follows. The second phase of illness is marked by high fever, chest-wall edema, hemorrhage, dyspnea, stridor, cyanosis, tissue necrosis, and septic shock. There may be meningeal involvement, with or without subarachnoid hemorrhage, in up to 50% of cases.18
Chest radiographs may show pleural effusions and a widened mediastinum, but there is typically no pneumonia seen, except in cases of preexisting pulmonary pathology.19 Blood smears from patients in the later stages of illness may contain the characteristic gram-positive, spore-forming bacilli.
Mortality has been reported to be nearly 100% in untreated cases,20 but it is important to note that this figure, still widely quoted today, is based on observations made during the early 1930s, before antibiotics or intensive care units were available. Inhalational anthrax is still a serious disease, but even though several patients who recently developed inhalational anthrax died, it is likely that, with the use of effective antimicrobial therapy and intensive care, the overall death rate may be much lower than 100%.
Early treatment of inhalational anthrax is essential. Death may still occur in the majority of treated cases if therapy is initiated too late (more than 48 hours after the onset of symptoms).20 This is why so many people with suspected or possible exposure to anthrax spores have been treated with ciprofloxacin and other antibiotics empirically.
GASTROINTESTINAL ANTHRAX
Gastrointestinal anthrax develops 2 to 5 days after eating undercooked meat from infected animals. It is believed that bacterial inoculation takes place at a breach in the mucosal lining of the gastrointestinal tract, but exactly where endospores germinate is unknown.10 Autopsy typically reveals hemorrhagic inflammation of the small intestine with bowel perforation. Microscopically, bacilli can be seen in the mucosal and submucosal lymphatic tissue, and there is evidence of mesenteric lymphadenitis.21
The clinical picture of gastrointestinal anthrax may be either that of a cholera-like gastroenteritis or that of an acute abdomen (severe abdominal pain, loss of appetite, nausea, fever, vomiting, bloody diarrhea, intestinal obstruction, and shock). Because of ulceration of the gastrointestinal mucosa, patients may vomit material that is blood-tinged or has a coffee-grounds appearance. Ascites, with a concomitant reduction in abdominal pain, may develop 2 to 4 days after the onset of symptoms.22 Cultured ascitic fluid often is positive for B. anthracis. Rarely, gastrointestinal anthrax is associated with cutaneous disease.22,23
Death results from intestinal perforation or anthrax toxemia. The reported mortality rate is extremely high (>50%),20 probably because, in the past, the gastrointestinal form of anthrax has been difficult to recognize in the absence of any obvious local lesion. If the patient survives, most of the symptoms subside in 10 to 14 days.23 Gastrointestinal anthrax has not been reported in the United States.
Anthrax Meningitis
Anthrax meningitis is a rare form of anthrax. The most common portal of entry is the skin, from which the organisms can spread to the central nervous system by hematogenous or lymphatic routes. Anthrax meningitis may also occur in association with cutaneous or inhalational anthrax.24,25 Clinically, anthrax meningitis is similar to other bacterial meningitides, although focal signs may occur more frequently. It is unusual in that the cerebrospinal fluid (CSF) is bloody in most cases.26 CSF cultures are usually positive for B. anthracis. Mortality approaches 100%, although survival has been reported.26,27
Anthrax Treatment
Antimicrobial therapy is the cornerstone of treatment. Early initiation of therapy is essential. Penicillin has traditionally been the drug of choice for naturally occurring anthrax. Only very rarely have naturally occurring strains been reported to be penicillin-resistant.28 With the emergence of anthrax as a weapon of biological terrorism/warfare, it is possible that strains of penicillin-resistant microorganisms will be cultivated. Fortunately, most strains of B. anthracis are susceptible to a number of antibiotics in addition to penicillin. These include ciprofloxacin and other fluoroquinolones, tetracycline, doxycycline, chloramphenicol, macrolides, aminoglycosides, vancomycin, imipenem, clindamycin, rifampin, and several first-generation cephalosporins.29-31 B. anthracis is generally resistant to third-generation cephalosporins.29-31
Ciprofloxacin is the most widely recognized treatment for anthrax. Although treatment of anthrax infection with ciprofloxacin has not been studied in humans, animal models suggest excellent efficacy.32 Anecdotal evidence from the recent cases associated with the US Postal Service also suggests excellent efficacy. In vitro data suggest that other fluoroquinolone antibiotics would have similar efficacy in treating anthrax, but no animal data exist for fluoroquinolones other than ciprofloxacin.31
For mild cutaneous cases, therapy can be given orally. For severe cutaneous cases and all inhalational and gastrointestinal cases, intravenous therapy is required, at least at the outset. Antibiotic therapy should be continued for at least 60 days.33
Prior to September 2001, few clinicians in the United States ever saw a case of anthrax. Because naturally occurring anthrax is such a rare disease, there has been no long-standing effort to develop and test the consensus recommendations or expert panel guidelines made for so many other infectious diseases. Nonetheless, recognizing the potential for the use of anthrax as a biological weapon, the Working Group on Civilian Biodefense (a panel composed of representatives from academic medical centers and research, government, military, public-health, and emergency-management institutions and agencies) published consensus-based recommendations for measures to be taken following the use of anthrax as a biological weapon against a civilian population.33
According to the Working Group on Civilian Biodefense, all individuals with fever or evidence of systemic disease in an area where anthrax cases are occurring should be treated for anthrax until the disease is excluded. See Table 1 for known or suspected inhalational anthrax recommendations.33
| Initial therapy* Ciprofloxacin, 400 mg intravenously every 12 hours (in vitro work suggests that ofloxacin, 400 mg intravenously every 12 hours, or levofloxacin, 500 mg intravenously every 24 hours, could be substituted for ciprofloxacin) Optimal therapy, if the strain is proven susceptible* *Duration of therapy=60 days; oral antibiotics should be substituted for intravenous antibiotics as soon as the patient’s clinical condition improves. |
| Table 1. Recommendations of the Working Group on Civilian Biodefense for medical therapy for adults with clinically evident inhalational anthrax infection in the contained casualty setting.35 |
The treatment for cutaneous anthrax historically has been oral penicillin. The Working Group recommends oral ciprofloxacin or tetracycline as suitable alternatives if antibiotic susceptibility is proven. Although previous groups have suggested treating cutaneous anthrax for 7 to 10 days,34 the group recommends treatment for 60 days in the setting of bioterrorism,33 given the patient’s presumed exposure to the primary aerosol. Treatment of cutaneous anthrax generally prevents progression to systemic disease, although it does not prevent the formation and evolution of the eschar. Topical therapy is not useful.
Post Exposure Prophylaxis
There are no specific antibiotic regimens approved by the US Food and Drug Administration for prophylaxis following exposure to a B. anthracis aerosol. The working group recommends the same antibiotic regimen as that for the treatment of mass casualties.34 Prophylaxis should be continued for 60 days (Table 2).33
| Initial therapy Ciprofloxacin, 500 mg by mouth every 12 hours for 60 days Optimal therapy, if strain is proven susceptible |
| Table 2. Recommendations of the Working Group on Civilian Biodefense for medical therapy for adults with clinically evident anthrax infection in the mass casualty setting or for postexposure prophylaxis.35 |
Vaccines
An anthrax vaccine is made for the US military, but none has been produced since 1998 because of manufacturing problems.35,36 The vaccine is an inactivated cell-free filtrate of a nonencapsulated, avirulent strain that expresses the protective antigen.35 It is given subcutaneously as six doses of 0.5 mL initially and again after 2 weeks, 4 weeks, 6 months, 12 months, and 18 months. Yearly boosters are required to maintain immunity. There is a high incidence (up to 30%) of local adverse reactions in people who receive the vaccine.38,39 Attempts are under way to produce an improved anthrax vaccine.39
The anthrax vaccine is not available for civilian use, although there have been recent reports of a movement to make the vaccine available to civilians who are occupationally exposed to anthrax spores (goat-hair, woolen-mill, tannery, veterinary, and laboratory workers).
Conclusion
Recent incidents, including the use of the US Postal Service to disseminate anthrax spores, underscore the need for all health care personnel to become familiar with anthrax and its treatment. Understanding the pathogenesis, signs, and symptoms of the different forms of clinical anthrax may aid in the provision of appropriate prophylaxis and therapy in the event of large-scale exposure to pathogenic endospores.
Phyllis C. Braun, PhD, is Professor, Department of Biology, Fairfield University, Fairfield, Conn. John D. Zoidis, MD, is a contributing writer for RT.
References
1. Pile JC, Malone JD, Eitzen EM, Friedlander AM. Anthrax as a potential biological warfare agent. Arch Intern Med. 1998;158:429-434.
2. Kaufmann AF. Observations on the occurrence of anthrax as related to soil type and rainfall. Salisbury Medical Bulletin. 1990;68(suppl):16-17.
3. Beck RW. A Chronology of Microbiology in Historical Context. Washington, DC: ASM Press; 2000.
4. Eurich FW. The history of anthrax in the wool industry of Bradford, and of its control. Lancet. 1926;I:57-58.
5. De Kruif P. Microbe Hunters. New York: Harbrace; 1953.
6. Turner M. Anthrax in humans in Zimbabwe. Cent Afr J Med. 1980;6:160-161.
7. Grinberg LM, Abramova FA, Yampolskaya OV, Walker DH, Smith JH. Quantitative pathology of inhalational anthrax. I: Quantitative microscopic findings. Mod Pathol. 2001;14:482-495.
8. Harrison LH, Ezzell JW, Abshire TG, Kidd S, Kaufmann AF. Evaluation of serologic tests for diagnosis of anthrax after an outbreak of cutaneous anthrax in Paraguay. J Infect Dis. 1989;160:706-710.
9. Baillie L. Bacillus anthracis, a bug with attitude. Curr Opin Microbiol. 2001;4:78-81.
10. Dixon TC, Meselson M, Guillemin J, Hanna PC. Anthrax. N Engl J Med. 1999;341:815-826.
11. Klimpel KR, Arora N, Leppla SH. Anthrax toxin lethal factor contains a zinc metalloproteinase consensus sequence which is required for lethal toxin activity. Mol Microbiol. 1994;13:1093-1100.
12. Hoover DL, Friedlander AM, Rogers LC, Yoon IK, Warren RL, Cross AS. Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP. Infect Immun. 1994;62:4432-4439.
13. Little SF, Ivins BE. Molecular pathogenesis of Bacillus anthracis infection. Microb Infect. 1999;1:131-139.
14. Ross JM. The pathogenesis of anthrax following the administration of spores by the respiratory route. Journal of Pathology and Bacteriology. 1957;73:485-494.
15. Franz DR, Jahrling PB, Friedlander AM, et al. Clinical recognition and management of patients exposed to biological warfare agents. JAMA. 1997;278:399-411.
16. Gold H. Anthrax: a report of 117 cases. Arch Intern Med. 1955;96:387-396.
17. Cieslak TJ, Eitzen EM. Clinical and epidemiologic principles of anthrax. Emerging Infectious Diseases. 1999;5:552-555.
18. Shafazand S, Doyle R, Ruoss S, Weinacker A, Raffin TA. Inhalational anthrax. Epidemiology, diagnosis, and management. Chest. 1999;116:1369-1376.
[AQ: cite in text]19. Eurich FW. Some notes on industrial anthrax: its diagnosis and treatment. BMJ. 1933;II:50-53.
20. Friedlander AM. Anthrax: clinical features, pathogenesis, and potential biological warfare threat. Curr Clin Top Infect Dis. 2000;20:335-349.
21. Dutz W, Kohout E. Anthrax. Pathology Annual. 1971;6:209-248.
[AQ: cite in text]22. MacDonald WD. Anthrax: report of a fatal case involving the cutaneous and gastrointestinal systems. N Engl J Med. 1942;226:949-951.
23. Alizad A, Ayoub EM, Makki N. Intestinal anthrax in a two-year-old child. Pediatr Infect Dis. 1995;14:394-395.
24. Abramova FA, Grinberg LM, Yampolskaya OV, Walker DH. Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci USA. 1993;90:2291-2294.
25. Tabatabaie P, Syadati A. Bacillus anthracis as a cause of bacterial meningitis. Pediatr Infect Dis J. 1993;12:1035-1037.
26. Lalitha MK, Mathai D, Thomas K, et al. Anthrax: a continuing problem in southern India. Salisbury Medical Bulletin. 1996;87(suppl):14-15.
27. Tahernia AC, Hashemi C. Survival in anthrax meningitis. Pediatrics. 1972;50:329-332.
30. Bradaric N, Punda-Polic V. Cutaneous anthrax due to penicillin-resistant Bacillus anthracis transmitted by an insect bite. Lancet. 1992;340:306-307.
31. Lightfoot NF, Scott RJD, Turnbull PCB. Antimicrobial susceptibility of Bacillus anthracis. Salisbury Medical Bulletin. 1990;68(suppl):95-98.
32. Odendaal MW, Pieterson PM, de Vos V, Botha AD. The antibiotic sensitivity patterns of Bacillus anthracis isolated from the Kruger National Park. Onderstepoort J Vet Res. 1991;58:17-19.
33. Doganay M, Aydin N. Antimicrobial susceptibility of Bacillus anthracis. Scand J Infect Dis. 1991;23:333-335.
34. Kelly D, Chulay JD, Mikesell P, Friedlander A. Serum concentrations of penicillin, doxycycline, and ciprofloxacin during prolonged therapy in rhesus monkeys. J Infect Dis. 1992;166:1184-1187.
35. Inglesby TV, Henderson DA, Bartlett JG, et al. Anthrax as a biological weapon. Medical and public health management. JAMA. 1999;281:1735-1745.
36. Penn C, Klotz SA. Anthrax. In: Gorbach SL, Bartlett JG, Blacklow NR, eds. Infectious Diseases. Philadelphia: WB Saunders; 1998:1575-1578.
[AQ: cite in text]37. Brachman P, Friedlander AM. Anthrax. In: Plotkin SA, Orenstein WA, eds. Vaccines. Philadelphia: WB Saunders; 1999:629-637.
38. US Food and Drug Administration. Report of the Inspection of Michigan Biologic Products Institute. Rockville, Md: US FDA; 1998.
[AQ: cite in text]39. Nass M. Anthrax vaccine. Model of a response to the biologic warfare threat. Infect Dis Clin North Am. 1999;13:187-208.
