Living in the soil are huge numbers of bacteria belonging to the genus Bacillus. This genus consists of aerobic, Gram-positive, spore-forming rods. All of the members share certain characteristics, including the ability to exist in two different forms. When conditions for growth are good, with plentiful nutrients and water available, they are rod-shaped organisms that grow and divide. When conditions are unfavorable, each forms a very resistant dormant spore that is able to survive extreme environmental conditions.

The spore is a dehydrated cell with thick walls and additional layers that form inside the cell membrane. It can remain inactive for many years, but if it comes into a favorable environment, it begins to grow again. It is sometimes called an endospore, because it initially develops inside the rod-shaped form. Features such as the location within the rod, the size and shape of the endospore, and whether or not it causes the wall of the rod to bulge out are characteristic of particular species of Bacillus. Depending upon the species, the endospores are round, oval, or occasionally cylindrical. They are highly refractile and contain dipicolinic acid. Electron micrograph sections show that they have a thin outer spore coat, a thick spore cortex, and an inner spore membrane surrounding the spore contents. The spores resist heat, drying, and many disinfectants (including 95% ethanol).¹

Most Bacillus species grow on dead and decaying organic matter, and are harmless to man and animals. One species, however, Bacillus anthracis, is a dangerous pathogen that causes the disease anthrax. It is a zoonosis, meaning that it affects domestic and wild animals, and, secondarily humans.

The ability of Bacillus anthracis to form spores makes it a difficult organism to control. Spores can exist in the soil for decades. They can drift gently in the wind, dormant until they find a place that has the temperature, nutrients, and other conditions to allow growth. When they find their new host (an animal or human) they change to the rod-like form and begin to multiply rapidly. While they are in the spore form they can survive boiling, freezing, or even suspension in alcohol. It takes special measures to kill them, such as steam under pressure, or chemicals known as sporicides. This ability to survive extreme conditions for long periods of time is one of the major reasons Bacillus anthracis has been used by terrorists.

For a microbiologist, growing Bacillus anthracis in the laboratory and causing it to form spores is an easy task. However, putting a culture containing millions of Bacillus anthracis spores into a form that makes an effective weapon is not easy.

Disease

Bacillus anthracis is typically a disease of herbivores (plant-eating mammals), although it can affect other animals as well. Among domestic animals, cattle, sheep, and goats have been the most frequent victims. In most industrialized countries, livestock are routinely vaccinated, and cases of anthrax are rare. In developing countries, however, where animal vaccination is not regularly practiced, anthrax in animals is a problem. This is especially so in tropical and sub-tropical environments. In the USA, anthrax cases among animals have been generally limited to the western plains.

Endospores can survive in the soil for years. Animals consume the spores along with grass as they graze. After the spores enter an animal, they germinate, changing from the resistant form into the growing and dividing vegetative form. The sporangium lyses, the spore germinates, and the bacilli multiply rapidly.

Anthrax is a very serious disease in animals, culminating in a fatal septicemia. The carcass of the animal should be burned where it lies. The carcass should never be opened, since doing this will cause the vegetative forms, which can be destroyed relatively easily, to form into the resistant spores that can survive for years.

Virulent Bacillus anthracis vegetative cells form capsules of poly-D-glutamic acid after they enter their host. The capsule has a negative charge which inhibits macrophages from engulfing and destroying the vegetative cells, impeding the hosts immune response. Thus the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host.²

Infection in humans traditionally has been much rarer than infection in animals. Anthrax occurred in people who came in contact with animals or animal products. It was frequently an occupational disease, affecting veterinarians, people who raised livestock, and people who prepared products from wool, hide, and hair of animals. The inhalation form of anthrax used to be known as woolsorters disease, because it affected people who worked with wool. Products from countries where anthrax in animals is prevalent are still a problem. Goat hair and handicrafts containing animal hides from the Middle East have been a repeated source of infection.³ The ordinary citizen in the USA is most likely to encounter anthrax from imported products that have not been treated sufficiently to destroy spores.

In humans there are three possible forms of the disease anthrax. Historically, the most common form has been cutaneous anthrax, in which the organism enters through a break in the skin. The cutaneous form begins as a papule at the entry site that progresses over several days to a vesicle and then ulcerates. Edema, sometimes massive, surrounds the lesions, which then develop a characteristic black eschar. The patient may have fever, malaise and headache.⁴ A small percentage of cutaneous infections become systemic, and these can be fatal.

A more serious form is inhalation anthrax. Here the victim breathes in the organism and develops a severe respiratory disease. Systemic infection resulting from inhalation of Bacillus anthracis has a mortality rate approaching 100%. Initial symptoms are vague and flu-like, progressing to hypotension, shock and massive bacteremia and toxemia. The severe symptoms are believed to be the result of the bacillis exotoxins. Early antibiotic treatment is an absolute necessity and should be started during the incubation period if a person has been exposed.⁵ After acute symptoms have appeared, antibiotics can kill the organisms, but will not destroy the powerful toxins that have already been formed, and the person commonly dies in 2-3 days from respiratory failure, sepsis and shock.

The third form, intestinal anthrax, is contracted from the consumption of contaminated meat. In industrialized countries this is not usually a risk, although rare exceptions have been described. In August 2000, the Minnesota Department of Health was notified that Bacillus anthracis had been isolated from a steer on a farm in Roseau County. The infected steer was one of five dead cattle found in a pasture. On the basis of identification of the bacteria by phage typing of isolates cultured from tissues and blood samples by the North Dakota State University Veterinary Diagnostic Laboratory, anthrax was confirmed. A report of this incident described the management of and public health response to human exposure to meat contaminated with anthrax.⁶

In countries where hunger is a serious problem, ingestion of contaminated meat is more of a risk than it is in the U.S. Oropharyngeal anthrax begins with severe sore throat or with an ulcer in the oropharyngeal cavity, accompanied by neck swelling and fever. Gastrointestinal anthrax begins with anorexia, nausea, vomiting and abdominal pain. There may be hemorrhagic diarrhea.⁷ Intestinal anthrax can become systemic and lead to death.

Anthrax can be treated with antibiotics, but it is essential that treatment begin early. If it is known that a person has been exposed, treatment should begin immediately, even before symptoms appear. In inhalation anthrax, the most serious form of the disease, the initial symptoms are general flu-like, respiratory symptoms. If treatment is delayed until specific symptoms appear, the fatality rate is extremely high. Ciprofloxacin and doxycycline are the drugs of choice. Penicillin can also be used. Because it is essential to completely eradicate the organism, treatment of anthrax must continue for an extended period, generally sixty days.

Because of current public health concerns, The New England Journal of Medicine has published three articles on the Web at www.nejm.org, several weeks prior to their appearance in print in the November 29, 2001, issue.

"Cutaneous anthrax infection" (published Nov. 6) by K.J. Roche, M.W. Chang and H. Lazarus describes the case of a seven-month old male infant who was hospitalized with a two-day history of swelling of the left arm and a weeping lesion at the left elbow. It was first diagnosed as a spider bite. He was treated with ampicillin-sulbactam and clindamycin. He had been at his mothers office at a television network three days before admission. After anthrax exposure was reported at another television network, two punch biopsies of the lesion were performed. Polymerase chain reaction and immunostaining for Bacillus anthracis were positive.

"Recognition and management of anthraxan update" (published Nov. 6) by M.N. Swarz reviews the characteristics of the organism and the diagnosis and treatment of the disease.

"Index case of fatal inhalational anthrax due to bioterrorism in the United States" (published Nov. 8) by L.M. Blush, B.H. Abrams, A. Beall, and C.C. Johnson describes in detail the first case of inhalation anthrax to occur in the United States since 1978. The patient was employed as a photo editor for a major tabloid newspaper in Florida, where he spent most of the day reviewing photographs submitted by mail or over the Internet. Coworkers report that the patient had closely examined a suspicious letter containing powder approximately eight days before the onset of illness. Bacillus anthracis was cultured from blood and from cerebrospinal fluid.

Research

It has been known for years that the part of the organism that causes the symptoms of disease is anthrax toxin, a very powerful poison. Much of the current research on anthrax involves examining the toxin in great detail, making small changes in its structure, and seeing how the changes affect its properties. Researchers are trying to understand every step in how the toxin exerts its effects. If the procedure is thoroughly understood, then researchers can look for specific steps where they can block the actions of the toxin. A recent article in Critical Reviews in Microbiology (vol. 27, no. 3, pp. 167-200) by R. Bhainagar and S. Batra reviews anthrax toxin.

Low levels of anthrax toxin induce release of cytokines such as tumor necrosis factor alpha. Dehydroepiandrosterone and melatonin have been found to inhibit the increased cytokine production of the anthrax lethal toxin and may have a role in therapy.⁸

It has been found that anthrax toxin actually consists of three proteins. Protective antigen (PA) binds to a cell receptor and mediates the entry of the other two components to the cytoplasm. Edema factor (EF), named for its ability to produce edema, is a calcium/calmodulin-dependent adenylate cyclase. Lethal factor (LF), the dominant virulence factor associated with the toxin, proteolytically inactivates mitogen-activated protein kinase kinases (MAP kinase kinases), which are important in intracellular signal transduction.⁹

The three separate proteins that make up anthrax toxin PA, EF and LF act in binary combinations to produce two distinct reactions in experimental animals: edema (PA+EF) and death (PA+LF).¹⁰

There has been a great deal of interest in PA, since it is essential for the activity of both EF and LF. It has been found that a proteolytically activated 63-kDa fragment of PA binds LF/EF and translocates them into the cytosol of mammalian cells. Domain II of PA has been implicated in membrane insertion and channel formation.

One study found that a PA mutant had considerably reduced toxicity in combination with LF, as well as decreased membrane insertion and translocation of LF into the cytosol.¹¹ In another study, mutations blocked the ability of PA to mediate pore formation and translocation in cells but had no effect on its receptor binding, proteolytic activation, or ability to oligomerize and bind the toxin's enzymes.¹² Another study identified mutants of PA that co-assemble with the wild-type protein and block its ability to translocate the enzymes across membranes. These mutants strongly inhibited toxin action in cell culture and in an animal intoxication model, suggesting they could be useful in therapy of anthrax.¹³

PA and LF acting together to produce death in animals are often referred to as lethal toxin. It has been shown that lethal toxin suppresses proinflammatory cytokine production in macrophages by inhibiting transcription of cytokine messenger RNA, even at extremely low levels of lethal toxin. Thus, one way lethal toxin causes the disease anthrax is by suppressing the inflammatory response.¹⁴

Another action of lethal toxin is to lyse macrophages, which are one of the body's important defense mechanisms against invading organisms. Lethal factor is a zinc-binding protein with metalloproteinase activity. The MAP kinase kinases Mek1 and Mek2 are macrophage proteins that interact with it. Lethal factor cleaves Mek1 and Mek2 and an additional related factor MKK3.¹⁵

Pretreatment of cultured peritoneal macrophages with inhibitors of intracellular calcium release protects against anthrax lethal toxin cytotoxicity. Calcium release from intracellular stores may be an essential step for the propagation of lethal toxin-induced cell damage in macrophages, suggesting a potential way to prevent the toxicity from anthrax lethal toxin.¹⁶

Two recent papers in Nature (vol. 414, 8 Nov 2001) are of interest. The first, by A.D. Pannifer, et al. (pp. 229-233), is "Crystal structure of the anthrax lethal factor". It identifies LF as a highly specific proteinase that cleaves MAP kinase kinases (MAPKK), and details its complex with the N terminus of MAPKK2.

The other paper, by K.A. Bradley, et al. (pp. 225-229) is "Identification of the cellular receptor for anthrax toxin". The cellular receptor to which PA binds, and cloning of this receptor, are described.

Researchers have found that EF is an adenylate cyclase that is activated by calmodulin at resting state calcium concentrations in infected cells.¹⁷ EF has been purified and studied to determine which residues are required for binding to anthrax PA.¹⁸

Another area of great interest is the development of rapid diagnostic tests for anthrax. Because it is so important to begin treatment early, it is important to know whether a person with general symptoms has anthrax or a less serious disease for which totally different treatments are indicated.

A polymerase chain reaction (PCR) amplification on a microarray of gel-immobilized oligonucleotides has been used for detection of bacterial toxins, including the anthrax toxin genes.¹⁹ Other authors have investigated the molecular characterization of Bacillus anthracis by use of multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), and random amplification of polymorphic DNA (RAPD).²⁰ The rpoB gene also has been used as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Variable region 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 Bacillus anthracis strains and 20 other related bacilli. Four nucleotides specific for Bacillus anthracis were identified. The assay was specific for 144 Bacillus anthracis strains from different geographical locations and did not cross-react with 175 strains of other related bacilli, with the exception of one strain.²¹ Such molecular methods, which examine the basic nature of the organism, could potentially be developed into rapid diagnostic methods, providing answers in minutes or hours instead of days.

For epidemiological work, as has been done with recent terrorist incidents, it is important to know whether anthrax bacilli isolated from different sources are the same or different strains. One method for differentiating strains of Bacillus anthracis used long-range repetitive element polymorphism-PCR. The authors examined five genetically distinct groups of diverse geographical origin. All strains produced fingerprints of seven to eight bands, referred to as skeleton bands, while one to three diagnostic bands differentiated between Bacillus anthracis strains. The fingerprints of Bacillus anthracis showed very little in common with those of related species such as Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides.²²

Immunoassays can also be useful in detection of Bacillus anthracis. An immunoaffinity-based phosphorescent sensor platform for the detection of bacterial spores has been developed. There is interest in the production of field portable sensors for use by non-specialists. The immunoaffinity column can capture spores. This is followed by the washing, elution and phosphorescent detection of the spores. Spores are generically detected via the extraction of dipicolinic acid followed by its chelation with terbium to yield a phosphorescent complex.²³

Vaccine

There is a human vaccine for anthrax, but it has been recommended only for people whose occupation puts them at risk of encountering anthrax. In recent years, because of the threat of bioterrorism, anthrax vaccine has been given to U.S. military personnel. It is not currently recommended for use by the general civilian population. There has been much controversy over the safety and effectiveness of the current vaccine.

Many people who receive the current vaccine may experience a mild flu-like illness and soreness at the injection site, but systemic reactions are rare. A recent study among military personnel estimated that 30% of recipients experience mild local reactions. One recipient experienced a delayed and potentially serious life-threatening adverse reaction.²⁴ There is great interest in developing new anthrax vaccines that would contain only the antigen(s) needed for protection, and not the portions of the cell that may cause reactions to the vaccine.

The Advisory Committee on Immunization Practices has issued recommendations concerning the use of aluminum hydroxide adsorbed cell-free anthrax vaccine (Anthrax Vaccine Adsorbed) in the United States.²⁵ This vaccine was studied in a rabbit model. At 6 and 10 weeks, the quantitative anti-protective antigen immunoglobulin G ELISA and the toxin-neutralizing antibody assays were used to measure antibody levels to protective antigen. Rabbits were challenged at 10 weeks with a lethal dose of anthrax spores by inhalation. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived. Antibody levels to protective antigen at both 6 and 10 weeks were significant predictors of survival.²⁶ Passive transfer of lymphocytes and sera from mice immunized using two different formulations of PA has been used to study the mechanism of protection against Bacillus anthracis infection. These results also showed that an antibody response may be important in protection against anthrax.²⁷

In another study, guinea-pigs were immunized with PA and then challenged with a lethal dose of anthrax spores. A direct correlation between survival and neutralizing antibody titer was found. Passive transfer of hyperimmune sera showed the same relationship between neutralizing antibody titers and protection. Such consistency was not found for antibody titers measured by ELISA.²⁸

Although most studies have concentrated on purification of PA for use as a vaccine, a study in mice immunized with a plasmid encoding the lethal factor protein provided protection against a challenge with anthrax lethal toxin.²⁹

For the average citizen today, protection for anyone exposed to anthrax is through treatment with doxycycline, ciprofloxacin, or penicillin. Vaccine is still in limited supply and is available for those whose occupations may bring them in contact with anthrax, including military personnel in locations where they are likely to encounter it.

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