Anthrax: History, Biology, Global Distribution, Clinical Aspects, Immunology, and Molecular Biology

This chapter deals with the early history of anthrax, its industrial incidence and association with animal products such a wool and hair, and effective methods for destroying anthrax spores. The global distribution and prevalence of anthrax along with geographic areas considered endemic for anthrax are presented. Major non natural anthrax incidents that have occurred during the past century are discussed in detail.

Bacillus anthracis, the causative organism of anthrax is a member of the B. cereus group of bacilli. The stained organism exhibits a unique and characteristic “Boxcar” appearance microscopically. The three forms of anthrax: (1) cutaneous, (2) inhalation, and (3) gastrointestinal are presented with clinical details. The bacteriology of B. anthracis is presented in terms of its minimal diagnostic characteristics, cultivation, colony appearance, along with the use of diagnostic bacteriophage. All of the major outer structural components of B. anthracis (capsule, cell wall, S layer) are pictorially illustrated and discussed. The extracellular enzymes such as hemolysins and phospholipases, in addition to the intracellular superoxide dismutases that presumably influence virulence are presented in detail. Studies on the long-term persistence of B. anthracis spores in soil are described along with stability of the 2 major virulence determining plasmids.

Systemic infections usually result in the patient reaching “the point of no return”. Among the three forms of anthrax: cutaneous, gastrointestinal, and inhalation, the cutaneous form constitutes 95% of all reported human cases. The clinical aspects of all three forms are presented in notable detail with illustrations. Meningeal anthrax infections resulting in the “red Cardinal's cap” are nearly always fatal. The major known virulence factors in anthrax include the cell-surface-associated antiphagocytic poly-Dglutamic acid capsule, the protective antigen (PA), and the edema (ET) and lethal (LT) toxins. Penicillin G, doxycycline, and the fluoroquinolone ciprofloxacin are considered the drugs of choice for the treatment of anthrax.

Both the lethal toxin (LT) and edema toxin (ET) of Bacillus anthracis are binary toxins and each for activity, must be bound to the protective antigen (PA) to enter and exert toxicity on eukaryotic cells. Eukaryotic cells harbor specific PA membrane receptors. Proteolytic activation of PA results in the formation of ring shaped heptamers which form ion-conductive pores in cell membranes with receptors. Each prepore binds a total of three EF and/or LF peptides competitively, and after endocytosis and transport of the complex to an acidic, vesicular compartment, it undergoes membrane insertion and mediates translocation of EF/LF to the macrophage cytosol where it exerts its cytotoxic effect. The transmembrane pore houses an aromatic iris or disk that creates a structural bottleneck in the pore, requiring that the LF and EF peptides unfold in order to be translocated into the cellular cytosol. This restricted site has been termed the “Φ-clamp” or phenylalanine clamp. The Φ-clamp or hydrophobic ratchet is thought to grasp hydrophobic sequences as they unfold from the molten peptide globule to direct the translocating chain through the channel. Treatment of macrophages with LT has been found to influence the expression of 103 genes. LT is proteolytic towards MAPKKs and thereby disrupts intracellular signaling.

A number of attenuated, partially attenuated, and fully virulent strains of B. anthracis have been utilized over the years for vaccine development and animal challenge studies which are fully described. A description of early veterinary vaccine development followed by the development of human vaccines and their relative safety and incidence of adverse reactions are presented in considerable detail. Studies on the elucidation of the major factors of virulence, the lethal and edema toxins and capsule formation and their encoding by plasmids constitutes a major advance in the understanding of the genetic factors influencing anthrax and the critical antigens involved in the development of vaccines. The development of a variety of different potential vaccines are discussed including attenuated whole cell vaccines, unattenuated dead cell vaccines, purified protein based vaccines, recombinant protein based vaccines, vaccines utilizing bacterial vectors, viral vectors, and eukaryotic vectors and the efficacy of various adjuvants are presented in detail. A notably comprehensive presentation of macrophage studies associated with various animal models of anthrax is used to facilitate a clarified presentation of the biochemical factors involved in anthrax infections and their influence on the innate immune system. Detection of B. anthracis in environmental samples is invariably based on detection of spores. A variety of immunological methods are described for the rapid detection of B. anthracis spores in environmental samples.

All isolates of Bacillus anthracis have been found to be genetically homogeneous and are considered to have arisen from a common clone derived from B. cereus. Major virulence factors are the protective antigen (PA), the edema toxin (ET), the lethal toxin (LT) and the poly-D-glutamic capsule. The plasmid pXO1 carries the noncontiguous toxin genes pagA, cya, and lef that encode the PA, ET, and LT proteins respectively. The capsule is produced at 37 °C but not at temperatures below 28 °C and only under condition of elevated CO₂. The plasmid pXO2 encodes the contiguous genes capBCA for capsule synthesis in addition to acpA which is a capsule gene activator. The gene acpA carried by the pXO2 plasmid encodes a positive trans-acting protein involved in the bicarbonate-mediated regulation of capsule synthesis. Molecular techniques used to distinguish isolates of B. anthracis include variable number tandem repeat (VNTR) loci, single nucleotide repeat (SNR) analysis, amplified fragment length polymorphism analysis (AFLP), random amplified polymorphic DNA (RAPD) analysis, multilocus sequence typing (MLST), and long-range repetitive element-PCR (LR REP-PCR), in addition to whole genome typing. Molecular techniques used for detection of B. anthracis include conventional PCR and real-time PCR and those used for detection and characterization include DNA microarrays and pulsed-field gel electrophoresis (PFGE).