In October 2001, the first inhalational anthrax case in the United States since 1976 was identified in a media company worker in Florida. A national investigation was initiated to identify additional cases and determine possible exposures to Bacillus anthracis. Surveillance was enhanced through health-care facilities, laboratories, and other means to identify cases, which were defined as clinically compatible illness with laboratory-confirmed B. anthracis infection. From October 4 to November 20, 2001, 22 cases of anthrax (11 inhalational, 11 cutaneous) were identified; 5 of the inhalational cases were fatal. Twenty (91%) case-patients were either mail handlers or were exposed to worksites where contaminated mail was processed or received. B. anthracis isolates from four powder-containing envelopes, 17 specimens from patients, and 106 environmental samples were indistinguishable by molecular subtyping. Illness and death occurred not only at targeted worksites, but also along the path of mail and in other settings. Continued vigilance for cases is needed among health-care providers and members of the public health and law enforcement communities.

Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that LT severely impairs the function of dendritic cells--which are pivotal to the establishment of immunity against pathogens--and host immune responses by disrupting the mitogen-activated protein (MAP) kinase intracellular signalling network. Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate antigen-specific T cells in vivo. Furthermore, injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity. These data suggest a role for LT in suppressing host immunity during B. anthracis infections, and represent an immune evasion strategy, where a microbe targets MAP kinases in dendritic cells to disarm the immune response.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

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In this report we describe the structure of the polysaccharide released from Bacillus anthracis vegetative cell walls by aqueous hydrogen fluoride (HF). This HF-released polysaccharide (HF-PS) was isolated and structurally characterized from the Ames, Sterne, and Pasteur strains of B. anthracis. The HF-PSs were also isolated from the closely related Bacillus cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared with those of B. anthracis. The structure of the B. anthracis HF-PS was determined by glycosyl composition and linkage analyses, matrix-assisted laser desorption-time of flight mass spectrometry, and one-and two-dimensional nuclear magnetic resonance spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of 36)-␣-GlcNAc- (134) Generally, the carbohydrate-containing components of the vegetative cell walls of Gram-positive bacteria consist of the extensive peptidoglycan layer, teichoic acids, lipoteichoic acids, capsular polysaccharides, and crystalline cell surface proteins known as S-layer proteins that are often glycosylated (2). However, the B. anthracis cell wall differs in several aspects from this generalized description. First, B. anthracis cells are surrounded by a poly-␥-D-glutamate capsule and not by a polysaccharide capsule. Second, their cell walls do not contain teichoic acid (3), and last, their S-layer proteins are not glycosylated (1, 4). However, glycosyl composition comparisons of the cell walls of B. anthracis, Bacillus cereus, and Bacillus thuringiensis show that they do contain glycosyl residues and that they differ from one another in their glycosyl compositions (5).To date, cell wall carbohydrates from the vegetative cells of members of the B. cereus group have been addressed only to a limited extent (6 -8). All of these carbohydrates are rich in amino glycosyl residues but have variations in the type and amounts of these residues. The study of Ekwunife et al. (6) focused on the glycosyl composition of a carbohydrate polymer released from the cell wall through hydrogen fluoride (HF) treatment (HF treatment releases wall polysaccharides covalently bound via a phosphate bond to the peptidoglycan) of B. anthracis (⌬ Sterne) and found that the HF-released polysaccharide (HF-PS) 3 contained Gal, GlcNAc, and ManNAc in an approximate ratio of 3:2:1. This HF-PS was also further investigated by Mesnage et al. (4). They reported the importance of a pyruvyl substituent with regard to the function of this polysaccharide in anchoring the S-layer proteins to the cell wall.Fox et al. (7) investigated a number of B. anthracis and B. cereus strains for their total cell glycosyl compositions, which showed interesting differences between the different strains. For example, in contrast to the B. anthracis strains, all B. cereus strains investigated contained GalNAc, suggesting possible differences in cell wall architecture in the different Bacillus species cell walls and, possibly, the occurrence of...

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

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The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.Anthrax is caused by infection with Bacillus anthracis, a sporeforming, Gram-positive bacterium. The dormant spore is resistant to extremes of temperature, desiccation, and a variety of chemical treatments. 1 The stability, ease of production, and infectious capacity of the spores confer B. anthracis with high potential as a biological weapon. 2 B. anthracis spores gain entry through a dermal abrasion or gastrointestinal lesion causing cutaneous or gastrointestinal anthrax, respectively, or are inhaled, causing pulmonary anthrax. Systemic infection from the progression of any of the three forms of anthrax frequently results in secondary shock, multiple organ failure, and death. 3 Early diagnosis is critical for effective treatment of pulmonary (inhalation) anthrax. 4 In the U.S. bioterrorism attacks of 2001, pulmonary anthrax had a 45% fatality rate despite antibiotic treatment and aggressive supportive care of the patients. 5,6 The two exotoxins of B. anthracis are binary combinations of protective antigen (PA) and either edema factor (EF) or lethal factor (LF). 7 The complex of PA and EF forms edema toxin (ETx) and PA complexed with LF forms lethal toxin (LTx). PA is secreted as an 83-kDa protein (PA83), which binds to known receptors TEM8 (tumor endothelium marker 8) 8 and CMG2 (capillary morphogenesis protein 2) 9 on the surface of target cells where it is cleaved by a furin-like cell surface protease to a 63-kDa protein (PA63). 10 PA83 may also be cleaved to the PA63 conformer by serum proteases. 11 Cleavage causes a conformational