Association of Bacillus anthracis capsule with lethal toxin during experimental infection.

Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA(63)) and the residual 20-kDa fragment (PA(20)) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA(63) was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-gamma-d-glutamic acid (gamma-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/gamma-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including gamma-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for gamma-DPGA in anthrax pathogenesis.

Production and validation of the use of gamma phage for identification of Bacillus anthracis.

Gamma phage specifically lyses vegetative cells of Bacillus anthracis and serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent B. anthracis strain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4 degrees C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial B. anthracis-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35 degrees C +/- 2 degrees C for 20 h. When tested with 51 B. anthracis strains and 49 similar non-B. anthracis Bacillus species, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible B. anthracis strains as well as a rare susceptible non-B. anthracis strain, B. cereus ATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with high- and low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.

Current laboratory methods for biological threat agent identification.

The authors present an integrated approach for the identification of biological threat agents. The methods used have been used extensively in field exercises and during response to incidents of biological terrorism. A diagnostic system, which integrates the clinical diagnosis or medical intelligence with immunodiagnostic tests, rapid gene amplification assays, and standard culture, provides results of the highest quality and confidence. In the future, selected reagents and technologies will be distributed through a network of civilian and military laboratories.

Antibiotic treatment of experimental pneumonic plague in mice.

A mouse model was developed to evaluate the efficacy of antibiotic treatment of pneumonic plague; streptomycin was compared to antibiotics with which there is little or no clinical experience. Infection was induced by inhalation of aerosolized Yersinia pestis organisms. Antibiotics were administered by intraperitoneal injection every 6 hours for 5 days, at doses that produced levels of drug in serum comparable to those observed in humans treated for other serious infections. These studies compared in vitro to in vivo activity and evaluated the efficacy of antibiotics started at different times after exposure. Early treatment (started 24 h after challenge, when 0 of 10 mice tested had positive blood cultures) with netilmicin, ciprofloxacin, ofloxacin, ceftriaxone, ceftazidime, aztreonam, ampicillin, and rifampin (but not cefazolin, cefotetan, or ceftizoxime) demonstrated efficacy comparable to streptomycin. Late treatment (started 42 h after exposure, when five of five mice tested had positive blood cultures) with netilmicin, ciprofloxacin, ofloxacin, and a high dose (20 mg/kg of body weight every 6 h) of gentamicin produced survival rates comparable to that with streptomycin, while all of the beta-lactam antibiotics (cefazolin, cefotetan, ceftriaxone, ceftazidime, aztreonam, and ampicillin) and rifampin were significantly inferior to streptomycin. In fact, all groups of mice treated late with beta-lactam antibiotics experienced accelerated mortality rates compared to normal-saline-treated control mice. These studies indicate that netilmicin, gentamicin, ciprofloxacin, and ofloxacin may be alternatives for the treatment of pneumonic plague in humans. However, the beta-lactam antibiotics are not recommended, based upon poor efficacy in this mouse model of pneumonic plague, particularly when pneumonic plague may be associated with bacteremia.

Pathology of experimental inhalation anthrax in the rhesus monkey.

BACKGROUND: The inhalation form of anthrax, although rare, is nearly always fatal because of the rapid progression of the disease with little host response until the terminal stages of the disease. The Gulf War heightened the concern that anthrax could be used as a biologic weapon. Past studies modeling pathologic changes in human inhalation anthrax have used the rhesus monkey. EXPERIMENTAL DESIGN: We studied pathologic changes in the rhesus monkey model of inhalation anthrax. Gross examination as well as light and electron microscopy were used to define pathologic alterations. Immunolabeling techniques were used to identify the anthrax bacillus by light and electron microscopy. RESULTS: Gross changes included hemorrhage in mesenteric (54%) and tracheobronchial (46%) lymph nodes, meninges (38%), lungs (31%), and small intestinal serosa (31%). Histopathologic changes included suppurative meningitis (77%); hemorrhages in the meninges (54%), neuropil (31%), and pulmonary alveoli (31%); and pneumonia (15%). Spleens and various lymph nodes from all monkeys had one or more of the following changes: hemorrhage, acute inflammation, extracellular bacilli, lymphocytic depletion, and histiocytosis. Spleens of two monkeys were devoid of extracellular bacilli, but degraded intrahistiocytic bacilli reacted with Ab to Bacillus anthracis cell wall polysaccharide. CONCLUSIONS: In our study, compared with previous reports, meningitis and mesenteric lymph node hemorrhages were more common, whereas mediastinal and tracheobronchial lymph node hemorrhages were less common. Immunostaining highlighted intracellular bacilli that would have been otherwise missed by light microscopic examination.

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1.

Many prokaryotic organisms possess surface layer (S-layer) proteins that are components of the outermost cell envelope. With immunogold labeling, it was demonstrated that the protein extractable antigen 1 (EA1) was localized on the outer surface and specifically to cell wall fragments from Bacillus anthracis which retained the S layer. When grown in rich medium under aerobic conditions, the avirulent strain Delta Sterne-1 released large amounts of EA1 into the medium. This EA1 had no higher-order structure initially but formed two-dimensional crystals under defined conditions. The released EA1 was purified in aqueous buffers with a three-step procedure and found to have a mass of 95 kDa when subjected to denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence data revealed exact identity to the first eight residues of the S-layer protein from B. thuringiensis 4045. Gel permeation chromatography of the purified EA1 under nondenaturing conditions revealed a single peak corresponding to a mass of approximately 400 kDa, suggesting that a tetramer or dimer of dimers was the primary species in solution. SDS-PAGE of EA1 purified in the absence of protease inhibitors revealed specific proteolytic processing to an 80-kDa form, which immunoreacted with polyclonal anti-EA1 antibodies. This proteolytic cleavage of EA1 to 80 kDa was duplicated with purified EA1 and the protease trypsin or pronase.

Use of immunoelectron microscopy to demonstrate Francisella tularensis.

Three immunoelectron microscopy (IEM) methods were employed to show laboratory-cultivated Francisella tularensis. By the IEM assays, F. tularensis was distinguished from four antigenically distinct gram-negative bacteria. IEM should be a valuable tool for confirming presumptive isolates of F. tularensis and may potentially be useful for demonstrating other medically important bacteria.

Postexposure prophylaxis against experimental inhalation anthrax.

Inhalation anthrax is a rare disease that is almost invariably fatal. This study determined whether a prolonged course of postexposure antibiotics with or without vaccination would protect monkeys exposed to a lethal aerosol dose of Bacillus anthracis when the antibiotic was discontinued. Beginning 1 day after exposure, groups of 10 animals were given penicillin, ciprofloxacin, doxycycline, doxycycline plus vaccination, vaccination alone, or saline. Antibiotics were administered for 30 days and then discontinued. Vaccine was given on days 1 and 15. Two animals died of causes other than anthrax and were not included in the statistical analysis. Nine of 10 controls and 8 of 10 animals given only vaccine died. Each antibiotic regimen completely protected animals while on therapy and provided significant long-term protection upon discontinuance of the drug (penicillin, 7 of 10 survived, P < .02; ciprofloxacin, 8 of 9 survived, P < .002; doxycycline, 9 of 10 survived, P < .002; doxycycline plus vaccination, 9 of 9 survived, P < .0002). Protection against rechallenge was provided by combining postexposure antibiotic treatment with vaccination.

Indigenous human cutaneous anthrax in Texas.

In December 1988 an indigenous case of cutaneous anthrax was identified in Texas. The patient, a 63-year-old male Hispanic from southwest Texas, was a sheep shearer and had a recent history of dissecting sheep that had died suddenly. He experienced an illness characterized by left arm pain and edema. A necrotic lesion developed on his left forearm, with cellulitis and lymphadenopathy. After treatment with oral and intravenous penicillins, the patient fully recovered. Western blot testing revealed a fourfold or greater rise in antibody titer to Bacillus anthracis protective antigen and lethal factor. This represents the first case of indigenous anthrax in Texas in more than 20 years.

Familial outbreak of agricultural anthrax in an area of northern Italy.

Three cases of cutaneous anthrax are reported which occurred in a farming family in northern Italy. Epidemiological studies revealed contact with an infected cow (delivery of a stillborn fetus and slaughter). The cow was slaughtered soon after the delivery; cultures of carcass specimens yielded growth of Bacillus anthracis. The origin of the animal infection was not known. Serum samples were obtained from all 11 members of the family group and randomly from 10 of the 75 cows on the farm, which appeared to be in good health. Tests for antibodies against protective antigen and lethal factor using EIA and Western blot techniques were positive in three subjects (in paired sera) with cutaneous anthrax and in one subject who neither had had direct contact with the infected cow nor showed any sign of anthrax.

Serum protease cleavage of Bacillus anthracis protective antigen.

The protective antigen component of anthrax lethal toxin, produced in vitro, has a molecular mass of 83 kDa. Cell-culture studies by others have demonstrated that upon binding of the 83 kDa protective antigen to cell-surface receptors, the protein is cleaved by an unidentified cell-associated protease activity. The resultant 63 kDa protein then binds lethal factor to form lethal toxin, which has been proposed to be internalized by endocytosis. We found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin. Conversion of protective antigen from 83 to 63 kDa was catalysed by a calcium-dependent, heat-labile serum protease. Except for being complexed to protective antigen, there was no apparent alteration of lethal factor during the course of anthrax infection. The protective antigen-cleaving protease appeared to be ubiquitous among a wide range of animal species, including primates, horses, goats, sheep, dogs, cats and rodents.

Identification of Bacillus anthracis by using monoclonal antibody to cell wall galactose-N-acetylglucosamine polysaccharide.

Guanidine extracts of crude Bacillus anthracis cell wall were used to vaccinate BALB/c mice and to develop monoclonal antibody (MAb) to vegetative cell surface antigens. Two hybridomas selected during this study produced immunoglobulin M immunoglobulins, which appear to be directed to an epitope associated with the galactose-N-acetyl-D-glucosamine polysaccharide. Both demonstrated specificity in their binding to purified B. anthracis cell wall, o-stearoyl-polysaccharide conjugates, and intact, nonencapsulated vegetative cells. The interaction of the MAbs with purified polysaccharide was inhibited by 0.5 M galactose and lactose but not by N-acetylglucosamine, glutamate, glycine, or glycerol. Inhibition by glucose or sucrose was approximately 75% of that seen with galactose. Electron microscopy showed that both MAbs interacted with the cell wall of vegetative cells as well as with the cortex of spores. Neither MAb reacted with encapsulated vegetative cells, such as those from infected guinea pigs, nor did they react with intact spores. After conjugation to fluorescein isothiocyanate, the MAbs stained intensely all B. anthracis strains tested, whereas with two exceptions, none of the strains of 20 other Bacillus spp. was stained. The exceptions, strains of Bacillus cereus, could be differentiated from B. anthracis by being beta-hemolytic on blood agar.

Evaluation of serologic tests for diagnosis of anthrax after an outbreak of cutaneous anthrax in Paraguay.

An outbreak of at least 21 cases of cutaneous anthrax occurred in rural Paraguay. A case-control study revealed that disease was associated with touching the raw meat of an ill cow (odds ration = 16.5, P = .02). Serum drawn from 12 cases and 16 colony and 2 noncolony controls 6 w after the outbreak were analyzed by electrophoretic-immunotransblots (EITB) to detect serum antibodies to the protective antigen (PA) and lethal factor components of anthrax toxin. Serum was also tested by enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to poly-D-glutamic acid capsule. Of 12 cases, 11 had a positive PA screen, for a sensitivity of 91.7% (76.1%-100%, 95% confidence interval [CI]) whereas none of the 18 controls was positive for a specificity of 100% (84.8%, one-sided binomial 95% CI). Only 6 (50%) of 12 cases (21.7%-78.3%, 95% CI) had positive lethal factor titers; all controls were negative. At a cutoff of greater than or equal to 1:32 for antibodies to capsule, 11 (91.7%) of 12 (76.1%-100%, 95% CI) were positive; 16 (88.9%) of 18 controls (74.5%-100%, 95% CI) were negative. These data suggest that the EITB for detection of antibody to PA, and ELISA for detection of anticapsule antibodies are both sensitive for the retrospective diagnosis of anthrax. Both tests were specific, but EITB may be more so than ELISA.

Efficacy of a commercial bacterin in protecting strain 13 guineapigs against Bordetella bronchiseptica pneumonia.

Bordetella bronchiseptica is known to be endemic in many guineapig (Cavia porcellus) colonies, and periodically is the aetiological agent of fatal epizootics of bronchopneumonia. A commercial, non-adjuvant B. bronchiseptica bacterin, which is approved for use in canines, was evaluated for induction of a protective immune response in Strain 13/N guineapigs against an airborne challenge of virulent B. bronchispeptica in small-particle aerosol. Seronegative animals were vaccinated on days 0 and 21 with intramuscular injections of 0.2 ml of bacterin. Humoral antibody titres of the vaccinated animals, as determined by ELISA, ranged from 128-1024 on day 49. On day 30 following the second dose of bacterin (study day 51), 12 vaccinated and 12 PBS sham-vaccinated animals were exposed to an inhaled dose of 4.3 x 10(5) CFU of B. bronchiseptica (325 LD50). Vaccinated, challenged animals remained clinically normal, although each guineapig did develop a localized upper respiratory infection. The rate of weight gain as well as rectal temperature of these animals were analogous to those exhibited by the control groups. Examination of 4 of the vaccinated, challenged animals on day 7 after exposure showed bacteria present in moderate to high numbers in the larynx and trachea but only minimally detectable in the lungs; by 30 days after exposure, the numbers of bacteria in the larynx and trachea were diminished, with none being detected in the lungs. Pathological alterations induced by B. bronchiseptica were not detected at either day 7 or day 30 after challenge in any of the vaccinated, challenged animals. Protection induced in Strain 13/N guineapigs by the commercial canine bacterin was sufficient to preclude the development of pulmonary disease, even in animals presented with a massive challenge of virulent bacteria in a small-particle aerosol.

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens.

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.

Serological studies of patients with cutaneous and oral-oropharyngeal anthrax from northern Thailand.

An outbreak of 52 cases of cutaneous anthrax and 24 cases of oral-oropharyngeal anthrax occurred in rural Northern Thailand in 1982, caused by contaminated water buffalo meat. Microbiologic diagnosis of many of these cases was hindered by delayed presentation for care and by prior antibiotic therapy. In a retrospective investigation, we used enzyme-linked immunosorbent assays to measure antibody titers to components of anthrax edema toxin (edema factor [EF] and protective antigen [PA]), lethal toxin (lethal factor [LF] and PA), and poly-D-glutamic acid capsule. Electrophoretic-immunotransblots (EITB, Western blot) were used to detect antibodies to PA and LF. Nine patients with cutaneous anthrax, 10 patients with oral-oropharyngeal anthrax, and 43 healthy unexposed Thai control villagers were studied. Over all, EITB was positive in 13/18 patients (sensitivity 72%) and 0/43 controls (specificity 100%). The sensitivity of the ELISA was 72% for PA, 42% for LF, 26% for EF, and 95-100% for capsule. Although a few control sera had apparent false positive titers to PA, the specificity of the ELISA confirmed by EITB (100%) demonstrated the applicability of these tests for the diagnosis of anthrax.

Immunological analysis of cell-associated antigens of Bacillus anthracis.

Sera from Hartley guinea pigs vaccinated with a veterinary live spore anthrax vaccine were compared with sera from guinea pigs vaccinated with the human anthrax vaccine, which consists of aluminum hydroxide-adsorbed culture proteins of Bacillus anthracis V770-NP-1R. Sera from animals vaccinated with the spore vaccine recognized two major B. anthracis vegetative cell-associated proteins that were either not recognized or poorly recognized by sera from animals that received the human vaccine. These proteins, termed extractable antigens 1 (EA1) and 2 (EA2), have molecular masses of 91 and 62 kilodaltons, respectively. The EA1 protein appeared to be coded by chromosomal DNA, whereas the EA2 protein was only detected in strains that possessed the pXO1 toxin plasmid. Both of the extractable antigen proteins were serologically distinct from the components of anthrax edema toxin and lethal toxin. Following vaccination with the live spore vaccine, the EA1 protein was the predominant antigen recognized, as determined by electrophoretic immunotransblots. Vaccine trials with partially purified EA1 demonstrated that it neither elicits protective antibody against anthrax nor delays time to death in guinea pigs challenged intramuscularly with virulent Ames strain spores. In addition, animals vaccinated with sterile gamma-irradiated cell walls had significant antibody titers to the N-acetylglucosamine-galactose polysaccharide of B. anthracis but were neither protected nor had a delay in time to death following challenge.