In 1993, the Aum Shinrikyo cult aerosolized Bacillus anthracis spores over Kameido, Japan. Spore samples were obtained from the release site, cultured, and characterized by molecular genetic typing. The isolates were consistent with strain Sterne 34F2, which is used in Japan for animal prophylaxis against anthrax.
Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the “gold standards,” doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log10 CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log10 CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log10 CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation.
We used unpublished reports, published manuscripts, and communication with investigators to identify and summarize 49 anthrax-related epidemiologic field investigations conducted by the Centers for Disease Control and Prevention from 1950 to August 2001. Of 41 investigations in which Bacillus anthracis caused human or animal disease, 24 were in agricultural settings, 11 in textile mills, and 6 in other settings. Among the other investigations, two focused on building decontamination, one was a response to bioterrorism threats, and five involved other causes. Knowledge gained in these investigations helped guide the public health response to the October 2001 intentional release of B. anthracis, especially by addressing the management of anthrax threats, prevention of occupational anthrax, use of antibiotic prophylaxis in exposed persons, use of vaccination, spread of B. anthracis spores in aerosols, clinical diagnostic and laboratory confirmation methods, techniques for environmental sampling of exposed surfaces, and methods for decontaminating buildings.
anthrax; Bacillus anthracis; bacterial infections; disease outbreaks; public health; bioterrorism; Centers for Disease Control and Prevention (U.S.); historical article (publication type); zoonoses
Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified α-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that α-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.
Bacillus anthracis is the bacterium that causes anthrax, a disease that can occur through natural infections and also through intentional release. B. anthracis makes spores, which are in a dormant state, similar to seeds of a plant, and are extremely resistant to the environment. B. anthracis spores can infect through the skin or the lung. Lung infections disseminate through the body and are lethal. In contrast, skin infections often remain localized, and patients survive even without treatment. It is not well understood why these bacteria cause a localized infection through the skin and a lethal disease through the lung.
Little is known about how B. anthracis is controlled. Neutrophils are the first white blood cells recruited to a site of infection and are specialized in killing microbes. Previous studies show that neutrophils are abundant in the skin form, but not in the lung form of anthrax. The researchers report that human neutrophils can take up B. anthracis spores. Once inside, the spores germinate to form vegetative bacteria. The vegetative bacteria are extremely susceptible to neutrophil-killing mechanisms. The B. anthracis virulence factors (molecules that make bacteria cause diseases) manipulate other human cells but do not deter neutrophils. B. anthracis is indeed exquisitely sensitive to the neutrophil protein α-defensin. These data support a new model where B. anthracis skin, but not lung, infections are controlled by the antimicrobial activity of neutrophils.
Live, attenuated strains of Bacillus anthracis lacking either the capsule plasmid pXO2, the toxin plasmid pXO1, or both were tested for their efficacy as vaccines against intravenous challenge with anthrax toxin in Fischer 344 rats and against aerosol or intramuscular challenge with virulent anthrax spores in Hartley guinea pigs. Animals immunized with toxigenic, nonencapsulated (pXO1+, pXO2-) strains survived toxin and spore challenge and demonstrated postimmunization antibody titers to the three components of anthrax toxin (protective antigen, lethal factor, and edema factor). Immunization with two nontoxigenic, encapsulated (pXO1-, pXO2+), Pasteur vaccine strains neither provided protection nor elicited titers to any of the toxin components. Therefore, to immunize successfully against anthrax toxin or spore challenge, attenuated, live strains of B. anthracis must produce the toxin components specified by the pXO1 plasmid.
Bacillusanthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 103 spores was achieved by incubating spores simultaneously with capture and detection antibodies (‘liquid-phase” assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.
Bacillus anthracis; anthrax; bioterrorism agent; immunological detection; spore germination; spore viability
While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.
Inhalation anthrax is a potentially lethal form of disease resulting from exposure to aerosolized Bacillus anthracis spores. Over the last decade, incidents spanning from the deliberate mailing of B. anthracis spores to incidental exposures in users of illegal drugs have highlighted the importance of developing new medical countermeasures to protect people who have been exposed to “anthrax spores” and are at risk of developing disease. The New Zealand White rabbit (NZWR) is a well-characterized model that has a pathogenesis and clinical presentation similar to those seen in humans. This article reports how the NZWR model was adapted to evaluate postexposure prophylaxis using a recombinant protective antigen (rPA) vaccine in combination with an oral antibiotic, levofloxacin. NZWRs were exposed to multiples of the 50% lethal dose (LD50) of B. anthracis spores and then vaccinated immediately (day 0) and again on day 7 postexposure. Levofloxacin was administered daily beginning at 6 to 12 h postexposure for 7 treatments. Rabbits were evaluated for clinical signs of disease, fever, bacteremia, immune response, and survival. A robust immune response (IgG anti-rPA and toxin-neutralizing antibodies) was observed in all vaccinated groups on days 10 to 12. Levofloxacin plus either 30 or 100 μg rPA vaccine resulted in a 100% survival rate (18 of 18 per group), and a vaccine dose as low as 10 μg rPA resulted in an 89% survival rate (16 of 18) when used in combination with levofloxacin. In NZWRs that received antibiotic alone, the survival rate was 56% (10 of 18). There was no adverse effect on the development of a specific IgG response to rPA in unchallenged NZWRs that received the combination treatment of vaccine plus antibiotic. This study demonstrated that an accelerated two-dose regimen of rPA vaccine coadministered on days 0 and 7 with 7 days of levofloxacin therapy results in a significantly greater survival rate than with antibiotic treatment alone. Combination of vaccine administration and antibiotic treatment may be an effective strategy for treating a population exposed to aerosolized B. anthracis spores.
Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. We previously described the biological activity of a nontoxic mutant of PA expressed in Bacillus subtilis. In the present study, we evaluated the efficacy of the purified mutant PA protein alone or in combination with the lethal factor and edema factor components of anthrax toxin to protect against anthrax. Both mutant and native PA preparations elicited high anti-PA titers in Hartley guinea pigs. Mutant PA alone and in combination with lethal factor and edema factor completely protected the guinea pigs from B. anthracis spore challenge. The results suggest that the mutant PA protein may be used to develop an effective recombinant vaccine against anthrax.
Bacillus anthracis is a pathogen that causes life-threatening disease--anthrax. B. anthracis spores are highly resistant to extreme temperatures and harsh chemicals. Inactivation of B. anthracis spores is important to ensure the environmental safety and public health. The 2001 bioterrorism attack involving anthrax spores has brought acute public attention and triggered extensive research on inactivation of B. anthracis spores. Single-walled carbon nanotubes (SWCNTs) as a class of emerging nanomaterial have been reported as a strong antimicrobial agent. In addition, continuous near infrared (NIR) radiation on SWCNTs induces excessive local heating which can enhance SWCNTs’ antimicrobial effect. In this study, we investigated the effects of SWCNTs coupled with NIR treatment on Bacillus anthracis spores.
Results and discussion
The results showed that the treatment of 10 μg/mL SWCNTs coupled with 20 min NIR significantly improved the antimicrobial effect by doubling the percentage of viable spore number reduction compared with SWCNTs alone treatment (88% vs. 42%). At the same time, SWCNTs-NIR treatment activated the germination of surviving spores and their dipicolinic acid (DPA) release during germination. The results suggested the dual effect of SWCNTs-NIR treatment on B. anthracis spores: enhanced the sporicidal effect and stimulated the germination of surviving spores. Molecular level examination showed that SWCNTs-NIR increased the expression levels (>2-fold) in 3 out of 6 germination related genes tested in this study, which was correlated to the activated germination and DPA release. SWCNTs-NIR treatment either induced or inhibited the expression of 3 regulatory genes detected in this study. When the NIR treatment time was 5 or 25 min, there were 3 out of 7 virulence related genes that showed significant decrease on expression levels (>2 fold decrease).
The results of this study demonstrated the dual effect of SWCNTs-NIR treatment on B. anthracis spores, which enhanced the sporicidal effect and stimulated the germination of surviving spores. SWCNTs-NIR treatment also altered the expression of germination, regulatory, and virulence-related genes in B. anthracis.
Single walled carbon nanotubes; Bacillus anthracis spores; Near infrared radiation; Germination; Dipicolinic acid; Gene expression
The CDC recommend 60 days of oral antibiotics combined with a three-dose series of the anthrax vaccine for prophylaxis after potential exposure to aerosolized Bacillus anthracis spores. The anthrax vaccine is currently not licensed for anthrax postexposure prophylaxis and has to be made available under an Investigational New Drug protocol. Postexposure prophylaxis based on antibiotics can be problematic in cases where the use of antibiotics is contraindicated. Furthermore, there is a concern that an exposure could involve antibiotic-resistant strains of B. anthracis. Availability of alternate treatment modalities that are effective in prophylaxis of inhalation anthrax is therefore highly desirable. A major research focus toward this end has been on passive immunization using polyclonal and monoclonal antibodies against B. anthracis toxin components. Since 2001, significant progress has been made in isolation and commercial development of monoclonal and polyclonal antibodies that function as potent neutralizers of anthrax lethal toxin in both a prophylactic and therapeutic setting. Several new products have completed Phase I clinical trials and are slated for addition to the National Strategic Stockpile. These rapid advances were possible because of major funding made available by the US government through programs such as Bioshield and the Biomedical Advanced Research and Development Authority. Continued government funding is critical to support the development of a robust biodefense industry.
antibiotic treatment; biodefense funding; inhalation anthrax; lethal factor; medical countermeasures; prophylactic antibodies; protective antigen; vaccination
Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming bacterium. The inhalational form of anthrax is the most severe and is associated with rapid progression of the disease and the outcome is frequently fatal. Transfer from the respiratory epithelium to regional lymph nodes appears to be an essential early step in the establishment of infection. This transfer is believed to occur by means of carriage within alveolar macrophages following phagocytosis. Therefore, the ability of B. anthracis to transit through the host macrophage or dendritic cell appears to be an early and critical step in B. anthracis pathogenesis. In this work, we examined the cytokine responses to spore infection in mouse primary peritoneal macrophages, in primary human dendritic cells, and during a spore aerosol infection model utilizing the susceptible A/J mouse strain. We demonstrated that both mouse peritoneal macrophages and human dendritic cells exhibited significant intracellular bactericidal activity during the first hours following uptake, providing the necessary time to mount a cytokine response prior to cell lysis. Strong tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) responses were seen in mouse peritoneal macrophages. In addition to TNF-α and IL-6, human dendritic cells produced the cytokines IL-1β, IL-8, and IL-12. A mixture of Th1 and Th2 cytokines were detected in sera obtained from infected animals. In this study, we provide further evidence of an acute cytokine response when cells in culture and mice are infected with B. anthracis spores.
The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores limit their practical use in early stages of product development. Conversely, the small size and low cost associated with using mice makes this animal model more practical for conducting experiments in which large numbers of animals are required. In addition, the availability of knockout strains and well-characterized immunological reagents makes it possible to perform studies in mice that cannot be performed easily in other species. Although we, along with others, have used the mouse aerosol challenge model to examine the outcome of B. anthracis infection, a detailed characterization of the disease is lacking. The current study utilizes a murine aerosol challenge model to investigate disease progression, innate cytokine responses, and histological changes during the course of anthrax after challenge with aerosolized spores. Our results show that anthrax disease progression in a complement-deficient mouse after challenge with aerosolized Sterne spores is similar to that described for other species, including rabbits and non-human primates, challenged with fully virulent B. anthracis. Thus, the murine aerosol challenge model is both useful and relevant and provides a means to further investigate the host response and mechanisms of B. anthracis pathogenesis.
Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 × 107 spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (≥100 μg/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.
The development of new approaches to combat anthrax requires that the pathogenesis and host response to Bacillus anthracis spores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection by B. anthracis in a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the animals were then infected intraperitoneally or by aerosol challenge with fully virulent, ungerminated B. anthracis strain Ames spores. The macrophage-depleted mice were significantly more susceptible to the ensuing infection than the saline-pretreated mice, whereas the differences observed between the neutropenic mice and the saline-pretreated controls were generally not significant. We also found that augmenting peritoneal neutrophil populations before spore challenge did not increase resistance of the mice to infection. In addition, the bacterial load in macrophage-depleted mice was significantly greater and appeared significantly sooner than that observed with the saline-pretreated mice. However, the bacterial load in the neutropenic mice was comparable to that of the saline-pretreated mice. These data suggest that, in our model, neutrophils play a relatively minor role in the early host response to spores, whereas macrophages play a more dominant role in early host defenses against infection by B. anthracis spores.
Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.
MyD88-deficient mice were previously shown to have increased susceptibility to Bacillus anthracis infection relative to wild-type animals. To determine the mechanism by which MyD88 protects against B. anthracis infection, knockout mice were challenged with nonencapsulated, toxigenic B. anthracis or with anthrax toxins. MyD88-deficient mice had increased susceptibility to B. anthracis and anthrax lethal toxin but not to edema toxin. Lethal toxin alone induced marked multifocal intestinal ulcers in the knockout animals, compromising the intestinal epithelial barrier. The resulting enteric bacterial leakage in the knockout animals led to peritonitis and septicemia. Focal ulcers and erosion were also found in MyD88-heterozygous control mice but with far lower incidence and severity. B. anthracis infection also induced a similar enteric bacterial septicemia in MyD88-deficient mice but not in heterozygous controls. We show that lethal toxin and B. anthracis challenge induce bacteremia as a result of intestinal damage in MyD88-deficient mice. These results suggest that loss of the intestinal epithelial barrier and enteric bacterial septicemia may contribute to sensitizing MyD88-deficient mice to B. anthracis and that MyD88 plays a protective role against lethal toxin-induced impairment of intestinal barrier.
Bacillus anthracis, the etiological agent of anthrax, is a spore-forming Gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs), sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A–dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deathly pathogen.
All mammals are susceptible to infection by Bacillus anthracis, the etiological agent of anthrax. Infection can occur either accidentally or as a potential consequence of a terrorism threat. Pulmonary infection is the most life-threatening form of the disease, causing a near 100% mortality. Despite appropriate therapy, all forms of infection may progress to fatal systemic anthrax, characterized by sepsis and respiratory failure. Thus, it is important to understand the mechanisms of host defense against B. anthracis. We have previously shown that alveolar macrophages produce an enzyme involved in innate defense that can kill B. anthracis: the enzyme is known as secreted phospholipase A2-IIA (sPLA2-IIA). The alveolar macrophage is one of the first cell types to come in contact with B. anthracis. In this study, we show that live B. anthracis spores stimulate the synthesis of sPLA2-IIA, this stimulation being counterbalanced by the inhibitory effect of the edema toxin produced by germinated spores and bacilli. Our study suggests that inhibition of sPLA2-IIA synthesis by edema toxin is a mechanism by which B. anthracis can escape innate host defense. These pioneering data provide new molecular targets for future intervention against this deadly pathogen.
Bacillus anthracis spores are the infectious form of the organism for humans and animals. However, the approved human vaccine in the United States is derived from a vegetative culture filtrate of a toxigenic, nonencapsulated B. anthracis strain that primarily contains protective antigen (PA). Immunization of mice with purified spore proteins and formalin-inactivated spores (FIS) from a nonencapsulated, nontoxigenic B. anthracis strain confers protection against B. anthracis challenge when PA is also administered. To investigate the capacity of the spore particle to act as a vaccine without PA, we immunized mice subcutaneously with FIS from nontoxigenic, nonencapsulated B. cereus strain G9241 pBCXO1−/pBC210− (dcG9241), dcG9241 ΔbclA, or 569-UM20 or with exosporium isolated from dcG9241. FIS vaccination provided significant protection of mice from intraperitoneal or intranasal challenge with spores of the virulent B. anthracis Ames or Ames ΔbclA strain. Immunization with dcG9241 ΔbclA FIS, which are devoid of the immunodominant spore protein BclA, provided greater protection from challenge with either Ames strain than did immunization with FIS from BclA-producing strains. In addition, we used prechallenge immune antisera to probe a panel of recombinant B. anthracis Sterne spore proteins to identify novel immunogenic vaccine candidates. The antisera were variably reactive with BclA and with 10 other proteins, four of which were previously tested as vaccine candidates. Overall our data show that immunization with FIS from nontoxigenic, nonencapsulated B. cereus strains provides moderate to high levels of protection of mice from B. anthracis Ames challenge and that neither PA nor BclA is required for this protection.
Animal species differ in their resistance both to infection by Bacillus anthracis and to anthrax toxin. A mouse model was developed to study the basis of the host differences and the pathogenesis of infection. When mice were infected with the virulent B. anthracis strain Vollum 1B, low 50% lethal dose (LD50) values (5 to 30 spores) were found for all 10 strains of inbred mice tested. However, analysis of time-to-death data revealed significant differences among the strains, which could be divided into three groups: most susceptible (A/J and DBA/2J); least susceptible (CBA/J, BALB/cJ, and C57BR/cdJ); and intermediate (the remaining five strains). In contrast, the mice were distinctly susceptible or resistant to lethal infection by the toxigenic, nonencapsulated Sterne vaccine strain. The LD50 for the susceptible A/J and DBA/2J mice was approximately 10(3) spores of the Sterne strain, whereas the remaining eight relatively resistant strains were killed only by 10(6) or more spores. F1 hybrid and backcross studies suggested that resistance to the Sterne strain is determined by a single dominant gene or gene complex. Mice lethally infected with B. anthracis showed an acute course of infection, characterized by extensive gelatinous edema and large concentrations of bacilli in the blood and organs (e.g., 10(9) CFU/g of spleen). The susceptibility of A/J and CBA/J mice to intravenously injected anthrax toxin components appeared to differ from their susceptibility to infection. The toxin LD50 values for both strains were similar. However, CBA/J mice died sooner than did A/J mice, with mean time to death of 0.9 and 3.7 days, respectively, in mice given 4 LD50 of toxin. The mouse model appears to be useful in studies on host resistance to anthrax and on the pathogenesis of the infection.
In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.
Anthrax; Bacillus anthracis; Bioterrorism; 16S rRNA gene
In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.
A quantitative immunofluorescence assay based on fiber optic microscopy was used to measure the reaction of formalized spores of Bacillus anthracis and Bacillus cereus isolates with fluorescein conjugates prepared by hyperimmunization with B. anthracis Vollum spores. The spores of 11 of the 20 B. cereus strains reacted with the anti-anthrax conjugate to such an extent that they were indistinguishable from the spores of the several B. anthracis isolates tested. However, absorption of the conjugate with spores of B. cereus NCTC 8035 and B. cereus NCTC 10320 greatly reduced the cross-reaction with the B. cereus preparations so that the mean specific fluorescence of samples of B. cereus spores was in no case higher than 14% of the fluorescence of a reference B. anthracis Vollum preparation.
Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Coinoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.
The lack of identified exposures in 2 of the 11 cases of bioterrorism-related inhalation anthrax in 2001 raised uncertainty about the infectious dose and transmission of Bacillus anthracis. We used the Wells-Riley mathematical model of airborne infection to estimate 1) the exposure concentrations in postal facilities where cases of inhalation anthrax occurred and 2) the risk for infection in various hypothetical scenarios of exposure to B. anthracis aerosolized from contaminated mail in residential settings. These models suggest that a small number of cases of inhalation anthrax can be expected when large numbers of persons are exposed to low concentrations of B. anthracis. The risk for inhalation anthrax is determined not only by bacillary virulence factors but also by infectious aerosol production and removal rates and by host factors.
Anthrax; Air microbiology; Infection; Risk; Inhalation exposure; Lethal Dose 50; Ventilation