Sterne Spores Research Articles

Resistance of Athymic Nude Mice to Experimental CutaneousBacillus anthracisInfection

Previous studies in a murine cutaneous anthrax model have demonstrated that hairless and haired HRS/J mice are extremely resistant to Bacillus anthracis. Because these mice are relatively thymus deficient, we used C57BL/6 athymic nude and euthymic mice to evaluate the relationship between T cell deficiency and this heightened resistance. Animals were epicutaneously inoculated with 1 X 10(7) B. anthracis (Sterne) spores onto abraded skin or injected with the spores intradermally or subcutaneously. The mice were then either monitored for survival or killed for quantitative histological experiments. Athymic mice were found to be markedly resistant to all 3 inoculation routes, compared with euthymic C57BL/6 mice. Athymic mice rendered leukopenic with cyclophosphamide became susceptible. Histological examination demonstrated increased inflammation and absence of organisms in the skin of athymic mice, compared with euthymic ones. The numbers of organisms in the athymic animals increased markedly after cyclophosphamide treatment. Superficial exudate fluids of inoculated skin showed many more neutrophils and ingested bacilli in the athymic mice. These experiments demonstrate that athymic nude C57BL/6 mice are markedly resistant to experimental cutaneous anthrax, apparently because of a superficial neutrophilic response that clears the inoculated organisms before they can invade the underlying skin.

Transcriptional and apoptotic responses of THP-1 cells to challenge with toxigenic, and non-toxigenic Bacillus anthracis

BackgroundBacillus anthracis secretes several virulence factors targeting different host organs and cell types during inhalational anthrax infection. The bacterial expression of a key virulence factor, lethal toxin (LeTx) is closely tied to another factor, edema toxin (EdTx). Both are transcribed on the same virulence plasmid (pXO1) and both have been the subject of much individual study. Their combined effect during virulent anthrax likely modulates both the global transcriptional and the phenotypic response of macrophages and phagocytes. In fact, responses brought about by the toxins may be different than each of their individual effects.ResultsHere we report the transcriptional and apoptotic responses of the macrophage-like phagocytic cell line THP-1 exposed to B. anthracis Sterne (pXO1+) spores, and B. anthracis Δ Sterne (pXO1-) spores. These cells are resistant to LeTx-induced cytolysis, a phenotype seen in macrophages from several mouse strains which are sensitive to toxigenic anthrax infection. Our results indicate that the pXO1-containing strain induces higher pro-inflammatory transcriptional responses during the first 4 hours of interaction with bacterium, evident in the upregulation of several genes relevant to Nf-κB, phosphatases, prostaglandins, and TNF-α, along with decreases in expression levels of genes for mitochondrial components. Both bacterial strains induce apoptosis, but in the toxigenic strain-challenged cells, apoptosis is delayed.ConclusionThis delay in apoptosis occurs despite the much higher level of TNF-α secretion induced by the toxigenic-strain challenge. Interestingly, CFLAR, an important apoptotic inhibitor which blocks apoptosis induced by large amounts of extracellular TNF-α, is upregulated significantly during toxigenic-strain infection, but not at all during non-toxigenic-strain infection, indicating that it may play a role in blocking or delaying TNF-α-mediated apoptosis. The suppression of apoptosis by the toxigenic anthrax strain is consistent with the notion that apoptosis itself may represent a protective host cell response.

Influence of Nisin on the Resistance of Bacillus anthracis Sterne Spores to Heat and Hydrostatic Pressure

The influence of nisin on the heat and pressure resistance of Bacillus anthracis Sterne spores was examined. The decimal reduction times (D-value) of spores in milk (2% fat) at 80, 85, and 90°C were determined. In the absence of nisin, the D-values were 30.09, 9.30, and 3.86 min, respectively. The D-values of spores heated in the presence of nisin (1 mg/ml) were not significantly different (P = 0.05). However, spores heated in the presence of nisin had a 1- to 2-log reduction in viability, after which the death kinetics became similar to those of spores in the absence of nisin. The z-values all were 11.2°C regardless of the presence or absence of nisin. The pressure sensitivity of B. anthracis Sterne spores in the presence and absence of nisin also was determined. Spores treated with nisin were 10 times more pressure sensitive than were spores subjected to pressure in the absence of nisin under the conditions used in this study.

Progressive and destructive hair follicle infections in a murine cutaneous anthrax model

Hair follicles may allow pathogen entry because they represent potential barrier defects and because there is immunological privilege within actively growing follicles. Experimental cutaneous Bacillus anthracis infections in mice have previously shown prominent organism invasion and proliferation within hair follicles. For the present study, C57BL/6 mice were inoculated with B. anthracis (Sterne) spores onto abraded skin with either anagen (actively growing) or telogen (inactive) hair follicles; skin samples were evaluated by histologic methods and electron microscopy. The infections were found to progress similarly in either anagen or telogen hair follicles, with bacilli occasionally invading deeper sites in anagen hair follicles. The infections progressed from the surface inward, rather than growing outward from within the follicles. Infecting bacilli destroyed the hair follicle keratinocytes and were initially not contacted by inflammatory cells within the follicles. However, at 3–4 days after inoculation, inflammatory cells did contact and disperse the massed follicle bacilli and led to apparent resolution of the follicle infections. Therefore, in this model system B. anthracis initially attacks superficial sites in active or inactive hair follicles and then progresses inward, producing destructive infections of the hair follicles; these infections clear when the massed bacilli are eventually contacted and dispersed by inflammatory cells.

Targeting Host Cell Furin Proprotein Convertases as a Therapeutic Strategy against Bacterial Toxins and Viral Pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.

Detection of Bacillus anthracis spores in liquid using phage-based magnetoelastic micro-resonators

This paper presents the results of an investigation to detect Bacillus anthracis spores in liquids using microfabricated magnetoelastic particles (MEP). Free standing, magnetoelastic resonating particles, as small as 500 μm × 100 μm × 4 μm, have been made using microelectronics fabrication techniques. These magnetoelastic resonators feature wireless signal transmission and are suitable for use in air and liquids. Affinity-selected filamentous bacteriophage was employed as the bio-molecular recognition agent and was immobilized onto all surfaces of the resonators. This phage was selected from a landscape phage library specifically for binding B. anthracis spores, the causative agent for the disease anthrax. The biosensors were tested in B. anthracis spore solutions with concentrations from 103 to 108 cfu/ml. A detection limit of 103 cfu/ml, with a sensitivity of 6.5 kHz/decade was observed. Scanning electron microscopy (SEM) micrographs of the tested sensors were used for visual correlation of the sensors’ responses. To investigate specificity, the biosensors were exposed to mixed spore solutions containing B. anthracis Sterne, Bacillus cereus and Bacillus megaterium spore strains. The phage-coated biosensors were proven to have better binding preference to B. anthracis spores than other strains. The study on the binding kinetics have shown that the dissociation constant (Kd) and the binding valency for the sensor in B. anthracis spores solutions are 193 cfu/ml and 2.32.

Human Lung Innate Immune Response toBacillus anthracisSpore Infection

Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1alpha/beta, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines.

Transcriptional stimulation of anthrax toxin receptors by anthrax edema toxin and Bacillus anthracis Sterne spore

We used quantitative real-time RT-PCR to not only investigate the mRNA levels of anthrax toxin receptor 1 (ANTXR1) and 2 (ANTXR2) in the murine J774A.1 macrophage cells and different tissues of mice, but also evaluate the effect of anthrax edema toxin and Bacillus anthracis Sterne spores on the expression of mRNA of these receptors. The mRNA transcripts of both receptors were detected in J774A.1 cells and mouse tissues such as the lung, heart, kidney, spleen, stomach, jejunum, brain, skeleton muscle, and skin. The ANTXR2 mRNA level was significantly higher than that of ANTXR1 in J774A.1 cells and all tissues examined. The mRNA expression of both receptors in the lung was the highest among the tissues evaluated. Interestingly, the mRNA expression of both receptors in J774A.1 cells was upregulated by edema toxin. In addition, ANTXR mRNA expression in the lung was downregulated after subcutaneous inoculation of B. anthracis Sterne spores as well as after intranasal administration of anthrax toxin-based vaccine BioThrax™. These results suggest that anthrax edema toxin and B. anthracis Sterne spore are involved in the ANTXR mRNA regulation in host cells.

Murine Aerosol Challenge Model of Anthrax

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.

28 ERK, P38, AND JNK SIGNALING PATHWAYS ARE IMPORTANT IN CHEMOKINE AND CYTOKINE INDUCTION BY BACILLUS ANTHRACIS SPORES IN A HUMAN LUNG SLICE MODEL.

<i>Bacillus anthracis</i>, the causative agent of inhalational anthrax, enters a host through the pulmonary system before disseminating throughout the body. Our previous work has shown that human alveolar macrophages play a critical role in the initial innate immune response to <i>B. anthracis</i> spores through cell signal-mediated cytokine release. We propose that the lung epithelia also play an important role in the innate immune response to pathogens, and we have developed a human lung slice model to study this process. Exposure of our lung slice model to <i>B. anthracis</i> (Sterne) spores caused rapid activation of the mitogen-activated protein kinase signaling pathways ERK, P38, and JNK. This was followed by an increase in mRNA of several cytokines and chemokines. This was reflected on a translational level with a peak fold increase of TNF-α, IL-6, IL-8, MIP-1α, and the MCP-1 protein of 25, 3, 9, 34, and 5, respectively, as determined by ELISA. Inhibition of individual pathways by the signaling inhibitors UO126, SP 600125, and SB 0203580 was not sufficient to block induction of chemokines and cytokines to background levels. When the three inhibitors were combined, induction of IL-6 and IL-8 was completely blocked and of MCP-1 and MIP-1α was partially blocked. Taken together, these data show activation of pulmonary epithelium in response to <i>B. anthracis</i> spore exposure. Thus, the lung epithelia actively participate in the innate immune response to <i>B. anthracis</i> infection through cell signal-mediated elaboration of cytokines and chemokines.

Characteristics of Spore Germination in a Mouse Model of Cutaneous Anthrax

Cutaneous infection is the most common form of human anthrax, but little is known about Bacillus anthracis spore germination in these infections. We used experimental inoculations of B. anthracis Sterne spores or vegetative bacilli onto intact or abraded mouse flank skin, followed by evaluation of the infections and enumeration of germinating spores and vegetative bacilli. Bacilli developed from a spore inoculum after application onto abraded, but not intact, skin of the mice. Germination appeared to occur extracellularly at the skin surface before the development of a phagocytic response; in fact, vegetative bacilli were seen after inoculation of the spores on top of a filter that separated them from the host phagocytic cells below. Malachite green staining demonstrated that spores began germinating 1-3 h after inoculation onto abraded skin. Vegetative bacilli were found not to be capable of initiating infection in the absence of cutaneous abrasion. The results indicate that epidermal damage is required for germination of B. anthracis spores in these infections; even so, spore germination by itself is not sufficient to produce infection of undamaged skin. In contrast to events in experimental inhalational anthrax, spore germination in these cutaneous infections appears to occur extracellularly.

Protection against anthrax by needle-free mucosal immunization with human anthrax vaccine

Human vaccination with BioThrax™ requires six injections followed by annual boosters. This makes it difficult for the compliance of the immunization program and underscores the need for development of a new and optimized vaccination protocol. Current research aims to demonstrate the proof of concept to develop a needle-free mucosal immunization protocol using a murine anthrax model. A/J mice were immunized with BioThrax™ via an intranasal route. Sera, saliva, vaginal, and nasal washes were evaluated for protective antigen (PA) specific antibody responses by ELISA. Antigen-specific, antibody-secreting lymphocytes were measured by ELISPOT. Sera neutralization antibody titers were determined by in vitro anthrax lethal toxin (Letx) neutralization assay. Immunized animals were challenged by a lethal dose of Bacillus anthracis Sterne spores to determine the efficacy of the vaccination. Nasal mucosal immunization with BioThrax™ elicited robust serum and mucosal antibody responses against PA. The antigen specific antibodies neutralized anthrax Letx, as demonstrated by in vitro neutralization assays. Two doses of intranasal BioThrax™ were sufficient to completely protect A/J mice against challenge with 100 × LD 50 B. anthracis Sterne spores. The data suggests that intranasal administration may be an effective immunization modality for an improved immunization program against anthrax.

Experimental cutaneous Bacillus anthracis infections in hairless HRS/J mice

Previous studies of experimental Bacillus anthracis cutaneous infections in mice have implicated hair follicles as a likely entry site. Hairless HRS/J mice were used to investigate this possibility because of their non-functional hair follicles that lack penetrating hair shafts. These mice also have diminished macrophage function, increased susceptibility to Listeria, and enhanced neutrophil responses. HRS/J and Balb/c mice were found to be resistant to epicutaneous inoculation with Bacillus anthracis (Sterne) spores onto abraded skin when compared with DBA/2 mice or leucopenic C57BL/6 mice. The HRS/J mice also resisted spore injections that bypassed hair follicles. Haired HRS/J heterozygote mice demonstrated similar reduced susceptibility to B. anthracis spores. Hairless HRS/J mice that were made leucopenic did become susceptible to the epicutaneous spore inoculations. Histologically, the hairless and haired HRS/J mice showed markedly reduced numbers of organisms in hair follicles and the interfollicular dermis when compared even with the resistant Balb/c mice; inflammatory cell infiltrates in the superficial dermis were increased in the HRS/J mice compared with more sensitive strains. Therefore, resistance in the HRS/J mice was apparent at the initial site of epicutaneous inoculation and seemed related to an accumulation of dermal neutrophils rather than to a lack of functional hair follicles.

Standard Practice for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents from Nonporous Surfaces: Collaborative Study

The draft ASTM Standard, "Standard Practice for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents from Nonporous Surfaces," was validated in a collaborative study consisting of 6 teams comprised of Civil Support personnel and First Responders, 2 levels of Bacillus anthracis Sterne and Bacillus thuringiensis Kurstaki spores, and 7 nonporous surfaces. The sample collection standard includes collection of the bulk sample (Method A) using a dry swab to push the sample onto a collection card and collection of residual sample (Method B) using an onsite test kit followed by a wet swab intended for additional onsite testing. Method A is to be performed prior to Method B in order to preserve unadulterated sample as potential criminal evidence. While statistical differences were observed between surfaces, between teams, and the interaction of surfaces and teams for the various sample types collected, these differences are due to the very low variability of the data and a much more narrow distribution than an ideal normal distribution, rather than to any practical differences. The data demonstrate that from both the 1.0 and 0.01 g powder samples, high levels of spores (mean >10(6) CFU) are recovered from the 7 surfaces by both the dry swab used in bulk sample collection (Method A) and the wet swab (Method B) sampling of the residual powder after bulk sample collection. Thus, after bulk sample collection, there is a high level of residual spores remaining for onsite biological testing and both Methods A and B should be performed in the field.

Synthesis and Antigenic Analysis of the BclA Glycoprotein Oligosaccharide from the Bacillus anthracis Exosporium

The glycoprotein BclA is an important constituent of the exosporium of Bacillus anthracis spores. This glycoprotein is substituted with an oligosaccharide composed of a beta-L-rhamnoside substituted with the previously unknown terminal saccharide, 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-D-glucopyranose, also referred to as anthrose. Anthrose has not been found in spores of B. cereus and B. thuringiensis, making it a potential species-specific marker for B. anthracis. In order to study the antigenicity of anthrose, efficient syntheses of an anthrose-containing trisaccharide and a series of structurally related analogues were developed. The analogues lacked either the methyl ether at C-2 or contained modified C-4 amino functionalities of anthrose. The synthetic compounds were equipped with an aminopropyl spacer to facilitate conjugation to the carrier proteins mariculture Keyhole Limpet Hemocyanin (mcKLH) and bovine serum albumin (BSA). Serum antibodies of rabbits immunized with live or irradiated spores of B. anthracis Sterne 34F(2) were able to recognize the synthetic trisaccharide-mcKLH conjugate. The specificity of the interaction was confirmed by competitive inhibition with the free- and BSA-conjugated trisaccharides. Inhibition using the trisaccharide analogues demonstrated that the isovaleric acid moiety of anthrose is an important structural motif for antibody recognition. These data demonstrate that 1) anthrose is a specific antigenic determinant of the B. anthracis Sterne spore; 2) this antigen is presented to the immune system of rabbits receiving the anthrax live-spore vaccine; 3) synthetic analogues of the oligosaccharide retain the antigenic structure; and 4) the antigenic region is localized to specific terminal groups of the oligosaccharide. Collectively these data provide an important proof-of-concept step in the synthesis and development of spore-specific reagents for detection and targeting of non-protein structures in B. anthracis.

Inactivation of Bacillus anthracis spores in murine primary macrophages

The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.

Evaluation of the Cepheid GeneXpertR system for detecting Bacillus anthracis

The Cepheid GeneXpert is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert to those run directly on the Cepheid SmartCycler without sample processing. The GeneXpert detection capability was three logs lower than the SmartCycler indicating the benefit of incorporating a nucleic acid extraction procedure. This study demonstrates that the GeneXpert is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. The GeneXpert is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.

Acceleration of epithelial cell syndecan-1 shedding by anthrax hemolytic virulence factors.

BackgroundIt has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice.ResultsThe isolated anthrax hemolytic proteins AnlB (sphingomyelinase) and AnlO (cholesterol-binding pore-forming factor), as well as ClnA (B. cereus homolog of B. anthracis phosphatidyl choline-preferring phospholipase C) cause accelerated shedding of Synd1 and E-cadherin from epithelial cells and compromise epithelial barrier integrity within a few hours. In comparison with hemolysins in a similar range of concentrations, anthrax lethal toxin (LT) also accelerates shedding albeit at slower rate. Individual components of LT, lethal factor and protective antigen are inactive with regard to shedding. Inhibition experiments favor a hypothesis that activities of tested bacterial shedding inducers converge on the stimulation of cytoplasmic tyrosine kinases of the Syk family, ultimately leading to activation of cellular sheddase. Both LT and AnlO modulate ERK1/2 and p38 MAPK signaling pathways, while JNK pathway seems to be irrelevant to accelerated shedding. Accelerated shedding of Synd1 also takes place in DBA/2 mice challenged with Bacillus anthracis (Sterne) spores. Elevated levels of shed ectodomain are readily detectable in circulation after 24 h.ConclusionThe concerted acceleration of shedding by several virulence factors could represent a new pathogenic mechanism contributing to disruption of epithelial or endothelial integrity, hemorrhage, edema and abnormal cell signaling during anthrax infection.

Rapid Chemical Digestion of Small Acid-Soluble Spore Proteins for Analysis of Bacillus Spores

A method for the rapid identification of Bacillus spores is proposed, based on the selective release and chemical digestion of small, acid-soluble spore proteins (SASPs). Microwave-assisted acid hydrolysis of SASPs from B. anthracis str. Sterne and B. subtilis str. 168 was accomplished in a single step requiring only 90 s of heating. The peptide products of the chemical digestion were identified by postsource decay sequencing with a MALDI-TOF-MS equipped with a curved-field reflectron. The specificity of the observed SASP peptides was evaluated using a cross-species sequence search. The incomplete nature of the acid digestion under these conditions allowed detection of the digest products along with the proteins from which they originated, which increased species identification confidence. The feasibility of this approach for the rapid identification of Bacillus species was further demonstrated by analyzing a mixture of B. subtilis str. 168 and B. anthracis str. Sterne spores.

Principal-components analysis of fluorescence cross-section spectra from pathogenic and simulant bacteria

Principal-components analysis of a new set of highly resolved (< 1 nm) fluorescence cross-section spectra excited at 354.7 nm over the 370-646 nm band has been used to demonstrate the potential ability of UV standoff lidars to discriminate among particular biological warfare agents and simulants over short ranges. The remapped spectra produced by this technique from Bacillus globigii (Bg) and Bacillus anthracis (Ba) spores were sufficiently different to allow them to be cleanly separated, and the Ba spectra obtained from Sterne and Ames strain spores were distinguishable. These patterns persisted as the spectral resolution was subsequently degraded in processing from approximately 1 to 34 nm. This is to the author's knowledge the first time that resolved fluorescence spectra from biological warfare agents have been speciated or shown to be distinguishably different from those normally used surrogates by optical spectroscopy.